I know that there is concern in the community about whether what is being published as FRET is really FRET or not. I think we are seeing FRET but want to know where I could be misleading myself. We have expressed the same actin binding domain in cells from two different plasmids. One is a fusion with GFP and one with mRFP-1. The population is not uniform in expression, so that there are cells expressing green only, cells expressing red only and cells expressing both. Examining the cell population with a Leica SP2, we tuned the two channels to acquire green and red signals. The green channel is tuned to see green cells only when the 488 laser is on and under these conditions there is no signal in the red channel for those green only cells. With only the 547 laser on, we see the red only cells and nothing in the green channel. When both lasers are on simultaneously, we see green, red and yellow cells. Now when we image with the 488 laser only and look in the red channel, the cells that were expressing both show a signal that is similar to but not identical to either red or green alone, and the single expressing cells show nothing. As far as I understand things, this must be FRET. Am I missing any controls to verify this? Thanks-Dave --
Dr. David Knecht Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. U-3125 Storrs, CT 06269-3125 knecht-at-uconn.edu 860-486-2200 860-486-4331 (fax) home page: http://www.sp.uconn.edu/~mcbstaff/knecht/knecht.html
Dear Tamara- I am presuming the samples are fixed and sort of infilterated with sucrose-PVP,they are better left at RT. This is before they are cryofixed for sectioning. Shashi
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-- [ From: Garber, Charles A. * EMC.Ver #3.1a ] --
Ling Pan wrote: ============================================================================ = When I do STEM/EDS analyses on my TEM specimens on 200- or 300-mesh Cu grids , I always find Cu peaks present no matter where the beam shoots. Sometimes they can even be more intense than the peaks from my specimen when I am working on PtRu nanoparticles (~ 2 nm in size). Can anyone tell me the cause of the Cu signals? If the EDS spectrum shows signals from Cu grids which is more than a micron away from the beam, how can one be sure about the probe size or spatial resolution then? ============================================================================ = There are several "solutions" to this problem:
1] Use Be grids, see URL www.2spi.com/catalog/grids/beryl.shtml
and eliminate the copper lines. If you are working in an institution where Be is not permitted, we hope to have our diamond (made from diamond) mesh grids available again later this year, after having been temporarily taken out of production.
2] Silicon nitride membrane window TEM grids http://www.2spi.com/catalog/instruments/silicon-nitride.shtml
These grids, especially at the 30 nm thickness level, should be contributing very little to any extraneous secondary fluorescence effects. I won't say zero but the problem will be far less bothersome than with a copper grid, especially if you are doing your work near the center of the window.
Disclaimer: SPI Supplies has been a long time global supplier of both beryllium TEM grids and also, silicon nitride membrane window TEM grids.
Chuck
============================================
Charles A. Garber, Ph. D. Ph: 1-610-436-5400 President 1-800-2424-SPI SPI SUPPLIES FAX: 1-610-436-5755 PO BOX 656 e-mail:cgarber-at-2spi.com West Chester, PA 19381-0656 USA Cust.Service: spi2spi-at-2spi.com
Look for us! ######################## WWW: http://www.2spi.com ######################## ============================================
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-- [ From: Garber, Charles A. * EMC.Ver #3.1a ] --
Chris Blanford wrote: ============================================================================ ====== First, has anyone noticed that the new tabs don't seem to be very conductive? I'm using Agar G3347 12mm conductive carbon tabs.
I've taken a multimeter to a tab on a stub and the resistance is off the scale. If I place a tab between two stubs and press them together as hard as possible, I still have a resistance of several megohms between the two Al stubs. Unfortunately I don't have any of the old, thinner ones to compare.
Second, has anybody else seen cases where the adhesive seems to have penetrated or collapsed a porous structure? We look at polymer films about 10-30 microns thick with holes ~100nm in diameter. On the thinner films especially, the bits of the film firmly in contact with the tabs are always less porous or non-porous than the bits that are suspended above. Could it be that the adhesive is that thick and that it's penetrating our films? ============================================================================ ======= At SPI Supplies, over the years, we have been contacted by customers who have had one problem or another with the use of not only their double sided conductive carbon tabs (we call them discs), but also essentially the same product in the form of either "carbon tape" or "carbon sheets". As a side comment, it has always been somewhat of an amazement to me that so many users of these products seem to forget where they actually did make their purchase, and once we press for further information, it turns out that the product of their frustration did not even necessarily have an SPI Supplies progeny.
But one aspect of these kinds of products (all of them, irrespective of the form, or vendor) is that they do have a finite shelf-life, meaning specifically, that with the passing of time, there is a loss of properties, the most noticeable one being loss of tac. This is also accompanied with a loss of electrical properties (the material becomes more resistive). So if you are interested in extending the shelf life of your stock of such products, they should be stored refrigerated since this will slow down significantly the changes (storing at 10 C deg. lower will slow down the ageing rate by roughly a factor of ten times). A carbon disc that is past its prime, because of the higher resistivity, can even seem to be "bubbling" or experiencing other strange behavior. But this is not a matter of thin vs. thick, but one of age.
As an aside, for some, especially those using these products in UHV systems, the off-gassing characteristics are as important if not more so than the resistivity characteristics. There are indeed differences in the vacuum compatibility between these types of products from different sources. I will avoid making comment about where the SPI Supplies origin products stand in this respect, but only to make the point that we have arrived at a system that is optimized for a number of properties other than just electrical conductivity.
Unfortunately, for the typical user, unless they are making their purchase from the "ultimate source", and they have some reason to believe they are indeed receiving "fresh" material, the important variable of remaining shelf -life is introduced. We have seen instances over the years where a customer purchases material from a stocking distributor but unbeknown to them, the material had sat in that inventory for some number of years. So while it might be a "fresh" purchase so far as the customer is concerned, the material they are actually receiving is far from being fresh. Should the problems someone might be having be with a product of SPI Supplies origin, send us the lot number on the maroon SPI Supplies label and at least as a starting point, we can establish how old or fresh is that particular material.
We have ourselves directly received reports supposedly comparing the so- called thinner material vs. the so-called thicker material. We have not ourselves actually run controlled experiments side by side to compare the thin vs. thick material but only because we have not had any reason to do so . The SPI offered material has always been what one would call the "thick" material, and we are not aware of anyone who has really had problems with the SPI product. Naturally if one was having some kind of problem, we would like to know about it so that we could better understand the cause of such a problem.
But when the material eventually gets into the hands of the final end user, in addition to the potential variation in remaining shelf life, other differences can be introduced such as for the die cut discs (when the die cutting involved too much pressure), some of the silicone release liner can be transferred to the disc, at times so much so that the disc becomes non- conductive (from an SEM perspective) and it could also result in "bubbling". One way to test out if this is indeed a problem you are experiencing, compare the result you get with the disc vs. the result you get with carbon tape (making sure it is of the same material). If the carbon tape "works" but the disc does not, all other factors such as age being the same, then it would be our own prediction that the problem is the result of release liner transferral to the disc during the die cutting.
So my point is, it is clear that this could be much more than just "where it came from" or whether it is "thin" vs. "thick" material. If you have had favorable experience with the carbon tabs from a particular vendor, then you are getting fresh and not stale dated material. If you want to extend the useful lifetime of your carbon adhesive products, store them refrigerated. If you have had a less-than-satisfactory experience with your double sided conductive carbon products, and are looking to make a change, consider the range of products on URL http://www.2spi.com/catalog/spec_prep/cond_adhes.html
If you have a UHV system, consider the 6 mm wide tape product, make expressly to minimize the amount of organics put into the vacuum system.
Disclaimer: SPI Supplies was one of the first in the world to offer double sided conductive carbon products for SEM applications so we would naturally have a vested interest in calling our range of products to everyone's attention.
Chuck
PS: Remember that we are striving to be 100% paperless, therefore there are no paper copies kept of this correspondence. Please be sure to always reply by way of "reply" on your software so that the entire string of correspondence can be kept in one place. ============================================
Charles A. Garber, Ph. D. Ph: 1-610-436-5400 President 1-800-2424-SPI SPI SUPPLIES FAX: 1-610-436-5755 PO BOX 656 e-mail:cgarber-at-2spi.com West Chester, PA 19381-0656 USA Cust.Service: spi2spi-at-2spi.com
Look for us! ######################## WWW: http://www.2spi.com ######################## ============================================
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Dear Sir/Madam,
NB: PLEASE REPLY ONLY TO ( mtakar-at-k.ro )
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My Committee is principally concerned with payment of all contract awarded from 2000 to date, in order of priority as regard capital projects of the NNPC.
We are positive and convince that you would provide us with solution to a money transfer deal valued at US$31 Million (Thirty One Million United States Dollars) and subsequently a joint business venture.
In the course of our duties as values, and project inspectors for the on-going liquefied Natural Gas (LNG) project, we have over-invoiced the value of some jobs done by foreign contractors for the NNPC to the tune of US$31M.
As follows: -
(1) Computer optimization and Installation 16,000.000.00.
(2) Installation of 250,000.00 Monax Turbine$10,000.000.00
(3) Turn Around Maintenance $5,000,000.00
This money is not related to drug or other violent crime. Our aim of over-invoicing this payment is to divert the excess amount to a discrete account abroad. This fund is now floating in suspense account at the Central Bank of Nigeria (CBN). This is the fund my colleagues and I have decided to transfer into your account since we, as civil servants are not allowed to operate or own foreign account.
The money will be shared as follows after transfer, 30% for you (Account Owner) 60% for me and my colleagues 10% to off-set both local and international expenses that would be incurred in the course of this transaction.
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If this proposal satisfies you, please call me immediately on +234-803-3433 283.
This transaction will last for 14 working days from the time we submit the required information, as all modalities concerning this transaction have been worked out and it is completely 100% risk free. Please be informed that this subject is classified sensitive. Therefore treat the transaction with utmost confidentiality and urgency.
NB: PLEASE REPLY ONLY TO ( mtakar-at-k.ro )
Yours Faithfully,
Engr Mamud Takar. Tel: +234-803-3433 283
----------
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.. And don't forget to focus the camera using the lowest power mag on your nosepiece. Then it will automatically be in focus for all other objectives.
Best regards, Barbara Foster Microscopy/Microscopy Education 125 Paridon Street, Suite 102 Springfield, MA 01118 PH: 413-746-6931 FX: 413-746-9311 Web: www.MicroscopyEducation.com
Will you be at M&M in San Antonio? If so, don't forget the Tuesday night seminar on Fluorescence Calibration. Also, join the tradition of over 10,000 microscopists: participate in our survey at any time during the meeting and receive a "sweet thank you". Booth #218 -at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at-
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Abidjan cote d'Ivoire. West Africa.
Dear Sir, I am miss Susan Buma, from Sierra Leone a country in west africa,I am 2oyrs old and a high school student.Please I would like you toassist me urgently as you can before they kill me please.
My father is a wealthy cocoa merchant.When Trouble began early last year my father's business associates start suspecting that my father is not giving proper accounts of all the tons of cocoa being cultivated by local farmers in some villages. As a result of this discovery and the enviness of the money ,he was poisioned by his business associates.At his hospital bed ,he revealed to me the reasons of his present predicament.He directed me where to get the documents of the said deposit, that he made me the next of kin and advice me to move immediately to Abidjan West Africa,where he deposited the money in a local bank.
Being the only child.He advise me that if he did not survival this illness,that I should not stay in the country because his business associates will equally distroy my life hence my mother died five years ago of breast cancer.
He directed me that I should look for a foreign partner who will help me to transfer the money into the person's or companies account and the person will help me to invest the money in their country. After all these,he finally died on 5th of November, my his genle soul rest in perfect peace Amen. And since then I was droped from school (University) and went into hiding for my life in Abidjan, because of my father's business associates. All the relevant documents of the $10.5 (ten million five hundred thousand dollars) that was deposited in the bank are with me now .
I am ready to give you 14% of this fund ,if you can help me transfer this fund into your account overseas for onward investment in your country.
Please contact me immediately with the above E-mail address and include your telephone and Fax number to enable me to forward it to the banker for the immediate transfer of the fund into your normited account, then you make an arrangement to remove me out from this country before they kill me.
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Abidjan cote d'Ivoire. West Africa.
Dear Sir, I am miss Susan Buma, from Sierra Leone a country in west africa,I am 2oyrs old and a high school student.Please I would like you toassist me urgently as you can before they kill me please.
My father is a wealthy cocoa merchant.When Trouble began early last year my father's business associates start suspecting that my father is not giving proper accounts of all the tons of cocoa being cultivated by local farmers in some villages. As a result of this discovery and the enviness of the money ,he was poisioned by his business associates.At his hospital bed ,he revealed to me the reasons of his present predicament.He directed me where to get the documents of the said deposit, that he made me the next of kin and advice me to move immediately to Abidjan West Africa,where he deposited the money in a local bank.
Being the only child.He advise me that if he did not survival this illness,that I should not stay in the country because his business associates will equally distroy my life hence my mother died five years ago of breast cancer.
He directed me that I should look for a foreign partner who will help me to transfer the money into the person's or companies account and the person will help me to invest the money in their country. After all these,he finally died on 5th of November, my his genle soul rest in perfect peace Amen. And since then I was droped from school (University) and went into hiding for my life in Abidjan, because of my father's business associates. All the relevant documents of the $10.5 (ten million five hundred thousand dollars) that was deposited in the bank are with me now .
I am ready to give you 14% of this fund ,if you can help me transfer this fund into your account overseas for onward investment in your country.
Please contact me immediately with the above E-mail address and include your telephone and Fax number to enable me to forward it to the banker for the immediate transfer of the fund into your normited account, then you make an arrangement to remove me out from this country before they kill me.
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Abidjan cote d'Ivoire. West Africa.
Dear Sir, I am miss Susan Buma, from Sierra Leone a country in west africa,I am 2oyrs old and a high school student.Please I would like you toassist me urgently as you can before they kill me please.
My father is a wealthy cocoa merchant.When Trouble began early last year my father's business associates start suspecting that my father is not giving proper accounts of all the tons of cocoa being cultivated by local farmers in some villages. As a result of this discovery and the enviness of the money ,he was poisioned by his business associates.At his hospital bed ,he revealed to me the reasons of his present predicament.He directed me where to get the documents of the said deposit, that he made me the next of kin and advice me to move immediately to Abidjan West Africa,where he deposited the money in a local bank.
Being the only child.He advise me that if he did not survival this illness,that I should not stay in the country because his business associates will equally distroy my life hence my mother died five years ago of breast cancer.
He directed me that I should look for a foreign partner who will help me to transfer the money into the person's or companies account and the person will help me to invest the money in their country. After all these,he finally died on 5th of November, my his genle soul rest in perfect peace Amen. And since then I was droped from school (University) and went into hiding for my life in Abidjan, because of my father's business associates. All the relevant documents of the $10.5 (ten million five hundred thousand dollars) that was deposited in the bank are with me now .
I am ready to give you 14% of this fund ,if you can help me transfer this fund into your account overseas for onward investment in your country.
Please contact me immediately with the above E-mail address and include your telephone and Fax number to enable me to forward it to the banker for the immediate transfer of the fund into your normited account, then you make an arrangement to remove me out from this country before they kill me.
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Abidjan cote d'Ivoire. West Africa.
Dear Sir, I am miss Susan Buma, from Sierra Leone a country in west africa,I am 2oyrs old and a high school student.Please I would like you toassist me urgently as you can before they kill me please.
My father is a wealthy cocoa merchant.When Trouble began early last year my father's business associates start suspecting that my father is not giving proper accounts of all the tons of cocoa being cultivated by local farmers in some villages. As a result of this discovery and the enviness of the money ,he was poisioned by his business associates.At his hospital bed ,he revealed to me the reasons of his present predicament.He directed me where to get the documents of the said deposit, that he made me the next of kin and advice me to move immediately to Abidjan West Africa,where he deposited the money in a local bank.
Being the only child.He advise me that if he did not survival this illness,that I should not stay in the country because his business associates will equally distroy my life hence my mother died five years ago of breast cancer.
He directed me that I should look for a foreign partner who will help me to transfer the money into the person's or companies account and the person will help me to invest the money in their country. After all these,he finally died on 5th of November, my his genle soul rest in perfect peace Amen. And since then I was droped from school (University) and went into hiding for my life in Abidjan, because of my father's business associates. All the relevant documents of the $10.5 (ten million five hundred thousand dollars) that was deposited in the bank are with me now .
I am ready to give you 14% of this fund ,if you can help me transfer this fund into your account overseas for onward investment in your country.
Please contact me immediately with the above E-mail address and include your telephone and Fax number to enable me to forward it to the banker for the immediate transfer of the fund into your normited account, then you make an arrangement to remove me out from this country before they kill me.
by sparc5.microscopy.com (8.9.3+Sun/8.9.3) id IAA05960 for dist-Microscopy; Mon, 2 Jun 2003 08:36:57 -0500 (CDT) Received: from njz_spm_filter (sparc5 [206.69.208.10]) by sparc5.microscopy.com (8.9.3+Sun/8.9.3) with SMTP id IAA05918 for "MicroscopyFilteredEmail3-at-msa.microscopy.com"; Mon, 2 Jun 2003 08:36:26 -0500 (CDT) Received: from inbox.net (aaron.visual.com [209.123.16.34]) by sparc5.microscopy.com (8.9.3+Sun/8.9.3) with SMTP id IAA05911 for {Microscopy-at-sparc5.Microscopy.Com} ; Mon, 2 Jun 2003 08:36:13 -0500 (CDT) Received: (qmail 20425 invoked by uid 99); 2 Jun 2003 12:28:37 -0000
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Abidjan cote d'Ivoire. West Africa.
Dear Sir, I am miss Susan Buma, from Sierra Leone a country in west africa,I am 2oyrs old and a high school student.Please I would like you toassist me urgently as you can before they kill me please.
My father is a wealthy cocoa merchant.When Trouble began early last year my father's business associates start suspecting that my father is not giving proper accounts of all the tons of cocoa being cultivated by local farmers in some villages. As a result of this discovery and the enviness of the money ,he was poisioned by his business associates.At his hospital bed ,he revealed to me the reasons of his present predicament.He directed me where to get the documents of the said deposit, that he made me the next of kin and advice me to move immediately to Abidjan West Africa,where he deposited the money in a local bank.
Being the only child.He advise me that if he did not survival this illness,that I should not stay in the country because his business associates will equally distroy my life hence my mother died five years ago of breast cancer.
He directed me that I should look for a foreign partner who will help me to transfer the money into the person's or companies account and the person will help me to invest the money in their country. After all these,he finally died on 5th of November, my his genle soul rest in perfect peace Amen. And since then I was droped from school (University) and went into hiding for my life in Abidjan, because of my father's business associates. All the relevant documents of the $10.5 (ten million five hundred thousand dollars) that was deposited in the bank are with me now .
I am ready to give you 14% of this fund ,if you can help me transfer this fund into your account overseas for onward investment in your country.
Please contact me immediately with the above E-mail address and include your telephone and Fax number to enable me to forward it to the banker for the immediate transfer of the fund into your normited account, then you make an arrangement to remove me out from this country before they kill me.
by sparc5.microscopy.com (8.9.3+Sun/8.9.3) id IAA06007 for dist-Microscopy; Mon, 2 Jun 2003 08:40:38 -0500 (CDT) Received: from njz_spm_filter (sparc5 [206.69.208.10]) by sparc5.microscopy.com (8.9.3+Sun/8.9.3) with SMTP id IAA06000 for "MicroscopyFilteredEmail3-at-msa.microscopy.com"; Mon, 2 Jun 2003 08:40:07 -0500 (CDT) by sparc5.microscopy.com (8.9.3+Sun/8.9.3) with ESMTP id IAA05993 for {microscopy-at-sparc5.microscopy.com} ; Mon, 2 Jun 2003 08:39:54 -0500 (CDT) Received: from [146.139.72.105] (aem105.amc.anl.gov [146.139.72.105]) by aaem.amc.anl.gov (8.12.8/8.12.2) with ESMTP id h52DULx7006326 for {microscopy-at-msa.microscopy.com} ; Mon, 2 Jun 2003 08:30:22 -0500 (CDT) Mime-Version: 1.0 Message-Id: {p05100302bb010259d477-at-[146.139.72.105]}
Colleagues
Yes I see the spam attack. I'm trying to mitigate it and find the "hole" in the filters.
Sure, now conductivity of the tabs could be very poor. On the tabs of one of the suppliers I even see charging artifacts (rectangle scanning area is visible after lowering of the magnification). And in some of the tabs I have found a lot of sulfur.
Vladimir Dusevich
} First, has anyone noticed that the new tabs don't seem to be very } conductive? I'm using Agar G3347 12mm conductive carbon tabs. } } I've taken a multimeter to a tab on a stub and the resistance is off } the scale. If I place a tab between two stubs and press them together } as hard as possible, I still have a resistance of several megohms } between the two Al stubs. Unfortunately I don't have any of the old, } thinner ones to compare. } } Second, has anybody else seen cases where the adhesive seems to have } penetrated or collapsed a porous structure? We look at polymer films } about 10-30 microns thick with holes ~100nm in diameter. On } the thinner } films especially, the bits of the film firmly in contact with } the tabs } are always less porous or non-porous than the bits that are suspended } above. Could it be that the adhesive is that thick and that it's } penetrating our films? } } Looking forward to hearing any insights. } } Regards, } } Chris Blanford } } }
I am Mr. Raphael Maloney and my sister is Miss Sofie Maloney, we are the children of late Chief John Maloney from Sierra Leone. I am writing you in absolute confidence primarily to seek your assistance to transfer our cash of eleven Million Dollars ($11,000.000.00) now in the custody of a private Security trust firm in Europe the money is in trunk box deposited and declared as family valuables by my late father as a matter of fact the company does not know the content as money, although my father made them to under stand that the box belongs to his foreign partner.
Source of the money:
My late father Chief John Maloney , a native of Makeni District in the Northerh province of Sierra Leone, was the General Manager of Sierra Leone Mining co-operation (S.L.M.C.) Freetown . According to my father, this money was the income accrued from Mining Co-operation's over draft and minor sales.
Before the peak of the civil war between the rebels forces of Major Paul Koroma and the combined forces of ECOMOG peace keeping operation that almost
destroyed my country, following the forceful removal from power of the civilian elected President Ahmed Tejan Kabbah by the rebels. My father had already made arrangement for his family thats talking about my mother, my little sister and myself to be evacuated to Cotonou Republic of Benin with the CERTIFICATE OFDEPOSIT he made with a security firm in Europe through the aid
of U.N evacuation team.
During the war in my country, and following the indiscriminate looting of Public and Government properties by the rebel forces, the Sierra Leone mining coop. Was one of the target looted and it was destroyed. My father including
other top Government functionaries were attaked and killed by the rebels in November 2000 because of his relationship with the civilian Government of Ahmed Tejan Kabbah.
As a result of my father's death , and with the news of my uncle's involvement in the air crash in January it dashed our hope of survival. The untimely deaths caused my mother's heart failure and other related complications of which she later died in the hospital after we must have spent a lot of money on her early this year . Now my 18 years old sister and myself are alone in this strange country suffering without any care or help. Without any relation, we are nowlike refugees and orphans.
Our only hope now is in you and the box deposited in the Security Firm To this effect, I humbly solicit your assistance in the followings ways.
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For your assistance, I have agreed with my younger sister that 15% of the total amount will be for your effort and another 5 % to cover all the expenses that may incur during the business transaction, Last, I urge you to
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Please as you show your willingness, Forward to us your full name, address and Tel/ Fax numbers, to me.
earnestly awaiting your response.
TO: raphmalon-at-slucia.com (MAY REPLY HERE)
Thanks.
May God bless you as you assist us.
Mr. RAPHAEL MALONEY.
Welcome to Sudan pioneer ISP http://www.sudanet.net
It is well known that copper grids will give a background of Cu during EDX measurements.
As Walck said, you can use Be grids (best but $$) but I use Ti grids to eliminate this Cu interference peak. I run Cu grids and if my customer or I suspect we have copper in the sample, then I also make up a Ti grid. It helps to keep some Ni grid handy too. Use whatever grid does not show a Ti or Ni peak with the Cu grid spectra. Ti has very few interferences. Be grids will show nothing by EDX.
You should always use a Be specimen holder to do EDX. I have a Philips CM 12 and the regular holder has a higher ridge on it than the Be analytical holder right at the grid edge. I use top hat apertures too, FYI. If I use the regular holder, then I get a Cu peak with a slight Zn peak. This is coming from the holder because it is close to the beam position. This has nothing to do with your beam spot size within the grid opening unless you are right near the grid bar. More on that later.
To address your concerns about higher Cu peaks than the sample, this does happen. I do a lot of thin sections of polymer coatings and different thickness' produce higher Cu peaks. Now I do not see the Cu peak when I use my Ti or Ni grids. Otherwise if I did, everything I run would have 'scope Copper' in it. If the Cu is coming from the scope, then Be grids would also show Cu. Where's the iron peak from the scope's pole pieces? I do not think the Cu is coming from the scope for these reasons. The Cu peaks can be higher for another reason. If the amount of carbon or polymer is high in the sample and somewhat lower in other elements, a higher Cu peak is observed. On the other hand, some ink coatings and ink jet pigment coatings I run that have a high pigment loadings, have higher 'metals' peaks than the copper peak.
In my opinion, the height of the Cu peak can be used to tell you that you should SUSPECT the material you are examining is highly loaded with organic. If I run a sample with a high Cu peak and nothing else shows up, then I conclude I have high organics, Be, Li, B or the geometry in the TEM is a problem. Philips has a lower EDX detector take off angle and geometry can be a problem.
So how is this Cu peak generated. From MY experience I have observed that it is caused by electrons from the sample being scattered around the holder. Take a pure formvar-carbon coated grid and run an EDX on it. The Cu peak should show up and it should be noisy. Then run a pure organic thin section and notice how the Cu peak signal to noise changes. You can also put more carbon on the formvar grid with an high vacuum old style evaporator to make a thicker carbon film. Use the formula in Electron Microscopy by John Bozzola on page 125 to determine the thickness you have added of rod. Instead of his density of materials (cm2*atomic thickness), use the regular density in a CRC handbook for bulk density. Correct his formula for this bulk density by multiplying John's formula by 10^(-8). I would use about 8 mgs minimum of carbon rod at a 10 CM height.
Carbon Mass = (4*Pi*distance2*thickness*rho)*(10^-8)/(sin beta) The units are grams, cm, A, gm/cm3, degrees; respectively. This can be derived by the integration of the surface area of revolution of a thin film of an atoms at distance, d, from a point source. For overhead evaporations, sin beta is 1.
You will see that the signal is proportional to the thickness of the carbon. Now this suggests to ME that the electrons impinge on your sample and are sprayed all around the areas of the holder. The thicker the thin film, the more electrons are scattered due to the increased path the electrons must travel. Some electron pass right through and hit your viewing screen. Right? Back scattered ones bounce right back up from the sample to your BS detector. Now there must be situations where the intermediate scattering exists and electrons do not miss the atoms or hit them dead center. These are the electrons that are scattered off to the sides of the sample and hit the holder and the grid material. They generate Cu, Ti, Ni or Be grid characteristic X-rays. These are the electron scatterings and interactions that are causing the metals to be seen in your spectra except Be will not show up.
Don't forget to remove your objective aperture during spectral collection. If you don't, the transmitted electrons will 'back scatter' and Pt will show up from the aperture material.
I hope my humble 'observations of the phenomena' help you understand what is going on here. You probably understand a lot of this but I wanted to make sure I covered most of what I have seen, just in case.
Paul Beauregard Senior Research Associate PPG Industries Monroeville Technical Center 440 College Park Drive Monroeville, PA 15146 800-446-3382 ext 5131
-----Original Message----- } From: LING PAN [mailto:lup2-at-psu.edu] Sent: Friday, May 30, 2003 5:09 PM To: Microscopy-at-sparc5.microscopy.com
When I do STEM/EDS analyses on my TEM specimens on 200- or 300-mesh Cu grids, I always find Cu peaks present no matter where the beam shoots. Sometimes they can even be more intense than the peaks from my specimen when I am working on PtRu nanoparticles (~ 2 nm in size). Can anyone tell me the cause of the Cu signals? If the EDS spectrum shows signals from Cu grids which is more than a micron away from the beam, how can one be sure about the probe size or spatial resolution then?
Thanks in advance for any comments.
Ling
------------------- Ling Pan, Ph.D. 248 MRL Building Materials Research Institute The Pennsylvania State University University Park, PA 16802 Tel: (814)865-6115 Fax: (814)865-2326
i'm having a bit of a problem with your question. it sounds to me more like you are discussing co-localization than FRET. while my knowledge of FRET is based on the molecular side of my life, i have discussed this with our technician who runs the confocal lab and one of the post-docs in his lab group. they concur.
the key is that you are co-expressing chimeric proteins, GFP and mRFP-1, which should fluoresce naturally when they are excited with the proper wavelength. as soon as they are excited with the proper wavelengths they will glow. they are not quenched. the first problem here is based upon the fact that they will fluoresce regardless of proximity to each other.
the second problem is that no matter how tightly you try to cut your channels, the signal given off at each point in the spectrum represents standard curves. they look like modified bell curves. as a result, there is, and always will be a small area of overlap between two different channels. if you excite for one and amplify the signal a lot you will see signal in this area in the other channel. in short, when you excite for green and look at red, you will get something in red that does not really look like green or red. if this is what is happening, you do not have FRET,
unless things have been defined differently in the con-focal area, and we are not aware of such a difference, the major point about FRET is Resonant Energy Transfer. you have something that does not fluoresce when you excite it. the label on the molecule being used to signal what you are looking for is marked with an inactive moiety. no matter how you look at it you will see nothing. your second marker molecule contains an activator. when the activator binds in close enough proximity you will see a transfer of resonant energy. the demonstration of this will be fluorescence. if there is no binding, or if they are too far apart, you will not see fluorescence. in molecular detection, binding must be within1-5 nucleotides, distance depending on with whom you speak.
your controls here are problematic. the negative control is simple. you put in your first and second detectors separately and see if either fluoresces. if either or both do, something is wrong, it is not fluorescent resonant energy transfer. the positive control requires your attachment of the detector in close enough proximity. how you design that in an experiment where you are looking at the expression of fluorescent marker chimeric proteins eludes me totally.
I'm happy to announce that the Project MICRO (Microscopy In Curriculum - Research Outreach) web page has been thoroughly revised; the URL is below. The text has been updated, and the reviewed bibliography of books, videos, CD-ROMs, and websites has been converted to a searchable database (THANK YOU, Nestor!). There's been a major increase in the number of CDs listed. If you know about missing items (nothing that's "out of print", please) contact me.
Are you making plans to attend M&M in San Antonio? Please note that there will be a MICRO workshop on Sunday afternoon. It's a unique opportunity; all 10 of the "Microscopic Explorations" learning stations will be set up, and the leader will be a Texas "GEMS Associate", an experienced teacher-trainer. Although the MICRO workshop is grouped with several others that are intended for Spanish-speaking attendees, it will be presented in English and everyone will be welcome. If you're interested, please help us plan by signing up for SC-11 (no charge) on your meeting reg form. Questions? Contact me directly. -- Caroline Schooley Project MICRO Coordinator Microscopy Society of America Box 117, 45301 Caspar Point Road Caspar, CA 95420 Phone/FAX (707)964-9460 Project MICRO: http://www.msa.microscopy.com/ProjectMicro/PMHomePage.html Intertidal invertebrates: http://www.fortbragg.k12.ca.us/AG/marinelab.html
On Friday, May 30, 2003, at 02:08 PM, LING PAN wrote:
} When I do STEM/EDS analyses on my TEM specimens on 200- or 300-mesh Cu } grids, I } always find Cu peaks present no matter where the beam shoots. } Sometimes they } can even be more intense than the peaks from my specimen when I am } working on } PtRu nanoparticles (~ 2 nm in size). Can anyone tell me the cause of } the Cu } signals? If the EDS spectrum shows signals from Cu grids which is more } than a } micron away from the beam, how can one be sure about the probe size or } spatial } resolution then? } } Thanks in advance for any comments. } Dear Ling, There are several possible sources for these peaks. 1) You could have either electrons or x-rays coming down the column outside the beam, arising from scattering from apertures, etc. 2) Electrons could be back-scattered from the lower objective pole piece. 3) Electrons scattered from your specimen could hit the grid bars. If the peaks are constant regardless of how far you are from a grid bar, you are likely dealing with either of the first two sources, and if the peaks are related to the position on the grid, the third source is significant. If you put a bare Cu grid in the scope, focus the beam on the middle of a grid square, and take a spectrum, do you get any peaks--especially a Cu peak? If so, you need to improve the shielding in the microscope. You can take a spectrum from one of your particles, then take a series of spectra from points displaced from the particle by various amounts. This will give you a background that you can subtract for quantitation, but that is less satisfactory than eliminating the spurious peaks. Yours, Bill Tivol EM Scientist and Manager Cryo-Electron Microscopy Facility Broad Center, Mail Code 114-96 California Institute of Technology Pasadena CA 91125 (626) 395-8833 tivol-at-caltech.edu
On Friday, May 30, 2003, at 05:17 PM, William P. Sharp wrote:
} We have been discussing the cryogens normally used for plunge freezing } - things like propane and ethane and (formerly) Freons of various } stripe. All have some rather significant downside, like potential } environmental damage with Freons and the potentially explosive } condensation of liquid oxygen in the hydrocarbons. This makes a rather } routine and quite useful protocol fraught with drama, especially when } it must be done in underground labs. } } Do any of you materials folks out there know of a compound or class of } compounds that have a rather large temperature spread between the } freezing and boiling points and which remain liquid at or near liquid } nitrogen temperatures (-196C) and which are also essentially inert } with regard to oxidation potential and which (as long as I'm dreaming) } have no real effect on human health? We were thinking that probably a } whole bunch of stuff has been synthesized in the last 20 or so years } that fit the bill (that is, since the pioneers of cryofixation did } their initial search for likely fluids), but that the people who know } about these compounds don't know why we would want to use them, and } those of us who need new, safe cryogens don't really know about the } possibility of these new substances. } Dear William, I can set your mind at ease on at least one thing. Since the freezing point of ethane is given as -183 C in the Handbook of Chemistry and Physics, and the boiling point of oxygen is given as -182.9 C in the same source, it is not a real concern that oxygen will condense in liquid ethane, especially since one always uses warm ethane gas to melt solid ethane during freezing. There may be some real danger of ethane exploding--our safety office was very concerned--but I have never heard of an actual occurrence of this. As far as other substances with low freezing points (and, of course, good thermal conductivity), in general, the larger the molecule, the higher the freezing point, so suitable cryogens may be restricted to small molecules, which can be rather easily enumerated. This last comment is strictly a guess on my part, but I think it unlikely that any suitable substance has been synthesized only in the last few years. Yours, Bill Tivol EM Scientist and Manager Cryo-Electron Microscopy Facility Broad Center, Mail Code 114-96 California Institute of Technology Pasadena CA 91125 (626) 395-8833 tivol-at-caltech.edu
Can anyone help me? I need to purchase a filter for viewing fluorescence but I don't know which is appropriate. I have a filter that transmits light to the eyepiece of our Wild-Heerbrugg M20 through which I am able to view both green and red fluorescence but I want to take digital images via the phototube so I need a filter for that too. Unfortunately I have no idea what type of filter it is. If possible, I would like to use two fluorophores simultaneously: FITC (ex495/em520 and FM 4-64 (ex560/em730). What should I buy?
