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From: michael-at-shaffer.net
Date: Sat, 31 Mar 2007 14:13:04 -0500
Subject: [Microscopy] Image Analysis: round robin measurement

Contents Retrieved from Microscopy Listserver Archives
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hello all :o)

I hope to publish and present a paper at the "Automated Mineralogy"
conference this year (Sept 1-2, Brisbane, Oz). Part of it will be a section
on error analysis, and I would like to include examples of similar
determinations as accomplished by other individuals. That is, there is no
independent check on this type of measurement, and even I judged the quality
of my measurements visually. It is a very simple task using tools as
provided by all image analysis softwares, which involves segmentation of a
specific mineral in the presence of other minerals. I have posted an
example image here:
http://www.micro-investigations.com/rrr.htm
(It is an JPEG example only, and I haven't yet determined the appropriate
image)

This message is only intended to gauge the interest is such a project, but I
do imagine that some of you will recognize immediately the problems
associated with this image (and tens of others like it acquired over a short
period of time with an SEM), and who may also like to contribute beyond
measuring a single image. For example, my script has measured several
thousand of these images, and for this paper I would surely be interested in
input beyond sampling as many as possible individuals' visual acuity with a
single image. I still need to consult my co-author, but significant
contributions could mean adding co-authors ... minimally being acknowledged.

Thank you in advance for your interest (or criticisms) ...
Genuinely, Michael Shaffer :o)

SEM/MLA Research Coordinator
http://www.mun.ca/creait/maf/
Inco Innovation Centre
Memorial University
St. John's, Newfoundland
Canada



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7, 18 -- From: "michael shaffer" {michael-at-shaffer.net}
7, 18 -- To: "MSA Microscopy list" {Microscopy-at-microscopy.com}
7, 18 -- Subject: Image Analysis: round robin measurement
7, 18 -- Date: Sat, 31 Mar 2007 16:40:51 -0230
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From: michael-at-shaffer.net
Date: Sat, 31 Mar 2007 14:15:53 -0500
Subject: [Microscopy] Image Analysis: round robin measurement (oops!)

Contents Retrieved from Microscopy Listserver Archives
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hello again :o)

Regarding my previous post to the list, I should have asked that you contact
me directly, except and unless you feel your reply is of general interest
...

Genuinely, Michael Shaffer :o)

SEM/MLA Research Coordinator
http://www.mun.ca/creait/maf/
Inco Innovation Centre
Memorial University
St. John's, Newfoundland
Canada



==============================Original Headers==============================
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6, 18 -- From: "michael shaffer" {michael-at-shaffer.net}
6, 18 -- To: "MSA Microscopy list" {Microscopy-at-microscopy.com}
6, 18 -- Subject: Image Analysis: round robin measurement (oops!)
6, 18 -- Date: Sat, 31 Mar 2007 16:43:45 -0230
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From: michael-at-shaffer.net
Date: Sun, 1 Apr 2007 05:58:13 -0500
Subject: [Microscopy] RE: Image Analysis: round robin measurement

Contents Retrieved from Microscopy Listserver Archives
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hello Paul, and thank you for your reply :o)

Your suggestion brings up an interesting point. That is, I haven't yet
created my "instructions" message that would outline what I want and in what
form, but it should surely invite other methods other than "grayscale
segmentation" as improperly suggested as the only method applicable.

Genuinely, Michael Shaffer :o)

SEM/MLA Research Coordinator
http://www.mun.ca/creait/maf/
Inco Innovation Centre
Memorial University
St. John's, Newfoundland



} -----Original Message-----
} From: Webster, Paul [mailto:PWebster-at-hei.org]
} Sent: March 31, 2007 6:29 PM
} To: michael-at-shaffer.net
} Subject: RE: [Microscopy] Image Analysis: round robin measurement
}
} Hi Micheal,
}
} It looks like you are seeking a way to get what biologists
} know as a volume density analysis. We do it very simply by
} placing cross-lattice overlays onto the iamges and then
} counting the number of points over the full structure and
} then counting points only over the strucuture of interest.
}
} Weibel first introduced the method and it has been verified
} by many people applying it to many applications. Hans
} Gundersen and Terry Mayhew have written a few reviews of the
} method. StereoInvestigator (MicroBrightField, in the US) and
} the CAST system (Olympus) have semi-automated it.
}
} It is a rapid, accurate way of estimating volume densities
} and if performed correctly gives a estimate with about a 5%
} error. At least is does seem to be that way for our
} biological samples.
}
} Regards,
}
} Paul Webster.
}
}
}
}
} -----Original Message-----
} From: michael-at-shaffer.net [mailto:michael-at-shaffer.net]
} Sent: Sat 3/31/2007 12:19 PM
} To: Webster, Paul
} Subject: [Microscopy] Image Analysis: round robin measurement
}
}
}
}
} --------------------------------------------------------------
} --------------
} The Microscopy ListServer -- CoSponsor: The Microscopy
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} --------------------------------------------------------------
} --------------
}
} hello all :o)
}
} I hope to publish and present a paper at the "Automated Mineralogy"
} conference this year (Sept 1-2, Brisbane, Oz). Part of it
} will be a section on error analysis, and I would like to
} include examples of similar determinations as accomplished by
} other individuals. That is, there is no independent check on
} this type of measurement, and even I judged the quality of my
} measurements visually. It is a very simple task using tools
} as provided by all image analysis softwares, which involves
} segmentation of a specific mineral in the presence of other
} minerals. I have posted an example image here:
} http://www.micro-investigations.com/rrr.htm
} (It is an JPEG example only, and I haven't yet determined the
} appropriate
} image)
}
} This message is only intended to gauge the interest is such a
} project, but I do imagine that some of you will recognize
} immediately the problems associated with this image (and tens
} of others like it acquired over a short period of time with
} an SEM), and who may also like to contribute beyond measuring
} a single image. For example, my script has measured several
} thousand of these images, and for this paper I would surely
} be interested in input beyond sampling as many as possible
} individuals' visual acuity with a single image. I still need
} to consult my co-author, but significant contributions could
} mean adding co-authors ... minimally being acknowledged.
}
} Thank you in advance for your interest (or criticisms) ...
} Genuinely, Michael Shaffer :o)
}
} SEM/MLA Research Coordinator
} http://www.mun.ca/creait/maf/
} Inco Innovation Centre
} Memorial University
} St. John's, Newfoundland
} Canada
}
}
}
} ==============================Original
} Headers==============================
} 7, 18 -- From michael-at-shaffer.net Sat Mar 31 14:13:04 2007 7,
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} 2007 14:13:04 -0500
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} n034.sc1.he.tucows.com (7.2.069.1) (authenticated as
} michael-at-shaffer.net)
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} Microscopy-at-microscopy.com; Sat, 31 Mar 2007 19:12:59 +0000
} 7, 18 -- From: "michael shaffer" {michael-at-shaffer.net} 7, 18
} -- To: "MSA Microscopy list" {Microscopy-at-microscopy.com} 7,
} 18 -- Subject: Image Analysis: round robin measurement 7, 18
} -- Date: Sat, 31 Mar 2007 16:40:51 -0230 7, 18 -- Message-ID:
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8, 21 -- References: {200703311919.l2VJJOd0017547-at-ns.microscopy.com} {87449E4A2B01DA47B29424CE5D6E0F8304178D7E-at-hi0sml1.hei.org}
8, 21 -- Subject: RE: [Microscopy] Image Analysis: round robin measurement
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From: gmartens-at-interchange.ubc.ca
Date: Mon, 2 Apr 2007 10:44:32 -0500
Subject: [Microscopy] viaWWW: Analoug video time lapse

Contents Retrieved from Microscopy Listserver Archives
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Hi Barbara,

We have successfully used an analog video camera and just recorded
onto a 8 hour VHS tape. From here we digitized the VHS tape using
iMovie.

Good luck,

Garnet

--
Garnet Martens

Research Manager
BioImaging Facility
University of British Columbia
6270 University Blvd.
Vancouver, B.C.
Canada
V6T 1Z4

phone 604-822-3354

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From: TindallR-at-missouri.edu
Date: Mon, 2 Apr 2007 13:26:20 -0500
Subject: [Microscopy] Free stuff

Contents Retrieved from Microscopy Listserver Archives
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Dear Listers,

We have a Varian 880 high-vac ionization gauge that works fine, as far
as I know. At least it did when we pulled it off the TEM it was on.

We also have an Arkay Gas Burst timer in basically mint condition. It
was a spare for one of our nitrogen-burst processors, so I can't say for
sure that it works, but it looks like it's right out of the box.

You payeth for shipping, they belongeth to you.

Cheers,
Randy

Randy Tindall
Senior EM Specialist
Electron Microscopy Core Facility---We Do Small Well!
W125 Veterinary Medicine
University of Missouri
Columbia, MO 65211
Tel: (573) 882-8304
Fax: (573) 884-2227
Email: tindallr-at-missouri.edu
Web: http://www.emc.missouri.edu
On-line calendar:
http://biotech.rnet.missouri.edu/cgi-bin/Calcium310.pl?Op=Splash&Amount=
Week&NavType=Both&Type=TimePlan


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From: TindallR-at-missouri.edu
Date: Mon, 2 Apr 2007 13:35:11 -0500
Subject: [Microscopy] Free stuff retraction

Contents Retrieved from Microscopy Listserver Archives
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Sorry, all. In my urge to clean up the lab, I typed before I thought.

It happens that these items in my previous post will need to go through
our surplus property system, rather than through an informal give-away.

My apologies for any disappointments.

Randy

Randy Tindall
Senior EM Specialist
Electron Microscopy Core Facility---We Do Small Well!
W125 Veterinary Medicine
University of Missouri
Columbia, MO 65211
Tel: (573) 882-8304
Fax: (573) 884-2227
Email: tindallr-at-missouri.edu
Web: http://www.emc.missouri.edu
On-line calendar:
http://biotech.rnet.missouri.edu/cgi-bin/Calcium310.pl?Op=Splash&Amount=
Week&NavType=Both&Type=TimePlan


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From: m_bouchaour-at-yahoo.fr
Date: Tue, 3 Apr 2007 08:47:19 -0500
Subject: [Microscopy] AskAMicroscopist: GaN/LiNbO3 in SEM

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Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (m_bouchaour-at-yahoo.fr) from http://www.microscopy.com/Ask-A-Microscopist/Ask-A-Microscopist.html on Tuesday, April 3, 2007 at 04:01:39
---------------------------------------------------------------------------

Email: m_bouchaour-at-yahoo.fr
Name: mama bouchaour

Organization: university of metz

Education: Graduate College

Location: metz, france

Question: while i scanned a surface of GaN/LiNbO3 in SEM, i saw, under the layer of GaN a white and brillant zone anb below the bulk of liNbO3. Why this white region appear. GaN is a semiconductor and LiNbO3 is very resistive.

---------------------------------------------------------------------------

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From: dieter.bosshardt-at-zmk.unibe.ch
Date: Tue, 3 Apr 2007 08:51:42 -0500
Subject: [Microscopy] viaWWW: TRAP staining in thick MMA ground sections

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This Question/Comment was submitted to the Microscopy Listserver
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Email: dieter.bosshardt-at-zmk.unibe.ch
Name: Dieter Bosshardt

Organization: School of Dental Medicine, University of Berne

Title-Subject: [Filtered] TRAP staining in thick MMA ground sections

Question: We would like to do tartrate-resistant acid phosphatase (TRAP) staining to visualize osteoclasts in methylmethacrylate (MMA)-embedded tissues. Most people do TRAP staining in paraffin sections or in about 5 micron thick MMA sections. In the past, we successfully did TRAP staining in about 100 micron thick MMA (ground) sections. However, at the moment we are unable to repeat the TRAP staining in thick MMA ground sections.
Is there anyone out there who has a protocol for TRAP staining in thick MMA ground sections? We fixed the tissues in 4% formalin. Thanks a lot in advance.

Best regards,
Dieter Bosshardt

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From: kraftpiano-at-gmail.com
Date: Tue, 3 Apr 2007 19:31:13 -0500
Subject: [Microscopy] JEOL JSM-840 donation is here!

Contents Retrieved from Microscopy Listserver Archives
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Well, we received the SEM today. We got it off of the truck and into
the cafeteria, which we are using as a staging area until we can get
it into its permanent home in my lab upstairs. That's right,
upstairs.

Despite their best efforts to the contrary, the shipping company did
not damage it too much. The main table holding the chamber had
shifted off of the springs, but was still fairly stable when we
unpacked it. The main transformer in the bottom of the control
console had leaked all over, so we are dismantling the console to get
all the oil out. Most of the screws had fallen out of the column
itself, so we just marked the location and order of the sections and
took them off as we moved it into the cafeteria. I found a good
number of the screws stuck to the side of the ion pump magnet which
had been set on the bottom of the main scope unit.

The vacuum system is a disaster, though. Whoever disconnected this
SEM from service did a real hack job. The water hoses are cut, and
the vacuum system was almost entirely dismantled. All of the
compressed air/nitrogen lines were disconnected. Anybody have a good
schematic as to which valves get connected where?

So, here is the order of the project priorities:

1: Get everything upstairs. Most will be in pieces when it gets up
there, but we have documented it to no end (About 180 photos of the
unit all taken down)

2: Re-assemble the column and microscope console. This will include
re-working the entire vacuum system. There is oil in the diffusion
pumps, but I don't know how much. I'll check and see if it pumps
before trying to add more. There is a measurable amount, so I'm not
too worried. It looks clean and good. I need to get all the water
and air lines connected again. Again- if anybody has a schematic of
this system I would greatly appreciate it.

3: Open and clean out the main transformer tank. Replace the oil,
since most of it is in the bottom of the truck and control console.

4: Re-connect everything in the control console. Luckily most of the
cables are labeled well.

5: Figure out the six wires that were cut. There are six wires that
were cut between the center triangular console and wherever they went.
Luckily, though, I can see that they have different numbers of wires
within the cables, so I should be able to get a decent idea where they
go when I find the other end. Then, the six conductor wire gets
spliced back to the six conductor, etc...

6: Obtain a vacuum pump. There was no pump box for this system.

7: Test the vacuum seals. Since we are having to re-seal most of the
vacuum connections, I will run several dry runs with the vacuum
system. I don't see an indicator for the vacuum level anywhere on the
console. Is there a test point I can read the value off of?

8: Order new filaments. None were included. I think I'll add a set
of apertures to that as well.

9: Try to power it up and see what happens at low accelerating voltages.

10: Explore the microscopic world! (We hope.)

It looks as though this was at one point hooked up to a digital
acquisition system, as there are soldered on BNC connectors labeled
for horizontal, vertical, and beam control.

One final question: In the control console, there are two boxes at the
bottom most level. The one on the left (As you are looking at the
rear) is the main HV transformer tank. The one on the right looks
like it has some plug-in modules in it, and there is a hose running
from each module to the next module. Is this for cooling water or
oil? There were no connections or hoses that lead to the system or
away from it.