Many thanks,
Patricia ---------------------- Patricia de Winter cdewi02-at-students.bbk.ac.uk
On Friday, May 30, 2003, at 05:17 PM, William P. Sharp wrote:
} We have been discussing the cryogens normally used for plunge } freezing - things like propane and ethane and (formerly) Freons of } various stripe. All have some rather significant downside, like } potential environmental damage with Freons and the potentially } explosive condensation of liquid oxygen in the hydrocarbons. This } makes a rather routine and quite useful protocol fraught with drama, } especially when it must be done in underground labs. } Do any of you materials folks out there know of a compound or class of compounds that have a rather large temperature spread between the freezing and boiling points and which remain liquid at or near liquid nitrogen temperatures (-196C) and which are also essentially inert with regard to oxidation potential and which (as long as I'm dreaming) have no real effect on human health? We were thinking that probably a whole bunch of stuff has been synthesized in the last 20 or so years that fit the bill (that is, since the pioneers of cryofixation did their initial search for likely fluids), but that the people who know about these compounds don't know why we would want to use them, and those of us who need new, safe cryogens don't really know about the possibility of these new substances.
We have long used a slurry of isopentane as a quenchant to produce amorphous samples of readily crystallizable polymers. The Tm is 113 K, so oxygen condensation should not be a problem and we found it gave a " better" quench than the refrigerant UCON-12 (Tm = 118) for material like linear polyethylene. We originally replaced the UCON with a slurry of nitrogen, but had to use liquid He or pumping to partially freeze it and the isopentane was simpler since it can be partially frozen in liquid N2. Refs. include Polymer, 20, 903 (1979), J. Macromol. Sci., Phys. B20, 37 (1981) and ibid, B21, 617 (1982) --
Phillip H. Geil; Ph. 217-333-0149 Fax 217-333-2736 Department of Materials Science and Engineering University of Illinois 1304 W. Green St. Urbana, IL 61801
I also use explosive liquid cryogens (usually propane) for freezing and would be interested in whether anyone else knows of cases of explosions occuring during use in this way. The two (anecdotal) cases of explosions I have heard were
1) an explosion when someone broke a light in the hood while using propane or ethane and
2) an explosion in a condensing coil being used to condense gaseous propane. The explanation of the latter was that oxygen condensed in the cold coil after the propane flow was turned off. I now condense the propane directly into a cold vessel set down in a styrofoam container of liquid nitrogen, which should keep the environment around the liquid propane purged of air. I thaw the propane when it freezes either with liquid propane or with a warm metal rod.
Any ideas about this? I am wondering whether there are other precautions.
Marie
Dr. Marie E. Cantino Director, Electron Microscopy Laboratory Associate Professor of Physiology and Neurobiology University of Connecticut Unit 3242 Storrs, CT 06269-3242 Phone: 860-486-3588 Fax: 860-486-6369
I think static electricity may be a real problem for liquid propane/ethane. Therefore, it's safer to use metal container and needles instead glass or plastic. Container itself should not be a big problem because of water condensation but plastic tubings. If dry propane/ethane travel through plastic tubing it may be enough to generate static electricity and therefore spark. Sergey
At 12:42 PM 6/2/2003, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
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Sergey Ryazantsev Ph. D. Electron Microscopy UCLA School of Medicine Department of Biological Chemistry 10833 Le Conte Ave, Room 33-089 Los Angeles, CA 90095
I'm looking for a couple of 10 litre dewars for liquid nitrogen and wondered if anyone could recommend a supplier? Alternatively if anyone in New Zealand has any second hand ones for sale I'd be interested.
Electron Microscope Facility School of Science and Technology The University of Waikato Ruakura Satellite Campus (RS5) Ruakura Road Hamilton New Zealand
To whom it may concern. If somebody has got a diagram for the Spectrum Monitor and related diagrams, could you please fax it to me. Thank you very much. Its urgent. Erik Sorbroden, Norway Center for materials Science fax +47 22 84 06 51
I would assume that you are correct, the issue is the length of your trinocular adapter.
Take a look at our webpage which lists solutions for adapting to the cameras to all the major microscope manufacturers as well as the 'off-brands' as well via a couple of mounting options: http://www.mvia.com/Coolpix/clpxadpt.htm
Our solutions are probably less expensive than other options you have looked at. Please feel free to contact me with your microscope model if you would like to go over the various options.
Thanks! Jim Haley
****************************** Jim Haley Applications Engineer MVIA, Inc. 125 Sherwood Drive Monaca, PA 15061 voice: (724) 728-7493 fax: (412) 291-1709 e-mail: haley-at-mvia.com webpage: http://www.mvia.com ******************************
michael shaffer wrote: } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } We have adapted a Nikon Coolpix 5000 to our microscope with trinocular } head. The adaptation is not specific to the microscope, and we are having a } problem with focus. That is, we can elevate the stage for vision's focus, } or we can elevate the stage for the camera's focus ... but neither are } coincident ... and we imagine this is because the adaptation is not specific } to the microscope (the adapter being quite expensive for this particular } brand & model). Judging from playing with the adapter, its tube-length is } slightly too long. } } I suppose we were hoping the camera could be manually focussed, so that } both could be made coincident, but for the Coolpix we cannot find a way of } manually focussing. Or, it may be impossible to focus because the adapter's } tube-length is too long rather than too short. Is this the correct } assumption? What have others done? } } cheerios ... shAf :o) } Avalon Peninsula, Newfoundland } www.micro-investigations.com
Thanks a lot for your information. I did collect "in-hole" spectra together with spectra on specimen. Actually I collected spectra both in complete hole area and on amorphous C film. The spectrum in complete hole area showed no peak at all, and the one collected on C film showed Cu peaks besides C peak. So the Cu peaks must be coming from the Cu grid instead of the column. (The double-tilt holder that I used was Be one.)
I did subtract the "in-hole" spectra from all other spectra. The Cu peaks were just a little weaker. I think mostly the heavy metals in my specimen accounted for that.
What I am thinking is, if the electrons scattering from the specimen could hit the Cu grid or whatever other grid, then they could hit other areas of the specimen too. Then how can one be sure the spectrum (except the Cu peaks) was really just the information of the beam-shooting area?
Sincerely,
Ling
On Tue, 03 Jun 2003 14:41:02, "CorneliuSarbu" wrote:
} } Dear colleague, } } yesterday I sent you a message about the "in-hole" X-EDS spectrum } acquisition. The bibliographical citation was not complete. Here is } the complete one: } Brent Fultz and James Howe, "Transmission Electron Microscopy } and Diffractometry of Materials", Springer-Verlag, Berlin and Heidelberg, } 2001. See page 209. } } Corneliu Sarbu, PhD } } } -----Original Message----- } From: LING PAN {lup2-at-psu.edu} } To: Microscopy-at-sparc5.microscopy.com {Microscopy-at-sparc5.microscopy.com} } Date: Saturday, May 31, 2003 1:09 AM } Subject: Cu peaks in STEM/EDS } } } } ------------------------------------------------------------------------ } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- } http://www.msa.microscopy.com/MicroscopyListserver } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } -----------------------------------------------------------------------. } } } } } } When I do STEM/EDS analyses on my TEM specimens on 200- or 300-mesh Cu } grids, I } } always find Cu peaks present no matter where the beam shoots. Sometimes } they } } can even be more intense than the peaks from my specimen when I am working } on } } PtRu nanoparticles (~ 2 nm in size). Can anyone tell me the cause of the Cu } } signals? If the EDS spectrum shows signals from Cu grids which is more than } a } } micron away from the beam, how can one be sure about the probe size or } spatial } } resolution then? } } } } Thanks in advance for any comments. } } } } Ling } } } } ------------------- } } Ling Pan, Ph.D. } } 248 MRL Building } } Materials Research Institute } } The Pennsylvania State University } } University Park, PA 16802 } } Tel: (814)865-6115 } } Fax: (814)865-2326 } } } } }
One of the listers wrote me off line, asking why the lowest mag rather than highest mag objective. I'm sure that many of you have the same question.
Here's the story: This is one of those weird idiosyncracies of microscopy imaging.
There are two planes of focus which are of interest: a. The depth in the specimen (properly called "depth of field") and b. The depth in the image - at the camera or detector (properly called "depth of focus")
The depth of field is directly related to the numerical aperture of the system. However, the depth of focus is directly related to the magnification(squared). Sooo... For a low power (ex: 5x scanner lens): 5(squared) = an image which is 25 "units" deep at the camera plane For a higher magnification (ex: 100x): 100(squared) = an image which is 10,000 "units" deep at the camera plane. Since "25 unit" slice is more shallow, it defines the depth of focus at the camera more precisely. That plane will always fall within the "10,000 unit" slice, so once the camera is "parfocal" (in focus for a plane matching the plane for the eyepieces) for your lowest magnification objective, it will parfocal for all other objectives.
Hope this is helpful.
Best regards, Barbara
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. And don't forget to focus the camera using the lowest power mag on your nosepiece. Then it will automatically be in focus for all other objectives.
Best regards, Barbara Foster Microscopy/Microscopy Education 125 Paridon Street, Suite 102 Springfield, MA 01118 PH: 413-746-6931 FX: 413-746-9311 Web: www.MicroscopyEducation.com
Will you be at M&M in San Antonio? If so, don't forget the Tuesday night seminar on Fluorescence Calibration. Also, join the tradition of over 10,000 microscopists: participate in our survey at any time during the meeting and receive a "sweet thank you". Booth #218 -at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at-
At 08:32 PM 5/30/03 -0700, David Burton wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
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-- [ From: Garber, Charles A. * EMC.Ver #3.1a ] --
Helen M Turner wrote: ======================================================= I'm looking for a couple of 10 litre dewars for liquid nitrogen and wondered if anyone could recommend a supplier? Alternatively if anyone in New Zealand has any second hand ones for sale I'd be interested. ======================================================= An excellent 10 liter dewar is shown on URL http://www.2spi.com/catalog/instruments/liquid-nitrogen-dewars.shtml of the SPI Supplies website. It can be purchased directly from SPI Supplies in the USA or via our distributor in New Zealand:
Charles A. Garber, Ph. D. Ph: 1-610-436-5400 President 1-800-2424-SPI SPI SUPPLIES FAX: 1-610-436-5755 PO BOX 656 e-mail:cgarber-at-2spi.com West Chester, PA 19381-0656 USA Cust.Service: spi2spi-at-2spi.com
Look for us! ######################## WWW: http://www.2spi.com ######################## ============================================
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Michael,
I would assume that you are correct, the issue is the length of your trinocular adapter.
Take a look at our webpage which lists solutions for adapting to the cameras to all the major microscope manufacturers as well as the 'off-brands' as well via a couple of mounting options: http://www.mvia.com/Coolpix/clpxadpt.htm
Our solutions are probably less expensive than other options you have looked at. Please feel free to contact me with your microscope model if you would like to go over the various options.
Thanks! Jim Haley
****************************** Jim Haley Applications Engineer MVIA, Inc. 125 Sherwood Drive Monaca, PA 15061 voice: (724) 728-7493 fax: (412) 291-1709 e-mail: haley-at-mvia.com webpage: http://www.mvia.com ******************************
michael shaffer wrote: } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } We have adapted a Nikon Coolpix 5000 to our microscope with trinocular } head. The adaptation is not specific to the microscope, and we are having a } problem with focus. That is, we can elevate the stage for vision's focus, } or we can elevate the stage for the camera's focus ... but neither are } coincident ... and we imagine this is because the adaptation is not specific } to the microscope (the adapter being quite expensive for this particular } brand & model). Judging from playing with the adapter, its tube-length is } slightly too long. } } I suppose we were hoping the camera could be manually focussed, so that } both could be made coincident, but for the Coolpix we cannot find a way of } manually focussing. Or, it may be impossible to focus because the adapter's } tube-length is too long rather than too short. Is this the correct } assumption? What have others done? } } cheerios ... shAf :o) } Avalon Peninsula, Newfoundland } www.micro-investigations.com
I need to do some experiments using a FEG SEM. I am using infected cells that I am planing to immunolabel with antibodies coupled to colloidal gold.
My questions are : is immunolabelling carried out in the same way as for labelling on grids ? critical point drying or not ? metallisation or carbon coating ? All answers will be helpfull, thanks in advance danile
Here's an update on the Kodak MDS 100 digital camera ...
Works pretty well on the Olympus SZH stereomicroscope. I had no focusing problems 'cuz I also got the correct Olympus C-mount camera adapter, the TVZ-M. It's got corrections for image distance (0.55 to 1.1) and fine focus. The fine focus is a boon 'cuz the SZH only has rack-and-pinion focus.
Color I dealt with by adding a heat-absorbing glass in front of the fiber-optic stalk(s) and with an IR cutoff filter in front of the CCD element. Both from Edmund Industrial Optics. Gets the red mostly out.
Resolution isn't quite what it seems to be - the camera will record images at 1280X960, but in the back of the instruction book the image sensor is only 640X480 pixels. And one can see an odd waffle pattern within uniformly colored areas sometimes. Looks like the image is being projected onto the back side of the sheet of Masonite hardboard, which is a product made on metal wire screens. These effects disappear when the image is presented at a reasonable size - say, 3 by 4 inches.
One aspect of the less-than-optimum resolution is that the effective magnification is boosted approximately 2.5 times by the small size of the image sensor compared to the coverage of the eyepiece lens. That stretches the resolving power of the stereomicroscope's objective lens.
Color corrects OK, once I read the instructions. Still, upon going to maximum magnification with the microscope, the image becomes very blue, probably because there's a color shift with light level, which of course goes down at high magnifications.
Installation is a breeze, as the camera gets its power and input/ouput from the USB port of the PC.
The software works well; and the images can be nicely edited with a full range of gamma, brightness, contrast, tint, RGB, etc. One can also annotate the image in the margins with the Kodak software. That annotation vanishes if one later edits with other software, of course. One "trick" to enhance performance is to let the camera work by itself without competition from other programs like MSWord, Netscape, and the like, which starve it of memory. However, I can leave a large number of images open during any one session without crashing the program. Haven't lost one yet.
For a total expenditure of ~$500 it's a good value and runs circles around my previous method of hand-holding a Sony 1.3 megapixel camera over the eyepiece of the stereomicroscope. Seventy-five images in a two-hour session, for example. I can still take Polaroids if all else fails, but that's really tedious now that I've been to the Big City.
Best regards, George Langford, Sc.D. Amenex Associates, Inc. amenex-at-amenex.com http://www.amenex.com/
Daniele Immunolabelling is the same as for grids. Gold sizes from ~5nm are suitable. Double labelling works fine, but don't overdo the size ratios. Mass increases with cube of diameter, so 25nm gold is 125 as massive as 5nm, and the supernova-like glare of the glare particles can obscure the smaller ones. Adjacent pairs in the table below should give sufficient size differential. Critical point drying is required. Coating is required either with carbon or chromium - you will need to experiment with coating thicknesses to suit your specimens and microscope parameters, but in general you will find optimised Cr coatings are thinner than C, give richer ultrastructural detail and excellent BSE signal. Noble metal coatings (Au, Au/Pd, Pt) are not suitable. Hope this helps Chris
C.E. Jeffree, H.W. McL. Rixon, G. Brown, J. Aitken, and R.J. Sugrue Distribution of the attachment (G) glycoprotein and GM1 within the envelope of mature respiratory syncytial virus filaments revealed using field emission scanning electron microscopy VIROLOGY 306 (2): 254-267 FEB 15 2003
I need to do some experiments using a FEG SEM. I am using infected cells that I am planing to immunolabel with antibodies coupled to colloidal gold.
My questions are : is immunolabelling carried out in the same way as for labelling on grids ? critical point drying or not ? metallisation or carbon coating ? All answers will be helpfull, thanks in advance danile
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Danile Spehner wrote: ============================================================================ = I need to do some experiments using a FEG SEM. I am using infected cells that I am planing to immunolabel with antibodies coupled to colloidal gold.
My questions are : is immunolabelling carried out in the same way as for labelling on grids ? critical point drying or not ? metallisation or carbon coating ? All answers will be helpfull, thanks in advance danile ============================================================================ == So far as I have always heard, it is done pretty much the way you would for labelling on grids. The sample is critical point dried.
When people talk about "metallization", they generally are talking about gold but you really can't coat with gold and expect to see with BSE the image of the gold distribution. But when one carbon coats, which has been the other alternative, you lose the sense of depth in the sample.
But now it has been shown that a thin layer of plasma deposited (from the vapor) osmium metal, in an extremely thin layer, such as can be done in the osmium plasma coaters gives you the best of all worlds: You have the needed conductivity, you get the sense of depth, while at the same time, you are not diminishing the signal from the surface tagged gold particles. This kind of deposition equipment is described on URL http://www.2spi.com/catalog/osmi-coat.html and I would call particular attention to the page URL http://www.2spi.com/catalog/osmium-plasma-coater-demonstration.html "Human astrocytoma cells, gold tagged, BSE imaging". If a picture is worth 10,000 words, I should be able to stop talking and let the micrographs speak for themselves.
Chuck
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Charles A. Garber, Ph. D. Ph: 1-610-436-5400 President 1-800-2424-SPI SPI SUPPLIES FAX: 1-610-436-5755 PO BOX 656 e-mail:cgarber-at-2spi.com West Chester, PA 19381-0656 USA Cust.Service: spi2spi-at-2spi.com
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Following my recent cooling water leak on a TEM we have come off 'mains' water and have a loan chiller. However this is incredibly noisy and of course throws out heat to the room. We need get our own system installed asap and engineering wise something to go in the same spot would be good. Does anyone know a system either chiller or heat exchanger which is particularly quiet?
Gillian Brown Histology Section, Asthma Biology RI CEDD, GlaxoSmithKline
We have just noticed that there was a typo in my previous posting for the TEM job in La Jolla (San Diego), CA
So here is the posting with the correct e-mail address.
Position Open: Electron Microscopist Research Assistance, to start June, 2003. (http://www.burnham-inst.org).
Requirements include working knowledge of high resolution TEM and a BA/BS or equivalent experience in biological or material science, or bioengineering. Responsibilities include negative stain sample preparation and electron microscopy, vitreous ice sample preparation and electron cryo-microscopy, some microscope maintenance and standard biochemical analyses.
Application should include a statement of career goals and addresses for three references. Salary is competitive and commensurate with experience. EOE.
Send applications to: Dr. Dorit Hanein, e-mail dorit-at-burnham.org
} I need to do some experiments using a FEG SEM. I am using infected } cells that I am planing to immunolabel with antibodies coupled to } colloidal gold. } } My questions are : } is immunolabelling carried out in the same way as for labelling on grids ?
Probably. The cells could also be labeled in their incubation medium in the wells or wherever they're grown, including in suspension. Post-label, fix with 1 or 1.25% glutaraldehyde + 1% tannic acid (helps preserve the cell membrane), in whatever buffer is appropriate for your cells.
} critical point drying or not ?
Yes, definitely.
} metallisation or carbon coating ?
High-resolution platinum coating, 2 to 4 nm thick. We use an ion-beam coater. Thinner, IF your cells *and* the mounting method allow it. Image at 5kV. Gold lights up nicely in the BSE at this accelerating voltage. (Your BSE detector will likely control what kV you have to use, though, but on a FEG SEM, you should have a high-resolution BSE detector anyway.)
Phil
} All answers will be helpfull, } thanks in advance } danile } -------------------------------------------- } Danile Spehner, INSERM EPI 99-08 - EFS-Alsace } 10 rue Spielmann - 67065 STRASBOURG } tel : 03 88 21 25 25 - fax : 03 88 21 25 44 } e-mail : daniele.spehner-at-efs-alsace.fr } ------------------------------------
-- Philip Oshel Supervisor, BBPIC microscopy facility Department of Animal Sciences University of Wisconsin 1675 Observatory Drive Madison, WI 53706 - 1284 voice: (608) 263-4162 fax: (608) 262-5157 (dept. fax)
We have an Aluminum sheet composite material that consists of Aluminum sheet/ polymer layer/ paper backing. The Aluminum sheet corroded through the paper and polymer layers in some areas. The Al corroded to the point that it contains large holes. We would like to cross section these samples for SEM keeping the paper layer intact.
We are looking for ideas on how to cross section the samples for SEM so all three layers can be imaged.
Kerry N. Siebein University of Florida Particle Engineering Research Center P. O. Box 116135 Gainesville, FL 32611-6135
We're looking for a 100x objective for our Zeiss Axiovert that will also be compatible with an Olympus scope (not that that should be an issue as our present objectives are interchangeable, but just in case). This will initially be used for H&E stain imaging but with later applications to follow.
E-mails are already into some reps, but does anybody have an "objective" view of quality, price, flexibility issues with different lens. Likes, dislikes...etc.
Thank you.
Gary
Gary S. Laevsky, Ph.D. Research Associate The Scripps Research Institute 10550 N. Torrey Pines Road/IMM-24 La Jolla, CA 92037 (858) 784-9372
I am not sure that you are going to get wafers that thin from them, but I have bought sapphire disks from Insaco in Philadelphia in the past. They also have the capability of machining ceramics. Their web site is http://www.insaco.com/index.html
-Scott
Scott D. Walck, Ph.D. PPG Industries, Inc. Glass Technology Center P. O. Box 11472 (letters) Guys Run Rd. (packages) Pittsburgh, PA 15238-0472
Walck-at-PPG.com
(412) 820-8651 (office) (412) 820-8515 (fax)
-----Original Message----- } From: Robb Westby [mailto:robb-at-ascendinstruments.com] Sent: Tuesday, June 03, 2003 5:50 PM To: Microscopy-at-sparc5.microscopy.com
Hello, I am trying to find a vendor for a piece of sapphire with the below criteria. Can anyone help in directing my search?
In our lab we routinely and effectively cryo-slice or cryo-cut papers/films. Freeze your material, and tweezers using LiqN2 in a mini dewar. To slice we use a variety of tools depending on what works best with the material in question. The tools can be micro scissors or straight blades. Be careful when using the blades, though! Be sure to freeze whatever slicing tool you choose.
EMS makes x-sect mounts for the SEM known as thin sample holders.
Good luck! -------------------------------- Jane Dowell Surface Science Labs WR Grace and Co. (T) 410-531-4115 (F) 410-531-4652 jane.e.dowell-at-grace.com
-----Original Message----- } From: ksiebein-at-erc.ufl.edu Sent: Wednesday, June 04, 2003 10:32 AM To: Microscopy-at-sparc5.microscopy.com
Check the paper below, in which SEM and immunogold is used to localize myosin on sperm cell surface.
Z Zhang, HQ Tian and SD Russell (1999) Localization of myosin on sperm-cell-associated membranes of tobacco. Protoplasma 208:123-128.
I'll be happy to send you a copy if you need.
Zhaojie Zhang Microscopy Core Facility University of Wyoming
-----Original Message----- } From: Spehner Daniele [mailto:daniele.spehner-at-efs-alsace.fr] Sent: Tuesday, June 03, 2003 5:14 PM To: Microscopy-at-sparc5.microscopy.com
Hi Listers,
I need to do some experiments using a FEG SEM. I am using infected cells that I am planing to immunolabel with antibodies coupled to colloidal gold.
My questions are : is immunolabelling carried out in the same way as for labelling on grids ? critical point drying or not ? metallisation or carbon coating ? All answers will be helpfull, thanks in advance danile
Thanks to all who replied, particularly Norm - your suggestion worked. I located the beamsplitter, unscrewed the head and dropped the filter in before the beamsplitter and hey presto - the fluorescence image was visible through both the eyepiece and the phototube. I've tried the filter with rhodamine and I can see red fluoresecence so thanks again - I don't need to buy a new filter!
Kind regards,
Patricia
---------------------- Patricia de Winter cdewi02-at-students.bbk.ac.uk
Dear listers, I received a call from an investigator at Univ. of Cinn who needs help with cryoultramicrotomy and TEM of nanotubes. He is willing to drive anywhere within a 4 or 5 hour drive from Cinn. If any of you would be able to help him please contact me. Thanks so much, Mary Gail Engle
Mary Gail Engle Sr. Research Laboratory Manager Electron Microscopy & Imaging Facility Health Sciences Research Bldg. 001 University of Kentucky Lexington, KY 40536-0305
Digital Image Capture and Management in Microscopy
October 16, 2003
A course on Digital imaging in light microscopy which will cover the following topics:
Optical Limitations in Light Microscopy...Photographic Imaging Strategies... Digital Imaging Strategies...Selection of Digital Capture (Camera vs. Scanner)...
Image Processing of Captured Images...Image File Formats...Printing Images... Color Management Systems...Database Management Software...Presentation Software for Oral Reports...Website Performance...Integration of Image Data with Sample Information, Calibration, Other Data & Reports...Acrobat and html Software for Written Reports and Archives...Examples of Efficient, Low Cost Image Handling Systems...
Examples of Electronic Microscopy Reports and Databases
The course instructors are Mary and John McCann of McCann Imaging.
WHEN: Thursday, October 16, 2003, from 9 A.M. to 5 P.M.
Never having done this, let me take a stab at it. This is free, so that it doesn't cost you anything.
Why don't you try to impregnate (vacuum impregnate?) the paper with epoxy and then try to polish it or cut it?
-Scott
Scott D. Walck, Ph.D. PPG Industries, Inc. Glass Technology Center P. O. Box 11472 (letters) Guys Run Rd. (packages) Pittsburgh, PA 15238-0472
Walck-at-PPG.com
(412) 820-8651 (office) (412) 820-8515 (fax)
-----Original Message----- } From: Siebein, Kerry [mailto:ksiebein-at-erc.ufl.edu] Sent: Wednesday, June 04, 2003 10:32 AM To: 'Microscopy-at-MSA.Microscopy.Com'
We have an Aluminum sheet composite material that consists of Aluminum sheet/ polymer layer/ paper backing. The Aluminum sheet corroded through the paper and polymer layers in some areas. The Al corroded to the point that it contains large holes. We would like to cross section these samples for SEM keeping the paper layer intact.
We are looking for ideas on how to cross section the samples for SEM so all three layers can be imaged.
Kerry N. Siebein University of Florida Particle Engineering Research Center P. O. Box 116135 Gainesville, FL 32611-6135
First, I must solicit your strictest confidence in this transaction, this is by virtue of it's nature as being utterly confidential and top secret .We are top officials from the Federal Ministry of Works and Housing,(FMWH),Federal Ministry of Finance and the Presidency, making up the Contract Review Panel (CRP) set up by the Federal Government of Nigeria to review contracts awarded by the past military administration. In the course of our work on the CRP, we discovered this fund which resulted from grossly over-invoiced contracts which were executed for the FMW&H during the last administration. The companies that executed the contracts have been duly paid and the contracts commissioned leaving the sum of US$35.4 Million floating in the escrow account of the Central Bank of Nigeria ready for payment. I have therefore been mandated as a matter of trust by my colleagues in the panel to look for an overseas partner to whom we could transfer the sum of US$35.4M legally subcontracting the entitlement to your company. This is bearing in mind that our civil service code of conduct forbids us from owning foreign company or running foreign account while in government service hence the need for an overseas partner. We have agreed that the funds will be shared thus after it has been paid into your account: (1) 30% of the money will go to you for acting as the beneficiary of the fund. (2) 10% has been set aside as an abstract projection for reimbursements to both parties for incidental expenses that may be incurred in the course of the transaction. (3) 60% to us the government officials (with which we wish to commence an importation business in conjunction with you). All logistics are in place and all modalities worked out for the smooth conclusion of the transaction within ten to fourteen days of commencement after receipt of the following information: Your company name, address, company's details, telephone & fax numbers. These information will enable us make the applications and lodge claims to the concerned ministries & agencies in favour of your company and it is pertinent to state here that this transaction is entirely based on trust as the solar bank draft or certified cheque draw able in any of the Central Bank of Nigeria correspondent bankers around the world is going to be made in your name.Please acknowledge the receipt of this letter using my e-mail address or the alternative email;tphector-at-ny.com, your interest and tel/fax number for further details. Yours faithfully, Mr. Hector Tunde Fax=234-9-2722017 Email:tphector-at-ny.com _________________________________________________ Votre mail gratuit ?ie avec http://lexpress.net
What are the thickness measurements of the constituent materials?
At first blush, a traditional Buehler polishing cross section should do the job. These are embedded in rigid epoxy. Very routine for microcircuit work. It may be applicable to your situation.
gary g.
At 07:31 AM 6/4/2003, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
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Maybe. Have not tried it. I would worry about the impact of this on ultrastructure. Can you tell me how/why HMDS works? Does the specimen get coated in the stuff, and if so is the residue of the material visible in a FEGSEM??
I have also wondered about osmium impregnation e.g. by OTO as an alternative to metal or carbon coating for immunogold labelled specimens. However, so far our experiences with Osmium /Tannic Acid /Osmium in another context have resulted in highly intrusive decoration of the specimen, so that all parts have the same, clearly artifactual ultrastructure. This is so fine that it would probably not be resolved by conventional SEM, but FEGSEM is less forgiving.
Chris
Dr. Chris Jeffree
----- Original Message ----- } From: "Dusevich, Vladimir" {dusevichv-at-umkc.edu} To: {Microscopy-at-sparc5.microscopy.com} Sent: Wednesday, June 04, 2003 6:33 PM
I am out of office until 16-06-2003. For urgent matters please contact micro-at-omnilabo.be or call 03 870 58 03
Hi, I was wondering which cover slips are in use for high resolution microscopy or has anyone reviewed cover slips that are commercially available? As it is possible for a user to inadvertently introduce error into a well-corrected system just by selecting the wrong cover slip, I have been instructing people who use the microscopes to utilise the correct thickness of the cover glass (0.17 millimeters) and try and move away from plastics.
Unfortunately with high quality cover glasses having a tolerance of +/- 10 micrometers, the FWHM is detrimentally altered by more than a factor of two if a cover slip is on the extreme of this tolerance.
Are there coverslips available with a finer degree of tolerance than the standard +/- 10 ? thanks
Steve
Steve Bagley Applied Imaging Facility Paterson Institute For Cancer Research Cancer Research UK Christie Hospital, Wilmslow Rd, Manchester. M20 9BX, UK
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Never had any trouble with HMDS affecting ultrastructure for SEM (even FEGSEM). As far as the OTOTO method goes, there was a paper about 20 years ago that discussed the need to find a consistent source for the TCH--they are not all created equal. Second, the authors recommended washing small amounts of the TCH crystals (can't remember the details) several times to remove impurities; this leaves TCH crystals that are white/semi-opaque, rather than the grayish color as received. Third, rigorous, thorough washing between steps is critical. I use 5x in dH2O between each and every step. This gives excellent impregnation and conductivity (plus secondary electron signal) as well as fine surface structure preservation in the FEGSEM. I have to look up the details for washing the TCH--I'll get back to you on that (it's obviously been awhile since I have done this).
Roger Moretz, Ph.d. Dept of Toxicology BI Pharmaceuticals, Inc. Ridgefield, CT -- Where the world is only slightly less weird than it actually is. } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Maybe. Have not tried it. I would worry about the impact of this on } ultrastructure. } Can you tell me how/why HMDS works? Does the specimen get coated in } the stuff, } and if so is the residue of the material visible in a FEGSEM?? } } I have also wondered about osmium impregnation } e.g. by OTO as an alternative to metal or carbon coating for } immunogold labelled specimens. However, so far our experiences with } Osmium /Tannic Acid /Osmium in another context have resulted in highly } intrusive decoration of the specimen, so that all parts have the same, } clearly artifactual ultrastructure. This is so fine that it would
} probably not be resolved by conventional SEM, but FEGSEM is less } forgiving. } } Chris } } Dr. Chris Jeffree } } ----- Original Message ----- } } From: "Dusevich, Vladimir" {dusevichv-at-umkc.edu} } To: {Microscopy-at-sparc5.microscopy.com} } Sent: Wednesday, June 04, 2003 6:33 PM } Subject: RE: SEM and Immunolabelling } } } } -------------------------------------------------------------------- } ---- } } The Microscopy ListServer -- Sponsor: The Microscopy Society of } America } } To Subscribe/Unsubscribe -- } http://www.msa.microscopy.com/MicroscopyListserver } } On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } -------------------------------------------------------------------- } ---. } } } } } } } } } } critical point drying or not ? } } } } } } Yes, definitely. } } } } What about HMDS? } } } } Vladimir } } } }
Kerry, I hope the University has a decent metallographic lab. There you will find the resources you need. I've often prepared similar samples for study.
If the sample is large, I would start by buttering the porous area with a prepared mixture of two-part epoxy. This epoxy is available from any metallographic supply house (Struers, Buehler, Leco, Lapmaster, MetLab, Mager), has excellent wetting properties, low viscosity for sample impregnation, and should be allowed to cure overnight. This epoxy will "fix" the porous and loose material in place for subsequent sectioning.
After the buttered area is hard, section slightly to the side of the area of interest. Rough cut the rest of the sample to fit in a metallographic mount. Mount the area - again using epoxy. After the mount has cured, rough polish the area of interest to remove cutting damage.
At this point, the sample should be "frozen" in place with a bit of porosity at the cut surface. Again, put some epoxy on the cut and rough polished surface. Use vacuum techniques to draw air out of the porosity and drive epoxy into the sample. After curing, prepare the sample metallographically for an optically flat surface. After sputter coating to make the sample conductive, it is ready for viewing in the SEM.
It takes a couple of days to prepare the sample, but this is the routine I use for samples of this type. If imaging is all you need and don't need EDS information, you may forego sputter coating and SEM examination and simply use a metallograph for your study.
Stu Smalinskas Metallurgist SKF USA Plymouth, Michigan (734) 414-6862 stu.smalinskas-at-skf.com
(I have no financial interest in the metallographic supply houses. I'm just a satisfied customer.)
We have an Aluminum sheet composite material that consists of Aluminum sheet/ polymer layer/ paper backing. The Aluminum sheet corroded through the paper and polymer layers in some areas. The Al corroded to the point that it contains large holes. We would like to cross section these samples for SEM keeping the paper layer intact.
We are looking for ideas on how to cross section the samples for SEM so all three layers can be imaged.
Kerry N. Siebein University of Florida Particle Engineering Research Center P. O. Box 116135 Gainesville, FL 32611-6135
You do not say how thick the material is but I have had success with a "cryo" method on similar materials.
The SEM is a clever tool, cut a material and it sees the cut, not the material interfaces! The only alternatives are embedding + mechanical sectioning + polishing or "cryo".
For cryo - cut a piece of the material into an egg timer shape being 1/2inch wide at top and bottom but only 1/4 inch wide in the neck. Lower this into liquid nitrogen and wait for the bubbling to subside. Remove with light weight pliers and, holding each end with the pliers, CRACK the material by bending it slightly. If the material does not crack do not repeat the bending but try again with another piece of material but make the neck 1/8 inch wide.
Hope this helps?
Steve Chapman Senior Consultant Protrain Electron Microscopy Training and Consultancy World Wide Tel +44 (0)1280 816512 Fax +44 (0)1280 814007 www.emcourses.com
----- Original Message ----- } From: "Siebein, Kerry" {ksiebein-at-erc.ufl.edu} To: "'Microscopy-at-MSA.Microscopy.Com'" {Microscopy-at-sparc5.microscopy.com} Sent: Wednesday, June 04, 2003 3:31 PM
Hi
I am on the point of buying a digital camera for microscope work.
I have seen a Japanese-made adapter for the Olympus Camedia C-3040 camera in which the end away from the camera was stepped in three different diameters, to suit a trinocular port plus the two common eyepiece tube diameters. The adapter had an internal lens, and was apparently made by a small non-English- speaking Japanese company without electronic communications, and was expensive. It would be useful for my purposes as the same adapter/camera can be easily and quickly put onto any one of a number of microscopes.
Adapter manufacturers' websites that I have seen seem to describe only adapters that suit only one microscope mounting point ie either C-mount OR 23mm eyepiece tube OR 30mm eyepiece tube.
Can someone either point me in the direction of someone who makes a similarly universal adapter or explain to me why such a device doesn't work as well as a less-flexible system does?
Also, does anyone have good grounds for a preference, for this kind of work, for the Coolpix 4500 over the Olympus 3040 or vice- versa? They seem pretty similar to me on specifications.
cheers
rtch
-- Ritchie Sims Ph D Phone : 64 9 3737599 ext 87713 Microanalyst Fax : 64 9 3737435 Department of Geology email : r.sims-at-auckland.ac.nz The University of Auckland Private Bag 92019 Auckland New Zealand
I sent out an email for 'help' regarding our aging 20 year-old Hitachi H-600 TEM on the 9th of April this year. We thought our HT cable had died and really could not justify spending in excess of $30,000 on a new gun/cable assembly as we were applying through a grant for a new TEM. I thought it may be beneficial to summarise the situation for Listserver members (see below). I would also like to extend a very BIG thank-you to all those who responded especially Joel McClintock, Steve Chapman,William Mushock, Terry Fitton, John Brealey, Sally Stowe, Noni Hudson and George Theodossiou. I really felt like I had a team helping me through this. Can you all now pray that my grant application is successful and we can replace the old girl sooner rather than later?
The symptoms were;
1. Unstable voltage readouts. When we set the voltage to, say, 50 Kv, it did not read 50 Kv but read 75Kv. 2. There was a 'hissing' sound when we tried to change from 75Kv to 100Kv. The hissing appeared to be coming from behind the gun. 3. Our filaments, when they broke, were showing signs of over-heating as evidenced by a round blob of tungsten on each of the broken ends. They were not being over-saturated. 4. When we opened the gun, it smelt. 5. When we disconnected the cable from the HT tank, the HT tank read-outs were OK.
Some of the people who responded to my cry for help suggested that the symptoms may indeed by caused by a dirty gun and/or poor vacuum.
What did we do? We cleaned the gun thoroughly. We did not dismantle the gun from the microscope but cleaned it in situ. We used diamond paste (wenol) sparingly on lint-free cloths to scrub off the pale toffee colored discoloration of the porcelain until it was white and shiny. We wiped any paste residue off with 10% quadralene (ammonia based) followed by water then ethanol (very dry) then acetone (very dry) . We then allowed it to pump down over the weekend. It was not a difficult procedure and one that, had I known how straight forward it would be, should have been done a lot earlier.
On Monday, we had trouble stabilising the HV at 100KV but after 3 goes, reached and held 100KV. I have taken a while to present this summary to the Listserver members as I wanted to make sure the dirty gun was the culprit. Now, 2 months down the track we remain clear of all the original symptoms. Of course there are other small problems that have sprung up associated with using a TEM that is 20 years old but they are all 'fixable'.
Again, thank-you list-server members
cheers
Sarah Ellis
Manager, Microscopy Imaging and Research Core Facility Peter MacCallum Cancer Centre Locked Bag #1 A'Beckett Street East Melbourne 8006
-----Message d'origine----- De : Dusevich, Vladimir [mailto:dusevichv-at-umkc.edu] Envoyé : mercredi 4 juin 2003 19:33 À : Microscopy-at-sparc5.microscopy.com Objet : RE: SEM and Immunolabelling
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} } critical point drying or not ? } } Yes, definitely.
Unless there is some way to adjust for tube length by a lens in the adapter, I would think you would have trouble establishing parfocality by using an adapter with a stepped outside to the tube. There have been another thread or two talking about adapters that allow for that. I know we don't have one of them.
I have posted some pictures on our ftp server to show what we had to go through with our Olympus Stereoscope. I modified the phototube so that I could use the same relay lens that worked just fine on our other two Olympus scopes (reflected and transmitted. I had to remove an adapter on the top of our tube that was used as the mechanical support for the Olympus camera. It prevented the relay lens from sliding in as far as it needed to. I then had to make a tube spacer to set the relay lens back off to the right length to obtain parfocality with the eyepieces.
I would suppose that you might be better off having your shop make some bushings for your larger phototubes to accomodate an adapter for the smaller tube. It might take a little doing to make sure you maintain the proper path length, but any machinist worth their pay should be able to rig up something. (Or maybe Edmund already stocks such bushings.) I just hope you don't run into any vignetting issues.
ftp://www.marl.iastate.edu/LabPix/Stereo_adapter/
Warren
At 02:15 PM 6/6/2003 +1200, you wrote:
} Hi } } I am on the point of buying a digital camera for microscope work. } } I have seen a Japanese-made adapter for the Olympus Camedia } C-3040 camera in which the end away from the camera was } stepped in three different diameters, to suit a trinocular port plus } the two common eyepiece tube diameters. The adapter had an } internal lens, and was apparently made by a small non-English- } speaking Japanese company without electronic communications, } and was expensive. } It would be useful for my purposes as the same adapter/camera } can be easily and quickly put onto any one of a number of } microscopes. } } Adapter manufacturers' websites that I have seen seem to } describe only adapters that suit only one microscope mounting } point ie either C-mount OR 23mm eyepiece tube OR 30mm } eyepiece tube. } } Can someone either point me in the direction of someone who } makes a similarly universal adapter or explain to me why such a } device doesn't work as well as a less-flexible system does? } } Also, does anyone have good grounds for a preference, for this } kind of work, for the Coolpix 4500 over the Olympus 3040 or vice- } versa? They seem pretty similar to me on specifications. } } cheers } } rtch } } -- } Ritchie Sims Ph D Phone : 64 9 } 3737599 ext 87713 } Microanalyst Fax : 64 9 } 3737435 } Department of Geology email : } r.sims-at-auckland.ac.nz } The University of Auckland } Private Bag 92019 } Auckland } New Zealand
I have acquired a surplus turbo pumped JEOL 845 SEM, but the documentation I have received doesn't seem to cover the turbo modification. Would anyone out there operating a turbo pumped JEOL 840 or 845 like to share their cold start instructions and operating instructions with me?
Sincerely,
Scott
F. Scott Miller, Ph.D. Advanced Materials Characterization Lab University of Missouri-Rolla 223 McNutt Hall Rolla, MO 65409 USA fax: 573 341 6934 voice: 573 341 4727
As Philip suggested a 2 - 4nm coating of platinum will allow you to examine the sample at 5Kv using the backscatter detector. In addition, it has been my experience that under those conditions the lower Se- detector provides some atomic number imaging, at least that's the case in my instrument. While I have not tried this technique on gold labeled cells I have had success looking for metal contaminants in organic crystal samples. The advantages offered by using the Se- detector certainly makes it worth a try with your equipment/samples.
John A. Robson Boehringer Ingelheim Pharmaceuticals, Inc. PO Box 368 900 Ridgebury Rd Ridgefield, CT 06877
-----Original Message----- } From: Philip Oshel [mailto:peoshel-at-wisc.edu] Sent: Wednesday, June 04, 2003 9:46 AM To: Microscopy-at-sparc5.microscopy.com
Daniele,
see below
} I need to do some experiments using a FEG SEM. I am using infected } cells that I am planing to immunolabel with antibodies coupled to } colloidal gold. } } My questions are : } is immunolabelling carried out in the same way as for labelling on grids ?
Probably. The cells could also be labeled in their incubation medium in the wells or wherever they're grown, including in suspension. Post-label, fix with 1 or 1.25% glutaraldehyde + 1% tannic acid (helps preserve the cell membrane), in whatever buffer is appropriate for your cells.
} critical point drying or not ?
Yes, definitely.
} metallisation or carbon coating ?
High-resolution platinum coating, 2 to 4 nm thick. We use an ion-beam coater. Thinner, IF your cells *and* the mounting method allow it. Image at 5kV. Gold lights up nicely in the BSE at this accelerating voltage. (Your BSE detector will likely control what kV you have to use, though, but on a FEG SEM, you should have a high-resolution BSE detector anyway.)
Phil
} All answers will be helpfull, } thanks in advance } danile } -------------------------------------------- } Danile Spehner, INSERM EPI 99-08 - EFS-Alsace } 10 rue Spielmann - 67065 STRASBOURG } tel : 03 88 21 25 25 - fax : 03 88 21 25 44 } e-mail : daniele.spehner-at-efs-alsace.fr } ------------------------------------
-- Philip Oshel Supervisor, BBPIC microscopy facility Department of Animal Sciences University of Wisconsin 1675 Observatory Drive Madison, WI 53706 - 1284 voice: (608) 263-4162 fax: (608) 262-5157 (dept. fax)
I suggest you take a look at our webpage for adapting the Coolpix series cameras to a microscope: http://www.mvia.com/Coolpix/clpxadpt.htm
1.) If you want to go through 23mm eyepieces, our optics adapter is a straight drop in.
2.) If you want to go through 30mm eyepieces, you use the exact same optics, and we have a sleeve to increase the diameter of the adapter to 30mm.
3.) If you want to go through a trinocular port, you use the exact same optics, and we have several clamps to fit to various microscopes.
4.) If you want to go through an existing 1X C-mount on your microscope, you use the exact same optics, and we have a clamp and thread adapter to convert our optics to a C-mount fitting.
In all of these cases, we are using the exact same optics system, the only thing we change is the clamp, sleeve, or thread fitting that will connect it to your microscope. All parts are interchangeable and part of our universal system.
We have an optics system specific to the Coolpix series cameras (4500 included) that will definitely work with the Coolpix 4500.
On the other hand, we also have a universal optics adapter for fitting to digital cameras, but the Olympus 3040 is an untested model for us and I am not sure how well it will work.
Thanks! Jim Haley
****************************** Jim Haley Applications Engineer MVIA, Inc. 125 Sherwood Drive Monaca, PA 15061 voice: (724) 728-7493 fax: (412) 291-1709 e-mail: haley-at-mvia.com webpage: http://www.mvia.com ******************************
Ritchie Sims wrote: } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Hi } } I am on the point of buying a digital camera for microscope work. } } I have seen a Japanese-made adapter for the Olympus Camedia } C-3040 camera in which the end away from the camera was } stepped in three different diameters, to suit a trinocular port plus } the two common eyepiece tube diameters. The adapter had an } internal lens, and was apparently made by a small non-English- } speaking Japanese company without electronic communications, } and was expensive. } It would be useful for my purposes as the same adapter/camera } can be easily and quickly put onto any one of a number of } microscopes. } } Adapter manufacturers' websites that I have seen seem to } describe only adapters that suit only one microscope mounting } point ie either C-mount OR 23mm eyepiece tube OR 30mm } eyepiece tube. } } Can someone either point me in the direction of someone who } makes a similarly universal adapter or explain to me why such a } device doesn't work as well as a less-flexible system does? } } Also, does anyone have good grounds for a preference, for this } kind of work, for the Coolpix 4500 over the Olympus 3040 or vice- } versa? They seem pretty similar to me on specifications. } } cheers } } rtch } } -- } Ritchie Sims Ph D Phone : 64 9 } 3737599 ext 87713 } Microanalyst Fax : 64 9 } 3737435 } Department of Geology email : } r.sims-at-auckland.ac.nz } The University of Auckland } Private Bag 92019 } Auckland } New Zealand
Unless specimen is mounted in direct contact with the underside of the cover glass, use No. 1 cover glasses.
Gary Gill
-----Original Message----- } From: Steve Bagley [mailto:SBagley-at-picr.man.ac.uk] Sent: Thursday, June 05, 2003 3:42 AM To: microscopy-at-sparc5.microscopy.com
Hi, I was wondering which cover slips are in use for high resolution microscopy or has anyone reviewed cover slips that are commercially available? As it is possible for a user to inadvertently introduce error into a well-corrected system just by selecting the wrong cover slip, I have been instructing people who use the microscopes to utilise the correct thickness of the cover glass (0.17 millimeters) and try and move away from plastics.
Unfortunately with high quality cover glasses having a tolerance of +/- 10 micrometers, the FWHM is detrimentally altered by more than a factor of two if a cover slip is on the extreme of this tolerance.
Are there coverslips available with a finer degree of tolerance than the standard +/- 10 ? thanks
Steve
Steve Bagley Applied Imaging Facility Paterson Institute For Cancer Research Cancer Research UK Christie Hospital, Wilmslow Rd, Manchester. M20 9BX, UK
This email is confidential and intended solely for the use of the person(s) ('the intended recipient') to whom it was addressed. Any views or opinions presented are solely those of the author and do not necessarily represent those of the Paterson Institute for Cancer Research or the Christie Hospital NHS Trust. It may contain information that is privileged & confidential within the meaning of applicable law. Accordingly any dissemination, distribution, copying, or other use of this message, or any of its contents, by any person other than the intended recipient may constitute a breach of civil or criminal law and is strictly prohibited. If you are NOT the intended recipient please contact the sender and dispose of this e-mail as soon as possible.
Applied Precision produces measurement and analysis hardware and software for the semiconductor and biotechnology industries.
We are currently seeking a success-oriented Applications Scientist to support Applied Precision's Life Science Sales Team. This is a technical support position both pre- and post- sale.
This person will be responsible for providing scientific and technical support during product demonstrations, tradeshow events, and workshops. Training users on hardware and software included in bio-imaging products is also a major constituent of the position.
The successful candidate will have excellent communication, organizational, and project management skills and be able to travel extensively. Must have a B.S. or higher in Cell or Molecular Biology or related field and must have expertise in several of the following areas: Widefield/Deconvolution or Confocal Microscopy, Fluorescence Imaging, Microarray Technology, Digital Image Analysis, UNIX based operating systems.
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-- [ From: Garber, Charles A. * EMC.Ver #3.1a ] --
Kerry N. Siebein wrote: ======================================================================== We have an Aluminum sheet composite material that consists of Aluminum sheet/ polymer layer/ paper backing. The Aluminum sheet corroded through the paper and polymer layers in some areas. The Al corroded to the point that it contains large holes. We would like to cross section these samples for SEM keeping the paper layer intact.
We are looking for ideas on how to cross section the samples for SEM so all three layers can be imaged. ======================================================================== Over the years we have had systems that sound similar, where the aluminum layer could have been as thin as aluminum foil and as thick as an old style aluminum lithographic printing plate. We have found that the following will work:
1] You need to have some kind of embedding to keep the paper intact but you won't want the paper to be swollen with the embedding resin to the extent that it loses all of it original dimensions. We like to apply a passivation layer of sputtered gold (if we did this now, we would use osmium metal in the osmium coater since you can get a much thinner effective barrier layer); the purpose of the passivation layer is to limit the amount of embedding resin infiltration and also to act as a decoration layer to more easily define the paper/embedding resin interface.
2] We have always found that our own (or that offered by at least some of our competitors) version of Epon 812 (ours is called SPI-Pon 812) works better than any of the other alternatives. It also gives reasonably good adhesion with the opposite aluminum layer.
3] The next step is to diamond knife thin section the block as if you were going to be doing TEM as well. We have found that when SEM views are really what are needed, we know that the best SEM "faced-off-piece" will be obtained when the best possible TEM sections are being taken. So even if TEM is not the main interest, we still evaluate the sections coming off to make sure they are of the quality needed to result in the best possible "faced-off-piece" for SEM.
4] At this point, and in order to give the faced-off-piece a bit more contrast, we subject it to a very slight plasma etching treatment in our SPI Plasma Prep II plasma etcher. Typically 10-20 seconds might be all that is needed but that is enough to etch down so that the aluminum layer "stands up" like a little micro wall. The etched structure of the polymer vs. paper is quite distinctively different. Inorganic additives are also prominently seen, often times like little "mesas" in Arizona.
5] The diamond knife ultramicrotomy is done using an SPI Supplies Materials Science Diamond Knife. To use anything "better" (e.g. a life science knife) is a waste of money since the inorganics typically found in the paper and polymer layers will impart striations to the knife edge with the first pass of the knife over the sample. We find that knifes with 45° angle are optimum and that if 55° angle knives are used, there are significant compression effects in the sections and the face-off-piece quality suffers as well.
Using this approach, one can indeed get a good view of the aluminum foil layer but it is not as good of a view if you are looking for incipient signs of aluminum corrosion (for that, the TEM is much better). The SEM view is good in terms of looking at the degree of penetration of the polymer into the paper structure or just looking at the uniformity of layer thickness of all three of the layers (because typically by SEM one is looking at a longer length of cross-section.
Disclaimer: SPI Supplies offers most of the products mentioned in this posting, as part of our SPI Supplies product line, including the embedding resin, plasma etcher, and diamond knives. We would have a vested interest in seeing more people viewing these kinds of samples this way since it uses up more of our products.......
Chuck
============================================
Charles A. Garber, Ph. D. Ph: 1-610-436-5400 President 1-800-2424-SPI SPI SUPPLIES FAX: 1-610-436-5755 PO BOX 656 e-mail:cgarber-at-2spi.com West Chester, PA 19381-0656 USA Cust.Service: spi2spi-at-2spi.com
Look for us! ######################## WWW: http://www.2spi.com ######################## ============================================
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Listers,
} From time to time we get a request here at Microscopy Today for an article that someone wants to read so as to learn about some aspect of microscopy.
Here's one: ====== Dear MT Editor,
I've been exchanging emails with a friend, and we've both decided we're mystified by coating. The physics of thin coats, how they actually form, what their real structures are, why are carbon coats conductive, those things, at the physical level. We both know how to use sputter coaters, vacuum evaporators and the like, and basically what they're doing, how they work, but ... what's *really* happening and why? One or more articles for MT? I don't know who to suggest, but with your connections, there has to be someone you know who could write them. And aren't evaporated C coats mostly bucky balls and nanotubes? ========
Is there anyone out there willing to write such an article? About 1,500 words plus pictures written in a tutorial style for a general, non-specialist, microscopy audience.
Please e-mail me off the listserver
Ron Anderson, Editor Microscopy Today microtoday-at-attglobal.net
The secondary electron detector does show the gold nicely, also. The gold particles don't light up with the SE like they do with the BSE, but they do stand out from the background of biological samples. I don't think this is atomic number imaging, though, as much as it is just that the gold particles are very good sources of secondary electrons, and much denser than the rest of the sample. If the instrument allows it, the best way to go is simultaneous acquisition of SE and BSE images.
Phil
} As Philip suggested a 2 - 4nm coating of platinum will allow you to examine } the sample at 5Kv using the backscatter detector. In addition, it has been } my experience that under those conditions the lower Se- detector provides } some atomic number imaging, at least that's the case in my instrument. } While I have not tried this technique on gold labeled cells I have had } success looking for metal contaminants in organic crystal samples. The } advantages offered by using the Se- detector certainly makes it worth a try } with your equipment/samples. } } John A. Robson } Boehringer Ingelheim Pharmaceuticals, Inc. } PO Box 368 } 900 Ridgebury Rd } Ridgefield, CT 06877 } } Phone (203)798-5640 } Fax (203)798-5698 } e-mail jrobson-at-RDG.boehringer-ingelheim.com } } } -----Original Message----- } } From: Philip Oshel [mailto:peoshel-at-wisc.edu] } Sent: Wednesday, June 04, 2003 9:46 AM } To: Microscopy-at-sparc5.microscopy.com } Subject: Re: SEM and Immunolabelling } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (bappooh-at-myexcel.com) from http://www.msa.microscopy.com/Ask-A-Microscopist.html on Saturday, June 7, 2003 at 21:15:39 ---------------------------------------------------------------------------
Email: bappooh-at-myexcel.com Name: Tammy
Organization: Northside Christian School
Education: K-8 Grade Grammar School
Location: City, State, Country Louisville,Ky.u.s.a.
Question: Can you use microscopy looking at human blood cells and determine any nutritional defiencies ?
Dear Sir As an exporter in China, We Ali trade Inc. can supply 12,000 kinds of products. Currently we are selling: pro audio connectors rca connectors audio equipments and mic leads Audio plugs&jacks wire cable electronic plug-in units fuse computer microphone multimedia sound sound/megaphone/switch indoor antenna piezo tweeter and horn available package demonstration manual reset circuit breakers fused power distribution blocks power distribution block braided screen power cables amplifier installation wiring kit series rac interconnect series optical fiber cables rca plugs/jacks cable drums for stage performance speakers&speaker boxes siren driver horn microphone headphone&mic multimedia speaker system woofer car audio amplifier car speaker component speaker package dome tweeter hi fi system boom box and etc. Fax:0086-574-87317472 Mr Qiu
Dear Sir As an exporter in China, We Ali trade Inc. can supply 12,000 kinds of products. Currently we are selling: pro audio connectors rca connectors audio equipments and mic leads Audio plugs&jacks wire cable electronic plug-in units fuse computer microphone multimedia sound sound/megaphone/switch indoor antenna piezo tweeter and horn available package demonstration manual reset circuit breakers fused power distribution blocks power distribution block braided screen power cables amplifier installation wiring kit series rac interconnect series optical fiber cables rca plugs/jacks cable drums for stage performance speakers&speaker boxes siren driver horn microphone headphone&mic multimedia speaker system woofer car audio amplifier car speaker component speaker package dome tweeter hi fi system boom box and etc. Fax:0086-574-87317472 Mr Qiu
Dear Sir As an exporter in China, We Ali trade Inc. can supply 12,000 kinds of products. Currently we are selling: pro audio connectors rca connectors audio equipments and mic leads Audio plugs&jacks wire cable electronic plug-in units fuse computer microphone multimedia sound sound/megaphone/switch indoor antenna piezo tweeter and horn available package demonstration manual reset circuit breakers fused power distribution blocks power distribution block braided screen power cables amplifier installation wiring kit series rac interconnect series optical fiber cables rca plugs/jacks cable drums for stage performance speakers&speaker boxes siren driver horn microphone headphone&mic multimedia speaker system woofer car audio amplifier car speaker component speaker package dome tweeter hi fi system boom box and etc. Fax:0086-574-87317472 Mr Qiu
Dear Sir As an exporter in China, We Ali trade Inc. can supply 12,000 kinds of products. Currently we are selling: pro audio connectors rca connectors audio equipments and mic leads Audio plugs&jacks wire cable electronic plug-in units fuse computer microphone multimedia sound sound/megaphone/switch indoor antenna piezo tweeter and horn available package demonstration manual reset circuit breakers fused power distribution blocks power distribution block braided screen power cables amplifier installation wiring kit series rac interconnect series optical fiber cables rca plugs/jacks cable drums for stage performance speakers&speaker boxes siren driver horn microphone headphone&mic multimedia speaker system woofer car audio amplifier car speaker component speaker package dome tweeter hi fi system boom box and etc. Fax:0086-574-87317472 Mr Qiu
} } } } Subject: re-freezing tissue in OCT for cryo-sectioning } } } } From: Angela welford {awelford-at-salud.unm.edu} } } } } To: microscopy-at-sparc5.microscopy.com } } } } Content-Transfer-Encoding: 7bit } } } } Message-Id: {121E4BB6-96D7-11D7-81B6-000A959AEFF4-at-salud.unm.edu} } } } } X-Mailer: Apple Mail (2.552) } } } } Content-Length: 660 } } } } } } } } } } } I have some unfixed kidney tissue that is currently in OCT at -70C. } } } } The pieces are quite large, as prepared for frozen sections for } } } } histology. My need is to cryo-section a portion of the tissue for } } } } immunolabelling for EM. Is there a recommended method to handle the } } } } tissue to minimize any damage from thawing and re-freezing, or do I } } } } need to be concerned about that since it is in OCT? My plan was to } } } } cut } } } } off a piece, let it thaw and cut it down for fixation in PF, then } } } } infiltrate with sucrose before freezing (plunge in LN2). If anyone } } } } has } } } } a protocol they can recommend, I would appreciate it! } } } } Angela Welford } } } } UNM, Albuquerque, NM } } } } (505)272-1445 } } } } } } } }
Edinburgh City Council are proposing to install a tramline network and I understand that this could have a severe impact upon the operation of our FEG SEM. The route for this network is still out to consultation but I'd be grateful of any other users' experience regarding proximity, magnetic fields, vibration etc so that I'm fully informed should it become an issue.
Thanks,
Frieda Christie, Electron Microscopist, Scientific & Technical Services, Royal Botanic Garden, 20A Inverleith Row, Edinburgh, EH3 5LR
I would suggest you touch base with Dougal Mcculloch {dougal.mcculloch-at-rmit.edu.au} at RMIT in Melbourne.
He had exactly this problem and as I recall he installed field canceling coils in one FEGTEM room which was closest to the TRAM line. As I vaguely remember in a second room the coils were not needed as the field was sufficiently low as not to affect the instrument.
Nestor Your Friendly Neighborhood SysOp
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The box said ... "This program requires Win 2K/XP or better..." So I bought a G3 Mac !
Someone has asked me to explain the way a sputtered coating of metal bonds to a surface.
I am a biologist, so the details are a little sketchy for me. In general terms, what is the mechanism for the bonding, and could it ever be considered 'covalent' bonding?
Thanks
Jonathan Krupp Microscopy & Imaging Lab University of California Santa Cruz, CA 95064 (831) 459-2477 jmkrupp-at-cats.ucsc.edu
At the request of one of the list members, herewith is a summary via cut and paste of all the replies I got about this subject.
try ultra darkfield first - I think Olympus makes an ultra darkfield condenser . Do you expect there to be some slight density differences? Maybe can you centrifuge them out in a sucrose gradient?
At ~2000g, all the cells will sediment. What about the particles? If the particles are less dense than hemoglobin, then layer a slightly less dense than particles but more dense than hemoglobin, hemolyze blood with distilled water, and spin. Your particles should 'fall' into 'dense' layer at bottom. On the other hand, your particles might be more buoyant than the cells, in which case you win that way. In the absence of answers to my questions, you are left with a manageable experiment anyway, especially if the particles start out in a jar on the shelf. If they are manufactured in the body, i.e., crystals, then you must do it the hard way. An Airfuge (Beckman) will sediment hemoglobin, but not certain mycoplasma-like organisms, and it will work with really small volumes. Even if the particles sediment faster than the hemoglobin, the hemoglobin isn't birefringent. With 1ml to start, you have a lot of flexibility.
Once you can use sedimentation, you can concentrate the stuff. In the end, by my estimate, the best bulk dehydrant of stuff in a dialysis bag is PEG (polyethylene glycol).
A hypotonic solution (hypotonic as compared to plasms, water or very dilute buffer) will cause the red cells (and the WBCs too I suppose) to swell and burst. Knowing the composition of the 'particles' you are looking for might help.
Interesting problem. SEM would indeed show your particles, but there are lots of things in the given size range, and it won't tell you which ones are birefringent (or even necessarily which ones are yours). Polarized light microscopy will, so that would be a good way to go. An old geological light microscope will do this. There might even be one of these around with a black light, so you could check for fluorescence. If your budget allows, McCrone Research in Chicago can do this -- they're big on polarized light microscopy. Then again ... thinking ... get a couple of 50 - 55 mm diameter polarization filters for a 35mm camera. Linear filters, not circular. Put one above the slide with your sample and the other below, and cross them. This would show birefringence. (Polarized sunglasses lenses would do this, too.) But I wonder ... what else in blood is birefringent? I think you're right about centrifugation, but why not filter first and eliminate everything larger than 3 microns, instead of lysing cells and all that. Then get tricky. What is the composition of the particles? Could they stand up to bleach, NaOH, enzyme detergents? If yes, then after filtering why not put the filtrate into something like one of these to digest the remaining cells and gunk, then spin down and wash the particles. Mind, if these are fluorescent, then I'd just throw the filtrate under a fluorescent microscope. Mind, I don't know if there is much in blood that is autofluorescent. Baxter doesn't have an ordinary fluorescent 'scope? ... our confocal uses a Nipkow disc and Hg lamps, so we can do UV easily. And we have DIC on it (meaning we have the polarizers, the Wollensten filters just need to be taken out of the light path). 8-)
This may be simplistic, but could you just dilute the blood with a very hypotonic solution - dilute saline for example - so the blood cells burst, and presumably things like macrophages would burst too. Then you could spin down the particles, I guess 100 nm particles would need a fairly high g spin, to concentrate them a bit. On a microscope with strain-free objectives, etc for polarised light you will be able to detect birefringent particles of this size and up. cheers,
Rupture the blood cells with osmotic pressure by adding distilled water and then centrifuging or filtering the results to get the partials. I doubt your can see and reliably be able to say what it is less than 500 nm with a light microscope.
I am not sure what sub wavelength birefringent partials do in polarized light but I wouldn't count on them acting like partials } 2 wavelengths in size. Unless you have some pretty good prior research you need see what happens with know bifringent specimens over these size rangers. Preferable the same compounds as you find in blood.
I would talk to your local hospital laboratory's hematology department. Thy will have an instrument for doing automated blood counts. This instrument requires a solution that lyses the red cells. They would very likely be willing to sell or maybe even give you a small amount of it, or you can get a hold of Beckman Coulter, their product is called Coulter Lyse. They also have a whole blood lysing kit in their flow cytometry e-catalog. The URL is http://www.beckman.com/products/instrument/flowcytometry/ecatalog/ProductDet ail.asp?partnumber=6602764. After you lyse the cells you could try filtering the blood through a 5um filter and dialyze.
Just regular, old fashioned polarized light would seem to be the most direct solution. Even if the particles are small, they should respond as bright against the dark background of crossed polars. Based on the size, I would suggest higher mags (60x or even 100x). I would also make sure that you do not use plan apos but use objectives especially suited for polarized light analysis. To test if the system itself might cause a problem, put a sample in place, establish Koehler illumination, then cross the polars to get the blackest background possible. Remove the sample and see if the background stays velvety black. If you do not get velvety black at all steps, the optics are exhibiting strain and will interfer with your imaging. You may also want to inquire about the lab-level microscopes that are built specifically for polarized light analysis. Leica's have been well known for decades and Nikon came out with a new series about 3 years ago.
Re: the size of your particles The good thing about "bright against dark background" types of imaging is that it is not resolution limited: you will be able to detect the particles, even at 100nm. You may not be able to size them at that level, or to get good edge or shape information, but you should be able to detect that they are. Of course, the higher the NA on the objective (and condenser), the better the imaging in general. Oil immersion may not be a bad idea. I had my hands on a 40x/1.3NA two weeks ago which was incredible!
Re: sample prep. You may want to dilute the blood with an isotonic solution, just to avoid overcrowding from red blood cells, but unless the red blood cells are just too thick, I don't see any need to sonicate or do anything else as drastic as you have described.
There you have it. Thanks again for all the suggestions.
Covalent bonding refers to actual chemical bonding or sharing of electrons between the substrate and the coating (e.g. epoxy). A coating of metal on a non-metal surface would stick because of adhesive forces. The strength of this bonding by adhesive forces is depending on surface roughness, percent of the surface in direct contact with the coating, and the relative surface energy of the surface substrate & coating (see contact angle measurements).
The physicists in our group can explain the details of adhesive forces in terms of quantum mechanics.
J. Roy Nelson Material Testing Lab. (609) 730-0575
Jon Krupp wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Hi: } } Someone has asked me to explain the way a sputtered coating of metal bonds } to a surface. } } I am a biologist, so the details are a little sketchy for me. In general } terms, what is the mechanism for the bonding, and could it ever be } considered 'covalent' bonding? } } Thanks } } Jonathan Krupp } Microscopy & Imaging Lab } University of California } Santa Cruz, CA 95064 } (831) 459-2477 } jmkrupp-at-cats.ucsc.edu
} ... } } Adapter manufacturers' websites that I have seen seem to } describe only adapters that suit only one microscope mounting } point ie either C-mount OR 23mm eyepiece tube OR 30mm } eyepiece tube. } } Can someone either point me in the direction of someone who } makes a similarly universal adapter or explain to me why such a } device doesn't work as well as a less-flexible system does? } } ...
Along these same lines, and with regard to my wanting to purchase something similar, how does a fixed adapter accommodate the the difference in stage 'Z' position, while one person will focus on the sample (specific for an interocular binoc setting, e.g., 62mm) and for another person's optical focus (e.g., interocular binoc setting, e.g., 70mm). It would seem, given trinocular head, for making camera focus coincident with optical focus, each user would first, personalize the the binocular, ... and second, fine tune the adapter with camera focus at infinity(???)
It seems to be a truism that subsidized academic SEM labs and microscopy core facilities flourish while unsubsidized labs generally languish and/or die.
Can those of you happily ensconsed in the subsidized facilities please let me know where your funding for staff salary and service contracts comes from? For my lab, at the moment, it's all got to come out of the user fees, helped somewhat by a small institutional supplement per billing hour.
Many thanks, Dee
*************************************************************** Please do not publicly post any of my correspondence without permission
Dee Breger Mgr. SEM/EDX Facility Lamont-Doherty Earth Observatory 61 Route 9W Palisades, NY 10964 USA T: 845/365-8640 F: 845/365-8155
http://www.ldeo.columbia.edu/micro http://www.lsc.org/antarctica/front.html Journeys in Microspace (Columbia University Press, 1995)
Johns Hopkins University in Baltimore Maryland has an opening for an Electron Microscopy Technician. If you are interested please contact me utilizing the indicated information.