Thanks,

Justin A. Kraft

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From: marilyn.lemieux-at-genzyme.com
Date: Tue, 3 Apr 2007 20:13:55 -0500
Subject: [Microscopy] AskAMicroscopist: Koehler illumination

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Email: marilyn.lemieux-at-genzyme.com
Name: Marilyn LeMieux

Organization: Genzyme Genetics

Education: Graduate College

Location: Orange, CA, USA

Title: Koehler illumination

Question: Some books say that this must be performed on both low and high power as you focus on an object to be imaged (digital image). Others say that you only need to perform the koehler steps on High power(images are taken only on high power).
Do those who say to do it on both low, then high power, is that to 'get you in the ballpark' prior to going to high, or is this step unnecessary?

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From: tttan-at-SIMTech.a-star.edu.sg
Date: Tue, 3 Apr 2007 20:18:46 -0500
Subject: [Microscopy] RE: Electron beam simulation

Contents Retrieved from Microscopy Listserver Archives
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Hello,
 
I am trying to simulate electron beam heating in the SEM. I am sure this is not a new topic and perhaps lots of people had done some work on it. I am totally new to this area so like to check if anyone has good journals to recommend?
 
In my simulation, I input a figure for the probe current density (got from some journals), I inevitably gets melting... I am still trying to verify this.
 
Can anyone point out to me a typical figure for probe size, current and perhaps even current distribution equation for a TEM probe?
 
Thank you very much
 
Regards
TT
 


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From: nizets2-at-yahoo.com
Date: Wed, 4 Apr 2007 02:20:05 -0500
Subject: [Microscopy] Re: AskAMicroscopist: Koehler illumination

Contents Retrieved from Microscopy Listserver Archives
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Hi!

I don't understand your expression, but performing a
Koehler takes less than 1 min, so why bother about NOT
doing it at lower mag if this can help at higher mag?
If you think this does not help, why do you care? The
purpose it to focus the beam on your object, in the
condition you take the picture.
That said, if you take all your pictures are the same
(high) magnification, you probably don't need to
perform a Koehler each and every time.

Stephane


--- marilyn.lemieux-at-genzyme.com wrote:

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} Email: marilyn.lemieux-at-genzyme.com
} Name: Marilyn LeMieux
}
} Organization: Genzyme Genetics
}
} Education: Graduate College
}
} Location: Orange, CA, USA
}
} Title: Koehler illumination
}
} Question: Some books say that this must be performed
} on both low and high power as you focus on an object
} to be imaged (digital image). Others say that you
} only need to perform the koehler steps on High
} power(images are taken only on high power).
} Do those who say to do it on both low, then high
} power, is that to 'get you in the ballpark' prior to
} going to high, or is this step unnecessary?
}
}
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} ==============================Original
} Headers==============================
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} 3 20:13:54 2007
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} of Ask-A-Microscopist)
} 8, 11 -- Subject: AskAMicroscopist: Koehler
} illumination
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From: keith.morris-at-ucl.ac.uk
Date: Wed, 4 Apr 2007 03:39:48 -0500
Subject: [Microscopy] AskAMicroscopist: Koehler illumination

Contents Retrieved from Microscopy Listserver Archives
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Hi Marilyn,

To quote: "The Koehler technique is recommended by all manufacturers of
modern laboratory microscopes because it can produce specimen illumination
that is uniformly bright and free from glare, thus allowing the user to
realize the microscope's full potential."

Find out more at:

http://micro.magnet.fsu.edu/primer/anatomy/kohler.html
http://www.aecom.yu.edu/aif/instructions/koehler/koehler.htm

Note that you should check Koehler illumination this every-time you change
objective on a microscope, and setting Koehler illumination is crucial if
you are using Phase Contrast (or DIC) optical contrast enhancement. So even
low power phase objectives require Koehler adjustment for good images via
transmission illumination. It is also required if you are capturing
transmission images via a camera (or they won't look that good at all).

For heavily stained sections at low magnifications you can get by without
bothering, but as Stephane points out it takes very little time to setup and
it is poor science not to check it every time you use the microscope
(particularly as you will have spent many hours preparing the specimen).
Previous users may have setup the optics incorrectly for various reasons.

Koehler illumination is irrelevant with epi-fluorescent imaging as with this
the light is backscattered into the objective, although often you will also
want a standard phase contrast or DIC transmission image as well. Koehler
illumination is essential for transmission images of unstained specimens
with limited contrast (where phase contrast or DIC optics is often also
required to enhance the specimens contrast by optical interference within
structures inside the specimen).

Poorly adjusted optics lead to very uneven illumination and the appearance
of dark shadows in the image. It will very badly affect contrast enhancement
optics (you won't get much enhancement). These problems are naturally best
avoided, particularly as setting the optics correctly is so easy. All
microscope manuals will tell you how to set up Kohler illumination with the
microscope (plus other important things like aligning illumination bulbs and
phase contrast rings). Expensive modern motorised microscopes can do much of
this automatically these days.

Regards

Keith

---------------------------
Dr Keith J Morris
Imaging Facilities Manager
Cell Biology
Institute of Ophthalmology
UCL, London EC1V 9EL




-----Original Message-----
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Sent: 04 April 2007 02:18
To: keith.morris-at-ucl.ac.uk

This Question was submitted to Ask-A-Microscopist by
(marilyn.lemieux-at-genzyme.com)
from http://microscopy.org/Ask-A-Microscopist/Ask-A-Microscopist.html on
Tuesday, April 3, 2007 at 12:33:59
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Question
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Please reply to both marilyn.lemieux-at-genzyme.com as well as to the
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Email: marilyn.lemieux-at-genzyme.com
Name: Marilyn LeMieux

Organization: Genzyme Genetics

Education: Graduate College

Location: Orange, CA, USA

Title: Koehler illumination

Question: Some books say that this must be performed on both low and high
power as you focus on an object to be imaged (digital image). Others say
that you only need to perform the koehler steps on High power(images are
taken only on high power).
Do those who say to do it on both low, then high power, is that to 'get you
in the ballpark' prior to going to high, or is this step unnecessary?

---------------------------------------------------------------------------

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From: rblyston-at-trinity.edu
Date: Wed, 4 Apr 2007 05:10:03 -0500
Subject: [Microscopy] AskAMicroscopist: Koehler illumination

Contents Retrieved from Microscopy Listserver Archives
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To all:

If you drive a car with a manual gear shift when you want to go, you
push in the clutch, put it into first gear and let out the clutch;
then you push in the clutch put it into second gear and let out the
clutch, then you push in the clutch and put it into third gear and
let out the clutch.

When you are driving a manual microscope, you click in the low power
objective, change the diaphragm and adjust the condenser; when you
want middle magnification (second gear) you change the diaphragm and
adjust the condenser; and when you want high power, etc.

I can continue to play with this analogy. I do not know why people
can accept moving the focus adjustment on a microscope but not the
condenser adjustment when magnification is changed. Perhaps these
poor souls did not do well in optics when they took college physics.

Bob Blystone


On Apr 4, 2007, at 2:21 AM, nizets2-at-yahoo.com wrote:

}
} Hi!
}
} I don't understand your expression, but performing a
} Koehler takes less than 1 min, so why bother about NOT
} doing it at lower mag if this can help at higher mag?
} If you think this does not help, why do you care? The
} purpose it to focus the beam on your object, in the
} condition you take the picture.
} That said, if you take all your pictures are the same
} (high) magnification, you probably don't need to
} perform a Koehler each and every time.
}
} Stephane
}
} }
} } Title: Koehler illumination
} }
} } Question: Some books say that this must be performed
} } on both low and high power as you focus on an object
} } to be imaged (digital image). Others say that you
} } only need to perform the koehler steps on High
} } power(images are taken only on high power).
} } Do those who say to do it on both low, then high
} } power, is that to 'get you in the ballpark' prior to
} } going to high, or is this step unnecessary?


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From: lcgould-at-med.cornell.edu
Date: Wed, 4 Apr 2007 08:02:42 -0500
Subject: [Microscopy] Re: AskAMicroscopist: Koehler illumination

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Dear Marilyn,
It is always good to "Kohler" every time you change lenses,
especially if you are going to take pictures (digital or otherwise).
"Kohlering" aligns the illumination system with the rest of the
microscope's optical axis, ensuring even illumination without odd
shadings or shadows. Once you get the hang of it, it only takes a few
seconds to do it, so it is certainly worth the effort.
Lee
--
Leona Cohen-Gould, M.S.
Sr. Staff Associate
Director, Electron Microscopy & Histology Core Facility
Manager, Optical Microscopy Core Facility
Weill-Cornell Medical College

voice (212)746-6146
fax (212)746-8175
http://www.cornellcelldevbiology.org
http://www.cornellbiochem.org

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From: lcgould-at-med.cornell.edu
Date: Wed, 4 Apr 2007 08:12:52 -0500
Subject: [Microscopy] Re: Koehler illumination

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Dear Marilyn,
It is always good to "Kohler" every time you change lenses,
especially if you are going to take pictures (digital or otherwise).
"Kohlering" aligns the illumination system with the rest of the
microscope's optical axis, ensuring even illumination without odd
shadings or shadows. Once you get the hang of it, it only takes a few
seconds to do it, so it is certainly worth the effort.
Lee
--
Leona Cohen-Gould, M.S.
Sr. Staff Associate
Director, Electron Microscopy & Histology Core Facility
Manager, Optical Microscopy Core Facility
Weill-Cornell Medical College

voice (212)746-6146
fax (212)746-8175
http://www.cornellcelldevbiology.org
http://www.cornellbiochem.org

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From: bfoster-at-mme1.com
Date: Wed, 4 Apr 2007 11:21:51 -0500
Subject: [Microscopy] AskAMicroscopist: Koehler illumination

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

To Marilyn and anyone who uses light microscopy:

I can't stress strongly enough the importance of establishing Koehler
illumination for all techniques ... and I agree strongly with several
of the responses which stress how easy and quick the process is, once
you have done it a few times.

Koehler illumination establishes the "baseline" for all other
imaging. Setting aside alignment of the lamp filament (which
typically only needs to be done when the lamp is changed), it
involves the simple setting of focus and apertures for three key lens
sets: objective, condenser, and eyepieces. On most microscopes,
each of these lenses has adjustment for focus. Also, it is important
to understand the appropriate setting for the field iris (which
controls scatter and glare) and aperture iris (which controls
coherence and has a major impact on edge fidelity as well as resolution).

Unfortunately, today's schedule doesn't permit a long discussion, but
for those of you who are interested in a brief anatomny and
physiology less regarding each of the three key lenses and their
apertures... Plus a short recipe for establishing Koehler, send me an
email with KOEHLER, PLEASE in the subject and I'll try to send you a
PDF early next week, when I am back in the office. Also, there is a
detailed section in Optimizing Light Microscopy (call Ken Piel here
at MME for ordering information).

The take away message: PLEASE...take a few minutes to become
familiar with Koehler illumination and use it daily and check it
whenever you move from one mag to another or one technique to
another. Your microscopy will improve dramatically.

Best regards,
Barbara Foster, President

Microscopy/Microscopy Education
313 S Jupiter Rd, Suite 100
Allen, TX 75002
P: 972-954-8011
W: www.MicroscopyEducation.com


MME is now scheduling customized, on-site courses through July. Call
us today for details.

P. S.
Need a good general reference or light microscopy text for next
semester? Call us today to learn more about "Optimizing LIght
Microscopy". Copies still available through MME... even for
class-room lots ... and we give quantity discounts. Call Ken Piel at
(972)954-8011.



At 11:31 AM 4/4/2007, keith.morris-at-ucl.ac.uk wrote:



} ----------------------------------------------------------------------------
} The Microscopy ListServer -- CoSponsor: The Microscopy Society of America


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From: frank.karl-at-degussa.com
Date: Wed, 4 Apr 2007 11:55:59 -0500
Subject: [Microscopy] Koehler illumination revisited

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Now, lets get one thing straight from the beginning. I use Koehler
illumination. I think Koehler illumination is the mark of the competent
microscopist. Don't use Koelhler illumination? As one of friends says
"Dude, that's just wrong!"

But in truth the only scope I have true Koehler illumination is a monocular
petrographic scope with a detached but focusable AO lamp with an iris.
This scope is my own at home in my lab. All the scope I have seem and used
in the last 20 years were missing some feature which prevented true Koehler
illumination.

Some were lacking centerable lamps, others immovable ground glass filters
while other did not have centerable objectives. Those that did had wire
filaments and not ribbon filaments.

I don't care whose brand...It seems impossible to set up true classic
Koehler illumination. (I don't even want to talk about focusable and
centerable Bertrand lens!)

That my story and I'm sticking to it

Frank


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From: larry.ackerman-at-ucsf.edu
Date: Wed, 4 Apr 2007 12:24:22 -0500
Subject: [Microscopy] histo staining voids

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

This is more of a histology question than EM but is related. One of the
labs I work for has had problems processing/staining sections on glass
light microscopy slides when they use the Shandon Sequenza Coverplate
technology* in a rack on a cooling plate in a microwave. The problem is
small circular voids in the staining/labeling. This happens with both
immuno and general tissue stains and is similar to a phenomena I
occasionally see when staining semi-thin epoxy sections with toluidine
blue on a hot plate. It appears as if there is some localized boiling
resulting in a bubble of gas thus displacing the stain reagent.

My question is: have you experienced this phenomena and do you have a
solution for eliminating it?

* This is a patented plastic device that fits over a standard microscope
slide allowing specimens to be immunostained with a minimum of reagent
(~ 80 microliters).

Thanks, Larry
--
Larry Ackerman, Associate Specialist
UCSF, Dept. of Anatomy, Rm S1347
513 Parnassus Ave., Box 0452
San Francisco, CA 94143

larry.ackerman-at-ucsf.edu


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From: wesaia-at-iastate.edu
Date: Wed, 4 Apr 2007 16:01:02 -0500
Subject: [Microscopy] Digital cameras and the TEM

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

This has been an interesting thread to follow. Each of the replies has brought out some valid points, but I don't know if they quite got to Frank's questions.
 
It is important to consider nanometers (or microns) per pixel. That will determine the ultimate resolution you have to work with. Of course Nyquist will tell you that you can't push things to the single-pixel dimensions - a couple of pixels is more likely your limit.
 
It is also important to remember that raw pixel count alone is meaningless. You have to consider the image formation process. The camera needs to be matched to the phosphor for optimum cost and performance. Excess pixels in the camera beyond the resolution of the phosphor will just waste money. Insufficient pixels will forego potential resolution. I am not a TEM fellow, so I don't know the specifics of the camera systems, but I would like to think that systems are fairly well matched by the designers, at least now that the costs of CCDs are coming down. I also won't address the limits of microscope resolution. It will enter into the picture at some magnification.
 
Frank's questions seemed to deal with appreciating or visualizing the resolution once the image was captured.
 
On the computer screen, much software seems to display the images, or portions thereof, at one pixel of image per one pixel of screen. Many screens are setup so that pixels are NOT terribly obvious to the eye from normal viewing distance. Therefore, it will be difficult to notice one pixel more or less to a dimension without zooming in on the image. The software will have full access to the image data and can make measurements down to the pixel level.
 
The printed image also raises visualization issues. Multiple dots are required to render a single pixel, at least for those printers (laser and many inkjets) where a dot is either there or not. A pattern of dots is needed together to represent shades. Therefore, the printed pixels per inch is practically an order of magnitude less than the dots per inch. And then there are the "truth in labeling" issues. What is the printer genuinely capable of?
Once again, the resolution of the eye comes into play. It is quoted at about 500 pixels (250 pixel pairs) per inch at 20 inches, but I don't think I would be appreciating one pixel more or less at that printed resolution. I have a hard time seeing jagginess in real-world, 1024-pixel-wide images printed at 5 inches. Zooming is necessary for me to clearly see individual pixels.
 
So it's time to get back to your original question about 3 megapixel cameras versus 1 megapixel cameras. My opinion is that you will only marginally appreciate the greater number of pixels on the screen or in a printed image. If your software maps images to the screen pixel by pixel, the image collected at a given magnification will be bigger on the screen and each pixel will represent a smaller dimension. The same software will probably print the image the same size and the question will be whether your eye will be able to appreciate the finer detail. It will offer larger prints (or more zoom) before pixel jagginess appears to the same degree.
 
For image analysis, the pixels will be finer at a given magnification (field of view). Therefore, you should be able to perform measurements on smaller features than before.
 
Warren Straszheim
----------------------------------------------------------------------------
The Microscopy ListServer -- CoSponsor:  The Microscopy Society of America

Let me float an question to the list.  What is the effect of changing a one
meg camera to a three meg camera on a TEM?

 I'm asked some fundamental questions, and I can't seem to answer them to
this person's satisfaction,  which implies I don't have a good grasp of the
question or answers.

 My immediate response is you would increase the resolution.  I can
envision the image size on the monitor changing,  but if the resolution of
the screen is lower than the captured image and if the computer/imaging
software wants to display all the image captured, will not any feature at a
specific magnification have the resolution of the monitor?   It seems the
same is true for the printer.  I can't simply expand the size of the paper
at will, so the software will either reduce the printed image magnification
or print just a smaller section of the total image.  Again, since the
printer has a fixed resolution will not the printed image resolution will
be limited by the printer's (This sounds like a Hi-Fi discussion from the
early 60's...  just change the words...) upper limit?

So why capture high resolution images?  My response is it allows you to
post process and expand the image to examine one feature and have
sufficient "stored" resolution to display the image without  empty
magnification.  This also implies (to me at least) if I want to measure
from point A to point B,  the more camera pixels I have the better I can
resolve where point A starts and point B ends which should allow me to have
better confidence in my measurements.  I believe that imaging software
works on the image in memory and not the image displayed on the screen so
size of the print or screen has little to do with data obtained. It's more
a function of the size of the captured image?

Lastly.....

If I feel the need to have at least 1000 pixels in an image feature, and
due to my camera I can only capture 500 at a magnification X, is there any
reason not to simply increase the magnification so I have a larger feature
which now occupies more pixels.  I realize I haven't increased the
resolution, but if my software need 999 pixels to "recognize"  a feature
haven't I met this requirement?

Thank in advance!
Frank (I miss film) Karl


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From: Edward.Calomeni-at-osumc.edu
Date: Wed, 4 Apr 2007 16:08:03 -0500
Subject: [Microscopy] histo staining voids

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Larry,

There may be two items happening. First is incomplete covering of the
sections by the reagents. I am assuming this is a capillary action type of
slide holder/stainer. Second is exactly what you mentioned, micro-boiling of
reagent.

Possible ways to help with the first is to add a surfactant/detergent to
reagents, i.e. triton/saponin in very dilute amounts.

Possible way to help with second is to lower the wattage of the microwave,
due multiple short times of microwaving, add more heat sinks to oven or go
back to the old tried and true method of overnight incubations at 4 degrees.

Ed


Edward P. Calomeni
Director EM Lab - Pathology
The Ohio State University
M018 Starling Loving Hall
320 W. 10th Ave.
Columbus, OH 43210
614-293-5580 (office)
614-293-8806 (lab)
edward.calomeni-at-osumc.edu
-----Original Message-----
X-from: larry.ackerman-at-ucsf.edu [mailto:larry.ackerman-at-ucsf.edu]
Sent: Wednesday, April 04, 2007 1:29 PM
To: Calomeni, Edward

This is more of a histology question than EM but is related. One of the labs
I work for has had problems processing/staining sections on glass light
microscopy slides when they use the Shandon Sequenza Coverplate
technology* in a rack on a cooling plate in a microwave. The problem is small
circular voids in the staining/labeling. This happens with both immuno and
general tissue stains and is similar to a phenomena I occasionally see when
staining semi-thin epoxy sections with toluidine blue on a hot plate. It
appears as if there is some localized boiling resulting in a bubble of gas
thus displacing the stain reagent.

My question is: have you experienced this phenomena and do you have a
solution for eliminating it?

* This is a patented plastic device that fits over a standard microscope
slide allowing specimens to be immunostained with a minimum of reagent (~ 80
microliters).

Thanks, Larry
--
Larry Ackerman, Associate Specialist
UCSF, Dept. of Anatomy, Rm S1347
513 Parnassus Ave., Box 0452
San Francisco, CA 94143

larry.ackerman-at-ucsf.edu


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From: TJJ-at-stowers-institute.org
Date: Wed, 4 Apr 2007 17:29:30 -0500
Subject: [Microscopy] histo staining voids

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Larry, we use this system and have not had this problem. However, in
another lab, in another galaxy, far, far away, our IHC lab had problems
with this. We deduced it was air bubble formation, and reduced the
concentration of detergent used in the rinse buffer. We currently use
0.05% Tween 20 and have no problems with it.

Careful application of the slides to the coverplate is also warranted!
If you wish, you can contact me off list and I'll give you our procedure
for mounting the slides.

Hope this helps!

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110


-----Original Message-----
X-from: larry.ackerman-at-ucsf.edu [mailto:larry.ackerman-at-ucsf.edu]
Sent: Wednesday, April 04, 2007 12:31 PM
To: Johnson, Teri

This is more of a histology question than EM but is related. One of the
labs I work for has had problems processing/staining sections on glass
light microscopy slides when they use the Shandon Sequenza Coverplate
technology* in a rack on a cooling plate in a microwave. The problem is
small circular voids in the staining/labeling. This happens with both
immuno and general tissue stains and is similar to a phenomena I
occasionally see when staining semi-thin epoxy sections with toluidine
blue on a hot plate. It appears as if there is some localized boiling
resulting in a bubble of gas thus displacing the stain reagent.

My question is: have you experienced this phenomena and do you have a
solution for eliminating it?

* This is a patented plastic device that fits over a standard microscope

slide allowing specimens to be immunostained with a minimum of reagent
(~ 80 microliters).

Thanks, Larry
--
Larry Ackerman, Associate Specialist
UCSF, Dept. of Anatomy, Rm S1347
513 Parnassus Ave., Box 0452
San Francisco, CA 94143

larry.ackerman-at-ucsf.edu


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From: kraftpiano-at-gmail.com
Date: Wed, 4 Apr 2007 21:11:10 -0500
Subject: [Microscopy] First pictures of the JEOL restoration.

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Ok. First of all, before I get yelled at for dismantling the column
and stage of the SEM when removing it from the truck, be aware that
the entire top portion of the console had shifted so badly during
transit that the center of gravity was so far off that it was going to
tip over if I even looked at it funny. The only way I saw to get it
out of the truck without a major catastrophe was to take the column
apart. Since the vacuum system was already pretty much dismantled, I
didn't see that this would cause too much more pain.

As far as dismantling the control console goes, I did it because I had
to get the transformer oil out of it. It was very slippery, and
getting it clean was priority one. Thanks to those who informed me of
the PCBs. I was aware of that, and I am taking as much precausion as
possible, given the extent of the leakage. Again, not to fan the
fires, I know that it is in the cafeteria, but it was pretty much
contained in the control console and cleaned out before any parts were
placed in the cafeteria. The table that the transformer is sitting on
was broken and about to be thrown out anyway, so I used it so I don't
have to heft the transformer off the floor.

So, for your perusal, here are the first sets of photos to come out of
the restoration.

http://www.jkraft.net/semproject

Enjoy!

--Justin.

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From: ewing.hr-at-bruker-axs.com
Date: Thu, 5 Apr 2007 08:10:39 -0500
Subject: [Microscopy] viaWWW: Job Opportunity at Bruker AXS

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

This Question/Comment was submitted to the Microscopy Listserver
using the WWW based Form at http://microscopy.com/MLFormMail.html
---------------------------------------------------------------------------
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Email: ewing.hr-at-bruker-axs.com
Name: Ted Juzwak

Organization: Bruker AXS

Title-Subject: [Filtered] Job Opportunity at Bruker AXS

Question: Ư
Microanalysis Applications Engineer

Bruker AXS, Inc., a leading manufacturer of Analytical Instrumentation, has a job opening for an Applications Engineer in their Ewing, NJ Microanalysis lab. This division specializes in Energy Dispersive X-ray (EDX) Analysis instrumentation for electron microscopes.

The position entails customer, service, sales and marketing support.

Primary Responsibilities:
* Demonstration of the equipment at Bruker and customer sites
* Analysis of customer samples and production of clear analysis reports
* Provide technical support to existing, and potential customers via e- mail and phone
* User training in the operation of the Bruker Microanalysis System at Bruker and customer sites
* Produce and maintain appropriate materials for customer training
* Preparation of technical sales materials
* Assist field service in interpreting and resolving customer problems
* Perform other tasks as assigned by manager or supervisor.

This position involves up to 30% travel, primarily in North America.

Candidates interested in this position require a minimum of an Associates degree in a Physical Science discipline with a Bachelors degree in Chemistry, Geology or Materials Science preferred. Two years SEM/X-ray microanalysis experience, including sample preparation, is preferred.

Requirements include excellent communication skills and proficiency with Microsoft Office.

Send resume and salary requirement to:

Ted Juzwak
Bruker AXS Microanalysis, Inc.
1239 Parkway Avenue, Suite 203
Ewing, NJ 08628

ewing.hr-at-bruker-axs.com

Bruker AXS Inc. offers a competitive salary and comprehensive benefits package including health and dental insurance, Flexible Spending Account, company sponsored life and disability insurance, 401(k) plan with company matching components, and vacation and sick/personal days.


---------------------------------------------------------------------------


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From: ewing.hr-at-bruker-axs.com
Date: Thu, 5 Apr 2007 08:22:01 -0500
Subject: [Microscopy] viaWWW:Position Open at Bruker AXS

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

This Question/Comment was submitted to the Microscopy Listserver
using the WWW based Form at http://microscopy.com/MLFormMail.html
---------------------------------------------------------------------------
Remember this posting is most likely not from a Subscriber, so when replying
please copy both ewing.hr-at-bruker-axs.com as well as the MIcroscopy Listserver
---------------------------------------------------------------------------

Email: ewing.hr-at-bruker-axs.com
Name: Ted Juzwak

Organization: Bruker AXS

Title-Subject: [Filtered] Job Opportunity at Bruker AXS

Question: *
Microanalysis Applications Engineer

Bruker AXS, Inc., a leading manufacturer of Analytical Instrumentation, has a job opening for an Applications Engineer in their Ewing, NJ Microanalysis lab. This division specializes in Energy Dispersive X-ray (EDX) Analysis instrumentation for electron microscopes.

The position entails customer, service, sales and marketing support.

Primary Responsibilities:
* Demonstration of the equipment at Bruker and customer sites
* Analysis of customer samples and production of clear analysis reports
* Provide technical support to existing, and potential customers via e- mail and phone
* User training in the operation of the Bruker Microanalysis System at Bruker and customer sites
* Produce and maintain appropriate materials for customer training
* Preparation of technical sales materials
* Assist field service in interpreting and resolving customer problems
* Perform other tasks as assigned by manager or supervisor.

This position involves up to 30% travel, primarily in North America.

Candidates interested in this position require a minimum of an Associates degree in a Physical Science discipline with a Bachelors degree in Chemistry, Geology or Materials Science preferred. Two years SEM/X-ray microanalysis experience, including sample preparation, is preferred.

Requirements include excellent communication skills and proficiency with Microsoft Office.

Send resume and salary requirement to:

Ted Juzwak
Bruker AXS Microanalysis, Inc.
1239 Parkway Avenue, Suite 203
Ewing, NJ 08628

ewing.hr-at-bruker-axs.com

Bruker AXS Inc. offers a competitive salary and comprehensive benefits package including health and dental insurance, Flexible Spending Account, company sponsored life and disability insurance, 401(k) plan with company matching components, and vacation and sick/personal days.


---------------------------------------------------------------------------

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From: kraftpiano-at-gmail.com
Date: Thu, 5 Apr 2007 17:51:42 -0500
Subject: [Microscopy] JEOL donation- desperate cry for a part!

Contents Retrieved from Microscopy Listserver Archives
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Ok. Today we concentrated on getting the column put back together.
So far so good. We got to the point where we're doing preliminary
vacuum checks- seeing if it holds a vacuum without powering it on, but
manually actuating the valves. It doesn't. After about an hour and a
half under the column checking every gasket, we discovered a gaping
hole where there was at one point an EDS system. Ok.

We need the blanking cover for the angled opening that heads down into
the chamber! If anybody has one lying around they aren't using, I
would greatly appreciate it!

We are also missing the little thingy that one uses to place the
specimen in the chamber through the airlock. I'm staring at an open
hole, and I know that there is some kind of rod that pushes the
specimen in, but we don't have it. If there is an extra somewhere,
I'd appreciate it. If you even have one that you could photograph
closely for me so I can see if I can make one, I would appreciate that
as well.

I've posted more pictures on my site- http://www.jkraft.net/semproject

Thanks,

Justin.

==============================Original Headers==============================
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From: snelson-at-wustl.edu
Date: Thu, 5 Apr 2007 21:36:01 -0500
Subject: [Microscopy] viaWWW: Fluorescence correlation spectroscopy question

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Email: snelson-at-wustl.edu
Name: Scott Nelson

Organization: Washington University

Title-Subject: [Filtered] Fluorescence correlation spectroscopy question

Question: I use a Zeiss Confocor 2 to look at diffusion of GFP in yeast cells. My plot of G(t) sometimes contains a very strong sine wave-like oscillation with a period of about 100 hertz, and it ruins the curve. There is no obvious oscillation in the raw fluorescence intensity plot.

We guess that there is some sort of mechanical vibration in the room, but the scope is on an air table. The yeast cells are physically stuck to my slide and don't move at all. I don't see the oscillation if I look at fluorophores in solution.

Does anyone have any idea what this is and how to eliminate it?