Lois Anderson Johns Hopkins University Dept. of Pathology Laboratory Manager Electron Microscopy/Immunofluorescence 600 N. Wolfe Street/Pathology 709 A Baltimore, MD 21287 (410) 955-2861/fax (410) 614-7110 landers-at-jhmi.edu
I am looking for an independent service contractor for microtomes. I have Leica UCT (with cryo), Ultracut E, LKB III, Sorvall MT-2, and microtomes for paraffin. It will be nice if the contractor can also take care of knife maker and cryostat. Our current service contracts are nearing the end.
Thanks,
Gang (Greg) Ning Director, Electron Microscopy Facility Huck Institute for Life Sciences The Pennsylvania State University 1 South Frear Lab University Park, PA 16802 Phone: 814-863-0994 Fax: 814-863-1357 Email: gxn7-at-psu.edu http://www.lsc.psu.edu/stf/em/home.html
A=sheet thickness B=thickness of sheet + pull tab + sticky tab C=Teflon pull tab thickness D=sticky tab thickness (calculated) E=resistance of tab from top to bottom sides F=resistance of tab from left side to right side on top (resistance readings taken under compression)
New Pella tab is stiff as a board. EMS is very similar to old film style Pella tab. Curiously, none of the new tabs are very conductive. Any reading lower than Infinity must be done under compression.
It may be worth comparing the Fullam thin film tab. Although, they are about $30 more per 200 than the EMS tabs.
I am running fluorescence time lapse investigations using a Zeiss Axiovert200M, with an oil immersion x63 lens (with PIFOC) and full environmental chamber. The time frame for collection of planes of focus are every three minutes for a period of twelve to sixteen hours.
My problem is that over this time frame there is a reduction of oil on the objective due to evaporation, hence the images deteriorate over time. To resolve the lens is lowered, addition of more oil, and then reposition the lens hoping that you have not knocked the slide in the process.
Is there an objective heater or similar device available, where oil can be put onto the objective with a minimum of disruption via a pump?
Many thanks,
Steve
Steve Bagley Associate Scientist Applied Imaging Facility Paterson Institute For Cancer Research Cancer Research UK Christie Hospital, Wilmslow Rd, Manchester. M20 9BX, UK
This email is confidential and intended solely for the use of the person(s) ('the intended recipient') to whom it was addressed. Any views or opinions presented are solely those of the author and do not necessarily represent those of the Paterson Institute for Cancer Research or the Christie Hospital NHS Trust. It may contain information that is privileged & confidential within the meaning of applicable law. Accordingly any dissemination, distribution, copying, or other use of this message, or any of its contents, by any person other than the intended recipient may constitute a breach of civil or criminal law and is strictly prohibited. If you are NOT the intended recipient please contact the sender and dispose of this e-mail as soon as possible.
Hi Dee, At the present time, my salary (split 50-50 between the EM and optical microscopy core facilities) and that of a half-time technician are paid by the medical college, from its research environment funding. I must generate enough income in the facility to cover costs of supplies, service contracts and other equipment maintenance costs, and other misc. expenses (phone, computer networking, etc). I do not know how long this relative Shangri La will last, but I will enjoy it while it does. Lee -- Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy Core Facility Manager, Optical Microscopy Core Facility Joan & Sanford I. Weill Medical College of Cornell University voice (212)746-6146 fax (212)746-8175
Can anyone tell me how to measure SEM/STEM beam diameter ("spot size") in the SEM?
I did this twenty years ago and still have the photo records but not the method! The method involved photographing latex spheres at e.g. x30,000.
I recall measuring edge fringes? But, the little grey cells don't want to make the connection anymore! Would it be simply the width of any blurred edge or fringes?
We want to get a "handle" on this in connection with x-ray microanalysis of compartments in frozen unicells in a cryoSEM. Then I want to try some Monte Carlo models (if applicable to biological specimens re. matrix etc) to get some idea of the volume from which the x-rays are derived. We are getting similar results for adjacent areas on area scans (to reduce damage? Ha!). I'm hoping to calculate and to reduce the excited volume - looking for strontium in pulse chase feeding experiments.
Thanks in advance for anything you can offer
Keith Ryan Marine Biological Association Plymouth, UK
I measured the beam diameter on my Dual-Beam FIB using a TEM type sample and a modified SEM mount. The sample is essentially a "low-background" version of the usual SEM resolution standard.
Take a C-coated formvar TEM grid and sputter a few seconds worth of gold on it so that you have gold islands.
SEM mount - Take a ~1/4 inch carbon rod (~3/4 inch long) and drill out the center. Use a 1/8 inch end mill to make a little recess in the end of the rod for the TEM grid. Attach the other end of the C-rod to an SEM stub using silver paint. You now have a vertical C-rod with a recess for the TEM grid. Drilling out the center of the rod makes an effective "Faraday cup" so that you don't have bulk scattering affecting your measurements.
I imaged the sample used the SE detector at up to 800kX (in-lens mode) and was able to look at the intensity profile across the edge of the Au particle. The profile will give you your beam diameter. It was pretty easy to show 1.5nm beam diameter (20% - 80% points on the profile). If I remember correctly, I used Dave Bright's "Lispix" software to extract the profile from the saved images.
cheers, Henk
At 04:54 PM 6/11/2003 +0100, K.P.Ryan wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Hendrik O. Colijn colijn.1-at-osu.edu Campus Electron Optics Facility Ohio State University (614) 292-0674 http://www.ceof.ohio-state.edu Time is that quality of nature which keeps events from happening all at once. Lately it doesn't seem to be working.
We have a JEOL 100S TEM scope (1974) that is in working condition and free to any taker. The scope has been under service contract since the beginning and is currently operating in our lab. Taker must pay any and all relocation fees and assume responsibility for the units upkeep. Please contact me at the e-mail address below if you are interested. Contact Terry at JEOL directly at 847-823-0306 if you want to know about costs of moving and assembly of the scope. Thanks!
Craig M. Klotz B.S., CT (ASCP) Neuromuscular Pathology cklotz-at-mcw.edu EM/Lab Tech. "Chance favors the prepared mind" - L. Pasteur
I think you will find that the spot size is not the critical dimension if you are doing x-ray analysis on thick samples. I would concentrate on a Monte Carlo simulation of the excitation volume given your matrix and beam conditions.
I routinely tell people that the volume is on the order of a micron at 20kV. Of course it is larger for low-Z materials like organics, but at least they get the idea that the volume is _much_ larger than the incident spot size.
Warren
At 04:54 PM 6/11/2003 +0100, you wrote:
} Hello Listers } } Can anyone tell me how to measure SEM/STEM beam diameter ("spot size") in } the SEM? } } I did this twenty years ago and still have the photo records but not the } method! The method involved photographing latex spheres at e.g. x30,000. } } I recall measuring edge fringes? But, the little grey cells don't want to } make the connection anymore! Would it be simply the width of any blurred } edge or fringes? } } We want to get a "handle" on this in connection with x-ray microanalysis of } compartments in frozen unicells in a cryoSEM. Then I want to try some Monte } Carlo models (if applicable to biological specimens re. matrix etc) to } get some idea of the volume from which the x-rays are derived. We are } getting similar results for adjacent areas on area scans (to reduce damage? } Ha!). I'm hoping to calculate and to reduce the excited volume - looking for } strontium in pulse chase feeding experiments. } } Thanks in advance for anything you can offer } } Keith Ryan } Marine Biological Association } Plymouth, UK
------------------------------------------- No files should be attached to this message ------------------------------------------- Warren E. Straszheim, Ph.D. Materials Analysis and Research Lab Iowa State University 46 Town Engineering Ames IA, 50011-3232
Does anyone have experiences using Evans Blue as a counterstain for Red Blood Cell in a cell suspension for confocal microscopy?
I am trying to locate bacterial cell in the infected red blood cells. Bacteria are stained with Alexa-488 similar to FITC. I tried to counterstain RBC with Evans Blue but I can not see them. Is this because the blood has been washed after Evans Blue staining?
I would particularly like to know the diluent, working concentration, length of staining. In addition, is it necessary to wash the RBC after Evans blue staining?
Are there any alternatives recommended for use as a counterstain for RBC?
I have an Anatech Hummer VII sputter coater that is being replaced. This unit is not working (no HV). Pump is OK and control electronics are OK. I suspect that one of the drive oscillator transistors are bad. The main feed voltage circuitry is OK.
I'm replacing this unit with a Denton Desk II and would like to find someone who can fix and use the Hummer. I have the Operation Manual and full schematics. This version is the better one with a single HV transformer rather than two. The HV driver transistors are TIP19 (TO-220). The pain is getting the main PC board out to access the transistors.
The unit comes with Au/Pd, Pt and Al targets (for plating and etching). I also can include a spare Edwards E2M1 pump. Seals are new, glass is perfect. This was recently acquired as a core exchange for an earlier unit that failed.
I can tell you how to "try" to measure it. Warren and others have done pretty much the same.
the method involves doing a line scan across a perfectly sharp edge into a bottomless pit (Faraday cup). I used a Pt aperture but found that it is not really all that sharp. I wound up using a #1 cover slip that was coated with about 200A of Au/Pd. The thickness is not all that important. The goal is to get a line that has good SE at the coating and nothing at the pit. Position the edge at the center of the screen and focus on it. Then up the mag to 200K or so. Then do a line scan and capture. You will get an output like:
----- \ \ \ \ \ \ \ ------- |-----| 20/80%
Do a measurement of 20% down from the top and 20% up from the bottom. Based on the mag, this 20/80% slope will represent the probe passing over the edge...or thereabouts.
Based on the SEMs I have tested, at 10A to 50A rez, I think I have to take the maker's word for this. Really tough to measure!!
gary g.
At 08:54 AM 6/11/2003, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Does anyone know a source (vendor, private person, researcher) for a Nikon 4.0 phase objective: Nikon Plan 4 DL 0.1 160/- PhL
Thank you very much! Michal
Dr. Michal Opas Department of Laboratory Medicine and Pathobiology University of Toronto 1 King's College Circle Medical Sciences Building, room 6326 Toronto, Ontario, M5S 1A8 Canada -------------- phone: (416) 978-8947 (laboratory) (416) 971-2140 (office) fax: (416) 978-5959 email: m.opas-at-utoronto.ca
I'd like to thank all those who passed on their thoughts regarding the possible effects on FEG SEM due to the proximity of tram lines. There certainly appear to be issues that may need consideration if our City Councillors go ahead with their plans.
Frieda Christie, Electron Microscopist, Scientific & Technical Services, Royal Botanic Garden, 20A Inverleith Row, Edinburgh, EH3 5LR
Our facility purchased a basic model XL30-TMP ESEM (tungsten filament) over two years ago. The Peltier cryo attachment was added last year and it appears we may (?) be adding an EDX system later this year.
Please send me your experiences with the different models of energy-dispersive X-ray systems integrated with ESEM microscopes offline. Thank you in advance!
Bruce F. Ingber, Biologist Electron Microscopy USDA-ARS, SRRC 1100 Robert E. Lee Blvd. New Orleans, LA 70124 bingber-at-srrc.ars.usda.gov 504-286-4270 phone 504-286-4419 fax
This is a question about doing surface analysis on a large telescope mirror.
The coating on the mirror is deteriorating after some 10 years in place. It will have to be recoated. Before recoating, we would like to look at the old coating to see if there are any clues as to why it is getting flakey. The mirror is too big to just slip into the SEM.
We can strip the coating off with 'Scotch' tape, but that leaves it 'upside down' for putting into an instrument like an SEM to see the 'top' of the coating. We suspect that there may be some elements, eg sulfur or others that should not be there, but we don't know how to check it out.
I thought of stripping the film with acetate replica tape and dissolving the tape to mount the film right side up and then doing some EDS in our SEM. Think it will work?
Alternatively, does any one have some ideas about some kind of portable device we could take to the mirror and get an idea of the elements present. I thought about a handheld XRF unit, but have no experience with such things.
Thanks, now put your thinking caps on.
Jonathan Krupp Microscopy & Imaging Lab University of California Santa Cruz, CA 95064 (831) 459-2477 jmkrupp-at-cats.ucsc.edu
Hi Liststers, I have a SEM project to do that involves making replicas of teeth. The replicas I have made were made with acetate replicating tape. The guy who gave me the teeth sez it is a kind of powder that one makes into a mixture that one coats the teeth and hardens much like a latex peel. Any advice as to how to make these and which would be better for SEM?
Barbara Plowman Univ. of the Pacific School of Dentistry ph: 415-929-6692 email: Bplowman-at-sf.uop.edu
Jonathan Krupp wrote: ====================================================== Dear Brainiacs:
This is a question about doing surface analysis on a large telescope mirror.
The coating on the mirror is deteriorating after some 10 years in place. It will have to be recoated. Before recoating, we would like to look at the old coating to see if there are any clues as to why it is getting flakey. The mirror is too big to just slip into the SEM.
We can strip the coating off with 'Scotch' tape, but that leaves it 'upside down' for putting into an instrument like an SEM to see the 'top' of the coating. We suspect that there may be some elements, eg sulfur or others that should not be there, but we don't know how to check it out.
I thought of stripping the film with acetate replica tape and dissolving the tape to mount the film right side up and then doing some EDS in our SEM. Think it will work?
Alternatively, does any one have some ideas about some kind of portable device we could take to the mirror and get an idea of the elements present. I thought about a handheld XRF unit, but have no experience with such things
Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (bop00rav-at-sheffield.ac.uk) from http://www.msa.microscopy.com/Ask-A-Microscopist.html on Thursday, June 12, 2003 at 15:30:13 ---------------------------------------------------------------------------
Email: bop00rav-at-sheffield.ac.uk Name: BOB VASEY
Organization: university of sheffield
Education: Graduate College
Location: sheffield, uk
Question: I am using LR White for light microscopy of plant roots. I want to stain with safranin then counterstain with either astra blue or fast green. I am interested in looking at cell walls/lignin etc.
I can stain with safranin ok, but trying to counterstain with Astra Blue or Fast Green does not work. Can anyone tell me why the counterstaining is not working in LR White - it is normally fine in parafin wax.
Does anyone have a protocol for staining with safranin then counterstaining with Astra Blue/Fast Green. Are there any other stains I could counterstain with.
I would be grateful for any suggestions anyone might have, as there is nobody in my lab who can help me.
Although I'm definitely no brainiac {grin} , I've worked with many telescope mirrors in the past as an amateur telescope maker (emphasis on "amateur", by the way). However, I've never encountered one that had the coating "get flakey". Generally what happens is that the coating loses some of its reflectivity (appears dull) due to microscopic pitting which results from long term exposure to air pollution. For Newtonian, Dobsonian, or Cassegrainian telescopes located in or near large urban areas it's not at all uncommon for the primary mirror to need to be recoated after 10 years. A Schmidt-Cassegrain will usually fare better due its design.
EDXS analysis of the coating will most likely yield a wide variety of elements, so it might be difficult to identify the actual culprit (AES or XPS techniques might be more useful). Today's aluminized mirrors typically have multilayer coatings consisting of titanium dioxide, silicon dioxide (or silicon monoxide), followed by magnesium fluoride as the outermost layer. For EDXS work, you might try to remove some of the flakes by using the adhesive side of a 3M "Sticky Note". For general EDXS work these are not considered to leave any significant residue. If you can remove some of the flakes this way then it should be easy to transfer them "right side up" to a piece of carbon tape, etc. It would also be a good idea to examine the underside of the flakes since this is where adhesion appears to be deteriorating, if I understand you correctly. I would not be surprised at all to detect sulfur on the flakes due to air pollution.
I'm also curious as to whether the aluminum is flaking off, too, or just the protective coatings? Either way, this is undesirable of course, but after 10 years of use in California I'm not so sure it would be considered particularly unusual (I'm assuming the telescope is located at or near the University).
I hope this helps! I'd love to hear more about the telescope and your analysis results if you'd like to write to me off-list.
Best regards,
Sue Kent Senior Staff Engineer Motorola, Inc. 21440 W. Lake Cook Road Deer Park, IL 60010 847-862-0216
Jonathan Plastic stripping could work. The traditional method of Juniper and Bradley (1958) Journal of Ultrastructure Research 2, 16-27 for stripping replicas from leaves (it works for other surfaces too) could be modified to suit your requirements. In this method, an acetone-soluble resin (Bedacryl - is it still available?) was employed as a separation layer between acetone-insoluble formvar-supported replica and the adhesive tape. The sequence was replica - formvar - bedacryl - tape - strip - dissolve bedacryl in acetone - dissolve formvar in chloroform.
To reverse the replica onto a stub you could do: bedacryl - tape - strip - formvar support the back of the stripped replica - dissolve bedacryl in acetone - attach formvar supported replica to stub.
However, a) the offending contaminant may be soluble b) the stripping method will only succeed in removing material that has poor adhesion to the glass. Your problem may be a tightly adherent contaminant film on the surface of the glass to which the coating doesn't stick. Stripping will leave it behind. On the other hand, if there is a contaminant layer that sticks to the coating but not too well to the glass your Scotch tape method puts it exactly where you want it.
Have you considered using Raman spectroscopy?
Chris
} } Dear Brainiacs: } } This is a question about doing surface analysis on a large telescope } mirror. } } The coating on the mirror is deteriorating after some 10 years in place. It } will have to be recoated. Before recoating, we would like to look at } the } old coating to see if there are any clues as to why it is getting } flakey. } The mirror is too big to just slip into the SEM. } } We can strip the coating off with 'Scotch' tape, but that leaves it } 'upside } down' for putting into an instrument like an SEM to see the 'top' of } the } coating. We suspect that there may be some elements, eg sulfur or } others } that should not be there, but we don't know how to check it out. } } I thought of stripping the film with acetate replica tape and dissolving } the tape to mount the film right side up and then doing some EDS in our } } SEM. Think it will work? } } Alternatively, does any one have some ideas about some kind of portable } device we could take to the mirror and get an idea of the elements present. } I thought about a handheld XRF unit, but have no experience with such } things. } } Thanks, now put your thinking caps on. } } Jonathan Krupp } Microscopy & Imaging Lab } University of California } Santa Cruz, CA 95064 } (831) 459-2477 } jmkrupp-at-cats.ucsc.edu } } }
I would think the interface of interest with a front surface coated mirror would be the one between the glass and the coating, particularly since it is flaking. Therefore, if you remove it using tape, that surface will be right- side up, as would be preferable. Since you mentioned that the surface, which I assume is aluminum, is flaking, can it simply be picked off with a pair of tweezers and both surfaces analyzed?
Regards,
Peter Tomic Agere Systems Allentown, PA
-----Original Message----- } From: jmkrupp-at-cats.ucsc.edu [mailto:jmkrupp-at-cats.ucsc.edu] Sent: Thursday, June 12, 2003 2:57 PM To: Microscopy-at-sparc5.microscopy.com
Dear Brainiacs:
This is a question about doing surface analysis on a large telescope mirror.
The coating on the mirror is deteriorating after some 10 years in place. It will have to be recoated. Before recoating, we would like to look at the old coating to see if there are any clues as to why it is getting flakey. The mirror is too big to just slip into the SEM.
We can strip the coating off with 'Scotch' tape, but that leaves it 'upside down' for putting into an instrument like an SEM to see the 'top' of the coating. We suspect that there may be some elements, eg sulfur or others that should not be there, but we don't know how to check it out.
I thought of stripping the film with acetate replica tape and dissolving the tape to mount the film right side up and then doing some EDS in our SEM. Think it will work?
Alternatively, does any one have some ideas about some kind of portable device we could take to the mirror and get an idea of the elements present. I thought about a handheld XRF unit, but have no experience with such things.
Thanks, now put your thinking caps on.
Jonathan Krupp Microscopy & Imaging Lab University of California Santa Cruz, CA 95064 (831) 459-2477 jmkrupp-at-cats.ucsc.edu
One has to consider both external and internal sources of potential destructive agents and you cannot exclude the glass itself. I assume this is not a hobbyist scope and that it is valuable enough to have a documented fabrication? Before proceeding too far you need to assemble the information about the materials that were used and any related experience on other scopes. You haven't indicated what the metallization is, whether is monometallic or layered, etc. The glass composition and whether other mirrors were made of the same lot, and how they have performed are all important. You should consider that the glass may be a source of either ionic contaminants or of alkali that may move the corrosion potential of the metal significantly away from the area of stability that would apply in their absence.
With the role of the glass in mind, you should consider that you might need to perform "control" analyses on non-metallized portions of the mirror. Defects related to glass breakdown may only be obvious where the metallization makes them stand out but they may be occurring everywhere.
Bear in mind that ionics often have a significant solublility in organic solvents. Therefore, attempts to replicate the defects, although a wise idea, may remove the causative agents.
Solvents themselves may also serve to introduce traces of ionics. This is particularly true of chloroform, mentioned in Chris' scheme to dissolve formvar, which can develop free chlorides if not properly stored (dry and dark). For this reason, if you expect to perform electron probe analyses on any replicas you probably should make the effort to use fresh, electronic grade solvents rather than reagent grades.
John Twilley Conservation Scientist
Jon Krupp wrote: } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Dear Brainiacs: } } This is a question about doing surface analysis on a large telescope mirror. } } The coating on the mirror is deteriorating after some 10 years in place. It } will have to be recoated. Before recoating, we would like to look at the } old coating to see if there are any clues as to why it is getting flakey. } The mirror is too big to just slip into the SEM. } } We can strip the coating off with 'Scotch' tape, but that leaves it 'upside } down' for putting into an instrument like an SEM to see the 'top' of the } coating. We suspect that there may be some elements, eg sulfur or others } that should not be there, but we don't know how to check it out. } } I thought of stripping the film with acetate replica tape and dissolving } the tape to mount the film right side up and then doing some EDS in our } SEM. Think it will work? } } Alternatively, does any one have some ideas about some kind of portable } device we could take to the mirror and get an idea of the elements present. } I thought about a handheld XRF unit, but have no experience with such } things. } } Thanks, now put your thinking caps on. } } Jonathan Krupp } Microscopy & Imaging Lab } University of California } Santa Cruz, CA 95064 } (831) 459-2477 } jmkrupp-at-cats.ucsc.edu
Your replicating idea and then dissolving the acetate film is probably the best bet. An XRF unit could give you some information, but the X-ray have a relatively long penetration depth and so most of the signal will come from the substrate for a thin film. You would need to measure substrate/film and subtract substrate only to get what your film is and there would be problems with statistics. Portable units are EDS type and I am not sure what the range of elements that can be analyzed with them. I thought that the range is limited, but I may be wrong.
Actually, using the Scotch(R) adhesive tape might be the best approach for analyzing the failure. The failure is probably at the interface of the substrate/film or at an interface of one of the layers. By pulling the film off with the tape, you have revealed the interface that has failed. You should then apply a surface analysis technique such as X-ray photoelectron spectroscopy or Auger electron spectroscopy to look at the chemistry of the surface. If it is peeling, see if you can get a sufficiently large piece without the tape or replicating tape.
-Scott
Scott D. Walck, Ph.D. PPG Industries, Inc. Glass Technology Center P. O. Box 11472 (letters) Guys Run Rd. (packages) Pittsburgh, PA 15238-0472
Walck-at-PPG.com
(412) 820-8651 (office) (412) 820-8515 (fax)
-----Original Message----- } From: jmkrupp-at-cats.ucsc.edu [mailto:jmkrupp-at-cats.ucsc.edu] Sent: Thursday, June 12, 2003 2:57 PM To: Microscopy-at-sparc5.microscopy.com
Dear Brainiacs:
This is a question about doing surface analysis on a large telescope mirror.
The coating on the mirror is deteriorating after some 10 years in place. It will have to be recoated. Before recoating, we would like to look at the old coating to see if there are any clues as to why it is getting flakey. The mirror is too big to just slip into the SEM.
We can strip the coating off with 'Scotch' tape, but that leaves it 'upside down' for putting into an instrument like an SEM to see the 'top' of the coating. We suspect that there may be some elements, eg sulfur or others that should not be there, but we don't know how to check it out.
I thought of stripping the film with acetate replica tape and dissolving the tape to mount the film right side up and then doing some EDS in our SEM. Think it will work?
Alternatively, does any one have some ideas about some kind of portable device we could take to the mirror and get an idea of the elements present. I thought about a handheld XRF unit, but have no experience with such things.
Thanks, now put your thinking caps on.
Jonathan Krupp Microscopy & Imaging Lab University of California Santa Cruz, CA 95064 (831) 459-2477 jmkrupp-at-cats.ucsc.edu
KeyMaster has a new handheld XRF system that goes down to Fl - see http://www.keymastertech.com
Quoting "Garber, Charles A." {cgarber-at-2spi.com} :
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- } http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } -- [ From: Garber, Charles A. * EMC.Ver #3.1 ] -- } } Jonathan Krupp wrote: } ====================================================== } Dear Brainiacs: } } This is a question about doing surface analysis on a large telescope mirror. } } The coating on the mirror is deteriorating after some 10 years in place. It } will have to be recoated. Before recoating, we would like to look at the old } coating to see if there are any clues as to why it is getting flakey. The } mirror is too big to just slip into the SEM. } } We can strip the coating off with 'Scotch' tape, but that leaves it 'upside } down' for putting into an instrument like an SEM to see the 'top' of the } coating. We suspect that there may be some elements, eg sulfur or others } that should not be there, but we don't know how to check it out. } } I thought of stripping the film with acetate replica tape and dissolving the } tape to mount the film right side up and then doing some EDS in our SEM. } Think it will work? } } Alternatively, does any one have some ideas about some kind of portable } device we could take to the mirror and get an idea of the elements present. } I thought about a handheld XRF unit, but have no experience with such things }
I have been using polystyrene beads (10-200 nm) to study uptake by cells. The product from polysciences has worked well, but these beads are almost impossible to see by TEM. Are there any suggestions as to what I can use in this size range that are electron dense? Is there a way to stain the polystyrene? I do not think gold is appropriate since it is hydrophobic and may interact in ways that I do not want.
Thanx
David ____________________
David Elliott
Yale University School of Medicine 810 LCI 333 Cedar Street New Haven, CT 06520-8022
A colleague of mine did her PhD thesis on the analysis of fossil teeth and discovered a super technique. Her name is Gina Semprebon and she is a professor at Bay Path College in Longmeadow, MA. I've left her a voice mail, asking for her email, so if you contact me off line early next week, I'll try to put the two of you together.
Best regards, Barbara Foster Microscopy/Microscopy Education 125 Paridon Street, Suite 102 Springfield, MA 01118 PH: 413-746-6931 FX: 413-746-9311 Web: www.MicroscopyEducation.com
Will you be at M&M in San Antonio? If so, don't forget the Tuesday night seminar on Fluorescence Calibration. Also, join the tradition of over 10,000 microscopists: participate in our survey at any time during the meeting and receive a "sweet thank you". Booth #218 -at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at-
At 03:10 PM 6/12/03 -0700, Barbara Plowman wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Our group is looking for a simple, conventional manual Rotation Microtome using metal blades, for paraplast and acrylat-embedded sections.
We are looking for an economic purchase, such as a second hand, demontration model, stock fade-out, etc. The microtome should be in good condition, requiring no or only minimal repairs.
If you can help us, reply to Matyas Buzgo, buzgo-at-systbot.unizh.ch Botany Department, University of Florida Gainesville 32611-8526 USA
I have a glass- nono-crystallites composite. Nanocrystallites may be ferroelectric in nature and I need to prove that nanocrystallites in the glassy phase are ferroelectric.
How can i prove using TEM that these nanocrystallites(probably single domain) are indeed ferroelectric?
Thanks Pradyumna N Gupta 5 East Packer Avenue, Whitaker Lab, MSE Lehigh University, Bethlehem, PA 18015 610 758 5590 Lab
Pradyumna N Gupta 5 East Packer Avenue, Whitaker Lab, MSE Lehigh University, Bethlehem, PA 18015 610 758 5590 Lab
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I have a glass- nono-crystallites composite. Nanocrystallites may be ferroelectric in nature and I need to prove that nanocrystallites in the glassy phase are ferroelectric.
How can i prove using TEM that these nanocrystallites(probably single domain) are indeed ferroelectric?
Pradyumna N Gupta MSE Lehigh University
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I am obtaining results on what influences people's purchasing decisions when they purchase scanning probe microscopes. If you have a moment, could you please fill out the following survey and return it to me via email? Your responses will remain anonymous. Thank you for your help.
Please, press reply, and on the following questions, please put the x near to the number, or bold or circle the number that most closely reflects your opinion when you purchase scanning probe microscopes and accessories. My e-mail address is Mickey-at-pnl.gov
How important to you is/are: Extremely Extremely Unimportant Important Price 1 2 3 4 5 Terms of delivery 1 2 3 4 5 Simple and intuitive user interface 1 2 3 4 5 Ergonomic instrument design 1 2 3 4 5 Open architecture and open software code 1 2 3 4 5 Igor Pro software Interface 1 2 3 4 5 Visual Basic interface 1 2 3 4 5 C/C++ library/interface 1 2 3 4 5 LabView interface 1 2 3 4 5 1 yr. warranty 1 2 3 4 5 3 yr. warranty 1 2 3 4 5 Annual service agreement 1 2 3 4 5 Detailed instruction manual (printed) 1 2 3 4 5 Multimedia training materials (DVD, web, video...)1 2 3 4 5 Prompt service and technical support 1 2 3 4 5 Knowledgeable customer and technical support 1 2 3 4 5 Instrument customization help and support 1 2 3 4 5 Web-based customer support 1 2 3 4 5 On-site product demonstration 1 2 3 4 5 Off-site product demonstration 1 2 3 4 5 On-site training 1 2 3 4 5 Off-site training 1 2 3 4 5 Web-based training 1 2 3 4 5 Product brochure 1 2 3 4 5 Previous publications utilizing particular product1 2 3 4 5 Instrument upgrade's capabilities 1 2 3 4 5 Reputation and previous experience 1 2 3 4 5 Resolution 1 2 3 4 5 Scan size 1 2 3 4 5 Environmental control 1 2 3 4 5 Integration with optical microscopes 1 2 3 4 5 STM 1 2 3 4 5 Contact mode AFM 1 2 3 4 5 AFM force spectroscopy 1 2 3 4 5 Tapping mode AFM or equivalent 1 2 3 4 5 Shear-Force mode 1 2 3 4 5 Pulsed-force mode or equivalent 1 2 3 4 5 Magnetic force mode 1 2 3 4 5 NSOM mode 1 2 3 4 5 E-chem SPM 1 2 3 4 5
On the following questions, please put the x near to the number, or bold or circle the number that most closely reflects your opinion.
How similar is: Extremely Extremely Similar Dissimilar
Veeco/DI to Asylum 1 2 3 4 5 Veeco/DI to Molecular Imaging 1 2 3 4 5 Veeco/DI to Omicron 1 2 3 4 5 Veeco/DI to WiTec 1 2 3 4 5 Veeco/DI to MT NDT 1 2 3 4 5 Asylum to Molecular Imaging 1 2 3 4 5 Asylum to Omicron 1 2 3 4 5 Asylum to WiTec 1 2 3 4 5 Asylum to MT NDT 1 2 3 4 5 Molecular Imaging to Omicron 1 2 3 4 5 Molecular Imaging to WiTec 1 2 3 4 5 Molecular Imaging to MT NDT 1 2 3 4 5 Omicron to WiTec 1 2 3 4 5 Omicron to MT NDT 1 2 3 4 5 Witec to MT NDT 1 2 3 4 5
Finally, please respond to the following questions:
Almost Almost Never Always
When I purchase SPM, I buy Veeco/DI 1 2 3 4 5 When I purchase SPM,, I buy Molecular Imaging 1 2 3 4 5 When I purchase SPM, I buy Asylum 1 2 3 4 5 When I purchase SPM, I buy Omicron 1 2 3 4 5 When I purchase SPM, I buy WiTec 1 2 3 4 5 When I purchase SPM, I buy MT NDT 1 2 3 4 5
Thank you for your time! The survey results will be available in three weeks !
Miodrag "Mickey" Micic mickey-at-pnl.gov ========================================================================== Miodrag Micic, Ph.D. Pacific Northwest National Lab, M/S K8-88, P.O.Box. 999 3335 Q. Avenue, Richland, WA 99352-999, USA. phone: 1-509-376-5394 fax: 1-509-376-6066 E-mail: Miodrag.Micic-at-pnl.gov
"But the fact that some geniuses were laughed at does not imply that all who are laughed at are geniuses. They laughed at Fulton, they laughed at the Wright brothers. But they also laughed at Bozo the Clown". Carl Sagan =========================================================================
We've used SEM of replicas for analysis of microwear on fossil teeth here. I was not involved in the specimen prep, but below is a reply from the researcher that was. (If you have further questions, let me know).
} Kevin, } } I don't know anything about the type of replicas described below. But for dental microwear analysis of fossil teeth, we use Colténe President Jet Plus (a polyvinylsiloxane material) to make tooth molds and then pour casts with epoxy. The epoxy is centrifuged to remove bubbles. The replicas look pretty precise in the SEM, even at thousands of times magnification.
------------------------------------------------ Kevin Frischmann Microscopy & Imaging Facility American Museum of Natural History Central Park West at 79th Street New York, NY 10024-5192 USA
At 03:10 PM 6/12/03 -0700, Barbara Plowman wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
OK...I too have been thinking about this interesting problem.
It seems to me that giving EDS a shot is a good start. Since the issue is more of a film or surface issue, EDS may give way to WDS or SIMS. Anyway, a first method is based on EDS. The problem (among others) is to reduce the volumetric interaction of a sample. I would suggest (naively?) that you use a SEM sample stub with a sticky tab to pull off some of the crud from the glass. Not elegant but potentially useful.
You can use an Aluminum stub or a Carbon stub. If you expect to find either of these elements in the material on the glass, volumetric interaction must be reduced. This can be done by coating the stub with a material that would not be thought to exist on the glass surface. One could use Au, Au/Pd or Pt. A reasonably thick coating would stop the beam from penetrating much into the substrate. A Monte Carlo simulation can be used to get a good idea of KV and film thickness relative to penetration. It is unlikely, I would think, that Al would be found on the glass. If so, an Al stub would work fine. Coat it with, say, Pt and then put the sticky tab on top. Do an EDS baseline of the stub and tab. Then, collect material from the glass. Re-do EDS and subtract the baseline from the specimen reading. That should allow C to be in the picture.
When you do the EDS analysis, also subtract/cancel out the noble metal(s) such that the contaminants are the only ones left.
Very interesting problem. let us know how it turns out!
gary g.
} Dear Brainiacs: } } } This is a question about doing surface analysis on a large telescope mirror. } } } The coating on the mirror is deteriorating after some 10 years in place. It } will have to be recoated. Before recoating, we would like to look at the } old coating to see if there are any clues as to why it is getting flakey. } The mirror is too big to just slip into the SEM. } } } We can strip the coating off with 'Scotch' tape, but that leaves it 'upside } down' for putting into an instrument like an SEM to see the 'top' of the } coating. We suspect that there may be some elements, eg sulfur or others } that should not be there, but we don't know how to check it out. } } } I thought of stripping the film with acetate replica tape and dissolving } the tape to mount the film right side up and then doing some EDS in our } SEM. Think it will work? } } } Alternatively, does any one have some ideas about some kind of portable } device we could take to the mirror and get an idea of the elements present. } I thought about a handheld XRF unit, but have no experience with such } things. } } } Thanks, now put your thinking caps on. } } } Jonathan Krupp } Microscopy & Imaging Lab } University of California } Santa Cruz, CA 95064 } (831) 459-2477 } jmkrupp-at-cats.ucsc.edu
Here are some additonal explnataion of my survey request. It is not intented for commercial purposes, and I appologise to Dr. Zaluzec for not informing him in advance. Besides being a postdoc, I am a part-time graduate student at the Washington State University, Tri-Cities campus, in the master of technology management program, see the link http://www.tricity.wsu.edu/business/mtm.html . This survey is for final project in my marketing class, and it do not have any commercial intention. Again,I do not have any product, neither I am promoting any product . This particular survey, is designed to provide 3 answers:
1. perceptual map of the current scanning probe microscopes brands (questions like compare, for example brand A with brand B), and are neutral, i.e. it cant be used to promote or degrade any mark, as I only ask similar/dissimilarity questions, to get clustering data.
2. attributes that drive consumer decision (how important it is to you)
3. correlation matrix questions, (when I buy AFM, I buy _____), which are use to correlate with the perceptual map, from the question 1.
Furthermore, I will make survey results publiclly available in a matter which is not promoting or derogating any particular product or brand. I will be glad to disclose results to you and subsequently the the list.
I am looking forward to hearing from you, and thanking you in advance for your participation,
I would kindly ask you to complete my survey quantiative survey on AFM, which is now on-line at http://www.AdvancedSurvey.com/default.asp?SurveyID=6098 . It takes about 4 minutes to complete it, and you can see current results at the end of the survey.
I would kindly ask you to complete my survey quantiative survey on AFM, which is now on-line at http://www.AdvancedSurvey.com/default.asp?SurveyID=6098 . It takes about 4 minutes to complete it, and you can see current results at the end of the survey.
Please take note of the updated page of The International School on Electron Microscopy of Powdered Nanostructured Materials to be hold in Wroc³aw (Poland), 19-20 September 2003. You will find there the program and the registration form. For complete information, please look at: http://celtam.int.pan.wroc.pl/ISEM/ Or Email: kepinski-at-int.pan.wroc.pl
Looking forward to seeing you in Wroc³aw, Organizing Committee,
Contact: Dr. Leszek Kepinski Institute of Low Temperature and Structure Research, Polish Academy of Sciences, P.O.Box 1410, 50-950 Wroclaw, Poland http://www.int.pan.wroc.pl/
Dear List, I will be attending MSA, and I would like to see if anyone would be willing to share a room with me to cut down on expenses. Please reply off-list. TIA. Yours, Bill Tivol EM Scientist and Manager Cryo-Electron Microscopy Facility Broad Center, Mail Code 114-96 California Institute of Technology Pasadena CA 91125 (626) 395-8833 tivol-at-caltech.edu
Dear Pradyumna The best way to detect and image ferroelectric inclusions in paraelectric matrix is to use a scanning probe microscopy techniques, namely piezoresponse force microscopy. This technique is specifically designed to distinguish piezoelectric and non-piezoelectric materials, and hence is probably the best choice in this case. It will be sensitive only to crystallites exposed on the surface or relatively shallow within the glass matrix (~10 nm) and will allow imaging of both in-plane and out-of-plane polarization components. You can find some examples in paper A.Y. Borisevich, S.V. Kalinin, D.A. Bonnell, and P.K. Davies, Analysis of phase distributions in the Li2O-Nb2O5-TiO2 system by piezoresponse imaging, J. Mater. Res. 16(2), 329-332 (2001). The extension of PFM, piezoresponse spectroscopy normally allows spatially resolved electromechanical hysteresis loop measurements, e.g. within single 50 nm grain, but it might not work for the crystal in the dielectric matrix. Yours Sergei
-- Sergei V. Kalinin, Oak Ridge National Laboratory, 1 Bethel Valley Rd, Bldg. 3025, MS6030, Oak Ridge, TN 37831 Phone: (865) 241-0236 FAX: (865) 574-4143 URL: sergei2.kalininweb.com
Dear Pradyumna The best way to detect and image ferroelectric inclusions in paraelectric matrix is to use a scanning probe microscopy techniques, namely piezoresponse force microscopy. This technique is specifically designed to distinguish piezoelectric and non-piezoelectric materials, and hence is probably the best choice in this case. It will be sensitive only to crystallites exposed on the surface or relatively shallow within the glass matrix (~10 nm) and will allow imaging of both in-plane and out-of-plane polarization components. You can find some examples in paper A.Y. Borisevich, S.V. Kalinin, D.A. Bonnell, and P.K. Davies, Analysis of phase distributions in the Li2O-Nb2O5-TiO2 system by piezoresponse imaging, J. Mater. Res. 16(2), 329-332 (2001). The extension of PFM, piezoresponse spectroscopy normally allows spatially resolved electromechanical hysteresis loop measurements, e.g. within single 50 nm grain, but it might not work for the crystal in the dielectric matrix. Yours Sergei
-- Sergei V. Kalinin, Oak Ridge National Laboratory, 1 Bethel Valley Rd, Bldg. 3025, MS6030, Oak Ridge, TN 37831 Phone: (865) 241-0236 FAX: (865) 574-4143 URL: sergei2.kalininweb.com
----- Original Message ----- } From: "Pradyumna N Gupta" {microprady-at-lehigh.edu} To: {Microscopy-at-sparc5.microscopy.com} Sent: Friday, June 13, 2003 4:05 PM
Hi, we have a problem with gun alignment of Cameca su-30...At such } mags 3000 or higher we can not get clear SEM images, we try to make } gun alignment in wobb mode by using the diaphragms...Does anyone know } the procedure which we have to follow(wobb, diaphragms, } astigmatism....)? Do we need to clean the apertures? But how? thanks...
Dear listers, Is there any way to quantify the lowest detection limit of SEM and compare it to ppm range?
I have a customer who would like to find out if chlorine from an ink marker diffused into the structure of 17-4 PH alloy? If it did, how deep? The customer is asking what is my detection limit for chlorine 100ppm? 200ppm?
Any advice is appreciated
Pavel Lozovyy ATC SEM Lab Phone: 216-692-6637 E-mail: atclabs-at-sbcglobal.net web: www.atclabs.com
We plan to use some internal fund to purchase a 2nd hand CM120 scope or the similar as soon as practical. Should you have one or know related info on this, please let me know. Thanks much!
**************************************** Chaoying Ni The W.M. Keck Electron Microscope Facility Facility location: 022 Spencer Laboratory Mailing address: 201 duPont Hall Dept of Matls Sci & Eng University of Delaware Newark, DE 19716 (302) 831-8354 (O); -2318(L); -4545(Fax) http://eml.masc.udel.edu *****************************************
Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (venu-at-purdue.edu) from http://www.msa.microscopy.com/Ask-A-Microscopist.html on Monday, June 16, 2003 at 17:45:28 ---------------------------------------------------------------------------
Email: venu-at-purdue.edu Name: Venu
Organization: Purdue University
Education: Graduate College
Location: City, State, Country
Question: Could you please tell me the imaging requirements (tools needed) and the proper technique to observe 30-40 nm gold particles using light microscopy?
This e-mail concerns long-wave ultraviolet stereomicroscope illumination and eye protection.
I recently inspected a long-wave UV ringlight stereomicroscope accessory (StockerYale Model 18 Superlight ringlight) at a trade show. Visual results for specimen inspection and preparation were remarkable, but the ringlight was not fitted with a barrier filter to block long-wave UV wavelengths from observation and did not appear sufficiently bright to tolerate a barrier filter. What eye safety precautions, if any, would be recommended or required to use such an illuminator?
We have being doing EM on neurons that were cultured on various coverslips, recently for a specific experiment, we have to use the poly-D-lysine coated coverslips. The problem is that it is very difficult to remove this type of coverslips as comparing to matrigel-coated ones at the end of polymerization, and we can not use Thermanox for this culture. The embedding medium we used is polybed/812 and we normally use liquid nitrogen to separate cells from the coverslips, and it works very well with matrigel-coverslips.
Any suggestions and thoughts will be highly appreciated.
Xinran
Xinran Liu, M.D., Ph.D. Center for Basic Neuroscience UT Southwestern Medical Center at Dallas Phone: 214-648-1830 Fax: 214-648-1801 E-mail: xinran.liu-at-utsouthwestern.edu
After waiting for a long time, we must now choose in hurry a new EDS system for our TEM. Our old Kevex died ((un)fortunately ?!) and after a few cycle monney-no monney-monney-no monney etc, we are again in a "monney" phase, and we must spend it fast ! So fast that we have no time to do test.
So any advises are wanted about the Noran Six, the PGT Avalon, the Oxford INCA and the EDAX Genesis EDS systems, to fit on a 200 keV TOPCON 02B TEM, and to be used in a material research lab, on magnetic materials, catalysts, bio-materials, etc. We know a little bit the PGT and the Oxford, I use personnaly the Noran Vantage, but the Six is new (only the GUI or the electronics too ?), and we know nothing (other than the manufacturer's doc) about the EDAX Genesis.
Any advise too about reparing/upgrading our old KEVEX detector, or buying a new one (much more monney). The old one worked fine before it died, and it is bellow seald, with the length and the transfer mechanism adapted to the TEM.
Answer please on the list, direct to me or to Jacques.Werckmann-at-ipcms.u-strasbg.fr
Many thanks
J. Faerber IPCMS-GSI (Institut de Physique et Chimie des Matériaux de Strasbourg Groupe Surface et Interfaces) 23, rue de Loess ; BP43 67034 Strasbourg CEDEX 2 France
I want order a LaB6 cathode from FEI Beam Tech and cannot get through to a person. Their automated phone system requires that you know who you want to talk to, no operator!! Duh!
Does anyone know a contact person there?
Owen
Owen P. Mills Electron Optics Engineer Materials Science & Engineering Michigan Technological University Rm 512 M&M Bldg. Houghton, MI 49931 PH 906-369-1875 FAX 906-487-2934 mailto:opmills-at-mtu.edu http://www.mm.mtu.edu/~opmills
Hi, everyone: An investigator here is looking at the changes in ribosomes with different chemical treatments. Can anyone recommend a fixation and/or staining procedure that will enhance the contrast of ribosomes at ultrastructural level? Thank you in advance and I look forward to hearing from you.
Hong Yi Emory School of Medicine EM Tel: (404) 727-8692 (Office), (404) 712-8491 (Lab) Fax: (404) 727-3157
It sounds like you may be already doing this but when we "pop" our cells off ECM coated glass coverslips in liquid nitrogen, we find that it is important to only polymerize for 6 hrs (if we go longer, they don't pop off well)- when we first take the coverslips out of the oven they can be slightly gooey but after they cool for 15-20 min, they are "tacky" but no longer gooey. we cross-hatch the surface with a razor blade and slowly immerse in liquid nitrogen. we have used this for matrigel but not in many years.
At 10:48 AM 6/17/2003 -0500, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Thomas E. Phillips, PhD Associate Professor of Biological Sciences Director, Molecular Cytology Core 3 Tucker Hall University of Missouri Columbia, MO 65211-7400
FEI is experiencing growing pains. They are in the process of moving out from one campus into their new facility. You can reach them at this number: 503-640-7500. Since they are local to us, I called and confirmed this is the number to reach them. Hope this helps!
John Quintero MikroMasch USA http://www.spmtips.com 7086 SW Beveland St. Portland, OR 97223 john-at-mikromasch.com Office: 503-598-9828 Fax: 503-598-9721
Dear Yi Ribosome (RS) is about 28 nm in diameter and wery fagile. You may not see the structure of RS on the conventional ultrathin sections with standard processing. You also could not see fine structure on the chemically fixed ribosomes. The only one way known to me to investigate the fine structure of the free ribosomes in vitro is cryo-EM with 3D-reconstruction. Please, made search for Joachim Frank works. Ribosome is extremely sensitive and complicated "machine": you have to perform state of the art biochemistry to isolate them intact. Usually we are using RNAase-free strains or thermophilic bacteria to isolate ribosomes. As far as I know, there is just a few places in US, where people is able to isolate intact ribosomes. Of coarse, "intact" is very subjective word. You may check Harry Noller works for RS isolation. Best wishes, Sergey
At 12:07 PM 6/17/2003, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
_____________________________________
Sergey Ryazantsev Ph. D. Electron Microscopy UCLA School of Medicine Department of Biological Chemistry 10833 Le Conte Ave, Room 33-089 Los Angeles, CA 90095
I'd say about 0.1% ie 1,000 ppm in the absence of any complicating factors, or 5000ppm with more confidence.
cheers
rtch
} From: "AtcSEM" {atcsem-at-sbcglobal.net} To: {MIcroscopy-at-sparc5.microscopy.com}
To all TEM facilities:
I need someone to help me with plant viruses/TEM research. I do have TEM background but do not have an electron microscope(none in Alaska). I need to look at purified viruses and viruses in plant tissue. I am looking for a EM facility that either contracts research projects or allows one to use their facility for a short segment of time. My immediate need is to have grids of purified virus or leaf dips looked at for virus particles.
Thank you,
Nancy Robertson
Nancy L. Robertson Research Plant Pathologist USDA, ARS 533 E. Fireweed Ave. Palmer Alaska 99645 907-746-9465 office 907-746-2898 lab 907-746-2677 fax ffnlr-at-uaf.edu
} } Question: Could you please tell me the imaging requirements (tools } needed) and the proper technique to observe 30-40 nm gold particles } using light microscopy? }
I don't think you have any chance with LM, but I'll be interested to hear if there is a way.
cheers
rtch
-- Ritchie Sims Ph D Phone : 64 9 3737599 ext 87713 Microanalyst Fax : 64 9 3737435 Department of Geology email : r.sims-at-auckland.ac.nz The University of Auckland Private Bag 92019 Auckland New Zealand
If you mean EDS analysis, then you can't get any lower then 2-3 %. ppm level isn't for EDS at all.
Regards,
Vladimir Igoshev, Ph.D. Toronto, Canada ----- Original Message ----- } From: "AtcSEM" {atcsem-at-sbcglobal.net} To: {MIcroscopy-at-sparc5.microscopy.com} Sent: Tuesday, June 17, 2003 8:49 AM
I have several EDAX and 2 Oxford systems, and like Oxford the better.
**************************************** Chaoying Ni, PhD The W.M. Keck Electron Microscope Facility Facility location: 022 Spencer Laboratory Mailing address: 201 duPont Hall Dept of Matls Sci & Eng University of Delaware Newark, DE 19716 (302) 831-8354 (O); -2318(L); -4545(Fax) http://eml.masc.udel.edu *****************************************
On Tue, 17 Jun 2003, Faerber Jacques wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } } Hi All } } After waiting for a long time, we must now choose in hurry a new EDS } system for our TEM. Our old Kevex died ((un)fortunately ?!) and after a } few cycle monney-no monney-monney-no monney etc, we are again in a } "monney" phase, and we must spend it fast ! So fast that we have no time } to do test. } } So any advises are wanted about the Noran Six, the PGT Avalon, the Oxford } INCA and the EDAX Genesis EDS systems, to fit on a 200 keV TOPCON 02B TEM, } and to be used in a material research lab, on magnetic materials, } catalysts, bio-materials, etc. We know a little bit the PGT and the } Oxford, I use personnaly the Noran Vantage, but the Six is new (only the } GUI or the electronics too ?), and we know nothing (other than the } manufacturer's doc) about the EDAX Genesis. } } Any advise too about reparing/upgrading our old KEVEX detector, or buying } a new one (much more monney). The old one worked fine before it died, and } it is bellow seald, with the length and the transfer mechanism adapted to } the TEM. } } Answer please on the list, direct to me or to } Jacques.Werckmann-at-ipcms.u-strasbg.fr } } Many thanks } } J. Faerber } IPCMS-GSI } (Institut de Physique et Chimie des Matériaux de Strasbourg } Groupe Surface et Interfaces) } 23, rue de Loess ; BP43 } 67034 Strasbourg CEDEX 2 } France } } Tel 00 33(0)3 88 10 71 01 } Fax 00 33(0)3 88 10 72 48 } E-mail Jacques.Faerber-at-ipcms.u-strasbg.fr } } }
I have a question regarding grids for TEM studies of nanoparticles (ferrites) with sizes from 40 - 100 nm. Can anyone help me with what kind of grids/films I can use to study my particles ? or if anyone knows if there is any good literature that I should take a look at? The particles are dispersed in ethanol and then a droplet of the ethanol with particles will be put placed on the grid/film. However, if there are other better methods, I am very interested.
Dear Faerber The important thing is that you can afford your choice and that the interfacing is being done well. What I mean is that the detector actually look at the sample. Our EDAX on the Technai 12 is a stuffed job and both EDAX and fei are keeping quiet hoping that one miracle morning the interfacing will work. Some manufacturers loos their flexibility in upgraded software versions. (Programmers write the software and are not actual operators!) Test drive the system and have a serious look at backup, downtime and poor service is expensive. The quality of after sale service differs at different parts of the world and a survey in your areas will be more helpful in that regard. Happy hunting.
-----Original Message----- } From: Faerber Jacques [mailto:Jacques.Faerber-at-ipcms.u-strasbg.fr] Sent: Tuesday, June 17, 2003 5:54 PM To: Microscopy Society of America
Hi All
After waiting for a long time, we must now choose in hurry a new EDS system for our TEM. Our old Kevex died ((un)fortunately ?!) and after a few cycle monney-no monney-monney-no monney etc, we are again in a "monney" phase, and we must spend it fast ! So fast that we have no time to do test.
So any advises are wanted about the Noran Six, the PGT Avalon, the Oxford INCA and the EDAX Genesis EDS systems, to fit on a 200 keV TOPCON 02B TEM, and to be used in a material research lab, on magnetic materials, catalysts, bio-materials, etc. We know a little bit the PGT and the Oxford, I use personnaly the Noran Vantage, but the Six is new (only the GUI or the electronics too ?), and we know nothing (other than the manufacturer's doc) about the EDAX Genesis.
Any advise too about reparing/upgrading our old KEVEX detector, or buying a new one (much more monney). The old one worked fine before it died, and it is bellow seald, with the length and the transfer mechanism adapted to the TEM.
Answer please on the list, direct to me or to Jacques.Werckmann-at-ipcms.u-strasbg.fr
Many thanks
J. Faerber IPCMS-GSI (Institut de Physique et Chimie des Matériaux de Strasbourg Groupe Surface et Interfaces) 23, rue de Loess ; BP43 67034 Strasbourg CEDEX 2 France
If you say SEM do you mean imaging with SE or BSE , EDS or WDS? If it is EDS I would say 0.5 atomic percent in a metal matrix at 25kV. Since it is a light element 15 kV would be sufficient to "see" all your elements in your alloy as well as Cl. The limit would be different since your interaction volume is different therefore determining the distance your EDS can see Cl (assuming you look at a cross section of the area of interest eg a fracture or a crack. Are the Cl real or from greasy hands handling the sample even the atmosphere close to the sea? Are you using theoretical standards, pure elements or a "real" 17-4 PH alloy standard that was verified by other more accurate means eg. a probe? All these factors do influence your detection limit and accuracy. The distance detectable in a trace scan is limited by your interaction volume thus density and kV combination. Remember that Cl is often driven away by the beam and the analysis should preferably not be carried out in spot mode and on a fresh area defeating the purpose of using a line or spot trace.
As You can see, this is not a simple question with a simple answer.
-----Original Message----- } From: Ritchie Sims [mailto:r.sims-at-auckland.ac.nz] Sent: Wednesday, June 18, 2003 1:47 AM To: AtcSEM; MIcroscopy-at-sparc5.microscopy.com
I'd say about 0.1% ie 1,000 ppm in the absence of any complicating factors, or 5000ppm with more confidence.
cheers
rtch
} From: "AtcSEM" {atcsem-at-sbcglobal.net} To: {MIcroscopy-at-sparc5.microscopy.com}
I appreciate all the answers I have received.
I have looked on the cross section of a sample and ran EDS and EDS line scan, but could not see anything. I did suggest the SIMS since they are looking to see how deep did the Cl diffused. Now I am trying to setup Cl standard for WDS and check the cross section, but I do not think that I would be successful.
Does any one knows if defused chlorine would cause any problems with 17-4PH, especially if the customer removes 3 mils from the surface that the parts were exposed to Cl (?) containing ink marker?
Pavel Lozovyy ATC SEM Lab Phone: 216-692-6637 E-mail: atclabs-at-sbcglobal.net web: www.atclabs.com
----- Original Message ----- } From: "Bill Miller" {microbill-at-mohawk.net} To: "AtcSEM" {atcsem-at-sbcglobal.net} Sent: Wednesday, June 18, 2003 7:23 AM
Ritchie writes ...
} I'd say about 0.1% ie 1,000 ppm in the absence of any } complicating factors, or 5000ppm with more confidence.
regarding ...
} } Is there any way to quantify the lowest detection limit of SEM and } } compare it to ppm range? } } } } I have a customer who would like to find out if chlorine from an ink } } marker diffused into the structure of 17-4 PH alloy? If it did, how } } deep? The customer is asking what is my detection limit for chlorine } } 100ppm? 200ppm?
I would think 1000ppm is a good approximation, assuming we are talking about SEM/EDX. However, I'd also comment ... a detection limit is difficult to evaluate for EDX, and indeed many EDX softwares do not even provide for it. The EDX background is relatively straight-forward with modern softwares, but the error associated with it is not ... and may also depend on parameters used for background modelling ... e.g., the absorption in the low energy end, any parameter used for the exponential high energy end, whether parameters are fixed or automatic (and an iterative function of composition), or if ROIs are used.
Not that EDX sensitivities are impossible, but rather impossible to address if a lot more is not known. It sounds like this "customer" wants not only quantitative wt%, but also wants the error quantified as well (my definition of "quantitative analysis"). This SEM operator, Mr. Pavel Lozovyy, should consult specifically with his EDX & software support.
We are only interested to see if there is Cl diffusion or not and how deep (if possible). We do not need to quantify it.
Pavel Lozovyy ATC SEM Lab Phone: 216-692-6637 E-mail: atclabs-at-sbcglobal.net web: www.atclabs.com ----- Original Message ----- } From: "michael shaffer" {michael-at-shaffer.net} To: "Ritchie Sims" {r.sims-at-auckland.ac.nz} ; "AtcSEM" {atcsem-at-sbcglobal.net} ; {MIcroscopy-at-sparc5.microscopy.com} Sent: Wednesday, June 18, 2003 8:20 AM
Pavel;
I agree with Ricth Sims about the lower limit of detection. However, there are some caveats you should be aware of such as excitation volume/interaction volume and the matrix of elements the Cl is in. The software program "Electron Flight Simulator" by Small World Systems, Inc. can give you an approximation of just how deep the interaction volume is for a given accelerating voltage. Also, you should know if any of the other elements in the matrix will absorb and of the Cl photon emissions [absorption edges].
If you send me the matrix off-line I'd be happy run the simulation for you.
If you are after a depth profile, it would be best to user AES [Auger Spectroscopy] or SIMS [Scanning Ion Mass Spectroscopy] SIMS would have the best sensitivity.
Peter Tomic Agere Systems
-----Original Message----- } From: AtcSEM [mailto:atcsem-at-sbcglobal.net] Sent: Tuesday, June 17, 2003 8:50 AM To: MIcroscopy-at-sparc5.microscopy.com
Dear listers, Is there any way to quantify the lowest detection limit of SEM and compare it to ppm range?
I have a customer who would like to find out if chlorine from an ink marker diffused into the structure of 17-4 PH alloy? If it did, how deep? The customer is asking what is my detection limit for chlorine 100ppm? 200ppm?
Any advice is appreciated
Pavel Lozovyy ATC SEM Lab Phone: 216-692-6637 E-mail: atclabs-at-sbcglobal.net web: www.atclabs.com
You need diffraction optics of some sort. Differential Interference Contrast (aka Normarski, DIC), Asymmetric Illumination Contrast (AIC), possibly Hoffman Modulation Contrast, although I haven't tried that one. Phase Contrast *might* work, although again, I haven't tried it. The idea is that these sub-resolution particles can be detected, although they cannot be resolved (or imaged). The "inflated diffraction image" is reasonably easy to see.
Phil
} Email: venu-at-purdue.edu } Name: Venu } } Organization: Purdue University } } Education: Graduate College } } Location: City, State, Country } } Question: Could you please tell me the imaging requirements (tools } needed) and the proper technique to observe 30-40 nm gold particles } using light microscopy? } } ---------------------------------------------------------------------------
-- Philip Oshel Supervisor, BBPIC microscopy facility Department of Animal Sciences University of Wisconsin 1675 Observatory Drive Madison, WI 53706 - 1284 voice: (608) 263-4162 fax: (608) 262-5157 (dept. fax)
You might also try "Lyman, Roger" {RLyman-at-FEICO.com} I haven't tried to contact anyone for some months, or anyone at all except Joe Race since the last round of buyouts (by Veeco [or whoever it was], if that's still the last round of buyouts), but I assume they're still there.
Phil
} Hi, } } I want order a LaB6 cathode from FEI Beam Tech and cannot get } through to a person. Their automated phone system requires that you } know who you want to talk to, no operator!! Duh! } } Does anyone know a contact person there? } } Owen } } Owen P. Mills } Electron Optics Engineer } Materials Science & Engineering } Michigan Technological University } Rm 512 M&M Bldg. } Houghton, MI 49931 } PH 906-369-1875 } FAX 906-487-2934 } mailto:opmills-at-mtu.edu } http://www.mm.mtu.edu/~opmills
-- Philip Oshel Supervisor, BBPIC microscopy facility Department of Animal Sciences University of Wisconsin 1675 Observatory Drive Madison, WI 53706 - 1284 voice: (608) 263-4162 fax: (608) 262-5157 (dept. fax)
} } I'd say about 0.1% ie 1,000 ppm in the absence of any } complicating factors, or } 5000ppm with more confidence. } } cheers } } rtch
I agree with this estimate, but nevertheless it is worth a try to examine the specimens with EDS. I suspect that a customer has a pitting corrosion of a stainless steel (Cl is a usual cause of a pitting). In that case Cl in the pits could (but not must) be detectable.
Vladimir
} } } } From: "AtcSEM" {atcsem-at-sbcglobal.net} } To: {MIcroscopy-at-sparc5.microscopy.com} } Subject: Fw: SEM detection limit of Cl } Date sent: Tue, 17 Jun 2003 08:49:33 -0400 } } } } ---------------------------------------------------------------------- } } -- The Microscopy ListServer -- Sponsor: The Microscopy Society of } } America To Subscribe/Unsubscribe -- } } http://www.msa.microscopy.com/MicroscopyListserver On-Line Help } } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } ---------------------------------------------------------------------- } } -. } } } } } } Dear listers, } } Is there any way to quantify the lowest detection limit of SEM and } } compare it to ppm range? } } } } I have a customer who would like to find out if chlorine from an ink } } marker diffused into the structure of 17-4 PH alloy? If it did, how } } deep? The customer is asking what is my detection limit for chlorine } } 100ppm? 200ppm? } } } } Any advice is appreciated } } } } Pavel Lozovyy } } ATC SEM Lab } } Phone: 216-692-6637 } } E-mail: atclabs-at-sbcglobal.net } } web: www.atclabs.com } } } } } } -- } Ritchie Sims Ph D Phone : 64 9 } 3737599 ext 87713 } Microanalyst Fax : } 64 9 3737435 } Department of Geology email : } r.sims-at-auckland.ac.nz } The University of Auckland } Private Bag 92019 } Auckland } New Zealand
Vladimir M. Dusevich, Ph.D. Electron Microscope Lab Manager 3127 School of Dentistry 650 E. 25th Street Kansas City, MO 64108-2784
Many thanks to all who responded to my query about subsidized SEM labs. As we all know, this is a chronic problem. Here's a rundown of the answers, edited for brevity and anonymity:
1. We get two full time staff positions, a faculty director and $10,000 per year from the Dean. The rest of our budget comes from user fees.
2. My salary (split 50-50 between the EM and optical microscopy core facilities) and that of a half-time technician are paid by the X college from its research environment funding. I must generate enough income in the facility to cover costs of supplies, service contracts and other equipment maintenance costs, and other misc. expenses (phone, computer networking, etc).
3. My salary is covered by the department. Our user fees pay for the service contract on our TEM and SEM. The user fee also covers incidental parts, gas, and LN2. Our greatest subsidy comes from outside users. They use our SEM more than 50% of the time.
4. In our case the director and one part time staff salary come from X college . Our computer specialist's 15% salary comes from the lab revenues . Our other part time staff's 50% salary comes from revenues and the other half comes from X college. The service contract for both SEM and TEM are fully paid by a pool of money coming from X, Y, and Z Colleges. We do not have service contracts for ancillary equipment.
5. I used to have my salary and the service contract funded by departmental contributions, in exchange for free scope time and technical assistance. There was some talk of subsidizing the facility solely through user fees, but we simply wouldn't survive that way, and many potential users couldn't afford the fees we would need to charge to keep going. For the upcoming year, I'll have to recover 1/3 of salary and contract through user fees, but I think that is do-able, and we will be able to keep user fees fairly low. The rest will come from the X Dept.
6. All the money - scope service contracts, my salary etc.- comes from (the lab's founder's) grant.
7. I would not survive here at my institution were it not for the commitment of the college. I now have a bare bones operation but I do not think I could ever recover all the costs to make these unsubsidized facilities.
8. Until this year state subsidies covered our salaries, now that this has been cut the University expects us to generate the money from recharge rates (user fees). As you know this doesn't cover the cost of running a lab. Could you please share any information you receive from 'those that are happily ensconced in subsidized facilities'! It would be wonderful to figure out how to keep our facilities alive.
*************************************************************** Please do not publicly post any of my correspondence without permission
Dee Breger Mgr. SEM/EDX Facility Lamont-Doherty Earth Observatory 61 Route 9W Palisades, NY 10964 USA T: 845/365-8640 F: 845/365-8155
http://www.ldeo.columbia.edu/micro http://www.lsc.org/antarctica/front.html Journeys in Microspace (Columbia University Press, 1995)
Venu, Ritchie Any fluorescence microscope equipped with a epi-polarization cube (Chroma #33001 or a 50/50 beam splitter and two polarizers from Omega) should work quite well. Tom
-----Original Message----- } From: Ritchie Sims [mailto:r.sims-at-auckland.ac.nz] Sent: Tuesday, June 17, 2003 7:38 PM To: venu-at-purdue.edu; MicroscopyListserver
} } Question: Could you please tell me the imaging requirements (tools } needed) and the proper technique to observe 30-40 nm gold particles } using light microscopy? }
I don't think you have any chance with LM, but I'll be interested to hear if there is a way.
cheers
rtch
-- Ritchie Sims Ph D Phone : 64 9 3737599 ext 87713 Microanalyst Fax : 64 9 3737435 Department of Geology email : r.sims-at-auckland.ac.nz The University of Auckland Private Bag 92019 Auckland New Zealand
I dont see hwo the differential interface contrast can work beyond diffraction limit ?
I will say only TEM or AFM can be used to succesfully image this small particle.
Just to detect them (like in the case of single molecular spectroscopy/imaging) a confocal imaging may work well, but you do not image them, just you see brighter spot, on the size of the half of the wavelenght.
Best regards,
Mickey
-----Original Message----- } From: Philip Oshel [mailto:peoshel-at-wisc.edu] Sent: Wednesday, June 18, 2003 6:30 AM To: Microscopy-at-sparc5.microscopy.com
Venu,
You need diffraction optics of some sort. Differential Interference Contrast (aka Normarski, DIC), Asymmetric Illumination Contrast (AIC), possibly Hoffman Modulation Contrast, although I haven't tried that one. Phase Contrast *might* work, although again, I haven't tried it. The idea is that these sub-resolution particles can be detected, although they cannot be resolved (or imaged). The "inflated diffraction image" is reasonably easy to see.
Phil
} Email: venu-at-purdue.edu } Name: Venu } } Organization: Purdue University } } Education: Graduate College } } Location: City, State, Country } } Question: Could you please tell me the imaging requirements (tools } needed) and the proper technique to observe 30-40 nm gold particles } using light microscopy? } } ---------------------------------------------------------------------------
-- Philip Oshel Supervisor, BBPIC microscopy facility Department of Animal Sciences University of Wisconsin 1675 Observatory Drive Madison, WI 53706 - 1284 voice: (608) 263-4162 fax: (608) 262-5157 (dept. fax)
Here is a simple method using cellulose acetate, possibly the kind of powder your "guy" had mentioned:
You add acetone (99%) to the cellulose acetate and stir quickly to make a finger nail polish thickness slurry. Then you simply paint it on the surface to be replicated and it will dry almost immediately. Then you peel it off and you have the replica - actually a "negative" of the surface.
You can then trim & mount that onto a SEM stub and sputter coat as usual.
We've done this here on plant leaf samples, for various reasons, sometimes to actually pick up fungal structures from the surface in order to look at them from underneath.
You could make a positive of the replica by coating it with a water based latex like bathroom caulking, then peel that off the replica. I've never tried it but what ever you do it must be water based material or you might dissolve or distort the acetate replica.
But most important in this case, before you start, don't forget to brush! - or not?
Good luck!
-- Gib Ahlstrand, Scientist Electron Optical Facility, University of Minnesota, CBS Imaging Center, 35 Snyder Hall, St. Paul, MN. USA. 55108 (612)624-3454 (612)625-5754 FAX, ahlst007-at-tc.umn.edu http://www.cbs.umn.edu/ic/
"You can learn a lot by observation - just by lookin'!" - Yogi Bera
} Hi Liststers, } I have a SEM project to do that involves making replicas of teeth. The } replicas I have made were made with acetate replicating tape. The guy who gave } me the teeth sez it is a kind of powder that one makes into a mixture that one } coats the teeth and hardens much like a latex peel. Any advice as to how to } make these and which would be better for SEM? } } Barbara Plowman } Univ. of the Pacific } School of Dentistry } ph: 415-929-6692 } email: Bplowman-at-sf.uop.edu
it's interesting that figures between about 0.1 to 0.5% and 2 are 3% are quoted for detectability in EDX and this appears to reinforce the fact that you can only be sure when you try it.