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From: roberta.burns-at-ametek.com
Date: Thu, 5 Apr 2007 21:36:50 -0500
Subject: [Microscopy] viaWWW: JOb Opportunity at EDAX

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Email: roberta. burns-at-ametek.com
Name: Roberta Burns

Organization: EDAX

Title-Subject: [Filtered] Job Opportunity at EDAX

Question: EDAX, Inc., a leading mnaufacturer of X-Ray Microanalysis Equipment has an opening for a Technical Product Manager for TEM Products.
The position will handle all matters pertaining to EDAX elemental analysis and crystalography products for the TEM to include stategic product planning, product specification, budget/financial planning, marketing programs and sales support.

The position requires a Masters Degree in Science/Engineering and 5 years of relevant work experience.

Plesas send resumes to:
Roberta Burns
Human Resources Manager
EDAX Inc.
91 McKee Drive
Mahwah, NJ 07430



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From: steguido-at-unina.it
Date: Fri, 6 Apr 2007 08:52:36 -0500
Subject: [Microscopy] LM - Time-lapse microscopy course announcement

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html


COURSE ANNOUNCEMENT

Time-Lapse Microscopy Short Course
Live Cell Imaging for Biomedical Applications

2-4 May 2007, Villa Orlandi (Capri)

The University of Napoli “Federico II”, with the patronage of the Italian Cell
Culture Association, is organizing a time-lapse microscopy course on May 2–4,
2007 in Capri. The course is addressed to both senior scientists and students
interested in live cell imaging techniques. Both lectures and practical
sessions on video microscopy workstations will be provided.

Course organizers
Stefano Guido and Marino Simeone (University of Napoli “Federico II”, Italy)

Course overview
The recent advances in microscopy techniques, coupled with the development of
fluorescence markers, have provided scientists with an array of experimental
tools to follow processes at the cellular level in a dynamic fashion. The
purpose of this course is to provide an intensive training on state-of-the-art
methods for live cell imaging by optical microscopy, with special emphasis on
time-lapse techniques. Starting with a basic introduction to optical
microscopy, the topics covered in the course program include microscope
incubation, 3D fluorescence and multidimensional microscopy,
computer-controlled sample scanning and optical sectioning, image processing
and archiving, cell tracking, specialized fluorescence techniques (FRAP, FLIP,
FRET, SPIM, TIRFM), and time-lapse applications.

Invited speakers:
- Alberto Diaspro (Università di Genova, Italy)
- Ernst H. K. Stelzer (EMBL, Heidelberg, Germany)
- Peter Evennett (Royal Microscopical Society, UK)
- Spencer L. Shorte (Institut Pasteur, Paris, France)
- M. Cristina Cardoso (Max Delbrück Center for Molecular Medicine MDC, Berlin,
Germany)
- Roman S. Polishchuk (Consorzio Mario Negri Sud, Chieti, Italy)
- Elena V. Polishchuk (Consorzio Mario Negri Sud, Chieti, Italy)
- Tony Collins (McMaster University, Hamilton, Canada)
- Dario Parazzoli (National Institute of Molecular Genetics INGM, Milano,
Italy)

MAIN SPONSOR: Okolab, Italy (http://www.oko-lab.com)
OTHER SPONSORS (to be updated): Zeiss, Hamamatsu, Coherent, Molecular Cytomics
Official media partner: Imaging & Microscopy.

Please find more information on the course website:
http://www.time-lapse-microscopy.unina.it or send an email to
segreteria-at-time-lapse-microscopy.unina.it


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From: jmastrangelo-at-ulbi.com
Date: Fri, 6 Apr 2007 20:51:53 -0500
Subject: [Microscopy] viaWWW: SEM preparation for insects, other recommendations

Contents Retrieved from Microscopy Listserver Archives
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Email: jmastrangelo-at-ulbi.com
Name: Joe Mastrangelo

Organization: Ultralife Batteries

Title-Subject: [Filtered] SEM preparation for insects, other recommendations

Question: Our company typically sponsors a tour for "National Bring your Child to Work Day". Now that we have a SEM in house, I hope to be able to make their visit to our lab a memorable one.

Since most of my imaging is done on powders, mixtures, coatings, etc. I figure something a little more interesting might be in order. I would like to appeal to the listers out there for suggestions that would have relatively simple preparation techniques. My initial thoughts go to spider "face", fly eyes/legs, etc.

My questions are:

1) Any suggestions besides spider "face", fly eye/leg that would really interest the kids and might be readily available for me to find? (please include preparation techniques)

2) What type of sample preparation should I use to dehydrate insects but maintain the structures I want to image? (I would hate to have their innards all over the inside of the chamber) I seem to remember some threads talking about dehydration with methanol?

Thanks in advance for any help you can provide.

Best regards,

Joe Mastrangelo

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From: dmayes-at-carilion.com
Date: Fri, 6 Apr 2007 20:52:37 -0500
Subject: [Microscopy] viaWWW: technical TEM services for renal biopsy service

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Email: dmayes-at-carilion.com
Name: daniel mayes

Organization: carilion clinical laboratories

Title-Subject: [Filtered] technical TEM services for renal biopsy service

Question: We are a laboratory serving the Carilion Health System, a network of hospitals and healthcare facilities located mainly in southwestern Virgina, and have a renal biopsy volume requiring electron microscopy of about one per month. We are looking for a facility to perform the technical service of TEM for these renal biopsies. The interpretation of the formalin fixed material and the immunofluorescence studies are performed here. The interpretation of the electron microscopic images could be performed either by us or by the performing facility. Please contact me at dmayes-at-carilion.com, or at 540-981-7889.

Thanks.

Daniel C. Mayes, M.D.

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From: kenconverse-at-qualityimages.biz
Date: Sat, 7 Apr 2007 11:10:59 -0500
Subject: [Microscopy] viaWWW: SEM preparation for insects, other

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Joe,
My experience has been that small, hard-bodied insects like small spiders,
ants, ticks, ladybugs, even bumblebees, can just be mounted on a stub and
sputtered. In fact fresh killed, or even live insects, don't even need to
be coated until they are fully dessicated. Small spiders are particularly
tough and may just walk away after an hour or 2 under vacuum! Pieces of
leaf (especially the stoma on the bottom side) or very small flowers can be
interesting with just sputtering. The more recognizable the object on the
screen, the better the impact.

I'm assuming this is not an FESEM. I might be a little less callous about
the vacuum if it were.

If you have TV scan rate available and can find an old wind-up watch, take
the back off and watch it run.

Ken Converse
owner

QUALITY IMAGES
Servicing Scanning Electron Microscopes
Since 1981
474 So. Bridgton Rd.
Bridgton, ME 04009
207-647-4348
Fax 207-647-2688
kenconverse-at-qualityimages.biz
qualityimages.biz



-----Original Message-----
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Sent: Friday, April 06, 2007 9:56 PM
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Email: jmastrangelo-at-ulbi.com
Name: Joe Mastrangelo

Organization: Ultralife Batteries

Title-Subject: [Filtered] SEM preparation for insects, other recommendations

Question: Our company typically sponsors a tour for "National Bring your
Child to Work Day". Now that we have a SEM in house, I hope to be able to
make their visit to our lab a memorable one.

Since most of my imaging is done on powders, mixtures, coatings, etc. I
figure something a little more interesting might be in order. I would like
to appeal to the listers out there for suggestions that would have
relatively simple preparation techniques. My initial thoughts go to spider
"face", fly eyes/legs, etc.

My questions are:

1) Any suggestions besides spider "face", fly eye/leg that would really
interest the kids and might be readily available for me to find? (please
include preparation techniques)

2) What type of sample preparation should I use to dehydrate insects but
maintain the structures I want to image? (I would hate to have their innards
all over the inside of the chamber) I seem to remember some threads talking
about dehydration with methanol?

Thanks in advance for any help you can provide.

Best regards,

Joe Mastrangelo

---------------------------------------------------------------------------

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==============================Original Headers==============================
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From: Mike.Bode-at-olympus-sis.com
Date: Sat, 7 Apr 2007 12:30:06 -0500
Subject: [Microscopy] viaWWW: SEM preparation for insects, other

Contents Retrieved from Microscopy Listserver Archives
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Hi Joe, this is an excellent oportunity to get kids excited about
science and microscopy. I have done this several times when I was
working as a scientist, and it's alwys fun.

I worked as a materials scientist, and we did not have any CPD
equipment, so I simply looked for dead insects in the nooks and crannies
of the lab and my home. Typically you find a good supply in corners of
the basement windows. I simply glued them to a sample holder stub (you
need to do this carefully, as they can be brittle and break. Then apply
a thin coating of gold and you have a sample that you can put in the
chamber. Put in a few. A fly, a spider, an ant, and a bug.

Perhaps a few pointers:

You can put in a sample of your powders, but the kids will most likely
not be interested in the details of grain structure or fractal
dimensions. They will lose interest quickly and become fidgety. Better
stick to flys and bugs.

Before the fun starts, put everything in your lab away that is small or
potentially dangerous. Depending on the size of the group, it is hard to
control the kids in the dark. I usually had a tape that I used to block
of an area where nobody was allowed to go, and everything that I could
not put in a cabinet was behind the tape out of reach.

The kids seemed to like to be engaged. Instead of "zooming in" on the
bug, I started out at high mag and had the kids guess at what they were
seeing. Ask them to be specific, not just "a bug". An interesting
starting point is the eye of an insect. You can zoom in between several
segments (sometimes there are hair-like structures that stick out from
there). Once you zoom out enough, the kids will identify it as a bug's
eye. The first one to give me a good answer was then allowed to "run"
the scope (change mag, and move the sample holder, slowly, of course).

Ask them what they would do if they had an SEM. Then use that to explain
some of the features. One kid, for example, told me he would use it to
watch the fish in his aquarium. Good hook to explain about vacuum, live
tissue in vacuum, why everything is black and white, etc.

Most importantly: have fun with it yourself. The kids react to your
engagement as much as they do to the pictures.

mike

Michael Bode

General Manager

OLYMPUS SOFT IMAGING SOLUTIONS CORP.
12596 West Bayaud Ave #300
Lakewood, CO 80228
USA
Tel.: +1 (303) 234-9270
Fax.: +1 (303) 234-9271
E-Mail: Mike.Bode-at-olympus-sis.net
www.olympus-sis.net

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This Question/Comment was submitted to the Microscopy Listserver
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Email: jmastrangelo-at-ulbi.com
Name: Joe Mastrangelo

Organization: Ultralife Batteries

Title-Subject: [Filtered] SEM preparation for insects, other
recommendations

Question: Our company typically sponsors a tour for "National Bring your
Child to Work Day". Now that we have a SEM in house, I hope to be able
to make their visit to our lab a memorable one.

Since most of my imaging is done on powders, mixtures, coatings, etc. I
figure something a little more interesting might be in order. I would
like to appeal to the listers out there for suggestions that would have
relatively simple preparation techniques. My initial thoughts go to
spider "face", fly eyes/legs, etc.

My questions are:

1) Any suggestions besides spider "face", fly eye/leg that would really
interest the kids and might be readily available for me to find? (please
include preparation techniques)

2) What type of sample preparation should I use to dehydrate insects but
maintain the structures I want to image? (I would hate to have their
innards all over the inside of the chamber) I seem to remember some
threads talking about dehydration with methanol?

Thanks in advance for any help you can provide.

Best regards,

Joe Mastrangelo

------------------------------------------------------------------------
---

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From: jbpawley-at-wisc.edu
Date: Sat, 7 Apr 2007 16:30:55 -0500
Subject: [Microscopy] RE: Electron beam simulation

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

--|
--|
--|Hello,
--|
--|I am trying to simulate electron beam heating in the SEM. I am sure
--|this is not a new topic and perhaps lots of people had done some
--|work on it. I am totally new to this area so like to check if anyone
--|has good journals to recommend?
--|
--|In my simulation, I input a figure for the probe current density
--|(got from some journals), I inevitably gets melting... I am still
--|trying to verify this.
--|
--|Can anyone point out to me a typical figure for probe size, current
--|and perhaps even current distribution equation for a TEM probe?
--|
--|Thank you very much
--|
--|Regards
--|TT

The current density may be high but the current itself is very low
because you have essentially a point source in the specimen.

You are right, it has been done many times. Except for really good
thermal insulators (Styrofoam?) the heating is negligible (|--1 deg C)
for beam currents of 10 to the minus 10 amps or lower. See Scanning
Electron Microscopy by Oliver Wells (1975).

Damage is usually due not to heating but to "radiation damage" cause
by the fact that most of the energy is deposited in "lumps" of more
than 20 eV each (i.e., large enough for one "lump" to break a
covalent bond). This is large and complex topic and is the reason
that it is VERY hard to make images showing better resolution than 3
nm of any covalently-bonded material (i.e., all biology). (See
Electron Crystallography on the web and authors such as Robert
Glaeser, Wah Chiu, and Ken Downing)

Cheers,

Jim Pawley

--
****************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building, FAX 608-262-9083
250 N. Mills St., Madison, WI, 53706 JBPAWLEY-at-WISC.EDU
"A scientist is not one who can answer questions but one who can
question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39
"He who can get you to believe absurdities, can get you to commit atrocities."
Voltaire.

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From: raharris-at-ucdavis.edu
Date: Mon, 9 Apr 2007 11:40:12 -0500
Subject: [Microscopy] viaWWW: SEM preparation for insects, other recommendations

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

I regularly had K1-12 students tour my facility at UCD (now
deconstructed by some very stupid and unethical faculty-oops, did I say
that out loud?) Anyhow, I had a group of stubs I prepared for the kids
to view and try to guess what they were seeing. I had a stub with both
a piece of feather and a moth wing on it. I had some very small bones
and teeth from a mouse (recovered from an owl pellet). I had a
beautiful fly prep but it was much too complicated too duplicate so I
suggest a fly wing. Most people are surprised that the wing is covered
with small hairs. Flies and spiders usually collapse upon drying but
beetles, wasps, etc. will do fine with air drying. As has been noted,
many insects can be mounted live and survive the ordeal and many are
even seen to move in the vacuum though usually they are immobilized by
the expansion of the gases in their haemolymph. I would notify the
class to bring in something to look at (within reason). Had to be no
bigger than a penny, hard not soft, clean as possible. Sure, you will
get scabs and buggers but you often get something worthwhile and the
kids love that. We looked at mechanical watch parts, isopods (pill
bugs), we compared hair from different animals (all on one stub), I had
some nice cross-sections of wood, interesting leaves, insect eggs
(Lepidoptera) with micropyle visible. Another good stub is an Argentine
ant, the little tiny guys you get in your home, mounted on the same stub
as a large ant like Pogonomyrmex or better yet, Camponotus, one of the
carpenter ants. If you are careful, you can mount the tiny ant on top
of the larger ant

I made a multi-stub holder and could arrange 6 stubs at once in our
Hitachi S3500N. Always a big hit with the kids.

Rick Harris
Informational and Educational Technology (no faculty in this division!!)
University of California at Davis



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From: gary-at-gaugler.com
Date: Tue, 10 Apr 2007 21:10:49 -0500
Subject: [Microscopy] FEI Sirion SFEG upgrade

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Has anyone been through an upgrade of Sirion SFEG
(XL-30 FESEM) to de-integrate the EDAX EDS and
to upgrade the PC hardware and the OS to WinXP?