Just to muddy the water a bit further I would be concerned if the chloride layer was much less that 1um thick because it could then potentially represent a smaller part of the interaction volume. I also understand that chlorine compounds can be a bit volatile in the beam and so some loss may occur.
But detectable levels can of course also be affected by other parameters such as the energy of the incident electrons, the amount of time of analysis, the relative composition of the matrix, whether the sample has to be coated and whether you believe the peak exists (and probably which way the wind's blowing).
Malcolm Haswell e.m. unit University of Sunderland UK
----- Original Message ----- } From: Vlad Igoshev {vladig-at-tht.net}
Dear Pavel, Detection limit in the EDS is not a simple issue and it varies considerably from element to element. I usually tell my customers that it is 0.5 wt. %, but it is better than that for the K lines of the metals and worse for L and M lines. Any overlapping element lines will worsen the detection limit considerably, as will large ZAF correction factors. I have detected and quantified 0.2 wt.% Mn in an aluminum alloy. For Cl it should be similar, but it is difficult to say for sure without testing it on a standard of known chloride content. 0.2 wt % is 2000ppm. Depth of penetration is another matter and is best answered by using one of the freeware Monte Carlo simulation programs available. Try the MSA Web site. To see the depth of the diffusion of the chloride, you will probably have to cross-section the metal and look for Cl from the surface down into the bulk, bearing in mind the size of the x-ray volume from the Monte Carlo. Regards, Mary Mager Electron Microscopist Metals and Materials Engineering University of British Columbia 6350 Stores Road Vancouver, B.C. V6T 1Z4 CANADA tel: 604-822-5648 e-mail: mager-at-interchange.ubc.ca ----- Original Message ----- } From: "AtcSEM" {atcsem-at-sbcglobal.net} To: {MIcroscopy-at-sparc5.microscopy.com} Sent: Tuesday, June 17, 2003 5:49 AM
Dear Colleagues Does anybody know what electrolyte to use for making TEM foils of TiNiPt alloys using jet polishing? TIA Anita
in addition to the suggestions, 'dark field' reflection modes may do the trick. Reflection Contrast Microscopy and Epipolarisation Microscopy, the latter using polarized light in epi-mode, used to detect lowl levels of silver in autoradiography and later on for detecting silver enhanced gold particles (from subnanometer to 10nm size as a particle to start of with before the enhancement). It is a very simple technique to use. I don't know exactly about the lower size limit for the particles, but we can figure that out fast.
Hope this helps, I would be interested to know what works best for you. Cheers.
================================ Jan Leunissen Aurion - http://www.aurion.nl present address: EM-Unit Dept. Anatomy and Struct Biology University of Otago PO Box 913 Dundedin, New Zealand
-- Jan Leunissen PhD Aurion Managing Director/Director R&D current address: EM-Unit Otago University Dunedin, New Zealand --
List, Any fluorescence microscope equipped with a epi-polarization cube (Chroma #33001 or a 50/50 beam splitter and two polarizers from Omega) should work quite well. Tom
-----Original Message----- } From: Philip Oshel [mailto:peoshel-at-wisc.edu] Sent: Wednesday, June 18, 2003 8:30 AM To: Microscopy-at-sparc5.microscopy.com
Venu,
You need diffraction optics of some sort. Differential Interference Contrast (aka Normarski, DIC), Asymmetric Illumination Contrast (AIC), possibly Hoffman Modulation Contrast, although I haven't tried that one. Phase Contrast *might* work, although again, I haven't tried it. The idea is that these sub-resolution particles can be detected, although they cannot be resolved (or imaged). The "inflated diffraction image" is reasonably easy to see.
Phil
} Email: venu-at-purdue.edu } Name: Venu } } Organization: Purdue University } } Education: Graduate College } } Location: City, State, Country } } Question: Could you please tell me the imaging requirements (tools } needed) and the proper technique to observe 30-40 nm gold particles } using light microscopy? } } ---------------------------------------------------------------------------
-- Philip Oshel Supervisor, BBPIC microscopy facility Department of Animal Sciences University of Wisconsin 1675 Observatory Drive Madison, WI 53706 - 1284 voice: (608) 263-4162 fax: (608) 262-5157 (dept. fax)
Pavel, Too often customers do not know what really do they need. Is this project a part of failure analysis? Than profiling with SIMS will be an overkill and a little bit too expensive.
} I appreciate all the answers I have received. } } I have looked on the cross section of a sample and ran EDS } and EDS line } scan, but could not see anything.
You do not need to do a line scan if spectra acquisition did not show a Cl peak. To get the best sensitivity possible with EDS you have to use the right geometry (WD and tilt angle) and highest possible current, so that dead time is about 30-50% with acquisition time 10 min (or more).
} I did suggest the SIMS since they are looking to see how deep } did the Cl } diffused. } } Now I am trying to setup Cl standard for WDS and check the } cross section, } but I do not think that I would be successful.
For the first step you do not need any standards. Just (again) use right geometry and high current and time for spectra acquisition. You will see whether you have a Cl peak.
} Does any one knows if defused chlorine would cause any } problems with 17-4PH, } especially if the customer removes 3 mils from the surface } that the parts } were exposed to Cl (?) containing ink marker?
I have not worked with exactly this steel, but I can assume that it could have the usual problems for this class of materials in chlorine environment - pitting and stress corrosion. Both these processes are surface initiated and I will not advice a customer from nonacademic organization to look for Cl diffusion profile.
Good luck,
Vladimir
Vladimir M. Dusevich, Ph.D. Electron Microscope Lab Manager 3127 School of Dentistry 650 E. 25th Street Kansas City, MO 64108-2784
} Pavel Lozovyy } ATC SEM Lab } Phone: 216-692-6637 } E-mail: atclabs-at-sbcglobal.net } web: www.atclabs.com } } } } ----- Original Message ----- } } From: "Bill Miller" {microbill-at-mohawk.net} } To: "AtcSEM" {atcsem-at-sbcglobal.net} } Sent: Wednesday, June 18, 2003 7:23 AM } Subject: Re: Fw: SEM detection limit of Cl } } } } sounds like a job for SIMS particularly if you want depth } profiles - low } Z } } detection in a high Z environment with EDX is tricky at best... } } } } At 08:49 AM 6/17/2003 -0400, you wrote: } } } } ------------------------------------------------------------- } ----------- } } } The Microscopy ListServer -- Sponsor: The Microscopy } Society of America } } } To Subscribe/Unsubscribe -- } } } http://www.msa.microscopy.com/MicroscopyListserver } } } On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } } ------------------------------------------------------------- } ----------. } } } } } } } } } Dear listers, } } } Is there any way to quantify the lowest detection limit of SEM and } compare } } } it to ppm range? } } } } } } I have a customer who would like to find out if chlorine } from an ink } marker } } } diffused into the structure of 17-4 PH alloy? If it did, } how deep? The } } } customer is asking what is my detection limit for chlorine 100ppm? } 200ppm? } } } } } } Any advice is appreciated } } } } } } Pavel Lozovyy } } } ATC SEM Lab } } } Phone: 216-692-6637 } } } E-mail: atclabs-at-sbcglobal.net } } } web: www.atclabs.com } } } } } } }
Uranyl acetate at pH 2.5 or below should help, it gives less general contrast but will be more specific towards nucleic acids. 2% solution en bloc for 3 h or overnight in the fridge, after usual GA-Os fixation and before dehydration. Then on sections 5-10 minutes, *not longer*, I used the same 2%, but 5% low pH may work even better. I would try with and without lead staining, depending on the specimen. Maybe even without Os, depending again on the purpose of investigation. And, of course, this calls for very thin sections, 40 nm and under.
Hope this is your only trouble now :) Best wishes, Vlad -- ------------------------------------------- Vladislav V. Speransky, Ph.D. Laboratory of Structural Biology NIAMS, National Institutes of Health 50 South Drive, Room 1504 Bethesda, MD 20892-8025 Phone: 301 496-3989 Fax: 301 480-7629 E-mail: Vladislav_Speransky-at-nih.gov
Hi, everyone: An investigator here is looking at the changes in ribosomes with different chemical treatments. Can anyone recommend a fixation and/or staining procedure that will enhance the contrast of ribosomes at ultrastructural level? Thank you in advance and I look forward to hearing from you.
Hong Yi Emory School of Medicine EM Tel: (404) 727-8692 (Office), (404) 712-8491 (Lab) Fax: (404) 727-3157
Dear Pavel, Did you detect Cl in the ink? The problem with Cl in stainless steel is that it may cause corrosion. You would see this on the cross-section, where it will cause intergranular cracks. Stainless steels with Mo added are more resistant to chloride corrosion. If you don't see any sign of intergranular cracking, a small amount of diffused Cl probably won't cause problems. It is relatively easy to calculate the detection limit of an element in WDX. I use the formula: C(l) = C(St)x(3 x square root B/P-B) where C(l) is detection limit in mass%, C(St) is mass concentration of the element of interest in the standard, P is the total peak counts in the standard and B is the total background counts in the standard. I use a mounted and polished KCl or NaCl crystal as a standard. Once you run the standard you can calculate the detection limit. It is usually much lower than in EDS. Good luck, Mary Mager Electron Microscopist Metals and Materials Engineering University of British Columbia 6350 Stores Road Vancouver, B.C. V6T 1Z4 CANADA tel: 604-822-5648 e-mail: mager-at-interchange.ubc.ca ----- Original Message ----- } From: "AtcSEM" {atcsem-at-sbcglobal.net} To: "Bill Miller" {microbill-at-mohawk.net} Cc: {Microscopy-at-sparc5.microscopy.com} Sent: Wednesday, June 18, 2003 4:51 AM
I would be very surprised if chlorine could diffuse into the stainless steel matrix. The diffusion coefficient for elements like carbon and nitrogen are extremely slow at temperatures below 1000F, and the chlorine atoms will be bigger and slower.
If the chlorine is present as a chloride ion, corrosion could occur. Then you may have pits that could penetrate into the stainless steel. Metallurgical changes due to corrosion by the chlorides should be readily visible in your cross sections.
Hope this helps.
-- Larry D. Hanke, P.E. Materials Evaluation and Engineering, Inc. Practical Solutions Through Technology and Innovation http://www.mee-inc.com (763) 449-8870
Hello Richard I am biological science guy, but it seems to me the grids with "holey film" covered with thin carbon film is sort of universal solution for high-resolution work. I think, major EM suppliers sells it. Personally, I am using my own home-made stuff. The biggest issue with hi-res work is stability and drift, so "holey film" should be permanently attached to the EM grid. My "holey film" is carbon "glued" to the 150 mesh copper Hex grid. The size of holes is about 1-2 mkm. The important things about "holey film" - the "bridges" between the holes should be uniform in thickness. It provides stability and eliminates the drift. Just before sample preparation I mount some carbon film over the grid. I usually use 1.2-1.8 nm thick carbon film evaporated on freshly cleaved mica by "electron gun" in the oil-free environment. Such films show extreme stability under the beam and they attached nicely to the carbon "holey film". You may not glow-discharge those films because of their thickness. From another hand, because those films produced in the very clean environment, I do find that they do work well in many applications where glow-discharge mandatory for ordinary films (DNA adsorption for instance). The advantage of thin film is that you have better noise-signal ratio and "electron gun" produced film is really stable. Have a good day, Sergey
At 08:29 PM 6/17/2003, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
_____________________________________
Sergey Ryazantsev Ph. D. Electron Microscopy UCLA School of Medicine Department of Biological Chemistry 10833 Le Conte Ave, Room 33-089 Los Angeles, CA 90095
The EDS limit is around 2% and the accelerating voltage doesn't affect it. Of cause, if you are dealing with a layer, then you can play with the voltage and the tilt, but the limit will be the same. I had to deal with a similar problem but a 'better" element - Ag. Its content in the alloy is around 2 - 2.5% and the alloy contains Sn, which makes everything much worse. I knew that the EDS limit around 2-3% but asked EDAX for a "formal" backup and they came back with the same numbers.
Regards,
Vladimir Igoshev, PhD.
Toronto, Canada ----- Original Message ----- } From: "Coetzee, Mr S. H Physics Science" {COETZEES-at-mopipi.ub.bw} To: {atcsem-at-sbcglobal.net} Cc: {Microscopy-at-sparc5.microscopy.com} Sent: Wednesday, June 18, 2003 6:01 AM
Dee,
Thanks for posting the summary of response on facility funding. I notice that no-one has explicitly mentioned depreciation. We have to bring enough from user fees to cover 1.75 full time salaries, running costs and depreciation. We don't see the depreciated funds again, they dissapear to the University Centre to be dispensed across the board. Each instrument is depreciated over ten years, so on top of fighting for funding for the purchase cost we have to find an additional 10% of the that cost for each year of the next decade. This situation makes it impossible to break even each year let alone imagine replacing some of our aging instruments. One could become rather jaded.
right the last sentence On Mercoledì, giu 18, 2003, at 15:29 Europe/Rome, Philip Oshel wrote:
} ----------------------------------------------------------------------- } - } The Microscopy ListServer -- Sponsor: The Microscopy Society of } America } To Subscribe/Unsubscribe -- } http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } ----------------------------------------------------------------------- } . } } } Venu, } } You need diffraction optics of some sort. Differential Interference } Contrast (aka Normarski, DIC), Asymmetric Illumination Contrast (AIC), } possibly Hoffman Modulation Contrast, although I haven't tried that } one. Phase Contrast *might* work, although again, I haven't tried it. } The idea is that these sub-resolution particles can be detected, } although they cannot be resolved (or imaged). The "inflated } diffraction image" is reasonably easy to see. } } Phil } } } Email: venu-at-purdue.edu } } Name: Venu } } } } Organization: Purdue University } } } } Education: Graduate College } } } } Location: City, State, Country } } } } Question: Could you please tell me the imaging requirements (tools } } needed) and the proper technique to observe 30-40 nm gold particles } } using light microscopy? } } } } ---------------------------------------------------------------------- } } ----- } } -- } Philip Oshel } Supervisor, BBPIC microscopy facility } Department of Animal Sciences } University of Wisconsin } 1675 Observatory Drive } Madison, WI 53706 - 1284 } voice: (608) 263-4162 } fax: (608) 262-5157 (dept. fax) } } } ....................................................................... ........................ Alberto Diaspro, Deptartment of Physics, University of Genoa Via Dodecaneso 33, 16146 Genoa, Italy voice: +39-0103536426/480/309 fax 010314218 e-mail: diaspro-at-fisica.unige.it URL: http://www.lambs.it ....................................................................... .....................
Is sulfur a bigger issue here? I'd be concerned about it more than chlorine. But that is just me. Stainless steel is differentiated by sulfur it seems to me, rather than chlorine. But of course, your SS may vary.
gary g.
} Date: Wed, 18 Jun 2003 17:25:24 -0500 } From: Larry Hanke {hanke-at-mee-inc.com} } Organization: Materials Evaluation and Engineering, Inc. } User-Agent: Mozilla/5.0 (Windows; U; Windows NT 5.0; en-US; rv:1.0.2) } Gecko/20030208 Netscape/7.02 } X-Accept-Language: en-us, en } To: Microscopy-at-sparc5.microscopy.com } Cc: AtcSEM {atcsem-at-sbcglobal.net} } Subject: Re: Fw: SEM detection limit of Cl } X-Spam-Status: No, hits=0.7 required=5.0 } tests=NOSPAM_INC,OUTLOOK_FW_MSG,RCVD_IN_RFCI,REFERENCES, } SIGNATURE_SHORT_DENSE,SPAM_PHRASE_00_01,USER_AGENT, } USER_AGENT_MOZILLA_UA,X_ACCEPT_LANG } version=2.43 } X-Spam-Level: } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
I made a service referral to someone out west who is having an Amray 1200C installed and who is now asking if I have a source for the operator's manual and schematics for it. I don't currently have those in my collection (I try to keep paper and digitized copies of old SEM documents handy for those who need them).
So, I thought I'd pass the request on to the kind citizens here. Can anyone help?
Allen R. Sampson Advanced Research Systems 317 North 4th. Street St. Charles, Illinois 60174
I was not able to detect any chlorine on the cross section that I was analyzing. WDS showed higher background than the peak. Of coarse it does not mean that we do not have any chlorine diffusion. It is entirely possible that I am not looking at the right plane of polish or in slightly different location, but there is no way of knowing where the chlorine would like to diffuse. The 17-4 PH parts marked with an ink, that possibly contains chlorine (according to my customer), were heat treated and do not meet a MIL spec due to the markings. My customer is trying to make a case that the chlorine containing ink on the parts ( value ~ $30K) would not have any effect on the parts, especially that they machine couple mils off.
I think for them to have a better case, they would have to go to SIMS.
Pavel Lozovyy ATC SEM Lab Phone: 216-692-6637 E-mail: atclabs-at-sbcglobal.net web: www.atclabs.com
----- Original Message ----- } From: "Mary Mager" {mager-at-interchange.ubc.ca} To: "AtcSEM" {atcsem-at-sbcglobal.net} Cc: "Microscopy" {microscopy-at-sparc5.microscopy.com} Sent: Wednesday, June 18, 2003 4:44 PM
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Hi Barbara,
Here is a simple method using cellulose acetate, possibly the kind of powder your "guy" had mentioned:
You add acetone (99%) to the cellulose acetate and stir quickly to make a finger nail polish thickness slurry. Then you simply paint it on the surface to be replicated and it will dry almost immediately. Then you peel it off and you have the replica - actually a "negative" of the surface.
You can then trim & mount that onto a SEM stub and sputter coat as usual.
We've done this here on plant leaf samples, for various reasons, sometimes to actually pick up fungal structures from the surface in order to look at them from underneath.
You could make a positive of the replica by coating it with a water based latex like bathroom caulking, then peel that off the replica. I've never tried it but what ever you do it must be water based material or you might dissolve or distort the acetate replica.
But most important in this case, before you start, don't forget to brush! - or not?
Good luck!
-- Gib Ahlstrand, Scientist Electron Optical Facility, University of Minnesota, CBS Imaging Center, 35 Snyder Hall, St. Paul, MN. USA. 55108 (612)624-3454 (612)625-5754 FAX, ahlst007-at-tc.umn.edu http://www.cbs.umn.edu/ic/
"You can learn a lot by observation - just by lookin'!" - Yogi Bera
} Hi Liststers, } I have a SEM project to do that involves making replicas of teeth. The } replicas I have made were made with acetate replicating tape. The guy who gave } me the teeth sez it is a kind of powder that one makes into a mixture that one } coats the teeth and hardens much like a latex peel. Any advice as to how to } make these and which would be better for SEM? } } Barbara Plowman } Univ. of the Pacific } School of Dentistry } ph: 415-929-6692 } email: Bplowman-at-sf.uop.edu
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Hi Pavel,
If you mean EDS analysis, then you can't get any lower then 2-3 %. ppm level isn't for EDS at all.
Regards,
Vladimir Igoshev, Ph.D. Toronto, Canada ----- Original Message ----- } From: "AtcSEM" {atcsem-at-sbcglobal.net} To: {MIcroscopy-at-sparc5.microscopy.com} Sent: Tuesday, June 17, 2003 8:49 AM
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Dear Pavel, Detection limit in the EDS is not a simple issue and it varies considerably from element to element. I usually tell my customers that it is 0.5 wt. %, but it is better than that for the K lines of the metals and worse for L and M lines. Any overlapping element lines will worsen the detection limit considerably, as will large ZAF correction factors. I have detected and quantified 0.2 wt.% Mn in an aluminum alloy. For Cl it should be similar, but it is difficult to say for sure without testing it on a standard of known chloride content. 0.2 wt % is 2000ppm. Depth of penetration is another matter and is best answered by using one of the freeware Monte Carlo simulation programs available. Try the MSA Web site. To see the depth of the diffusion of the chloride, you will probably have to cross-section the metal and look for Cl from the surface down into the bulk, bearing in mind the size of the x-ray volume from the Monte Carlo. Regards, Mary Mager Electron Microscopist Metals and Materials Engineering University of British Columbia 6350 Stores Road Vancouver, B.C. V6T 1Z4 CANADA tel: 604-822-5648 e-mail: mager-at-interchange.ubc.ca ----- Original Message ----- } From: "AtcSEM" {atcsem-at-sbcglobal.net} To: {MIcroscopy-at-sparc5.microscopy.com} Sent: Tuesday, June 17, 2003 5:49 AM
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Thank you for the reply.
We are only interested to see if there is Cl diffusion or not and how deep (if possible). We do not need to quantify it.
Pavel Lozovyy ATC SEM Lab Phone: 216-692-6637 E-mail: atclabs-at-sbcglobal.net web: www.atclabs.com ----- Original Message ----- } From: "michael shaffer" {michael-at-shaffer.net} To: "Ritchie Sims" {r.sims-at-auckland.ac.nz} ; "AtcSEM" {atcsem-at-sbcglobal.net} ; {MIcroscopy-at-sparc5.microscopy.com} Sent: Wednesday, June 18, 2003 8:20 AM
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Ritchie writes ...
} I'd say about 0.1% ie 1,000 ppm in the absence of any } complicating factors, or 5000ppm with more confidence.
regarding ...
} } Is there any way to quantify the lowest detection limit of SEM and } } compare it to ppm range? } } } } I have a customer who would like to find out if chlorine from an ink } } marker diffused into the structure of 17-4 PH alloy? If it did, how } } deep? The customer is asking what is my detection limit for chlorine } } 100ppm? 200ppm?
I would think 1000ppm is a good approximation, assuming we are talking about SEM/EDX. However, I'd also comment ... a detection limit is difficult to evaluate for EDX, and indeed many EDX softwares do not even provide for it. The EDX background is relatively straight-forward with modern softwares, but the error associated with it is not ... and may also depend on parameters used for background modelling ... e.g., the absorption in the low energy end, any parameter used for the exponential high energy end, whether parameters are fixed or automatic (and an iterative function of composition), or if ROIs are used.
Not that EDX sensitivities are impossible, but rather impossible to address if a lot more is not known. It sounds like this "customer" wants not only quantitative wt%, but also wants the error quantified as well (my definition of "quantitative analysis"). This SEM operator, Mr. Pavel Lozovyy, should consult specifically with his EDX & software support.
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Guys,
The EDS limit is around 2% and the accelerating voltage doesn't affect it. Of cause, if you are dealing with a layer, then you can play with the voltage and the tilt, but the limit will be the same. I had to deal with a similar problem but a 'better" element - Ag. Its content in the alloy is around 2 - 2.5% and the alloy contains Sn, which makes everything much worse. I knew that the EDS limit around 2-3% but asked EDAX for a "formal" backup and they came back with the same numbers.
Regards,
Vladimir Igoshev, PhD.
Toronto, Canada ----- Original Message ----- } From: "Coetzee, Mr S. H Physics Science" {COETZEES-at-mopipi.ub.bw} To: {atcsem-at-sbcglobal.net} Cc: {Microscopy-at-sparc5.microscopy.com} Sent: Wednesday, June 18, 2003 6:01 AM
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right the last sentence On Mercoledì, giu 18, 2003, at 15:29 Europe/Rome, Philip Oshel wrote:
} ----------------------------------------------------------------------- } - } The Microscopy ListServer -- Sponsor: The Microscopy Society of } America } To Subscribe/Unsubscribe -- } http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } ----------------------------------------------------------------------- } . } } } Venu, } } You need diffraction optics of some sort. Differential Interference } Contrast (aka Normarski, DIC), Asymmetric Illumination Contrast (AIC), } possibly Hoffman Modulation Contrast, although I haven't tried that } one. Phase Contrast *might* work, although again, I haven't tried it. } The idea is that these sub-resolution particles can be detected, } although they cannot be resolved (or imaged). The "inflated } diffraction image" is reasonably easy to see. } } Phil } } } Email: venu-at-purdue.edu } } Name: Venu } } } } Organization: Purdue University } } } } Education: Graduate College } } } } Location: City, State, Country } } } } Question: Could you please tell me the imaging requirements (tools } } needed) and the proper technique to observe 30-40 nm gold particles } } using light microscopy? } } } } ---------------------------------------------------------------------- } } ----- } } -- } Philip Oshel } Supervisor, BBPIC microscopy facility } Department of Animal Sciences } University of Wisconsin } 1675 Observatory Drive } Madison, WI 53706 - 1284 } voice: (608) 263-4162 } fax: (608) 262-5157 (dept. fax) } } } ..................................................................... ...................... Alberto Diaspro, Deptartment of Physics, University of Genoa Via Dodecaneso 33, 16146 Genoa, Italy voice: +39-0103536426/480/309 fax 010314218 e-mail: diaspro-at-fisica.unige.it URL: http://www.lambs.it ..................................................................... ...................
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Venu, Ritchie Any fluorescence microscope equipped with a epi-polarization cube (Chroma #33001 or a 50/50 beam splitter and two polarizers from Omega) should work quite well. Tom
-----Original Message----- } From: Ritchie Sims [mailto:r.sims-at-auckland.ac.nz] Sent: Tuesday, June 17, 2003 7:38 PM To: venu-at-purdue.edu; MicroscopyListserver
} } Question: Could you please tell me the imaging requirements (tools } needed) and the proper technique to observe 30-40 nm gold particles } using light microscopy? }
I don't think you have any chance with LM, but I'll be interested to hear if there is a way.
cheers
rtch
-- Ritchie Sims Ph D Phone : 64 9 3737599 ext 87713 Microanalyst Fax : 64 9 3737435 Department of Geology email : r.sims-at-auckland.ac.nz The University of Auckland Private Bag 92019 Auckland New Zealand
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} } Question: Could you please tell me the imaging requirements (tools } needed) and the proper technique to observe 30-40 nm gold particles } using light microscopy? }
I don't think you have any chance with LM, but I'll be interested to hear if there is a way.
cheers
rtch
-- Ritchie Sims Ph D Phone : 64 9 3737599 ext 87713 Microanalyst Fax : 64 9 3737435 Department of Geology email : r.sims-at-auckland.ac.nz The University of Auckland Private Bag 92019 Auckland New Zealand
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Hi,
in addition to the suggestions, 'dark field' reflection modes may do the trick. Reflection Contrast Microscopy and Epipolarisation Microscopy, the latter using polarized light in epi-mode, used to detect lowl levels of silver in autoradiography and later on for detecting silver enhanced gold particles (from subnanometer to 10nm size as a particle to start of with before the enhancement). It is a very simple technique to use. I don't know exactly about the lower size limit for the particles, but we can figure that out fast.
Hope this helps, I would be interested to know what works best for you. Cheers.
================================ Jan Leunissen Aurion - http://www.aurion.nl present address: EM-Unit Dept. Anatomy and Struct Biology University of Otago PO Box 913 Dundedin, New Zealand
-- Jan Leunissen PhD Aurion Managing Director/Director R&D current address: EM-Unit Otago University Dunedin, New Zealand --
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I appreciate all the answers I have received.
I have looked on the cross section of a sample and ran EDS and EDS line scan, but could not see anything. I did suggest the SIMS since they are looking to see how deep did the Cl diffused. Now I am trying to setup Cl standard for WDS and check the cross section, but I do not think that I would be successful.
Does any one knows if defused chlorine would cause any problems with 17-4PH, especially if the customer removes 3 mils from the surface that the parts were exposed to Cl (?) containing ink marker?
Pavel Lozovyy ATC SEM Lab Phone: 216-692-6637 E-mail: atclabs-at-sbcglobal.net web: www.atclabs.com
----- Original Message ----- } From: "Bill Miller" {microbill-at-mohawk.net} To: "AtcSEM" {atcsem-at-sbcglobal.net} Sent: Wednesday, June 18, 2003 7:23 AM
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I have several EDAX and 2 Oxford systems, and like Oxford the better.
**************************************** Chaoying Ni, PhD The W.M. Keck Electron Microscope Facility Facility location: 022 Spencer Laboratory Mailing address: 201 duPont Hall Dept of Matls Sci & Eng University of Delaware Newark, DE 19716 (302) 831-8354 (O); -2318(L); -4545(Fax) http://eml.masc.udel.edu *****************************************
On Tue, 17 Jun 2003, Faerber Jacques wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } } Hi All } } After waiting for a long time, we must now choose in hurry a new EDS } system for our TEM. Our old Kevex died ((un)fortunately ?!) and after a } few cycle monney-no monney-monney-no monney etc, we are again in a } "monney" phase, and we must spend it fast ! So fast that we have no time } to do test. } } So any advises are wanted about the Noran Six, the PGT Avalon, the Oxford } INCA and the EDAX Genesis EDS systems, to fit on a 200 keV TOPCON 02B TEM, } and to be used in a material research lab, on magnetic materials, } catalysts, bio-materials, etc. We know a little bit the PGT and the } Oxford, I use personnaly the Noran Vantage, but the Six is new (only the } GUI or the electronics too ?), and we know nothing (other than the } manufacturer's doc) about the EDAX Genesis. } } Any advise too about reparing/upgrading our old KEVEX detector, or buying } a new one (much more monney). The old one worked fine before it died, and } it is bellow seald, with the length and the transfer mechanism adapted to } the TEM. } } Answer please on the list, direct to me or to } Jacques.Werckmann-at-ipcms.u-strasbg.fr } } Many thanks } } J. Faerber } IPCMS-GSI } (Institut de Physique et Chimie des Matériaux de Strasbourg } Groupe Surface et Interfaces) } 23, rue de Loess ; BP43 } 67034 Strasbourg CEDEX 2 } France } } Tel 00 33(0)3 88 10 71 01 } Fax 00 33(0)3 88 10 72 48 } E-mail Jacques.Faerber-at-ipcms.u-strasbg.fr } } }
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I would be very surprised if chlorine could diffuse into the stainless steel matrix. The diffusion coefficient for elements like carbon and nitrogen are extremely slow at temperatures below 1000F, and the chlorine atoms will be bigger and slower.
If the chlorine is present as a chloride ion, corrosion could occur. Then you may have pits that could penetrate into the stainless steel. Metallurgical changes due to corrosion by the chlorides should be readily visible in your cross sections.
Hope this helps.
-- Larry D. Hanke, P.E. Materials Evaluation and Engineering, Inc. Practical Solutions Through Technology and Innovation http://www.mee-inc.com (763) 449-8870
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I'd say about 0.1% ie 1,000 ppm in the absence of any complicating factors, or 5000ppm with more confidence.
cheers
rtch
} From: "AtcSEM" {atcsem-at-sbcglobal.net} To: {MIcroscopy-at-sparc5.microscopy.com}
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Pavel;
I agree with Ricth Sims about the lower limit of detection. However, there are some caveats you should be aware of such as excitation volume/interaction volume and the matrix of elements the Cl is in. The software program "Electron Flight Simulator" by Small World Systems, Inc. can give you an approximation of just how deep the interaction volume is for a given accelerating voltage. Also, you should know if any of the other elements in the matrix will absorb and of the Cl photon emissions [absorption edges].
If you send me the matrix off-line I'd be happy run the simulation for you.
If you are after a depth profile, it would be best to user AES [Auger Spectroscopy] or SIMS [Scanning Ion Mass Spectroscopy] SIMS would have the best sensitivity.
Peter Tomic Agere Systems
-----Original Message----- } From: AtcSEM [mailto:atcsem-at-sbcglobal.net] Sent: Tuesday, June 17, 2003 8:50 AM To: MIcroscopy-at-sparc5.microscopy.com
Dear listers, Is there any way to quantify the lowest detection limit of SEM and compare it to ppm range?
I have a customer who would like to find out if chlorine from an ink marker diffused into the structure of 17-4 PH alloy? If it did, how deep? The customer is asking what is my detection limit for chlorine 100ppm? 200ppm?
Any advice is appreciated
Pavel Lozovyy ATC SEM Lab Phone: 216-692-6637 E-mail: atclabs-at-sbcglobal.net web: www.atclabs.com
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Pavel, Too often customers do not know what really do they need. Is this project a part of failure analysis? Than profiling with SIMS will be an overkill and a little bit too expensive.
} I appreciate all the answers I have received. } } I have looked on the cross section of a sample and ran EDS } and EDS line } scan, but could not see anything.
You do not need to do a line scan if spectra acquisition did not show a Cl peak. To get the best sensitivity possible with EDS you have to use the right geometry (WD and tilt angle) and highest possible current, so that dead time is about 30-50% with acquisition time 10 min (or more).
} I did suggest the SIMS since they are looking to see how deep } did the Cl } diffused. } } Now I am trying to setup Cl standard for WDS and check the } cross section, } but I do not think that I would be successful.
For the first step you do not need any standards. Just (again) use right geometry and high current and time for spectra acquisition. You will see whether you have a Cl peak.
} Does any one knows if defused chlorine would cause any } problems with 17-4PH, } especially if the customer removes 3 mils from the surface } that the parts } were exposed to Cl (?) containing ink marker?
I have not worked with exactly this steel, but I can assume that it could have the usual problems for this class of materials in chlorine environment - pitting and stress corrosion. Both these processes are surface initiated and I will not advice a customer from nonacademic organization to look for Cl diffusion profile.
Good luck,
Vladimir
Vladimir M. Dusevich, Ph.D. Electron Microscope Lab Manager 3127 School of Dentistry 650 E. 25th Street Kansas City, MO 64108-2784
} Pavel Lozovyy } ATC SEM Lab } Phone: 216-692-6637 } E-mail: atclabs-at-sbcglobal.net } web: www.atclabs.com } } } } ----- Original Message ----- } } From: "Bill Miller" {microbill-at-mohawk.net} } To: "AtcSEM" {atcsem-at-sbcglobal.net} } Sent: Wednesday, June 18, 2003 7:23 AM } Subject: Re: Fw: SEM detection limit of Cl } } } } sounds like a job for SIMS particularly if you want depth } profiles - low } Z } } detection in a high Z environment with EDX is tricky at best... } } } } At 08:49 AM 6/17/2003 -0400, you wrote: } } } } ------------------------------------------------------------- } ----------- } } } The Microscopy ListServer -- Sponsor: The Microscopy } Society of America } } } To Subscribe/Unsubscribe -- } } } http://www.msa.microscopy.com/MicroscopyListserver } } } On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } } ------------------------------------------------------------- } ----------. } } } } } } } } } Dear listers, } } } Is there any way to quantify the lowest detection limit of SEM and } compare } } } it to ppm range? } } } } } } I have a customer who would like to find out if chlorine } from an ink } marker } } } diffused into the structure of 17-4 PH alloy? If it did, } how deep? The } } } customer is asking what is my detection limit for chlorine 100ppm? } 200ppm? } } } } } } Any advice is appreciated } } } } } } Pavel Lozovyy } } } ATC SEM Lab } } } Phone: 216-692-6637 } } } E-mail: atclabs-at-sbcglobal.net } } } web: www.atclabs.com } } } } } } }
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I am a bit lost.
If you say SEM do you mean imaging with SE or BSE , EDS or WDS? If it is EDS I would say 0.5 atomic percent in a metal matrix at 25kV. Since it is a light element 15 kV would be sufficient to "see" all your elements in your alloy as well as Cl. The limit would be different since your interaction volume is different therefore determining the distance your EDS can see Cl (assuming you look at a cross section of the area of interest eg a fracture or a crack. Are the Cl real or from greasy hands handling the sample even the atmosphere close to the sea? Are you using theoretical standards, pure elements or a "real" 17-4 PH alloy standard that was verified by other more accurate means eg. a probe? All these factors do influence your detection limit and accuracy. The distance detectable in a trace scan is limited by your interaction volume thus density and kV combination. Remember that Cl is often driven away by the beam and the analysis should preferably not be carried out in spot mode and on a fresh area defeating the purpose of using a line or spot trace.
As You can see, this is not a simple question with a simple answer.
-----Original Message----- } From: Ritchie Sims [mailto:r.sims-at-auckland.ac.nz] Sent: Wednesday, June 18, 2003 1:47 AM To: AtcSEM; MIcroscopy-at-sparc5.microscopy.com
I'd say about 0.1% ie 1,000 ppm in the absence of any complicating factors, or 5000ppm with more confidence.
cheers
rtch
} From: "AtcSEM" {atcsem-at-sbcglobal.net} To: {MIcroscopy-at-sparc5.microscopy.com}
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Dear Pavel, Did you detect Cl in the ink? The problem with Cl in stainless steel is that it may cause corrosion. You would see this on the cross-section, where it will cause intergranular cracks. Stainless steels with Mo added are more resistant to chloride corrosion. If you don't see any sign of intergranular cracking, a small amount of diffused Cl probably won't cause problems. It is relatively easy to calculate the detection limit of an element in WDX. I use the formula: C(l) = C(St)x(3 x square root B/P-B) where C(l) is detection limit in mass%, C(St) is mass concentration of the element of interest in the standard, P is the total peak counts in the standard and B is the total background counts in the standard. I use a mounted and polished KCl or NaCl crystal as a standard. Once you run the standard you can calculate the detection limit. It is usually much lower than in EDS. Good luck, Mary Mager Electron Microscopist Metals and Materials Engineering University of British Columbia 6350 Stores Road Vancouver, B.C. V6T 1Z4 CANADA tel: 604-822-5648 e-mail: mager-at-interchange.ubc.ca ----- Original Message ----- } From: "AtcSEM" {atcsem-at-sbcglobal.net} To: "Bill Miller" {microbill-at-mohawk.net} Cc: {Microscopy-at-sparc5.microscopy.com} Sent: Wednesday, June 18, 2003 4:51 AM
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Dee,
Thanks for posting the summary of response on facility funding. I notice that no-one has explicitly mentioned depreciation. We have to bring enough from user fees to cover 1.75 full time salaries, running costs and depreciation. We don't see the depreciated funds again, they dissapear to the University Centre to be dispensed across the board. Each instrument is depreciated over ten years, so on top of fighting for funding for the purchase cost we have to find an additional 10% of the that cost for each year of the next decade. This situation makes it impossible to break even each year let alone imagine replacing some of our aging instruments. One could become rather jaded.
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To all TEM facilities:
I need someone to help me with plant viruses/TEM research. I do have TEM background but do not have an electron microscope(none in Alaska). I need to look at purified viruses and viruses in plant tissue. I am looking for a EM facility that either contracts research projects or allows one to use their facility for a short segment of time. My immediate need is to have grids of purified virus or leaf dips looked at for virus particles.
Thank you,
Nancy Robertson
Nancy L. Robertson Research Plant Pathologist USDA, ARS 533 E. Fireweed Ave. Palmer Alaska 99645 907-746-9465 office 907-746-2898 lab 907-746-2677 fax ffnlr-at-uaf.edu
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Hi Hong,
Uranyl acetate at pH 2.5 or below should help, it gives less general contrast but will be more specific towards nucleic acids. 2% solution en bloc for 3 h or overnight in the fridge, after usual GA-Os fixation and before dehydration. Then on sections 5-10 minutes, *not longer*, I used the same 2%, but 5% low pH may work even better. I would try with and without lead staining, depending on the specimen. Maybe even without Os, depending again on the purpose of investigation. And, of course, this calls for very thin sections, 40 nm and under.
Hope this is your only trouble now :) Best wishes, Vlad -- ------------------------------------------- Vladislav V. Speransky, Ph.D. Laboratory of Structural Biology NIAMS, National Institutes of Health 50 South Drive, Room 1504 Bethesda, MD 20892-8025 Phone: 301 496-3989 Fax: 301 480-7629 E-mail: Vladislav_Speransky-at-nih.gov
Hi, everyone: An investigator here is looking at the changes in ribosomes with different chemical treatments. Can anyone recommend a fixation and/or staining procedure that will enhance the contrast of ribosomes at ultrastructural level? Thank you in advance and I look forward to hearing from you.
Hong Yi Emory School of Medicine EM Tel: (404) 727-8692 (Office), (404) 712-8491 (Lab) Fax: (404) 727-3157
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Is sulfur a bigger issue here? I'd be concerned about it more than chlorine. But that is just me. Stainless steel is differentiated by sulfur it seems to me, rather than chlorine. But of course, your SS may vary.
gary g.
} Date: Wed, 18 Jun 2003 17:25:24 -0500 } From: Larry Hanke {hanke-at-mee-inc.com} } Organization: Materials Evaluation and Engineering, Inc. } User-Agent: Mozilla/5.0 (Windows; U; Windows NT 5.0; en-US; rv:1.0.2) } Gecko/20030208 Netscape/7.02 } X-Accept-Language: en-us, en } To: Microscopy-at-sparc5.microscopy.com } Cc: AtcSEM {atcsem-at-sbcglobal.net} } Subject: Re: Fw: SEM detection limit of Cl } X-Spam-Status: No, hits=0.7 required=5.0 } tests=NOSPAM_INC,OUTLOOK_FW_MSG,RCVD_IN_RFCI,REFERENCES, } SIGNATURE_SHORT_DENSE,SPAM_PHRASE_00_01,USER_AGENT, } USER_AGENT_MOZILLA_UA,X_ACCEPT_LANG } version=2.43 } X-Spam-Level: } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
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} } I'd say about 0.1% ie 1,000 ppm in the absence of any } complicating factors, or } 5000ppm with more confidence. } } cheers } } rtch
I agree with this estimate, but nevertheless it is worth a try to examine the specimens with EDS. I suspect that a customer has a pitting corrosion of a stainless steel (Cl is a usual cause of a pitting). In that case Cl in the pits could (but not must) be detectable.
Vladimir
} } } } From: "AtcSEM" {atcsem-at-sbcglobal.net} } To: {MIcroscopy-at-sparc5.microscopy.com} } Subject: Fw: SEM detection limit of Cl } Date sent: Tue, 17 Jun 2003 08:49:33 -0400 } } } } ---------------------------------------------------------------------- } } -- The Microscopy ListServer -- Sponsor: The Microscopy Society of } } America To Subscribe/Unsubscribe -- } } http://www.msa.microscopy.com/MicroscopyListserver On-Line Help } } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } ---------------------------------------------------------------------- } } -. } } } } } } Dear listers, } } Is there any way to quantify the lowest detection limit of SEM and } } compare it to ppm range? } } } } I have a customer who would like to find out if chlorine from an ink } } marker diffused into the structure of 17-4 PH alloy? If it did, how } } deep? The customer is asking what is my detection limit for chlorine } } 100ppm? 200ppm? } } } } Any advice is appreciated } } } } Pavel Lozovyy } } ATC SEM Lab } } Phone: 216-692-6637 } } E-mail: atclabs-at-sbcglobal.net } } web: www.atclabs.com } } } } } } -- } Ritchie Sims Ph D Phone : 64 9 } 3737599 ext 87713 } Microanalyst Fax : } 64 9 3737435 } Department of Geology email : } r.sims-at-auckland.ac.nz } The University of Auckland } Private Bag 92019 } Auckland } New Zealand
Vladimir M. Dusevich, Ph.D. Electron Microscope Lab Manager 3127 School of Dentistry 650 E. 25th Street Kansas City, MO 64108-2784
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Dear listeners,
I have a question regarding grids for TEM studies of nanoparticles (ferrites) with sizes from 40 - 100 nm. Can anyone help me with what kind of grids/films I can use to study my particles ? or if anyone knows if there is any good literature that I should take a look at? The particles are dispersed in ethanol and then a droplet of the ethanol with particles will be put placed on the grid/film. However, if there are other better methods, I am very interested.
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List, Any fluorescence microscope equipped with a epi-polarization cube (Chroma #33001 or a 50/50 beam splitter and two polarizers from Omega) should work quite well. Tom
-----Original Message----- } From: Philip Oshel [mailto:peoshel-at-wisc.edu] Sent: Wednesday, June 18, 2003 8:30 AM To: Microscopy-at-sparc5.microscopy.com
Venu,
You need diffraction optics of some sort. Differential Interference Contrast (aka Normarski, DIC), Asymmetric Illumination Contrast (AIC), possibly Hoffman Modulation Contrast, although I haven't tried that one. Phase Contrast *might* work, although again, I haven't tried it. The idea is that these sub-resolution particles can be detected, although they cannot be resolved (or imaged). The "inflated diffraction image" is reasonably easy to see.
Phil
} Email: venu-at-purdue.edu } Name: Venu } } Organization: Purdue University } } Education: Graduate College } } Location: City, State, Country } } Question: Could you please tell me the imaging requirements (tools } needed) and the proper technique to observe 30-40 nm gold particles } using light microscopy? } } ---------------------------------------------------------------------------
-- Philip Oshel Supervisor, BBPIC microscopy facility Department of Animal Sciences University of Wisconsin 1675 Observatory Drive Madison, WI 53706 - 1284 voice: (608) 263-4162 fax: (608) 262-5157 (dept. fax)
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Hi Barbara,
Here is a simple method using cellulose acetate, possibly the kind of powder your "guy" had mentioned:
You add acetone (99%) to the cellulose acetate and stir quickly to make a finger nail polish thickness slurry. Then you simply paint it on the surface to be replicated and it will dry almost immediately. Then you peel it off and you have the replica - actually a "negative" of the surface.
You can then trim & mount that onto a SEM stub and sputter coat as usual.
We've done this here on plant leaf samples, for various reasons, sometimes to actually pick up fungal structures from the surface in order to look at them from underneath.
You could make a positive of the replica by coating it with a water based latex like bathroom caulking, then peel that off the replica. I've never tried it but what ever you do it must be water based material or you might dissolve or distort the acetate replica.
But most important in this case, before you start, don't forget to brush! - or not?
Good luck!
-- Gib Ahlstrand, Scientist Electron Optical Facility, University of Minnesota, CBS Imaging Center, 35 Snyder Hall, St. Paul, MN. USA. 55108 (612)624-3454 (612)625-5754 FAX, ahlst007-at-tc.umn.edu http://www.cbs.umn.edu/ic/
"You can learn a lot by observation - just by lookin'!" - Yogi Bera
} Hi Liststers, } I have a SEM project to do that involves making replicas of teeth. The } replicas I have made were made with acetate replicating tape. The guy who gave } me the teeth sez it is a kind of powder that one makes into a mixture that one } coats the teeth and hardens much like a latex peel. Any advice as to how to } make these and which would be better for SEM? } } Barbara Plowman } Univ. of the Pacific } School of Dentistry } ph: 415-929-6692 } email: Bplowman-at-sf.uop.edu
You might also try "Lyman, Roger" {RLyman-at-FEICO.com} I haven't tried to contact anyone for some months, or anyone at all except Joe Race since the last round of buyouts (by Veeco [or whoever it was], if that's still the last round of buyouts), but I assume they're still there.
Phil
} Hi, } } I want order a LaB6 cathode from FEI Beam Tech and cannot get } through to a person. Their automated phone system requires that you } know who you want to talk to, no operator!! Duh! } } Does anyone know a contact person there? } } Owen } } Owen P. Mills } Electron Optics Engineer } Materials Science & Engineering } Michigan Technological University } Rm 512 M&M Bldg. } Houghton, MI 49931 } PH 906-369-1875 } FAX 906-487-2934 } mailto:opmills-at-mtu.edu } http://www.mm.mtu.edu/~opmills
-- Philip Oshel Supervisor, BBPIC microscopy facility Department of Animal Sciences University of Wisconsin 1675 Observatory Drive Madison, WI 53706 - 1284 voice: (608) 263-4162 fax: (608) 262-5157 (dept. fax)
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Hello
it's interesting that figures between about 0.1 to 0.5% and 2 are 3% are quoted for detectability in EDX and this appears to reinforce the fact that you can only be sure when you try it.
Just to muddy the water a bit further I would be concerned if the chloride layer was much less that 1um thick because it could then potentially represent a smaller part of the interaction volume. I also understand that chlorine compounds can be a bit volatile in the beam and so some loss may occur.
But detectable levels can of course also be affected by other parameters such as the energy of the incident electrons, the amount of time of analysis, the relative composition of the matrix, whether the sample has to be coated and whether you believe the peak exists (and probably which way the wind's blowing).
Malcolm Haswell e.m. unit University of Sunderland UK
----- Original Message ----- } From: Vlad Igoshev {vladig-at-tht.net}
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Phil and Venu,
I dont see hwo the differential interface contrast can work beyond diffraction limit ?
I will say only TEM or AFM can be used to succesfully image this small particle.
Just to detect them (like in the case of single molecular spectroscopy/imaging) a confocal imaging may work well, but you do not image them, just you see brighter spot, on the size of the half of the wavelenght.
Best regards,
Mickey
-----Original Message----- } From: Philip Oshel [mailto:peoshel-at-wisc.edu] Sent: Wednesday, June 18, 2003 6:30 AM To: Microscopy-at-sparc5.microscopy.com
Venu,
You need diffraction optics of some sort. Differential Interference Contrast (aka Normarski, DIC), Asymmetric Illumination Contrast (AIC), possibly Hoffman Modulation Contrast, although I haven't tried that one. Phase Contrast *might* work, although again, I haven't tried it. The idea is that these sub-resolution particles can be detected, although they cannot be resolved (or imaged). The "inflated diffraction image" is reasonably easy to see.
Phil
} Email: venu-at-purdue.edu } Name: Venu } } Organization: Purdue University } } Education: Graduate College } } Location: City, State, Country } } Question: Could you please tell me the imaging requirements (tools } needed) and the proper technique to observe 30-40 nm gold particles } using light microscopy? } } ---------------------------------------------------------------------------
-- Philip Oshel Supervisor, BBPIC microscopy facility Department of Animal Sciences University of Wisconsin 1675 Observatory Drive Madison, WI 53706 - 1284 voice: (608) 263-4162 fax: (608) 262-5157 (dept. fax)
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Dear Yi Ribosome (RS) is about 28 nm in diameter and wery fagile. You may not see the structure of RS on the conventional ultrathin sections with standard processing. You also could not see fine structure on the chemically fixed ribosomes. The only one way known to me to investigate the fine structure of the free ribosomes in vitro is cryo-EM with 3D-reconstruction. Please, made search for Joachim Frank works. Ribosome is extremely sensitive and complicated "machine": you have to perform state of the art biochemistry to isolate them intact. Usually we are using RNAase-free strains or thermophilic bacteria to isolate ribosomes. As far as I know, there is just a few places in US, where people is able to isolate intact ribosomes. Of coarse, "intact" is very subjective word. You may check Harry Noller works for RS isolation. Best wishes, Sergey
At 12:07 PM 6/17/2003, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
_____________________________________
Sergey Ryazantsev Ph. D. Electron Microscopy UCLA School of Medicine Department of Biological Chemistry 10833 Le Conte Ave, Room 33-089 Los Angeles, CA 90095
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Venu,
You need diffraction optics of some sort. Differential Interference Contrast (aka Normarski, DIC), Asymmetric Illumination Contrast (AIC), possibly Hoffman Modulation Contrast, although I haven't tried that one. Phase Contrast *might* work, although again, I haven't tried it. The idea is that these sub-resolution particles can be detected, although they cannot be resolved (or imaged). The "inflated diffraction image" is reasonably easy to see.
Phil
} Email: venu-at-purdue.edu } Name: Venu } } Organization: Purdue University } } Education: Graduate College } } Location: City, State, Country } } Question: Could you please tell me the imaging requirements (tools } needed) and the proper technique to observe 30-40 nm gold particles } using light microscopy? } } ---------------------------------------------------------------------------
-- Philip Oshel Supervisor, BBPIC microscopy facility Department of Animal Sciences University of Wisconsin 1675 Observatory Drive Madison, WI 53706 - 1284 voice: (608) 263-4162 fax: (608) 262-5157 (dept. fax)
It depends on what you mean by "can work". It can't image the particles, or resolve 2 particles close together, but the way the particles diffract light does make them detectable. They appear to be larger than they really are.
Phil
} Phil and Venu, } } I dont see hwo the differential interface contrast can work beyond } diffraction limit ? } } I will say only TEM or AFM can be used to succesfully image this } small particle. } } Just to detect them (like in the case of single molecular } spectroscopy/imaging) a confocal imaging may work well, but you do } not image them, just you see brighter spot, on the size of the half } of the wavelenght. } } Best regards, } } Mickey } } -----Original Message----- } } From: Philip Oshel [mailto:peoshel-at-wisc.edu] } Sent: Wednesday, June 18, 2003 6:30 AM } To: Microscopy-at-sparc5.microscopy.com } Subject: Re: Ask-A-Microscopist:LM 30 nm gold } } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
In response to the request for information about procedures for operating a turbo pump on a JEOL 845 microscope. If you can't obtain explicit instructiuons for this particular instrument, you might refer to Section 6.18 in my book, 'Vacuum Methods in Electron Microscopy' (see:http://www.2spi.com/catalog/books/book48.html/) for a description), where general operating procedures for instruments with turbo pumps are discussed. This discussion should give you the necessary information to devise the necessary procedures for yopur instrument. Even if you do obtain such instructions, Chapter 6 contains a rather detailed description of the construction and operating characteristics of turbomolecular pumps that should help you in understanding the performance of your instrument.
Good luck , WCB
-- Wilbur C. Bigelow, Prof. Emeritus Materials Sci. & Engr., University of Michigan 3062 Dow Bldg.; 2300 Hayward St. Ann Arbor, MI 48109-2136 e-mail: bigelow-at-umich.edu; Fx:734-763-4788; Ph:734-662-5237
That rather sounds like double-dipping. If you received an instrumentation grant from outside the university to purchase the equipment, it seems that depreciation should not be an issue. However, if you are fighting internally for part of a university-wide pot of money, then I could see how the university might insist on the depreciation. It seems that they never really gave you the money for the instrument, they are letting you "buy" it according to the depreciation schedule. That way, they can replenish the pot for the next pieces of instrumentation.
Is that a fair assessment? If not, please clarify.
Warren
At 01:27 PM 6/19/2003 +1200, you wrote:
} Dee, } } Thanks for posting the summary of response on facility funding. I } notice that no-one has explicitly mentioned depreciation. We have to } bring enough from user fees to cover 1.75 full time salaries, running } costs and depreciation. We don't see the depreciated funds again, they } dissapear to the University Centre to be dispensed across the board. } Each instrument is depreciated over ten years, so on top of fighting } for funding for the purchase cost we have to find an additional 10% of } the that cost for each year of the next decade. This situation makes it } impossible to break even each year let alone imagine replacing some of } our aging instruments. } One could become rather jaded. } } Regards } Bryony
------------------------------------------- No files should be attached to this message ------------------------------------------- Warren E. Straszheim, Ph.D. Materials Analysis and Research Lab Iowa State University 46 Town Engineering Ames IA, 50011-3232
Scanning electron microscopy, x-ray analysis, and image analysis of materials Computer applications and networking
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That rather sounds like double-dipping. If you received an instrumentation grant from outside the university to purchase the equipment, it seems that depreciation should not be an issue. However, if you are fighting internally for part of a university-wide pot of money, then I could see how the university might insist on the depreciation. It seems that they never really gave you the money for the instrument, they are letting you "buy" it according to the depreciation schedule. That way, they can replenish the pot for the next pieces of instrumentation.
Is that a fair assessment? If not, please clarify.
Warren
At 01:27 PM 6/19/2003 +1200, you wrote:
} Dee, } } Thanks for posting the summary of response on facility funding. I } notice that no-one has explicitly mentioned depreciation. We have to } bring enough from user fees to cover 1.75 full time salaries, running } costs and depreciation. We don't see the depreciated funds again, they } dissapear to the University Centre to be dispensed across the board. } Each instrument is depreciated over ten years, so on top of fighting } for funding for the purchase cost we have to find an additional 10% of } the that cost for each year of the next decade. This situation makes it } impossible to break even each year let alone imagine replacing some of } our aging instruments. } One could become rather jaded. } } Regards } Bryony
------------------------------------------- No files should be attached to this message ------------------------------------------- Warren E. Straszheim, Ph.D. Materials Analysis and Research Lab Iowa State University 46 Town Engineering Ames IA, 50011-3232
Scanning electron microscopy, x-ray analysis, and image analysis of materials Computer applications and networking
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Thanks for the reply,
my material is a glass containing nano-size ferroelectric crystallites 100- 200 nm. I want to make sure that these nano-crystallites are ferroelectic. I read your reply to use PFM and i have some questions after reading some papers on PFM.
QUE1- What is the minimum d33 coefficient to get a recognizable contrast because i read that the piezoresponse depends upon the d33 coefficient? the d33 coefficient of the single crystals same as nanocrystallites in my material is 1.3 pm/V, which is very small compared to BaTiO3.
Question2- In response to Dr Sergei's letter (below), He said that since crystallites are inside the dielectric medium this technique might not work. My question is-- in this technique, should upper and lower both srfaces of the crystallites should touch conductive surface?
QUE2- Is there any book describing this technique?
Thanks Pradyumna
Quoting "Sergei V. Kalinin" {sergei2-at-ornl.gov} :
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- } http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Dear Pradyumna } The best way to detect and image ferroelectric inclusions in } paraelectric matrix is to use a scanning probe microscopy techniques, } namely piezoresponse force microscopy. This technique is specifically } designed to distinguish piezoelectric and non-piezoelectric materials, } and hence is probably the best choice in this case. It will be } sensitive only to crystallites exposed on the surface or relatively } shallow within the glass matrix (~10 nm) and will allow imaging of both } in-plane and out-of-plane polarization components. You can find some } examples in paper } A.Y. Borisevich, S.V. Kalinin, D.A. Bonnell, and P.K. Davies, } Analysis of phase distributions in the Li2O-Nb2O5-TiO2 system by } piezoresponse imaging, J. Mater. Res. 16(2), 329-332 (2001). } The extension of PFM, piezoresponse spectroscopy normally allows } spatially resolved electromechanical hysteresis loop measurements, e.g. } within single 50 nm grain, but it might not work for the crystal in the } dielectric matrix. } Yours } Sergei }
Hi, } } I have a glass- nono-crystallites composite. Nanocrystallites may be } ferroelectric in nature and I need to prove that nanocrystallites in the } glassy phase are ferroelectric. } } How can i prove using TEM that these nanocrystallites(probably single domain) } are indeed ferroelectric?
} -- } Sergei V. Kalinin, } Oak Ridge National Laboratory, } 1 Bethel Valley Rd, } Bldg. 3025, MS6030, } Oak Ridge, TN 37831 } Phone: (865) 241-0236 } FAX: (865) 574-4143 } URL: sergei2.kalininweb.com } } }
610 7585590 Lab Pradyumna N Gupta MSE Lehigh University
610 7585590 Lab Pradyumna N Gupta MSE Lehigh University
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} } Guys, } } The EDS limit is around 2% and the accelerating voltage
OK. To get real numbers from real system I just performed test on XL30 with field emission gun at 15 kV, 40-45% dead time, 10 min acquisition time. I measured Cl peak from Au/Pd coated crystals of table salt and background in Cl peak position on a stainless steel razor blade. With ZAF correction detection limit was 0.09%. If Cl could diffuse in steel it would be very convenient element for trace analysis.
} doesn't affect it.
It does affect. It changes background, peak, correction coefficients.
} Of cause, if you are dealing with a layer, then you can play with the } voltage and the tilt, but the limit will be the same. I had } to deal with a } similar problem but a 'better" element - Ag. Its content in } the alloy is } around 2 - 2.5% and the alloy contains Sn, which makes everything much } worse. I knew that the EDS limit around 2-3% but asked EDAX } for a "formal" } backup and they came back with the same numbers.
I am very surprised. But did you asked service technician or application lab?
Vladimir Dusevich
} Regards, } } Vladimir Igoshev, PhD. } } Toronto, Canada } ----- Original Message ----- } } From: "Coetzee, Mr S. H Physics Science" {COETZEES-at-mopipi.ub.bw} } To: {atcsem-at-sbcglobal.net} } Cc: {Microscopy-at-sparc5.microscopy.com} } Sent: Wednesday, June 18, 2003 6:01 AM } Subject: RE: SEM detection limit of Cl } } } } } -------------------------------------------------------------- } ---------- } } The Microscopy ListServer -- Sponsor: The Microscopy } Society of America } } To Subscribe/Unsubscribe -- } http://www.msa.microscopy.com/MicroscopyListserver } } On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } -------------------------------------------------------------- } ---------. } } } } } } I am a bit lost. } } } } If you say SEM do you mean imaging with SE or BSE , EDS or WDS? } } If it is EDS I would say 0.5 atomic percent in a metal } matrix at 25kV. } Since it is a light element 15 kV would be sufficient to } "see" all your } elements in your alloy as well as Cl. The limit would be } different since } your interaction volume is different therefore determining } the distance your } EDS can see Cl (assuming you look at a cross section of the } area of interest } eg a fracture or a crack. Are the Cl real or from greasy } hands handling the } sample even the atmosphere close to the sea? } } Are you using theoretical standards, pure elements or a } "real" 17-4 PH } alloy standard that was verified by other more accurate means } eg. a probe? } All these factors do influence your detection limit and accuracy. The } distance detectable in a trace scan is limited by your } interaction volume } thus density and kV combination. } } Remember that Cl is often driven away by the beam and the } analysis should } preferably not be carried out in spot mode and on a fresh } area defeating the } purpose of using a line or spot trace. } } } } As You can see, this is not a simple question with a simple answer. } } } } CHEMICAL COMPOSITION of an typical 17-4 PH alloy } } } } CARBON. 0.07% MAX. } } CHROMIUM. 15.O - 17.5%. } } MANGANESE. 1.00% } } MAX. NICKEL. 3.00- 5.00%. } } PHOSPHORUS. 0.04% MAX. } } COPPER. 3.00- 5.00%. } } SULPHUR. 0.03% MAX. } } } } -----Original Message----- } } } From: Ritchie Sims [mailto:r.sims-at-auckland.ac.nz] } } Sent: Wednesday, June 18, 2003 1:47 AM } } To: AtcSEM; MIcroscopy-at-sparc5.microscopy.com } } Subject: Re: Fw: SEM detection limit of Cl } } } } } } } -------------------------------------------------------------- } ---------- } } The Microscopy ListServer -- Sponsor: The Microscopy } Society of America } } To Subscribe/Unsubscribe -- } http://www.msa.microscopy.com/MicroscopyListserver } } On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } -------------------------------------------------------------- } ---------. } } } } } } } } I'd say about 0.1% ie 1,000 ppm in the absence of any complicating } factors, or } } 5000ppm with more confidence. } } } } cheers } } } } rtch } } } } } } } } } From: "AtcSEM" {atcsem-at-sbcglobal.net} } } To: {MIcroscopy-at-sparc5.microscopy.com} } } Subject: Fw: SEM detection limit of Cl } } Date sent: Tue, 17 Jun 2003 08:49:33 -0400 } } } } } } ---------------------------------------------------------------------- } } } -- The Microscopy ListServer -- Sponsor: The Microscopy } Society of } } } America To Subscribe/Unsubscribe -- } } } http://www.msa.microscopy.com/MicroscopyListserver On-Line Help } } } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } } ---------------------------------------------------------------------- } } } -. } } } } } } } } } Dear listers, } } } Is there any way to quantify the lowest detection limit of SEM and } } } compare it to ppm range? } } } } } } I have a customer who would like to find out if chlorine } from an ink } } } marker diffused into the structure of 17-4 PH alloy? If } it did, how } } } deep? The customer is asking what is my detection limit } for chlorine } } } 100ppm? 200ppm? } } } } } } Any advice is appreciated } } } } } } Pavel Lozovyy } } } ATC SEM Lab } } } Phone: 216-692-6637 } } } E-mail: atclabs-at-sbcglobal.net } } } web: www.atclabs.com } } } } } } } } } } -- } } Ritchie Sims Ph D Phone : 64 9 3737599 ext 87713 } } Microanalyst Fax : 64 9 3737435 } } Department of Geology email : r.sims-at-auckland.ac.nz } } The University of Auckland } } Private Bag 92019 } } Auckland } } New Zealand } } } } } } }
We have a SEM Phillips XL30. All the images taken at extra high resolution (3232 x 2420 pixels) come with faint vertical bands spaced 11 pixels. The bands are about 4 pixels wide, and they are apparent by having a greater variance in gray levels (i.e., they look more grainy).
Does anyone have an idea of why this occur or how to solve it?
Martin
Martín J. Ramírez División Aracnología Museo Argentino de Ciencias Naturales Av. Angel Gallardo 470 C1405DJR Buenos Aires Argentina tel +54 11 4982-8370 fax +54 11 4982-4494
A DAKO Autostainer and a Shandon Consul Automatic Coverslipper are available for a reasonable price. These automated systems were purchased 3 years ago and have been maintained under service contract through the manufacturer. Since the equipment was primarily designed for labs routinely with large volume of histology and immunohistochemistry/cytochemistry work, they have never been heavily used in our EM research lab. In another word, they are almost new and in a very good condition. The EM Facility will provide manual and maintenance records, and training if required.
Please contact Jocelyn Torcolini in the Electron Microscopy Facility at Penn State (814-865-0212 or jmt175-at-psu.edu) for more information.
Gang (Greg) Ning Director, Electron Microscopy Facility Huck Institute for Life Sciences The Pennsylvania State University 1 South Frear Lab University Park, PA 16802 Phone: 814-863-0994 Fax: 814-863-1357 Email: gxn7-at-psu.edu http://www.lsc.psu.edu/stf/em/home.html
Well, here are another numbers. I collected an EDS spectrum from 1010 steel (Jeol/Kevex/tungsten, 20 kV, 30% dead time, 2 minutes), forced the software to account for Cl and Ar peaks and ran quantification. What I've got was 0.11at%Cl and 0.26at%Ar. I like the "trick" and use it once in a while to demonstrate my customers that if you don't see a peak then the EDS isn't the right technique for the analysis and some other methods should be used (like SIMS), particularly for light elements. Of cause, if you know the element is there then it's easier to "believe" the numbers.
Regards,
Vladimir ----- Original Message ----- } From: "Dusevich, Vladimir" {dusevichv-at-umkc.edu} To: "Vlad Igoshev" {vladig-at-tht.net} ; {Microscopy-at-sparc5.microscopy.com} Sent: Thursday, June 19, 2003 5:39 PM
Did you try your question on the XL30 list?
gary g.
At 06:19 AM 6/20/2003, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
I could use a little help with overlaying images. On our Zeiss LSM-310, we can scan a reflected laser image of a sample (computer chip, in our case) into the Green plane, and then maybe an OBIC image into the Red plane, and then select the RGB mode. This will display our OBIC image overlaid on the reflected light image, showing where the OBIC signal is coming from on the sample.
Now the problem. We have a Roper Scientific CCD mounted on the Zeiss, which we use for reflected light images, and for capturing images of the light emitted from the computer chip while it is powered up (photo emission microscopy - PEM). We are using the WinView32 software that came with the CCD, and I have tried all of the math functions, etc., that I can find in WinView32. I have been unable to do an "overlay" of two images.
Does anyone know how to do a simple overlay with WinView32, or if there is another application that will work? (Hopefully cheap or free) I don't need a lot of image processing/analysis power. Just the ability to overlay. WinView32 can do pseudo coloring of the images to emulate the Red and Green planes of the Zeiss.
Thanks for your attention, Darrell IBM Microelectronics
I don't know WinView32 at all, but Adobe Photoshop has a variety of techniques for merging and overlaying images either using Layers with effects such as muliplication, difference or exclusion, or by pasting images into separate RGB or CMYK colour channels.
Dr. Chris Jeffree Inveresk Cottage 26, Carberry Road Inveresk Musselburgh Midlothian EH21 8PR Tel: +44 131 665 6062 FAX +44 131 653 6248 Mobile 07710 585 401 ----- Original Message ----- } From: "Darrell Miles" {milesd-at-US.ibm.com} To: {Microscopy-at-sparc5.microscopy.com} Sent: Saturday, June 21, 2003 1:54 AM
You can either use a conventional photo program such as photoshop, or look into ImageJ, a pc port of NIH-Image (http://rsb.info.nih.gov/ij/). Both of these can do overlays. If you have to adjust the sizes to match, both programs do have ways of doing that, as well.
If you need specifics, feel free to contact me off list. Joel
You should be able to do it with ImagJ http://rsbweb.nih.gov/ij/ It is pubic domain so the price is right. It is written in Java and runs on virtually any computer and has support for user written plugins and a very large collection growing of them. There is an active users group on developing plugins.
I think ImageJ is a tool everyone that works with images should have in their tool box since it an open ended growing project and not tied to any platform, computer of software. You can have familiar tools on Windows, Mac, Unix and Linux.
It sounds like all you need to do is use ImageJ's combine RGB plugin on the files you have. you might have to massaged he files a little first to get them in the correct format.
If I can help let me know.
Good luck Gordon Couger gcouger-at-couger.com
I collect links on information related to light microscopes. http://www.couger.com/microscope/links/gclinks.html Please forward any links or information you think might be useful to others. ----- Original Message ----- } From: "Darrell Miles" {milesd-at-US.ibm.com} To: {Microscopy-at-sparc5.microscopy.com} Sent: Friday, June 20, 2003 7:54 PM
On Wednesday, June 18, 2003, at 04:51 AM, AtcSEM wrote:
} I have looked on the cross section of a sample and ran EDS and EDS line } scan, but could not see anything. } Dear Pavel, Something that has not yet been mentioned is that, since Cl is volatile, working at low temperature will be an advantage. If you have cryo-SEM available to you, try it. Yours, Bill Tivol EM Scientist and Manager Cryo-Electron Microscopy Facility Broad Center, Mail Code 114-96 California Institute of Technology Pasadena CA 91125 (626) 395-8833 tivol-at-caltech.edu
Paint Sho Pro from Jasc Software (www.jasc.com) has similar basic features to those in Photosphop for lower price (around $100). The layer function will alloy you to overlay two images and adjust the relative intensity of each.