If you did, how did it go and what was the
ballpark cost? Any hidden gotchas?

tnx,
gary g.


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From: dsherman-at-purdue.edu
Date: Tue, 10 Apr 2007 21:27:09 -0500
Subject: [Microscopy] Plant microtubules

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Hi all,

Have any of you had success imaging higher plant microtubules? If so than I
would really appreciate the method used.

I would also appreciate a fixation protocol for microbes, yeast, or animal
microtubules that is particularly successful. This would be a starting point
if we cannot find one used on higher plants.

Although high pressure freezing and FS is an option (protocol?) I would like
to start with a chemical fixation as it would permit faster turn-around to
possibly see if the experiment is sufficiently promising to go further.

Thanks,
Debby

Debby Sherman, Director 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
http://www.agriculture.purdue.edu/microscopy


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From: kraftpiano-at-gmail.com
Date: Wed, 11 Apr 2007 16:34:55 -0500
Subject: [Microscopy] SEM: Odds and ends from JSM-840 re-assembly.

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Ok. We've got it put together, but there are a couple of things that I'm
dwelling on. First of all, there is an ion pump but no ion pump
controller. Do I need the ion pump if I'm not running a LaB6 cathode?

Second, there are a couple of as-yet unidentified modules in the control
console. These are labeled "MSI" "STB" and "SDU." Can anyone tell me what
these modules are for? I can't make it out from following the connecting
wires.

Finally, we have an optical microscope and WDS spectrometers on the
instrument. I really don't have the ability to use these. I would love to
do some X-ray spectroscopy, however we have no controller or software to use
with these detectors.

As far as what I need now, here's the list:

1: Any sample prep equipment: Sputter coater, carbon coater, freeze dryer,
etc...

2: Any type of digital image acquisition system. We have the photographic
system, but we really can't afford the film for the number of students that
will want pictures of everything that they see. We're using this with
middle school students as well as high school students.

3: Specimen holders. Right now the stage just has the opening that goes all
the way through the rotation gear. There is no holder, and no sample
insertion rod, etc... Anything along those lines would be great.

4: Ion pump controller. (This is fairly low on the priority list, and I
would probably be willing to part with the ion pump in exchange towards
something on our wish-list.)

5: Better vacuum pump. We have a small vacuum pump that kind-of works, but
doesn't work well. I would like to get a better two-stage pump that is
actually made for SEM work, as opposed to our A/C serviceman's pump.

We have some extra items that we can sell/trade for anything you might have
extras of. First, the WDS detectors. (I can supply detailed pictures and
such if you want them) All I would ask extra for the WDS detectors are the
blanking plates- if I take them off, I'd need the plates.

I also have a Denton Desk II Carbon accessory, as well as a spare Desk II
glass cylinder for the sputter coater, (But no coater to use it with).

If we don't need the ion pump, or if we don't get a controller, I'd trade
that too.

Also, if anybody has broken unusable parts in the line of detectors and
such (I have a broken PMT) that they would be willing to part with, I would
love to have some disassembled stuff to demonstrate the different parts of
the SEM. For instance, I would love to cut a diffusion pump in half to show
how that works and make the connection to how much more of a vacuum is
needed than say, their vacuum cleaner at home.

And finally, I would like to take a moment to thank everyone on this
list for their insightful and helpful comments, as well as your
support of this project over the last couple of
weeks. I really could not have done any of this without the wonderful
advice and well-wishes from the members of this group.


--Justin A. Kraft

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From: petrov-at-mrl.uiuc.edu
Date: Wed, 11 Apr 2007 17:06:03 -0500
Subject: [Microscopy] In-Situ Electron Microscopy at AVS54

Contents Retrieved from Microscopy Listserver Archives
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Dear colleagues,

This is to inform you and to encourage you to consider participating in
the topical conference on In-Situ Electron Microscopy organized within
the 54th AVS international symposium
(http://www2.avs.org/call/2007/main.html) October 14-19, 2007, Seattle.

The topical conference call for papers is at

http://www2.avs.org/call/2007/ie.html

AVS provides a unique opportunity of electron microscopists to interact
with a wide circle of scientists who study the dynamics of materials by
in-situ techniques.

The confirmed invited speakers are:

U. Dahmen, Lawrence Berkeley National Laboratory
J.M. Howe, University of Virginia
B. Kabius, Argonne National Laboratory
I. Robertson, University of Illinois, Urbana-Champaign
R. Sharma, Arizona State University
E. Stach, Purdue University
S. Takeda, Osaka University, Japan, "In-situ Environmental TEM of the
Nucleation and Growth of One-Dimensional Nanostructures"
J.M. Zuo, University of Illinois, Urbana-Champaign

If you have suggestion for names of other invited speakers please
contact the topical conference organizers:

Suneel Kodambaka (kodambaka-at-ucla.edu)
Ivan Petrov (petrov-at-uiuc.edu)





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From: nholson-at-ucsd.edu
Date: Wed, 11 Apr 2007 19:15:25 -0500
Subject: [Microscopy] Postdoc or JR Staff position at UC San Diego

Contents Retrieved from Microscopy Listserver Archives
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--
A postdoctoral or junior staff position, with demonstrated experience
in electron cryo-microscopy, is open in the laboratory of Dr. Timothy
S. Baker at the University of California - San Diego in the Chemistry
and Biochemistry Department.

Applications will be accepted from individuals who possess a degree
in the Natural Sciences (Ph. D. for postdoctoral position only). The
salary and level of the position will be commensurate with the
candidate's experience and skills.

The research focuses on structure-function studies of macromolecular
assemblies (primarily viruses) with cryo-TEM and 3D image
reconstruction techniques. Highly-motivated candidates with
significant experience in cryo-TEM and/or cryo-tomography, along with
experience in image reconstruction and molecular image analysis, are
encouraged to apply. See http://cryoem.ucsd.edu/facilities.shtm for
a description of facilities. Image processing is performed in-house
on a three-node Linux cluster housed at the San Diego Supercomputer
Center, an organized research unit of UCSD. Additional computations
are performed using the NSF supported TeraGrid resources.

San Diego is a vibrant, cosmopolitan city, known for its beautiful,
year-round climate. The area includes about 70 miles of beaches, and
water sports such as surfing, snorkeling, kayaking and fishing are
all very popular. Winter alpine skiing and the deserts are within a
couple of hours drive. The city is home to both the San Diego
Chargers and the San Diego Padres. Mexico is close enough for an
afternoon trip.

Qualified applicants should send a CV that identifies areas of
expertise and interest, employment history, a publications list, and
the names and contact information of three, professional references
to Dr. Timothy S. Baker, Professor of Chemistry/Biochemistry _and
Molecular Biology, _University of California, San Diego_, 9500 Gilman
Drive MC-0378, La Jolla, California 92093-0378. Alternatively, CVs
may be sent to tsb-at-ucsd.edu. See http://cryoem.ucsd.edu for more
information about the laboratory.

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From: gstrout-at-ou.edu
Date: Thu, 12 Apr 2007 12:05:47 -0500
Subject: [Microscopy] Reichert Ultracut motor drive belt

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

I have a Reichert Ultracut that is badly in need of a new motor drive
belt. I have changed these belts on other microtomes, but not an
Ultracut. It is not immediately obvious how the belt comes off the
pully located behind the flywheel/handcrank. Does anyone have
instructions for changing out this belt?

Who is the current technical representative for the older Reichert
machines and does Leica Microsystems currently hold the Reichert name?

Thanks,
Greg

--
--
==================================================================
Greg Strout
Electron Microscopist, University of Oklahoma
WWW Virtual Library for Microscopy:
http://www.ou.edu/research/electron/www-vl/
e-mail: gstrout-at-ou.edu
Opinions expressed herein are mine and not necessarily those of
the University of Oklahoma
==================================================================



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From: mraderma-at-physiology.med.uvm.edu
Date: Thu, 12 Apr 2007 13:36:43 -0500
Subject: [Microscopy] 3rd UVM course on 3D reconstruction of single particles

Contents Retrieved from Microscopy Listserver Archives
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Dear all,

We are happy to announce the

3rd UVM Practical Course
on Three-dimensional Cryo Electron Microscopy of Single Particles
August 13-19 2007,
University of Vermont, Burlington, VT


This international course will teach the principles of three-dimensional
reconstruction of single particles from cryo electron micrographs. The course
will include a lecture series teaching in-depth the basic theoretical principles
of single particle analysis and discussions and demonstrations of experimental
procedures. Six hours per day are allocated for hands-on processing of data sets
from electron micrographs of single particles.

Participants will work in groups of two. Each group will have one instructor,
who will provide step-by-step guidance through the complete three-dimensional
reconstruction process and offer additional explanations and support. The course
aims to provide participants with a strong practical and theoretical background
that will enable them later to use these techniques in their home laboratory.

For details please visit:

http://physioweb.med.uvm.edu/Cryo_Practical/


______________________________________________
Michael Radermacher, Assoc. Prof.
University of Vermont
Dept. Molecular Physiology and Biophysics
HSRF 120 / 149 Beaumont Ave
Burlington, VT 05405






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From: wcrgs-at-aol.com
Date: Fri, 13 Apr 2007 08:12:50 -0500
Subject: [Microscopy] viaWWW: Potential difference across a microscope slide

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

This Question/Comment was submitted to the Microscopy Listserver
using the WWW based Form at http://www.microscopy.com/MLFormMail.html
---------------------------------------------------------------------------
Remember this posting is most likely not from a Subscriber, so when replying
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Email: wcrgs-at-aol.com
Name: Ronald Obie

Organization: WCRG

Title-Subject: [Filtered] Potential difference across a microscope slide

Question: Hello.
Does anyone know of a microscope slide available which has the capability for a potential difference to be placed across it? We would like to evaluate the charge of various particles by looking at the direction to which they move relative to the charge placed at the edge of a cover slip. Has anyone ever tried this? Are there references available?
Thank you for your response.
Ronald Obie
WCRG
336-841-0264


---------------------------------------------------------------------------

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From: cervantes-at-bendres.com
Date: Fri, 13 Apr 2007 14:55:46 -0500
Subject: [Microscopy] Reference for EM of Biological Tissues and Cells

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Can anyone recommend a good reference for electron microscopy images of
biological tissues and cells? I have Bozzola's Electron Microscopy:
Principles and Techniques for Biologists, but would like a reference
focused on identification of tissues/cells, rather than technique.
Thanks and TGIF,
Jessica Cervantes
Bend Research, Inc
Bend, OR



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From: cervantes-at-bendres.com
Date: Fri, 13 Apr 2007 15:12:47 -0500
Subject: [Microscopy] TEM: Alternative to Uranyl Acetate Stain

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Due to some bureaucratic mumbo-jumbo, I am not able to use Uranyl
Acetate (nuclear warheads, blah, blah, blah). I would like to stain
some osmium-fixed tissue thin-sections for TEM; the procedure I'm
following has a UA/Sato's lead stain step for the sections. Does anyone
know of a suitable alternative? I did a quick google and listserver
archive search and didn't find anything, but I'm hoping someone has run
into this problem before and can suggest something.

Crossing my fingers,
Jessica Cervantes
Bend Research, Inc
Bend, OR


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From: lcgould-at-med.cornell.edu
Date: Fri, 13 Apr 2007 15:19:28 -0500
Subject: [Microscopy] Re: Reference for EM of Biological Tissues and Cells

Contents Retrieved from Microscopy Listserver Archives
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Dear Jessica,
If you can find it, you can't do much better than HISTOLOGY: A Text
and Atlas by Johannes AG Rhodin. The copy I have is copyrighted
1974. It was published by Oxford University Press.
Lee
--
Leona Cohen-Gould, M.S.
Sr. Staff Associate
Director, Electron Microscopy & Histology Core Facility
Manager, Optical Microscopy Core Facility
Weill-Cornell Medical College

voice (212)746-6146
fax (212)746-8175
http://www.cornellcelldevbiology.org
http://www.cornellbiochem.org

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From: Walter.Bobrowski-at-pfizer.com
Date: Fri, 13 Apr 2007 15:33:18 -0500
Subject: [Microscopy] Re: Reference for EM of Biological Tissues and

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Histology: A text and Atlas was updated 2006 (paperback) with CD-ROM and
is available from Barnes & Noble.
Another excellent choice is Color Atlas of Cytology, Histology and
Microscopic Anatomy by Wolfgang Kuehnel, also available from B&N. The
paperback edition is excellent!

Best regards,

Walter F. Bobrowski
Sr. Scientist
Pfizer Global R&D
2800 Plymouth Rd.
Ann Arbor, MI 48105

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-----Original Message-----
X-from: lcgould-at-med.cornell.edu [mailto:lcgould-at-med.cornell.edu]
Sent: Friday, April 13, 2007 4:24 PM
To: Bobrowski, Walter

Dear Jessica,
If you can find it, you can't do much better than HISTOLOGY: A Text
and Atlas by Johannes AG Rhodin. The copy I have is copyrighted
1974. It was published by Oxford University Press.
Lee
--
Leona Cohen-Gould, M.S.
Sr. Staff Associate
Director, Electron Microscopy & Histology Core Facility
Manager, Optical Microscopy Core Facility
Weill-Cornell Medical College

voice (212)746-6146
fax (212)746-8175
http://www.cornellcelldevbiology.org
http://www.cornellbiochem.org

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==============================Original Headers==============================
16, 31 -- From Walter.Bobrowski-at-pfizer.com Fri Apr 13 15:33:10 2007
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From: neerajg-at-clemson.edu
Date: Sat, 14 Apr 2007 15:51:38 -0500
Subject: [Microscopy] viaWWW: Animations for Tomography

Contents Retrieved from Microscopy Listserver Archives
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This Question/Comment was submitted to the Microscopy Listserver
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Email: neerajg-at-clemson.edu
Name: Neeraj V. Gohad

Organization: Clemson University

Title-Subject: [Filtered] Animations for Tomography

Question: Dear all,

I will be giving a seminar on Electron Tomography in our department as a part of our departmental seminar series. I am searching for animations illustrating the process of tomography, I do have schematics and animations showing the weighted backprojections and aligned stacks. I am looking for an animations which shows the electrons hitting the specimen in the column as the tilt series is being taken. Does any one know a source where I can find an animation and or schematic?

As always I appreciate everyoneís help ,

Regards,

Raj.

Neeraj V. Gohad
Graduate Research Assistant
Department of Biological Sciences
Clemson University,
Clemson, SC-29634



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From: phillipst-at-missouri.edu
Date: Sat, 14 Apr 2007 21:04:03 -0500
Subject: [Microscopy] Re: Reference for EM of Biological Tissues and

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Don Fawcett's The Cell (publisher = Saunders) has superb TEM views of most
organelles. don't know if it is still in print.

At 02:57 PM 04/13/07, you wrote:



} ----------------------------------------------------------------------------
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Thomas E. Phillips, PhD
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400

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573-882-0123 (fax)
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From: sally.stowe-at-anu.edu.au
Date: Sun, 15 Apr 2007 18:25:39 -0500
Subject: [Microscopy] RE: Reichert Ultracut motor drive belt

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Does anyone have a service manual for Ultracuts and /or Ultracut Es ?

Thanks
Sally


Dr SJ Stowe
Facility Coordinator
ANU Electron Microscopy Unit
ANU CRICOS#00120C



} -----Original Message-----
} From: gstrout-at-ou.edu [mailto:gstrout-at-ou.edu]
} Sent: Friday, 13 April 2007 3:06 AM
} To: sally.stowe-at-anu.edu.au
} Subject: [Microscopy] Reichert Ultracut motor drive belt
}
}
}
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From: cgarber-at-2spi.com
Date: Sun, 15 Apr 2007 22:25:09 -0500
Subject: [Microscopy] Special microscope slide request

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Ronald Obie wrote:
==============================================================
Does anyone know of a microscope slide available which has the capability
for a potential difference to be placed across it? We would like to
evaluate the charge of various particles by looking at the direction to
which they move relative to the charge placed at the edge of a cover slip.
Has anyone ever tried this? Are there references available?
=======================================================
We at SPI Supplies have offered a line of ITO (indium tin oxide) coated
microscope slides, see URL
http://www.2spi.com/catalog/standards/ITO-coated-slides-resistivities.html

If you want microscope slide sizes substrates, then at the bottom, click on
"Microscopy Slides". They are also available with busbars which makes for
an easier connection for electrical contacts. The slides are available at
different resistivities.

Note that we also offer ITO coated cover slips, either with or without
busbars.

Chuck

===================================================
Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400
President
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: rcmoretz-at-gmail.com
Date: Mon, 16 Apr 2007 07:22:27 -0500
Subject: [Microscopy] Re: TEM: Alternative to Uranyl Acetate Stain

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Jessica:

You can get depleted (or at least you used to be able to get this) UAc
so that your safety people (or whoever) can be assured that you are
not getting into making nuclear warheads, etc. Of course, I suppose
that trying to reason with them by noting the very small amount you
would be receiving could not be used for such purposes, plus it is not
enriched U but natural isotopes.... Probably too much to hope that
reason and logic has any place in such discussions.

Roger Moretz, Ph.D.
Dept. of Toxicology
Boehringer Ingelheim Pharmaceuticals, Inc.

Disclaimer: The comments and opinions are those of the author alone,
and do not reflect any official position by his employer.

On 4/13/07, cervantes-at-bendres.com {cervantes-at-bendres.com} wrote:
}
}
}
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}
} Due to some bureaucratic mumbo-jumbo, I am not able to use Uranyl
} Acetate (nuclear warheads, blah, blah, blah). I would like to stain
} some osmium-fixed tissue thin-sections for TEM; the procedure I'm
} following has a UA/Sato's lead stain step for the sections. Does anyone
} know of a suitable alternative? I did a quick google and listserver
} archive search and didn't find anything, but I'm hoping someone has run
} into this problem before and can suggest something.
}
} Crossing my fingers,
} Jessica Cervantes
} Bend Research, Inc
} Bend, OR
}
}
} ==============================Original Headers==============================
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==============================Original Headers==============================
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5, 28 -- Date: Mon, 16 Apr 2007 08:22:24 -0400
5, 28 -- From: "Roger Moretz" {rcmoretz-at-gmail.com}
5, 28 -- To: cervantes-at-bendres.com, "Microscopy Listserv" {Microscopy-at-microscopy.com}
5, 28 -- Subject: Re: [Microscopy] TEM: Alternative to Uranyl Acetate Stain
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From: carnahan-at-edison-labs.com
Date: Mon, 16 Apr 2007 07:58:52 -0500
Subject: [Microscopy] RE: Potential difference across microscope slide.

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Ronald,



We prepare slides for micro-electrochemistry from standard 1X3 glass slides
which are masked with a strip of tape over the region that will hold the
sample then sputter coated with gold using 3-5X the exposure used for SEM
sample prep. Be sure to wrap the tape all the way around the slide and also
along the back to avoid conductive paths along the edges and to avoid
conduction paths to the stage. We typically use tape cut to 3mm for our
cell width and also mask the entire back and edges of the slide. After
sputtering, we clean the slide and use a drop of conductive silver paint (
the usual SEM suppliers) to attach flexible lead wires. The samples are
applied and covered with a square cover slip.



Our own applications are all low voltage but if you intend to run at high
voltages you need to prepare very carefully for microscopist safety. If you
are intending to use high voltages, especially electrophoresis supplies, you
need to get competent design and audit help from electrical engineers with
high voltage safety experience.



James Carnahan

Edison Analytical Laboratories, Inc.
301 Nott Street
Schenectady, NY 12305

(518) 393-2112



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From: cervantes-at-bendres.com
Date: Mon, 16 Apr 2007 10:57:44 -0500
Subject: [Microscopy] TEM:Alternative to Uranyl Acetate Stain

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Thanks to everyone who responded. I have indeed brought up the fact
that the UA is from depleted uranium, and will continue to try to reason
with people. I have managed to get sodium cacodylate (it contains
arsenic!) and OsO4 (can blacken your eyeball!) in the door, so there is
some hope . . . in the meantime at least now I have some alternatives to
try.

Thanks again.

Jessica Cervantes
Bend Research, Inc
Bend, OR



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From: cervantes-at-bendres.com
Date: Mon, 16 Apr 2007 10:58:04 -0500
Subject: [Microscopy] Re: Reference for EM of Biological Tissues and Cells

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Thanks to everyone who provided references. I've got quite the list now
. . .

Jessica Cervantes
Bend Research Inc
Bend, OR


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From: tbargar-at-unmc.edu
Date: Mon, 16 Apr 2007 10:58:42 -0500
Subject: [Microscopy] cell cultures and aldehyde fixatives

Contents Retrieved from Microscopy Listserver Archives
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Dear listers,

I'm processing some monolayer cell cultures grown on Thermanox coverslips
for SEM. I seem to recall that aldehyde fixatives can cause an artifact
that results in small holes in the cell membrane. If I remember, Picric
Acid is added to the fixative and that it helps prevent the artifact. I
don't remember where I originally read this. Am I off base on this? If it
is correct, does anyone out there remember the formulation of the fixative
using Picric Acid? I currently use 2% glutaraldehyde, 2% paraformaldehyde
and 0.5% acrolein in 0.1M Sorensen's phosphate buffer 7.2pH.

Also during processing the thin cytoplasmic extensions of the cells break.
I assume this is due to shrinkage during the dehydration steps and critical
point drying. Anyone know of a way to prevent this?

All help is appreciated. Thanks.

Tom Bargar
University of Nebraska Medical Center
Core Electron Microscopy Research Facility
986395 Nebraska Medical Center
Omaha, NE 68198-6395
402-559-7347
tbargar-at-unmc.edu


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From: oshel1pe-at-cmich.edu
Date: Mon, 16 Apr 2007 11:44:18 -0500
Subject: [Microscopy] Re: cell cultures and aldehyde fixatives

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Tom,

For the membrane holes, try 1% tannic acid in the glut. You want the
monomeric form, Mallinckrodt 1674 - or 1764, I keep transposing those
digits. 1% T.A. can also be used in an OsO4 post-fix.
I would also suggest cutting the glut to 1 or 1.25%, and don't bother
with the formalin, it's not neeed for cell monolayers.
Acrolein I haven't used, so can't comment. I've just done 1-1.25%
glut + 1% tannic acid.
I don't know about picric acid, and would be interested to hear if it works.

Phil

} Dear listers,
}
} I'm processing some monolayer cell cultures grown on Thermanox coverslips
} for SEM. I seem to recall that aldehyde fixatives can cause an artifact
} that results in small holes in the cell membrane. If I remember, Picric
} Acid is added to the fixative and that it helps prevent the artifact. I
} don't remember where I originally read this. Am I off base on this? If it
} is correct, does anyone out there remember the formulation of the fixative
} using Picric Acid? I currently use 2% glutaraldehyde, 2% paraformaldehyde
} and 0.5% acrolein in 0.1M Sorensen's phosphate buffer 7.2pH.
}
} Also during processing the thin cytoplasmic extensions of the cells break.
} I assume this is due to shrinkage during the dehydration steps and critical
} point drying. Anyone know of a way to prevent this?
}
} All help is appreciated. Thanks.
}
} Tom Bargar
} University of Nebraska Medical Center
} Core Electron Microscopy Research Facility
} 986395 Nebraska Medical Center
} Omaha, NE 68198-6395
} 402-559-7347
} tbargar-at-unmc.edu
--
Philip Oshel
Microscopy Facility Supervisor
Biology Department
024C Brooks Hall
Central Michigan University
Mt. Pleasant, MI 48859
voice: (989) 774-3576
dept. fax: (989) 774-3462

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From: lcgould-at-med.cornell.edu
Date: Mon, 16 Apr 2007 11:54:22 -0500
Subject: [Microscopy] Re: cell cultures and aldehyde fixatives

Contents Retrieved from Microscopy Listserver Archives
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HI Tom-
I use picric acid in my primary fix to preserve membranes. My
"recipe" is 2.5% glut, 4% pfa in 0.1M Na-cacod. with 0.02% picric
acid. I did the math years ago, and this worked out to be:
1 vial of 10% glut + 1 vail of 16% pfa in 20 ml of 0.2m cacod with 2
ml of saturated aqueous picric acid added.
I've had the same 100g bottle of picric acid for over 15 years. I
just keep it saturated with the liquid well above the level of the
crystals. When I draw some off, I add more water. Our Life Safety
guys here are just thrilled with me: osmium, uranium picric acid,
suspected carcinogens ...all the fun stuff we EM folk play with. As
long as you are careful about keeping the crystals fully under water,
and not letting any accumulate around the rim or anywhere where they
could dry out, you're fine.

As for your broken processes: they may be getting beaten up during
your CPD run. Be sure to do your CO2 exchanges gently, never letting
the fluid level drop below your sample (this will mean extra
exchanges), and then, at the end, vent very slowly...100 psi/minute
or less.

Good luck!
Lee
--
Leona Cohen-Gould, M.S.
Sr. Staff Associate
Director, Electron Microscopy & Histology Core Facility
Manager, Optical Microscopy Core Facility
Weill-Cornell Medical College

voice (212)746-6146
fax (212)746-8175
http://www.cornellcelldevbiology.org
http://www.cornellbiochem.org

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From: drk-at-SHCC.org
Date: Mon, 16 Apr 2007 12:05:52 -0500
Subject: [Microscopy] fixation of lysosome membranes

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Greetings Microscopists,

We are trying to stabilize lysosomes for section surface immunocytochemisty
and are searching for a fixation method which might best preserve the
membranes without destroying antigenicity. We are working with cultured
cells embedded in LR White. Our usual method of 4% paraformaldehyde with
1.0% glut (buffered with culture media) appears to allow stabilization thru
approximately 70% EtOH, but the lysosomes appear to break afterwards,
spilling their contents into the cytoplasm. Fixation and dehydration were
done on ice. We are considering a PLT procedure, perhaps also including a
little Uranyl acetate in the fixative. Are there any suggestions for
preserving these delicate membranes?

Many thanks,

Doug

Douglas R. Keene
Assistant Investigator
Micro-Imaging Center
Shriners Hospital for Children
3102 S.W. Sam Jackson Park Road
Portland, Oregon 97239
503-221-3434
drk-at-shcc.org



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From: cervantes-at-bendres.com
Date: Mon, 16 Apr 2007 12:57:05 -0500
Subject: [Microscopy] TEM:Alternatives to Uranyl Acetate Stain - Responses to the Original Query

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

A couple of you have asked what the responses were to my query for
alternatives to UA. Here's the list:

1. Bismuth Staining - from Mike Nesson

Quote: There are several references that deal with Bismuth staining. I
tried some of these years ago and was quite satisfied with the results.

Locke, M and Huie, P. "Bismuth staining for light and electron
microscopy" Tissue and Cell: 9; 347-371 (1977).


2. Straight lead citrate - from Ted, Managing Director, The EMscope
Company Ltd., Thailand.


3. Nanovan (a negative stain)- from David Gene Morgan, University of
California at Davis

Quote: There is a product called nanovan (sold by Nanoprobes) which is a
methylamine vanadate stain that has similar features to uranly acetate.
The manufacturers claim finer grain size and some other benefits, if I
remember correctly. The only uses I am aware of have been to replace
simple uranyl acetate staining of isolated biological materials, and I
have no idea if it would even work as a TEM post-stain.


4. Reduced Osmium - from Rachid

Quote: There is no need for UA if your sample is treated with reduced
osmium (osmium + potassium ferrocyanide). You will just have to
contrast 2 min with lead citrate ... the contrast you will get will be
really good. You can check on this web site that I am putting together
... all pictures in the gallery are produced as I mentioned.
http://rsougrat.googlepages.com/


5. KMnO4 and/or Tannic Acid - from Mike Reedy

Quote: You will get even more contrast if you use KMnO4 followed by
Sato's, as we have since 1964 (when Pb was was Pb cittrate, not Sato's).
And, you can get good contrast on thinner sections. Using Tannic acid
in the block stain, before OSO4, or after but followed by UrAc, adds to
contrast, improves preservation. We have had excellent results (see
attached).

Some reports of using TA as a section stain before Pb stain can also be
found with google help. I've never tried it yet.

KMnO4 section stain won't work if NMA is in your Epon mix. End quote.

Mike included attachments in his reply, which can't be posted.


6. Tell the safety people to get a brain - from several people


Thanks again to everybody who responded. This is a great resource.

Jessica Cervantes
Bend Research Inc
Bend, OR



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From: lcgould-at-med.cornell.edu
Date: Mon, 16 Apr 2007 15:46:45 -0500
Subject: [Microscopy] cell cultures and aldehyde fixatives

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

In 1964, I published a small note about the potential use of Indium
Trichloride as a stain during embedding (J de Microscopie, 3: pp575-
578). As I recall, it added some contrast, specifically to virus
structures. It might be worth considering.

Joel

Date sent: Mon, 16 Apr 2007 12:57:11 -0500
To: jbs-at-temple.edu
X-from: cervantes-at-bendres.com
Send reply to: cervantes-at-bendres.com


Hi All,
Jan Leunissen pointed out that I neglected to include the original
volumes of the vials of pfa and glut...sorry about that. I buy 10ml
vials of both.
Lee
--
Leona Cohen-Gould, M.S.
Sr. Staff Associate
Director, Electron Microscopy & Histology Core Facility
Manager, Optical Microscopy Core Facility
Weill-Cornell Medical College

voice (212)746-6146
fax (212)746-8175
http://www.cornellcelldevbiology.org
http://www.cornellbiochem.org

==============================Original Headers==============================
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From: rpowell-at-nanoprobes.com
Date: Mon, 16 Apr 2007 18:21:33 -0500
Subject: [Microscopy] viaWWW: TEM:Alternatives to Uranyl Acetate Stain

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Email: rpowell-at-nanoprobes.com
Name: Rick Powell

Organization: Nanoprobes, Incorporated

Title-Subject: [Microscopy] TEM:Alternatives to Uranyl Acetate Stain

Question: Response from commercial vendor (Nanoprobes):

Hello Everyone:

We do offer "NanoVan" as a commercial product. It is based on methylamine vanadate, which has a lower atomic number than uranium, and will give a ligher stain (since we make small gold particles, this is helpful).