-- Larry D. Hanke, P.E. Materials Evaluation and Engineering, Inc. Practical Solutions Through Technology and Innovation http://www.mee-inc.com (763) 449-8870
} Hi all... } } } } I could use a little help with overlaying images. On our Zeiss } } LSM-310, we } } } } can scan a reflected laser image of a sample (computer chip, in our } } case) } } } } into the Green plane, and then maybe an OBIC image into the Red } } plane, } } } } and then select the RGB mode. This will display our OBIC image } } overlaid } } } } on the reflected light image, showing where the OBIC signal is } } coming } } } } from on the sample. } } } } } } Now the problem. We have a Roper Scientific CCD mounted on the } } Zeiss, } } } } which we use for reflected light images, and for capturing images of } } the } } } } light emitted from the computer chip while it is powered up (photo } } emission } } } } microscopy - PEM). We are using the WinView32 software that came } } with } } } } the CCD, and I have tried all of the math functions, etc., that I } } can find } } } } in } } } WinView32. I have been unable to do an "overlay" of two images. } } } } } } Does anyone know how to do a simple overlay with WinView32, or if } } there } } } } is another application that will work? (Hopefully cheap or free) I } } don't } } } } need a lot of image processing/analysis power. Just the ability to } } } overlay. } } } WinView32 can do pseudo coloring of the images to emulate the Red } } and } } } } Green planes of the Zeiss. } } } } } } Thanks for your attention, } } } Darrell } } } IBM Microelectronics }
If you can't actually see the peak then you can't trust what the software says.
I bet the detection limit of 0.09% was generated by the software from consideration of counting statistics. These may well be OK for WDS, but for EDS it's not the counting stats that limit detectability, it's the overlaps and other spectral interferences.
Even if you do 10 replicate measurements and look at the stats of the actual wt % results you're not getting the full story as precision is not the same as accuracy.
However, the 2% figure is unnecessarily pessimistic and I find it difficult to believe that it originated from an EDS detector manufacturer.
If anyone wishes to email me, I will send them the spectrum of volcanic glass containing 0.36% Cl, obtained on my JEOL 840/PGT Prism/Moran Scientific system in 130 seconds.
cheers
rtch
} } Well, here are another numbers. I collected an EDS spectrum from 1010 } steel (Jeol/Kevex/tungsten, 20 kV, 30% dead time, 2 minutes), forced } the software to account for Cl and Ar peaks and ran quantification. } What I've got was 0.11at%Cl and 0.26at%Ar. I like the "trick" and use } it once in a while to demonstrate my customers that if you don't see a } peak then the EDS isn't the right technique for the analysis and some } other methods should be used (like SIMS), particularly for light } elements. Of cause, if you know the element is there then it's easier } to "believe" the numbers. }
} } } } } } The EDS limit is around 2% and the accelerating voltage } }
} } OK. To get real numbers from real system I just performed } } test on XL30 with field emission gun at 15 kV, 40-45% dead time, 10 } } min acquisition time. I measured Cl peak from Au/Pd coated crystals } } of table salt and background in Cl peak position on a stainless } } steel razor blade. With ZAF correction detection limit was 0.09%.
-- Ritchie Sims Ph D Phone : 64 9 3737599 ext 87713 Microanalyst Fax : 64 9 3737435 Department of Geology email : r.sims-at-auckland.ac.nz The University of Auckland Private Bag 92019 Auckland New Zealand
I want to embed a layer of epithelium growing on a lens explant. The whole thing is only a thin collagenous matrix with a single layer of cells on top, about 4mm in diameter. Rather fragile. Does anyone have a suggestion as to the best way to keep this flat without losing it or mashing it? It's currently sitting in fixative. The aim is to cut sections perpendicular to the plane of growth.
Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (sfontouris-at-hotmail.com) from http://www.msa.microscopy.com/Ask-A-Microscopist.html on Monday, June 23, 2003 at 08:00:10 ---------------------------------------------------------------------------
Question: I would appreciate it very much if someone explained the differences between i)phase contrast, ii)bright field, iii) Normanski and iv) Hoffmann optics. Many thanks in advance
If you are EXTREMELY careful, and check with your Life Safety office first, you can do what I do...I etch the glass away with HF. It is extremely hazardous and you must be very careful about handling it, etc., but it works beautifully. A couple of hours on a stir plate in HF and all the glass is gone. You then retrieve the blocks (with plastic forceps) and wash them like mad. Lee -- Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy Core Facility Manager, Optical Microscopy Core Facility Joan & Sanford I. Weill Medical College of Cornell University voice (212)746-6146 fax (212)746-8175
I have an old JEOL JXA-840 SEM, late 80s vintage in storage. I need to clear out all storage areas to prepare for upcoming renovations to the building. I am offering the SEM free to a good home. It was working when it was replaced with a newer, donated JSM-840A about 2 years ago. The old scope has occasionaly been used as a source of parts for troubleshooting the newer scope. At this time all of the parts have been returned, but it has not been powered up in two years. One module in the newer scope (magnification power amp) had a hard failure and was replaced with the module from the old scope. The power amp will probably require repair or replacement by JEOL. The water chiller runs too warm and needs a refrigerant recharge. There is no EDS or computer image acquisition with the scope, as the one we had was transferred to the newer scope.
Preference given to academic labs. If you want it, you need to make arrangements for shipping, or pick it up in Medford, MA. Contact me offline for further information.
Dave Wilbur
-- __________________________________ David J. Wilbur, Ph.D. Instrumentation Specialist Department of Chemistry Tufts University 62 Talbot Ave. Medford, MA 02155 voice: 617-627-2163 Fax: 617-627-3443 email: david.wilbur-at-tufts.edu __________________________________
On Sunday, June 22, 2003, at 02:11 PM, Ritchie Sims wrote:
} If you can't actually see the peak then you can't trust what the } software } says.
} I bet the detection limit of 0.09% was generated by the software from } consideration of counting statistics. These may well be OK for WDS, but } for EDS it's not the counting stats that limit detectability, it's the } overlaps } and other spectral interferences. } Dear Richie, Overlaps should not be a problem for Cl and steel. When I was looking for Cl in sediment, I saw what were very small peaks that were only slightly larger than the noise. However, in several examples there were small amounts of Ti such that the Ti k-alpha peaks were clearly present and the k-beta peaks were the same size as the Cl k-alpha. Since I knew that the Ti k-beta peaks were real, that gave me confidence that the Cl peaks were also. Yours, Bill Tivol EM Scientist and Manager Cryo-Electron Microscopy Facility Broad Center, Mail Code 114-96 California Institute of Technology Pasadena CA 91125 (626) 395-8833 tivol-at-caltech.edu
We would like to match two images, ideally pixel-to-pixel, obtained from a single sample under two different conditions. The sample may have changed a little (small tears, streaks) between the acquisition of the two images and the re-loading onto the microscope may change the orientation slightly. The extent of differences is expected to be less than 10% of the total number of pixels.
I'll be grateful if anyone could please suggest an algorithm we could use to program these tasks. Commercial software that can accomplish this would also be useful and pointers to those are welcome.
Thanks for your help, Rohit
Rohit Bhargava National Institutes of Health Building 5, B1-38W LCP/NIDDK Bethesda, MD 20892-0520
Greetings, All microscopies involve a probe, a sample, and a detector. They generate contrast by virtue of a specific interaction between the probe and the sample. In the case of light microscopies, the probe is light. The different types of microscopy you list differ in the property of the sample used to generate contrast.
1) Brightfield would be much more usefully called 'absorption contrast': Contrast is generated based on differences in absorption. That is why it is good for stained samples, which absorb specfic wavelengths strongly.
2) Phase contrast depends on the optical path through the sample (refractive index times thickness). It is tricky for generating contrast based on optical path differences and F. Zernike (spelling??) got a Nobel prize for figuring out how.
3) Nomarski (also called differential interference contrast) depends on the gradient of optical path in a sample (this was invented by George Nomarski who should have gotten a Nobel prize for this work too).
4) I am not sure about Hoffman.
You can find further details in any good book about microscopy.
Tobias Baskin
} Below is the result of your feedback form (NJZFM-ultra-55). It was } submitted by (sfontouris-at-hotmail.com) from } http://www.msa.microscopy.com/Ask-A-Microscopist.html on Monday, } June 23, 2003 at 08:00:10 } --------------------------------------------------------------------------- } } Email: sfontouris-at-hotmail.com } Name: Yannis Sfontouris } } Organization: University of Nottingham } } Education: Graduate College } } Location: Nottingham, UK } } Question: I would appreciate it very much if someone explained the } differences between i)phase contrast, ii)bright field, iii) } Normanski and iv) Hoffmann optics. } Many thanks in advance } } Yannis } } ---------------------------------------------------------------------------
Please put the answer on the list server for those of us who have been so immersed in EM of various types. I've always wanted a better explanation than I got way back in high school. Thanks Ken Converse owner Quality Images third party SEM service Delta, PA
by way of Ask-A-Microscopist wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (pgan-at-ap.ansell.com) from http://www.msa.microscopy.com/Ask-A-Microscopist.html on Tuesday, June 24, 2003 at 05:58:42 ---------------------------------------------------------------------------
Email: pgan-at-ap.ansell.com Name: Phay Fang Gan
Organization: Ansell Shah Alam Sdn Bhd
Education: Graduate College
Location: Shah Alam,Selangor, Malaysia
Question: We are having a unit of Hitachi S3000N SEM in our lab.
It would be nice if anyone out there who can tell me where I could get local agent to provide Preventive Maintenance on our SEM in Malaysia.
The article in Microscopy Today, May/June 2003 by O'Neil et al. appears to be exactly what you want. Title: "Use of a New Imaging Technique to Document Deformations Recorded in the ESEM."
I see that you have a subscription. The article is on page 36.
Ron Anderson, MT Editor
-----Original Message----- } From: Rohit Bhargava [mailto:rxb29-at-po.cwru.edu] Sent: Monday, June 23, 2003 5:02 PM To: Microscopy-at-sparc5.microscopy.com
Hello:
We would like to match two images, ideally pixel-to-pixel, obtained from a single sample under two different conditions. The sample may have changed a little (small tears, streaks) between the acquisition of the two images and the re-loading onto the microscope may change the orientation slightly. The extent of differences is expected to be less than 10% of the total number of pixels.
I'll be grateful if anyone could please suggest an algorithm we could use to program these tasks. Commercial software that can accomplish this would also be useful and pointers to those are welcome.
Thanks for your help, Rohit
Rohit Bhargava National Institutes of Health Building 5, B1-38W LCP/NIDDK Bethesda, MD 20892-0520
On Lunedì, giu 23, 2003, at 23:49 Europe/Rome, Tobias Baskin wrote:
} ----------------------------------------------------------------------- } - } The Microscopy ListServer -- Sponsor: The Microscopy Society of } America } To Subscribe/Unsubscribe -- } http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } ----------------------------------------------------------------------- } . } } } Greetings, } All microscopies involve a probe, a sample, and a detector. They } generate contrast by virtue of a specific interaction between the } probe and the sample. In the case of light microscopies, the probe is } light. The different types of microscopy you list differ in the } property of the sample used to generate contrast. } } 1) Brightfield would be much more usefully called 'absorption } contrast': Contrast is generated based on differences in absorption. } That is why it is good for stained samples, which absorb specfic } wavelengths strongly. } } 2) Phase contrast depends on the optical path through the sample } (refractive index times thickness). It is tricky for generating } contrast based on optical path differences and F. Zernike (spelling??) } got a Nobel prize for figuring out how. } } 3) Nomarski (also called differential interference contrast) depends } on the gradient of optical path in a sample (this was invented by } George Nomarski who should have gotten a Nobel prize for this work } too). } } 4) I am not sure about Hoffman. } } You can find further details in any good book about microscopy. } } Tobias Baskin } } } } } Below is the result of your feedback form (NJZFM-ultra-55). It was } } submitted by (sfontouris-at-hotmail.com) from } } http://www.msa.microscopy.com/Ask-A-Microscopist.html on Monday, June } } 23, 2003 at 08:00:10 } } ---------------------------------------------------------------------- } } ----- } } } } Email: sfontouris-at-hotmail.com } } Name: Yannis Sfontouris } } } } Organization: University of Nottingham } } } } Education: Graduate College } } } } Location: Nottingham, UK } } } } Question: I would appreciate it very much if someone explained the } } differences between i)phase contrast, ii)bright field, iii) Normanski } } and iv) Hoffmann optics. } } Many thanks in advance } } } } Yannis } } } } ---------------------------------------------------------------------- } } ----- } } } -- } _ ____ __ ____ Tobias I. Baskin } / \ / / \ / \ \ Biology } Department } / / / / \ \ \ University of } Massachusetts } /_ / __ /__ \ \ \__ Amherst, MA, 01003 } / / / \ \ \ / / } / \ \ \ Voice: 413 - 545 - 1533 / } / ___ / \ \__/ \ ____ Fax: } 413 - } http://www.biosci.missouri.edu/Baskin/baskin_lab__home_page.htm } } } ....................................................................... ........................ Alberto Diaspro, Deptartment of Physics, University of Genoa Via Dodecaneso 33, 16146 Genoa, Italy voice: +39-0103536426/480/309 fax 010314218 e-mail: diaspro-at-fisica.unige.it URL: http://www.lambs.it ....................................................................... .....................
Dear colleagues, in our lab of Electron Microscopy, we have an "Ultramicrotomy TOP 170 Pabish". Until nobody has used it, but we want to try preparation for TEM observations with it. Unfortunately we have lost its manual, please could anybody with the same equipment help us and send us a copy of the manual. My e-mail is marilena.re-at-brindisi.enea.it Thanks in advance Marilena Re
Our address is: ENEA C.R. Brindisi - Lab. of Electron Microscopy S.S. 7 - Appia km 712,700 72100 Brindisi - Italy
I have just received the good news that I have been approved for new SEM equipment to replace our old SEM. The new SEM will go in the room where the old SEM is now.
We have had problems with stray fields in the past and would like to remedy this situation before installation of the new SEM.
The 2 solutions to deal with the stray fields problem, of which I am aware, are either a field cancellation device or room shielding.
I need to determine the cost of eliminating the stray fields from the room and submit it to Management within the next day or so.
Could any companies which deal with either field cancellation devices or room sheilding materials please contact me offline as soon as possible?
Thanks in advance for your help.
Paula.
Paula M. Allan-Wojtas Research Scientist - Food Microstructure / Chercheur scientique - microstructure des aliments Food Safety and Quality Team / Salubrité et qualité des aliments Agriculture and Agri-Food Canada /Agriculture et Agroalimentaire Canada Telephone / Téléphone: 902-679-5566 Facsimile / Télécopieur: 902-679-2311 32 Main Street / 32 rue Main Kentville, Nova Scotia / Kentville (Nouvelle-Écosse) B4N 1J5 allanwojtasp-at-agr.gc.ca
} } Return-Path: {bfostermme-at-earthlink.net} } } Received: from rly-zd03.mx.aol.com (rly-zd03.mail.aol.com } } [172.31.33.227]) by air-zd02.mail.aol.com (v94.1) with ESMTP id } } MAILINZD24-37f83ef7accf282; Mon, 23 Jun 2003 21:43:43 -0400 } } Received: from postoffice.lightshipmail.net } } (postoffice.lightshipmail.net [216.204.0.41]) by rly-zd03.mx.aol.com } } (v94.27) with ESMTP id MAILRELAYINZD37-3a93ef7acab2cf; Mon, 23 Jun 2003 } } 21:43:07 -0400 } } Received: (qmail 32505 invoked by uid 888); 24 Jun 2003 01:43:04 -0000 } } Delivered-To: mme1-com-cbarnack-at-mme1.com } } Received: (qmail 32481 invoked from network); 24 Jun 2003 01:43:04 -0000 } } Received: from exodus.lightshipmail.net (216.204.0.47) } } by postoffice.lightshipmail.net with SMTP; 24 Jun 2003 01:43:04 -0000 } } Received: (qmail 34894 invoked from network); 24 Jun 2003 01:43:04 -0000 } } Received: from albatross.mail.pas.earthlink.net (207.217.120.120) } } by exodus.lightshipmail.net with SMTP; 24 Jun 2003 01:43:04 -0000 } } Received: from daisy.psp.pas.earthlink.net ([207.217.78.223]) } } by albatross.mail.pas.earthlink.net with esmtp (Exim 3.33 #1) } } id 19Ucpq-0007bH-00; Mon, 23 Jun 2003 18:43:02 -0700 } } Received: from [207.217.78.15] by EarthlinkWAM via HTTP; Mon Jun 23 } } 18:43:02 PDT 2003 } } Message-ID: } } {4663833.1056418982409.JavaMail.nobody-at-daisy.psp.pas.earthlink.net} } } Date: Mon, 23 Jun 2003 18:45:21 -0500 (CDT) } } From: bfostermme-at-earthlink.net } } To: by way of Ask-A-Microscopist {sfontouris-at-hotmail.com} , } } Microscopy-at-sparc5.microscopy.com } } Subject: Re: Ask-A-Microscopist: phase contrast vs bright field } } Mime-Version: 1.0 } } Content-Type: text/plain; charset=us-ascii } } Content-Transfer-Encoding: 7bit } } X-Mailer: Earthlink Web Access Mail version 3.0 } } } } Dear Yannis, } } } } This question is extremely complicated to answer quickly, but here are } } several guidelines. } } } } Brightfield is the type of imaging you create when you align the } } microscope by setting up what is called Koehler illumination. For an } } object to be visible in brightfield, it has to present either signficant } } color and/or intensity difference from the background. Many objects in } } both biological and materials science do not possess either } } characteristic and,as a result, are not very visible against the } } background. That is to say, they "lack contrast". } } } } The other three techniques, Phase, Hoffman Modulation, and Nomarski, are } } all contrast enhancement techniques. Phase is used to enhance the body } } of a material while Hoffman and Nomarski detect gradients or } } slopes. Objects which respond well to Phase appear as darker against a } } soft gray background. Objects which respond well to HMC and Nomarski } } (aka Differential Interference Contrast or DIC), will appear three-dimensional. } } } } Each technique has special components engineered into the microscope to } } carefully control the interaction of the light and matter. A full } } discussion of all four techniques is well beyond the scope of the } } listserver. I would recommend the following: } } } } Both the Nikon and Olympus sites have great educational resources. I } } would start there. } } } } Alternatively, I would suggest that you get a copy of my book, } } "Optimizing Light Microscopy". It has detailed discussions of these } } topics as well as pictures of one, standard sample (cheek cells) } } in about 7 different contrast techniques, along with simple line } } drawings and instructions for setting up the microscope for each. There } } are also simple experiments so that you can try each at your own } } microscope. Details are on our website (www.MicroscopyEducation.com) } } } } Also, I would be remiss, as a long-time Fellow of the Royal Microscopical } } Society, not to mention their summer courses which, if memory serves, are } } coming up in July. They also publish a set of monographs which you would } } find helpful. } } } } } } Finally, I may have the manuscripts of some course notes back in the } } office. I will be back there later this week and, if you contact me } } directly, I will see what is available. There will not be as many } } pictures (I typically show 35mm slides to bring home that point), but at } } least you would get some of the diagrams and a simple explanation of the } } theory. } } } } Good hunting! } } } } Best regards, } } Barbara Foster } } Microscopy/Microscopy Education } } } } } } } } -------Original Message------- } } From: by way of Ask-A-Microscopist {sfontouris-at-hotmail.com} } } Sent: 06/23/03 08:05 AM } } To: Microscopy-at-sparc5.microscopy.com } } Subject: Ask-A-Microscopist: phase contrast vs bright field } } } } } } } } ------------------------------------------------------------------------ } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Here's my quick and dirty attempt to work through this.
First, all three contrast systems are based on the phenomenon that light can interact with regions of different refractive index by (a) slowing down, and (b) changing its path. (Some of the people I have talked to suggest that the change in path is really a diffraction effect, rather than refraction. Maybe theyre actually the same phenom.)
The three microscope systems you mention take advantage of this in different ways. All, however, have to face the challenge of how to distinguish the altered light from the wash of unaffected light -the background.
This is accomplished by constricting the illumination. For phase contrast, a hollow cone of light is projected through the sample onto a ring in the objective lens. For Hoffman, a narrow beam is projected through the sample obliquely to a small region on the side of the objective lens. For Nomarski, the system is a bit more complex. The illuminating system is set up between crossed polarizers, so that no undeviated light should get through at the end. The light passes through the first polarizer, and then through a special prism system (Wollaston) that splits the light into two beams whose vibrations are perpendicular. This pair of beams passes through the sample.
Now, what happens in the sample? I am referring only to the light that interacts, not the background. In the phase contrast system, the light is bent out of the path of the background light, and ends up passing through a different part of the ring in the objective. For Hoffman, the light is again bent to a different part of the Hoffman plate in the objective. In Nomarski, if (and this is critical) the two beams interact differently, they end up out of phase with each other.
As we follow the light up in the microscope, we reach the back focal plane of the objective lens. This is where the Phase and Hoffman systems do their dirty work. In the phase contrast system, there is a phase plate to which the cone of light is projected. Remember that the background light is projected to the ring itself. The light from the sample will be bent away from this ring, and elsewhere on this plate. Ziernike realized that not only was the light bent, but it had been slowed down so that it was 1/4 wavelength delayed compared to the background. He reasoned that if that difference could be expanded to 1/2 a wavelength, the light from the two sources could be made to interfere destructively --i.e. cancel each other out, and one would see a dark region--this would emphaize contrast.
In the Hoffman system, the deviated light passes through a region that is different in transmission from the plate to which most of the light is projected. As a result, it appears bright, compared to the background.
For Nomarski, the two perpendicular beams passs through a second Wollaston prism, which serves to reunite them. However, if one of the two beams is shifted in phase compared to the other, the resulting beam has an altered polarization, and passes through the final polarizer, while the background does not. --or vice versa, depending on how the system is set up.
The result of all three systemss is that one obtains contrast from refractive index differences within a sample. The consequence is that stains are not necessary, and details of living cells can be visualized.
You can get much more detail, with beautiful illustrations and animations at the microscopy web site:
http://micro.magnet.fsu.edu Look for the "primer" section.
} ---------------------------------------------------------------------- } -- The Microscopy ListServer -- Sponsor: The Microscopy Society of } America To Subscribe/Unsubscribe -- } http://www.msa.microscopy.com/MicroscopyListserver On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } ---------------------------------------------------------------------- } -. } } } Below is the result of your feedback form (NJZFM-ultra-55). It was } submitted by (sfontouris-at-hotmail.com) from } http://www.msa.microscopy.com/Ask-A-Microscopist.html on Monday, June } 23, 2003 at 08:00:10 } ---------------------------------------------------------------------- } ----- } } Email: sfontouris-at-hotmail.com } Name: Yannis Sfontouris } } Organization: University of Nottingham } } Education: Graduate College } } Location: Nottingham, UK } } Question: I would appreciate it very much if someone explained the } differences between i)phase contrast, ii)bright field, iii) Normanski } and iv) Hoffmann optics. Many thanks in advance } } Yannis } } ---------------------------------------------------------------------- } ----- }
Joel B. Sheffield, Ph.D. Biology Department, Temple University 1900 North 12th Street Philadelphia, PA 19122 jbs-at-temple.edu (215) 204 8839, fax (215) 204 0486 http://astro.temple.edu/~jbs
the easiest way to match two similar images would be to use a cross correlation between the two images. It uses Fourier algorithms to come up with a "most likely" match of the two images by allowing a shift in X and Y (typically no rotation).
In your case it might already be sufficient. In case the images need to further adjusted manually, I would suggest looking at fluorescence software. Many of the available programs allow to shift two or more images against each other manually to compensate for small shifts between acquisition of the various fluorescence channels.
If you want further information how our analySIS software does this, or if you want to send me a couple of images for testing, please contact me off-line.
Thanks.
mike
Michael Bode, Ph.D. Soft Imaging System Corp. 12596 West Bayaud Avenue Suite 300 Lakewood, CO 80228 =================================== phone: (888) FIND SIS (303) 234-9270 fax: (303) 234-9271 email: mailto:info-at-soft-imaging.com web: http://www.soft-imaging.com ===================================
-----Original Message----- } From: Rohit Bhargava [mailto:rxb29-at-po.cwru.edu] Sent: Monday, June 23, 2003 3:02 PM To: Microscopy-at-sparc5.microscopy.com
Hello:
We would like to match two images, ideally pixel-to-pixel, obtained from a single sample under two different conditions. The sample may have changed a little (small tears, streaks) between the acquisition of the two images and the re-loading onto the microscope may change the orientation slightly. The extent of differences is expected to be less than 10% of the total number of pixels.
I'll be grateful if anyone could please suggest an algorithm we could use to program these tasks. Commercial software that can accomplish this would also be useful and pointers to those are welcome.
Thanks for your help, Rohit
Rohit Bhargava National Institutes of Health Building 5, B1-38W LCP/NIDDK Bethesda, MD 20892-0520
Below are some rather useful resources when trying to get some light microscopy answers. Here are some short answers from me (please understand the complexity of these systems, these are VERY abbreviated answers);
There are three basic ways that light interacts with non fluorescent samples. They are; 1) absorption of light 2) phase shift of light 3) diffraction of light
Only the first can be seen by the eye. There are different modes of microscopy that permit us to take advantage of 2 and 3.
i) When light passes through something with a different optical density, the phase of the light is changed. (More dense samples will slow the light down more). The human eye can not see these changes in phase. In a phase contrast system, these changes in phase are changed into changes in brightness (contrast), which the eye can see. The image then becomes a gray scale image where the differences in intensity are representations in the differences in the optical density of the sample. This is done with a phase ring (in the objective) and a phase annulus (in the condenser). This system is lower resolution than bright field, but permits viewing samples that absorb almost no light (such as living cells). ii) Bright field is the most common way to look at a sample slide. The information is generated by differences in the absorption of light. This is seen as color and darkness. This is normally used for samples that have great differences in the amount of light absorbed (non biological samples) or samples that have been stained. Bright field also takes advantage of diffracted light. The condenser focuses light on the sample in such a way that the light diffracted by the sample is focused by the objective. This diffracted information dramatically increases the resolution of the system. A stained cell preparation on a slide would be a standard biological sample. iii) Nomarski (or DIC for Differential Interference Contrast microscopy) is a method that used differences in optical density to create differences in brightness. Phase contrast shows the observer the absolute optical density of the sample. DIC shows the local CHANGES in optical density. This is done by an optical system that employs two closely spaced beams of light that start in phase with each other. They then pass through the sample close to each other. The beams are then recombined in such a way that any differences in the optical path is seen as a difference in intensity. This is accomplished with polarized light and a pair of prisms that split the light before the sample and recombine the light after the sample. iv) Hoffmann will give results like DIC but is cheeper to implement and can be used to image through plastic. It is lower resolution (I assume that this will be argued by the company reps. that sell the system :-) but has made great strides recently.
I recommend the book "Fundamentals of Light Microscopy and Electronic imaging" by Doug Murphy. This is a great book that starts from simple principals and goes into great depth in all of these questions and more. If running a class, this should be the text. It is available on Amazon. I have no financial interest in the book or the publisher, I just think it is the best.
On Monday, June 23, 2003, at 09:05 AM, by way of Ask-A-Microscopist wrote:
} ----------------------------------------------------------------------- } - } The Microscopy ListServer -- Sponsor: The Microscopy Society of } America } To Subscribe/Unsubscribe -- } http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } ----------------------------------------------------------------------- } . } } } Below is the result of your feedback form (NJZFM-ultra-55). It was } submitted by (sfontouris-at-hotmail.com) from } http://www.msa.microscopy.com/Ask-A-Microscopist.html on Monday, June } 23, 2003 at 08:00:10 } ----------------------------------------------------------------------- } ---- } } Email: sfontouris-at-hotmail.com } Name: Yannis Sfontouris } } Organization: University of Nottingham } } Education: Graduate College } } Location: Nottingham, UK } } Question: I would appreciate it very much if someone explained the } differences between i)phase contrast, ii)bright field, iii) Normanski } and iv) Hoffmann optics. } Many thanks in advance } } Yannis } } ----------------------------------------------------------------------- } ---- } }
____________________
David Elliott
Yale University School of Medicine 810 LCI 333 Cedar Street New Haven, CT 06520-8022
I would like to thank those of you who kindly shared the experience regarding removing coverslips off EM blocks.
Since some of you are also interested in this issue, here I post the following summary of the responses.
1. Use chemicals, such as: hydrofluoric acid or hydrogen fluoride, the potential problem is that it may also cause damage to the cell or remove osmium, and they are hazardous.
2. Play with a hot plate instead of liquid nitrogen.
3. Use a jeweler's saw to carefully remove glass coverslips.
4. This method maybe is the cleanest way to get rid of the glass off blocks. I have tried myself and it worked quite well. Polymerize the coverslips with a glass slide together and place on a hotplate or dip into liquid nitrogen. The coverslip will come off with glass slide and leave the cells on the top of the blocks.
We are doing this type of things on a routine basis, so if someone needs more information, please let me know.
Xinran
Xinran Liu, M.D., Ph.D. Center for Basic Neuroscience UT Southwestern Medical Center at Dallas Phone: 214-648-1830 Fax: 214-648-1801 E-mail: xinran.liu-at-utsouthwestern.edu
Does anyone on the list have possession of a Cressington 208HR or Emitech K575X high resolution sputter coater for FEG-SEM with a working IRIDIUM target?
Thanks in advance for any help.
Paul Beauregard PPG Industries Senior Research Associate Monroeville Technical Center 440 College Park Drive Monroeville, PA 15146
I was referring in a general sense to detection limits and/or precision estimates that are delivered by the EDS spectrum-processing software.
My point is that while statistical considerations may well give a realistic estimate of the precision of determination of higher concentrations, they don't RELIABLY provide meaningful detection limits for EDS, in which overlaps and other spectral artefacts such as coincidence peaks and escape peaks can play a large part.
Do you really believe the quoted 'detection limit' of 0.09%?
Even without entering the discussion of exactly what is meant by 'detection limit'.
It would be interesting to hear something on this subject from the EDS system manufacturers. My personal suspicion is that a lot of 'quantitative' results, obtained from standardless EDS packages, are quoted to unjustifiable precision, even to two decimal places!
cheers
rtch
} Dear Richie, } Overlaps should not be a problem for Cl and steel. When I was } looking } for Cl in sediment, I saw what were very small peaks that were only } slightly larger than the noise. However, in several examples there } were small amounts of Ti such that the Ti k-alpha peaks were clearly } present and the k-beta peaks were the same size as the Cl k-alpha. } Since I knew that the Ti k-beta peaks were real, that gave me } confidence that the Cl peaks were also. Yours, Bill Tivol EM Scientist } and Manager Cryo-Electron Microscopy Facility Broad Center, Mail Code } 114-96 California Institute of Technology Pasadena CA 91125 (626) } 395-8833 tivol-at-caltech.edu }
} } } On Sunday, June 22, 2003, at 02:11 PM, Ritchie Sims wrote: } } } If you can't actually see the peak then you can't trust what the } } software says. } } } I bet the detection limit of 0.09% was generated by the software } } from consideration of counting statistics. These may well be OK for } } WDS, but for EDS it's not the counting stats that limit } } detectability, it's the overlaps and other spectral interferences. } }
-- Ritchie Sims Ph D Phone : 64 9 3737599 ext 87713 Microanalyst Fax : 64 9 3737435 Department of Geology email : r.sims-at-auckland.ac.nz The University of Auckland Private Bag 92019 Auckland New Zealand
Hi all, I have an investigator whose lab is in another building, making it inconvenient for them to come over to use our light microscopes routinely. They have an older Zeiss fluorescent microscope and a Coolpix 900 digital camera that they want to set up to quick-check samples. Do you know of any software that would let them preview the images on a computer screen and capture directly to the computer? Using the small camera view screen is not nearly as desirable an option.
Another, probably more expensive option, would be to purchase a new camera. Please let me know if anyone has a camera option for under $2000. Remember that this is for screening only and is not intended to replace the more sophisticated microscope-camera systems available.
Thanks in advance, Debby
Debby Sherman, Manager Phone: 765-494-6666 Life Science Microscopy Facility FAX: 765-494-5896 Purdue University E-mail: dsherman-at-purdue.edu S-052 Whistler Building 170 S. University Street West Lafayette, IN 47907
Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (nmorehouse-at-asu.edu) from http://www.msa.microscopy.com/Ask-A-Microscopist.html on Tuesday, June 24, 2003 at 14:54:17 ---------------------------------------------------------------------------
Question: Hi! I'm looking to measure 100-150 nm features (microstructures on the surface of butterfly wing scales) in a low Z number matrix using a secondary electron signal on a JEOL 840A SEM (LaB 6). The specimens are dehydrated but not fixed. Any suggestions on specimen prep and/or techniques for conducting spatial measurements?
Dear Colleagues, The Organizing Committee has the honor to announce that:
1. The 4th ASEAN Microscopy Conference and The 3rd Vietnam Conference on Electron Microscopy will be continuously held in Hanoi on 05-06 January 2004.
2. The deadline for article submission will be on 30 August 2003.
3. The submitted and approved articles will be put in the Proceedings of the Conference.
Chairman of the Organizing Committee
Nguyen Van Man M.D., D.M.Sc
If you need more informations, please contact at: Assoc. Prof Nguyen Kim Giao Electron Microscopy Unit National Institute of Hygiene and Epidemiology 1- Yersin Str - HaiBaTrung Distr - HaNoi-VietNam Tel: 84.4.9715434 Fax: 84.4.8210853 Email: emlad-at-hn.vnn.vn or emunihe-at-vol.vnn.vn
We do not have either of those instruments, but we routinely sputter iridium with our IBS/e system. Is there something we can do to help or perhaps refer you to one of our customers for some assistance?
Please let me know.
DISCLAIMER: South Bay Technology produces equipment and supplies as described above and, therefore, has a vested interest in promoting their use.
Best regards-
David
"Beauregard, Paul A." wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.msa.microscopy.com/MicroscopyListserver } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Hi, } } Does anyone on the list have possession of a Cressington 208HR or Emitech K575X high resolution sputter coater for FEG-SEM with a working IRIDIUM target? } } Thanks in advance for any help. } } Paul Beauregard } PPG Industries } Senior Research Associate } Monroeville Technical Center } 440 College Park Drive