We also offer Nano-W, an alternative based on tungsten which produces a denser stain. The two may be combined for intermiediate stain densities.

Catalog and information:

http://www.nanoprobes.com/Nstain.html

Newsletter article:

http://www.nanoprobes.com/Vol8_Iss3.html#4

Hope this is helpful,

Rick Powell

Nanoprobes, Incorporated
www.nanoprobes.com

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From: DLowry-at-asu.edu
Date: Mon, 16 Apr 2007 18:21:56 -0500
Subject: [Microscopy] viaWWW: Mycobacterium prep for immuno-label

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Email: DLowry-at-asu.edu
Name: David Lowry

Organization: Arizona State University

Title-Subject: [Filtered] Mycobacterium prep for immuno-label

Question: I am attempting to do thin section immuno-labeling experiments with Mycobacterium tuberculosis. I have used standard procedures: fixation with 4% paraformaldehyde/0.1% glut, then enmeshed cells in ~ 1% agarose prior to EtOH dehydration and LR White resin. Very basic stuff.

Unfortunately, in 2 separate trials I have encountered a problem with very large holes in the resin around and between the clusters of cells. It seems to be either incomplete dehydration or poor penetration of resin, although in both attempts I used 100% EtOH dehydration and extensive time in 100% LR.

Are there special considerations to take when working with this organism? I have searched on-line for information but could not locate any specific indications. I suspect there may be something unusual about the outer cell boundary that is causing this problem, and would like to know if anyone may have experience in dealing with this organism.

Thank you

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From: nizets2-at-yahoo.com
Date: Tue, 17 Apr 2007 04:46:03 -0500
Subject: [Microscopy] Hoechst Vs DAPI staining

Contents Retrieved from Microscopy Listserver Archives
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Thanks Vlad. I did realize that some of these alternatives would not
work for my application, but wanted to list them for others (ie, Nanovan
is an alternative to UA as a negative stain, not for sections, as you
point out). I do plan on continuing to educate people here on UA, but
sometimes these things are political, rather than scientific.

Thanks again,
Jessica Cervantes

-----Original Message-----
X-from: Vlad Speransky [mailto:vladislav_speransky-at-nih.gov]
Sent: Monday, April 16, 2007 11:59 AM
To: Cervantes, Jessica

Dear colleagues,

Usually to observe apopototic patterns in fluorescence
microscopy the most straigthforward method is to label
the cell nuclei with Hoechst. I very rarely noticed
that DAPI was used. Why?
Is there a reason why Hoechst should be preferred to
DAPI?

Stephane, epifluorescer not confocaler (or is it
confocaliser?)


__________________________________________________
Do You Yahoo!?
Tired of spam? Yahoo! Mail has the best spam protection around
http://mail.yahoo.com

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From: oshel1pe-at-cmich.edu
Date: Tue, 17 Apr 2007 07:25:57 -0500
Subject: [Microscopy] Hayat & Miller "Negative Staining"

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List,

Anyone have a copy in good or better condition of Hayat & Miller,
"Negative Staining" that they want to sell? I can only find one on
the web at an outrageous price. This is for the lab out of my pocket,
so price is also important.
Be nice if they put out a 2nd edition (hint, hint).
Thanks.

Phil
--
Philip Oshel
Microscopy Facility Supervisor
Biology Department
024C Brooks Hall
Central Michigan University
Mt. Pleasant, MI 48859
voice: (989) 774-3576
dept. fax: (989) 774-3462

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From: eschumacher-at-mccrone.com
Date: Tue, 17 Apr 2007 07:30:13 -0500
Subject: [Microscopy] Meeting: M3S Optical Techniques Workshop

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Greetings Colleagues,

The next meeting of the Midwest Microscopy and Microanalysis Society, an
Optical Techniques Workshop, will be held on Thursday, May 17, at the
College of Microscopy in Westmont, IL (The McCrone Group). Please
follow the link below and click on Meetings for program details and
registration information.

www.midwestmicroscopy.org

We look forward to seeing you there.

Elaine Schumacher
President, Midwest Microscopy and Microanalysis Society
elaine-at-midwestmicroscopy.org



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From: rpowell-at-nanoprobes.com
Date: Tue, 17 Apr 2007 08:25:44 -0500
Subject: [Microscopy] viaWWW: TEM:Alternatives to Uranyl Acetate Stain

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Email: rpowell-at-nanoprobes.com
Name: Rick Powell

Organization: Nanoprobes, Incorporated

Title-Subject: [Filtered] TEM:Alternatives to Uranyl Acetate Stain

Question: Hello Everyone:

In my somewhat hasty response to Jessica Cervantes, I had overlooked the fact that the original post referred to poststaining (positive staining) of sections. To clarify, our NanoVan and Nano-W products are intended as negative stains for protein complexes, viruses, etc.; we are not aware of any references that describe their use on thin sections.

If anyone has tried this, or has seen any references, we would of course very much like to know.

Regards,

Rick Powell

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From: gwe-at-ufl.edu
Date: Tue, 17 Apr 2007 09:19:16 -0500
Subject: [Microscopy] Video Catalog

Contents Retrieved from Microscopy Listserver Archives
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The MSA video catalog is currently unavailable online . It had been
hosted at the University of Florida, Biotech Center. They are totally
revamping their web site and many things have not yet been restored to
active status. I have arranged with Nestor to have the catalog hosted
on the MSA server. AS soon as I clean up the file a bit, Nestor will be
able to get it up and running and accessible from the MSA main page. In
the meantime, if you need any info about the video collection, please
feel free to email me.
The "Tips & Tricks" page that was hosted by the EM Core Lab at Florida
is also offline. I am told that it will eventually be restored.
Stay tuned.

Greg
--
Greg Erdos
University of Florida, Retired
Micanopy, Florida

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From: gmartens-at-interchange.ubc.ca
Date: Tue, 17 Apr 2007 11:22:48 -0500
Subject: [Microscopy] FISH followed by EM

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Hi everyone,

I have a request from a client who wants to localize a gene locus
within the nucleus using FISH but then wants to determine better
resolution with TEM. My first reaction would be that the FISH
protocol would destroy the ultrastructure but thought I would ask the
bigger brain if anyone out there has done something similar. I have
read a couple of papers that have done this but I was not impressed
with the ultrastructure. All thoughts are appreciated.

Garnet

--
Garnet Martens

Research Manager
BioImaging Facility
University of British Columbia
6270 University Blvd.
Vancouver, B.C.
Canada
V6T 1Z4

phone 604-822-3354

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From: bfoster-at-mme1.com
Date: Tue, 17 Apr 2007 12:11:16 -0500
Subject: [Microscopy] Re: FISH followed by EM

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Hi, Garnet

There is a intriguing new technique that combines AFM with
ultramicrotomy that images from the block face. Some of the early
work, done by Dr. Anton Efimov of NT-MDT (efimov-at-ntmdt.ru) shows very
delicate ultrastructure (~2-6nm structures) . The advantage is that
this system uses local differences in elasticity to image, rather
than heavy metal staining. You can do serial sections and, while the
AFM images from the block face, the slices are available for
conventional imaging with any sort of microscopy.

Might be an interesting alternative. Suggest that you write directly
to Dr. Efimov for further information.

Best regards,
Barbara Foster, President

Microscopy/Microscopy Education
313 S Jupiter Rd, Suite 100
Allen, TX 75002
P: 972-954-8011
W: www.MicroscopyEducation.com


MME is now scheduling customized, on-site courses through July. Call
us today for details.

P. S.
Need a good general reference or light microscopy text for next
semester? Call us today to learn more about "Optimizing LIght
Microscopy". Copies still available through MME... even for
class-room lots ... and we give quantity discounts. Call Ken Piel at
(972)954-8011.


At 12:05 PM 4/17/2007, gmartens-at-interchange.ubc.ca wrote:



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From: mike.reedy-at-cellbio.duke.edu
Date: Tue, 17 Apr 2007 19:40:31 -0500
Subject: [Microscopy] RE: TEM: Alternative to Uranyl Acetate Stain

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Jessica
Don't know about KMnO4 staining compatibility
with LR White; please let me know. The easy test
is to put a blank block or chunk or flake of
cured LR White in 1-2% KMnO4 and soak it for an
hour or a day to see if resin surface turns light
brown, dark brown, or black and charred looking.
No reaction is good news for section staining.

The more stringent assay for reaction is to put
the test tube of KMnO4 solution with the cured
embedding resin in a beaker of water and then
heat the water to boiling for an hour or so, cool
it off and examine the resin surface for such
changes. Araldite 506 (lowest viscosity one I
know of) and other Araldites used for embedding
are remarkable resistant to surface discoloration
when this is done. Epon DDSA (no MNA; fiddle
the mix until it is hard enough to please you,
and forget the epoxy:anhydride ratio lore) turns
moderately browner, as I recall, but is still
acceptable for section staining, with some
tendency to be more granular and maybe show some
hard-to-eliminate nano-pepper stain deposit in
contrast to Araldite.

LR White is an acrylic says EMS:
LR White is a polar monomer polyhydroxylated
acromatic acrylic resin. It can be cured by heat
or by UV light. Sections of polymerized LR White
resin are hydrophilic
I think Lawn's 1960 JCB paper introducing KMnO4
section staining used sections of methacrylate,
the mix of methyl and butyl methacrylate we all
used in the late 1950s before epoxies, especially
Epon, turned up and became dominant.

BTW-- tannic acid is a fixative and a mordant,
and magically capable of superior structure
preservation and assuring really strong uniform
staining in our hands-- see the evidence of 13Å
preservtion in fiber x-ray diffraction from
fixed-embedded fibers in Sader et al 2007 in J
Struct Biol (Articles In Press on line), and
look at EMs in some reprints I sent you for
evidence of the good morphology. The latter is
a wondrous gift of TA I had no idea of until I
learned it from David Begg et al, J. Cell Biol.
79:846-852, an all-time key paper in my book of
methodological turning points.. The other key
was the observation by Hirose and Wakabayashi (J.
Mol. Biol. 204:797-801) that TA followed by OsO4
or UrAc give great morphological fixation without
aldehydes. They used it for freeze-substitution,
as we have, but we found it also worked very well
as a general fix (we termed this TAURAC) for
permeabilized cells in aqueous buffers so long as
we exluded TA blockers like PVP, Triton X 100 etc.

Your UA police might be interested in a demo of
how completely tannins, esp tannic acid, can
convert a UrAc solution into a flocculent brown
precipitate at the bottom of a UrAc-free
supernatan (it LOOKS UrAc free! I made no
measurements.). TA is cheap in non-EM grades;
maybe they'd accept precipitation as a way of
rendering it safely bio-inactive. From the
information online at EMS ib their
datasheet/22400, I've estimated that the 10,400
counts/secin depleted uranium is reduced to about
-2 ct/sec in a very small bundle of muscle fibers
containing 1 ug of protein and binding maybe 5 ug
of UrAc. But it still requires health police and
special containment if such a specimen is to be
transported into or within the DOE facility at
Argonne National Laboratory. The inconvenience
has so far discouraged me from pursuing some
experiments that would require such a specimen,
but one day I will do it, legally and according
to their regulations. I doubt a Geiger counter
could detect any rise above background in the
presence of that small an amount of UrAc.

-mike-


} Thanks for this Mike. I think KMnO4 followed by Sato's is one of the
} better alternatives I've seen so far. Your results are very impressive.
}
}
} Any idea if KMnO4 will work with LR White embedded tissues?
}
} Thanks,
} Jessica Cervantes
} Bend Research Inc
}
} -----Original Message-----
} From: Mike Reedy [mailto:mike.reedy-at-cellbio.duke.edu]
} Sent: Saturday, April 14, 2007 5:19 PM
} To: Cervantes, Jessica
} Subject: Re: [Microscopy] TEM: Alternative to Uranyl Acetate Stain
}
} You will get even more contrast if you use KMnO4 followed by Sato's,
} as we have since 1964 (when Pb was was Pb cittrate, not Sato's).
} And, you can get good contrast on thinner sections. Using Tannic
} acid in the block stain, before OSO4, or after but followed by UrAc,
} adds to contrast, improves preservation. We have had excellent
} results (see attached).
}
} Some reports of using TA as a section stain before Pb stain can also
} be found with google help. I've never tried it yet.
}
} KMnO4 section stain won't work if NMA is in your Epon mix. see
} attached.
} -mike reedy-
} } -----------------------------------------------------------------------
} -----
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} America


--
-mike reedy-

************************
Michael K. Reedy, M.D.
Duke Univ. Med. Center
Dept. Cell Biology, Box 3011 (for U.S. Mail)
458 Alex Sands Bldg, Research Dr (courier)
Durham, NC 27710

Office 919-668-2534
Lab 919-684-5674
Fax 919-681-9929
mike.reedy-at-cellbio.duke.edu
http://note.cellbio.duke.edu/Faculty/Research/Reedy.html


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From: mike.reedy-at-cellbio.duke.edu
Date: Tue, 17 Apr 2007 19:43:17 -0500
Subject: [Microscopy] RE: TEM: Alternative to Uranyl Acetate Stain

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Jessica
sorry to resend all that. I am trying to see if I can calm the HTML
detector to break through into the list server,
--
-mike reedy-

************************
Michael K. Reedy, M.D.
Duke Univ. Med. Center
Dept. Cell Biology, Box 3011 (for U.S. Mail)
458 Alex Sands Bldg, Research Dr (courier)
Durham, NC 27710

Office 919-668-2534
Lab 919-684-5674
Fax 919-681-9929
mike.reedy-at-cellbio.duke.edu
http://note.cellbio.duke.edu/Faculty/Research/Reedy.html

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From: Niraj.Trivedi-at-childrens.harvard.edu
Date: Tue, 17 Apr 2007 20:15:20 -0500
Subject: [Microscopy] viaWWW: NESM 2007 Woods Hole Meeting

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Email: Niraj.Trivedi-at-childrens.harvard.edu
Name: Niraj Trivedi

Organization: New England Society of Microscopy

Title-Subject: [Filtered] NESM 2007 Woods Hole Meeting

Question: Please join us for the New England Society of Microscopy's (NESM)24th Annual Woods Hole Meeting, where we will be celebrating NESM's 40th birthday!

Expect very exciting talks and a special presentation from most of the past presidents.

Please see the link to the newsletter for more information:

http://nesm.cims.harvard.edu/Newsletters/2007_April_Newsletter.pdf

--Niraj

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From: allan.mitchell-at-stonebow.otago.ac.nz
Date: Tue, 17 Apr 2007 22:11:32 -0500
Subject: [Microscopy] RE: TEM: Alternative to Uranyl Acetate Stain

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Hi all

Been away so missed the original question but has any one suggested p-
Phenylenediamine?

Numerous references to using p-phenylenediamine but one to start
with is "The use of p-phenylenediamine in the block to enhance
osmium staining for electron microscopy" Stain Technology, Vol 47,
No5 pp 239 - 243.

Added in the 70% ethanol dehydration step from memory.

Regards

Allan



Allan Mitchell
technical Manager
Otago Centre for Electron Microscopy
Department of Anatomy and Structural Biology
School of Medical Sciences
P.O. Box 913
Dunedin
New Zealand

Phone (03) 479 5642 or 479 7301
Fax (03) 479 5086 or 479 7254

EM Centre: http://ocem.otago.ac.nz/
Confocal Centre: http://occm.otago.ac.nz/
Department: http://anatomy.otago.ac.nz/


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From: nizets2-at-yahoo.com
Date: Wed, 18 Apr 2007 08:04:09 -0500
Subject: [Microscopy] aluminosilicate and BSE

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Dear Listers,

I want to detect aluminosilicate particles in the
middle of organic material. The particles are expected
to be too small for light microscopy and too dilute
for TEM. The solution would be to dry everything flat
on a SEM stub and to find a way to differentiate
organic particles for aluminosilicate particles.
Our EDX doesn't want to start so I wondered if I could
see something with BSE?
X-from the atomic weight of the elements, Al and Si are
not particularly heavy but they are of course very
dense in the particles.
Do you think it would be possible? Any remark?

Stephane



__________________________________________________
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From: petra.wahlbring-at-goodyear.com
Date: Wed, 18 Apr 2007 08:23:11 -0500
Subject: [Microscopy] Re: aluminosilicate and BSE

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Dear Stephane,

if you own a decent BSE detector, I'm sure that you will be able to see a
contrast between the two types of particles. Average atomic number is well
apart from each other. To make the observation easier, I would recommend to
use a low-Z SEM stub like graphite or a graphite plate on top of a standard
holder. Then you will be able to see your silicates with a bright contrast
with respect to the background and the other particles.

Best regards,

Petra
---------------------------------------
Dr. Petra Wahlbring
Goodyear S.A. Technical Center
Analytical Test Laboratories
L-7750 Colmar-Berg
Luxembourg
Tel +352 8199 3725
Fax +352 8199 3905
e-mail: petra.wahlbring-at-goodyear.com

- May Contain Confidential and/or Proprietary Information. May not be
copied or disseminated without the expressed written consent of The
Goodyear Tire & Rubber Company. -





nizets2-at-yahoo.com

04/18/07 03:09 PM To
petra.wahlbring-at-goodyear.com
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Please respond to
nizets2-at-yahoo.com Subject
[Microscopy] aluminosilicate and
BSE














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Dear Listers,

I want to detect aluminosilicate particles in the
middle of organic material. The particles are expected
to be too small for light microscopy and too dilute
for TEM. The solution would be to dry everything flat
on a SEM stub and to find a way to differentiate
organic particles for aluminosilicate particles.
Our EDX doesn't want to start so I wondered if I could
see something with BSE?
X-from the atomic weight of the elements, Al and Si are
not particularly heavy but they are of course very
dense in the particles.
Do you think it would be possible? Any remark?

Stephane



__________________________________________________
Do You Yahoo!?
Tired of spam? Yahoo! Mail has the best spam protection around
http://mail.yahoo.com

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From: tauria-at-hotmail.com
Date: Wed, 18 Apr 2007 08:33:16 -0500
Subject: [Microscopy] AskAMicroscopist: OPERATING MANUAL FOR A PERKIN ELMER INFRARED

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Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (tauria-at-hotmail.com) from http://www.microscopy.com/Ask-A-Microscopist/Ask-A-Microscopist.html on Wednesday, April 11, 2007 at 12:33:00
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Email: tauria-at-hotmail.com
Name: DR. FRANCIS J. PRONESTI

Organization: WORLD ENERGY SERVICES, LTD.

Education: Graduate College

Location: castellana grotte, bari, italy

Question: HELLO EVERYONE,
I NEED URGENTLY AN OPERATING MANUAL FOR A PERKIN ELMER INFRARED MICROSCOPE,MODEL FT-IR, S/N 144235 MANUFACTURED IN 1991. PART NO. N187-3065.
THANKS AND KIND REGARDS TO ALL.
DR. FRANCIS J. PRONESTI


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From: akoorts-at-medic.up.ac.za
Date: Wed, 18 Apr 2007 08:33:50 -0500
Subject: [Microscopy] AskAMicroscopist: count gold particles

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Email: akoorts-at-medic.up.ac.za
Name: Alida Koorts

Organization: University of Pretoria

Education: Graduate College

Location: Pretoria, South Africa

Question: Are there any software available to count gold particles (immunolocalization) on TEM photo's?

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From: mcauliff-at-umdnj.edu
Date: Wed, 18 Apr 2007 08:58:20 -0500
Subject: [Microscopy] Re: Alternative to Uranyl Acetate Stain/p-phenylene

Contents Retrieved from Microscopy Listserver Archives
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Allan Micthell suggested para-phenylene diamine as an osmium
'enhancer' and I agree. I have used p-pd in 70% ethanol during
dehydration for years, it chemically reduces osmium bound to the tissue
and increases contrast. The only drawback is that tissue so treated is
difficult to stain with the usual toluidine blue + borax solution.

Geoff


allan.mitchell-at-stonebow.otago.ac.nz wrote:

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8, 36 -- diamine
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From: mike.reedy-at-cellbio.duke.edu
Date: Wed, 18 Apr 2007 10:11:53 -0500
Subject: [Microscopy] RE: TEM: Alternative to Uranyl Acetate Stain

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Jessica
Don't know about KMnO4 staining compatibility
with LR White; please let me know. The easy test
is to put a blank block or chunk or flake of
cured LR White in 1-2% KMnO4 and soak it for an
hour or a day to see if resin surface turns light
brown, dark brown, or black and charred looking.
No reaction is good news for section staining.

The more stringent assay for reaction is to put
the test tube of KMnO4 solution with the cured
embedding resin in a beaker of water and then
heat the water to boiling for an hour or so, cool
it off and examine the resin surface for such
changes. Araldite 506 (lowest viscosity one I
know of) and other Araldites used for embedding
are remarkable resistant to surface discoloration
when this is done. Epon DDSA (no MNA; fiddle
the mix until it is hard enough to please you,
and forget the epoxy:anhydride ratio lore) turns
moderately browner, as I recall, but is still
acceptable for section staining, with some
tendency to be more granular and maybe show some
hard-to-eliminate nano-pepper stain deposit in
contrast to Araldite.

LR White is an acrylic says EMS:
LR White is a polar monomer polyhydroxylated
acromatic acrylic resin. It can be cured by heat
or by UV light. Sections of polymerized LR White
resin are hydrophilic
I think Lawn's 1960 JCB paper introducing KMnO4
section staining used sections of methacrylate,
the mix of methyl and butyl methacrylate we all
used in the late 1950s before epoxies, especially
Epon, turned up and became dominant.

BTW-- tannic acid is a fixative and a mordant,
and magically capable of superior structure
preservation and assuring really strong uniform
staining in our hands-- see the evidence of 13Å
preservtion in fiber x-ray diffraction from
fixed-embedded fibers in Sader et al 2007 in J
Struct Biol (Articles In Press on line), and
look at EMs in some reprints I sent you for
evidence of the good morphology. The latter is
a wondrous gift of TA I had no idea of until I
learned it from David Begg et al, J. Cell Biol.
79:846-852, an all-time key paper in my book of
methodological turning points.. The other key
was the observation by Hirose and Wakabayashi (J.
Mol. Biol. 204:797-801) that TA followed by OsO4
or UrAc give great morphological fixation without
aldehydes. They used it for freeze-substitution,
as we have, but we found it also worked very well
as a general fix (we termed this TAURAC) for
permeabilized cells in aqueous buffers so long as
we exluded TA blockers like PVP, Triton X 100 etc.

Your UA police might be interested in a demo of
how completely tannins, esp tannic acid, can
convert a UrAc solution into a flocculent brown
precipitate at the bottom of a UrAc-free
supernatan (it LOOKS UrAc free! I made no
measurements.). TA is cheap in non-EM grades;
maybe they'd accept precipitation as a way of
rendering it safely bio-inactive. From the
information online at EMS ib their
datasheet/22400, I've estimated that the 10,400
counts/secin depleted uranium is reduced to about
-2 ct/sec in a very small bundle of muscle fibers
containing 1 ug of protein and binding maybe 5 ug
of UrAc. But it still requires health police and
special containment if such a specimen is to be
transported into or within the DOE facility at
Argonne National Laboratory. The inconvenience
has so far discouraged me from pursuing some
experiments that would require such a specimen,
but one day I will do it, legally and according
to their regulations. I doubt a Geiger counter
could detect any rise above background in the
presence of that small an amount of UrAc.

-mike-


} Thanks for this Mike. I think KMnO4 followed by Sato's is one of the
} better alternatives I've seen so far. Your results are very impressive.
}
}
} Any idea if KMnO4 will work with LR White embedded tissues?
}
} Thanks,
} Jessica Cervantes
} Bend Research Inc
}
} -----Original Message-----
} From: Mike Reedy [mailto:mike.reedy-at-cellbio.duke.edu]
} Sent: Saturday, April 14, 2007 5:19 PM
} To: Cervantes, Jessica
} Subject: Re: [Microscopy] TEM: Alternative to Uranyl Acetate Stain
}
} You will get even more contrast if you use KMnO4 followed by Sato's,
} as we have since 1964 (when Pb was was Pb cittrate, not Sato's).
} And, you can get good contrast on thinner sections. Using Tannic
} acid in the block stain, before OSO4, or after but followed by UrAc,
} adds to contrast, improves preservation. We have had excellent
} results (see attached).
}
} Some reports of using TA as a section stain before Pb stain can also
} be found with google help. I've never tried it yet.
}
} KMnO4 section stain won't work if NMA is in your Epon mix. see
} attached.
} -mike reedy-
} } -----------------------------------------------------------------------
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--
-mike reedy-

************************
Michael K. Reedy, M.D.
Duke Univ. Med. Center
Dept. Cell Biology, Box 3011 (for U.S. Mail)
458 Alex Sands Bldg, Research Dr (courier)
Durham, NC 27710

Office 919-668-2534
Lab 919-684-5674
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From: drk-at-SHCC.org
Date: Wed, 18 Apr 2007 12:43:19 -0500
Subject: [Microscopy] stabilization ofmembranes

Contents Retrieved from Microscopy Listserver Archives
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That would be a good place to start. I have posted some quite images of
inclusions in an organic goo on our web site
(ftp://www.marl.iastate.edu/Interesting/Residue/) . The mineral
inclusions show up nicely.

Another poster mentioned doing this on a carbon substrate. In fact, I
prepared this sample twice - once on a carbon stub and once on an
aluminum stub. I wanted to see how much signal was coming from below.
That was mostly for EDS and did appreciably affect the images. If you
had a mixture of particles only, the situation might be a little
different and I would recommend the dark background of a carbon
substrate.

You may run up against resolution limits for BSE depending on particle
size. The images may not be particularly sharp due to the sizeable
interaction volume and the high currents typically required for BSE.
These images were collected at 25mm WD with a sizeable current for
simultaneous EDS. You could improve resolution by cutting the working
distance and reducing current as much as possible while still
maintaining signal strength.

Warren Straszheim
Materials Analysis and Research Lab
Iowa State University

-----Original Message-----
X-from: nizets2-at-yahoo.com [mailto:nizets2-at-yahoo.com]
Sent: Wednesday, April 18, 2007 8:05 AM
To: wesaia-at-iastate.edu

Fellow Microscopists,


A few days ago I posted a query for suggestions on how to stabilize
lysosomal membranes in cultured cells prior to immunocytochemistry. We have
tried several of the tips but without success. Thanks to those who replied!
We have been following the condition of the lysosomes using a GFP tag that
we know to be specifically compartmentalized. We note that following
fixation and a rinse in TRIS-Glycine the GFP is still compartmentalized.
However, perhaps in 90% EtOH and definitely in 1:1 LR White:90% EtOH we see
the GFP moves from the lysosomes to the cytoplasm and nucleus. It seems that
ethanol and certainly LR White contribute to the deterioration of membranes.
Can anyone suggest an embedding media that might be a little gentler on
membranes and still possibly work for surface label immunocytochemistry? We
are thinking of trying Nanoplast, a water soluble media but any suggestions
are welcome.


Thanks! Doug

Douglas R. Keene
Assistant Investigator
Micro-Imaging Center
Shriners Hospital for Children
3102 S.W. Sam Jackson Park Road
Portland, Oregon 97239
503-221-3434
drk-at-shcc.org


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From: mnesta-at-ebsciences.com
Date: Wed, 18 Apr 2007 13:59:20 -0500
Subject: [Microscopy] SEM Accessory Sales Position Opening

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Energy Beam Sciences, Inc. is seeking a Technical Sales Representative
with a background in Scanning Electron Microscopy to represent an
exciting line of imaging, measurement and analysis software that is new
to our Company. This is a full time position that will entail
significant travel and frequent Customer interaction. For detailed
information about this great opportunity please visit us on the web at
http://www.ebsciences.com/jobs.html.

Sincerely,

Michael R. Nesta
Managing Director
Energy Beam Sciences, Inc.
Tel: 860 653-0411
Fax: 860 653-0422
MNesta-at-ebsciences.com
www.ebsciences.com
“ADDING BRILLIANCE TO YOUR VISION”



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From: David.Patton-at-uwe.ac.uk
Date: Thu, 19 Apr 2007 05:29:37 -0500
Subject: [Microscopy] TEM: Alternative to Uranyl Acetate Stain

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

We get nice contrast with 1% potassium permanganate (aq) followed by
lead citrate. It is good for membranes. In the past we have made it up
in 0.1M phosphate buffer at pH below 6.5 to avoid precipitates.

Dave

-----Original Message-----
X-from: cervantes-at-bendres.com [mailto:cervantes-at-bendres.com]
Sent: 13 April 2007 21:16
To: David Patton

Due to some bureaucratic mumbo-jumbo, I am not able to use Uranyl
Acetate (nuclear warheads, blah, blah, blah). I would like to stain
some osmium-fixed tissue thin-sections for TEM; the procedure I'm
following has a UA/Sato's lead stain step for the sections. Does anyone
know of a suitable alternative? I did a quick google and listserver
archive search and didn't find anything, but I'm hoping someone has run
into this problem before and can suggest something.

Crossing my fingers,
Jessica Cervantes
Bend Research, Inc
Bend, OR


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From: David.Patton-at-uwe.ac.uk
Date: Thu, 19 Apr 2007 09:51:25 -0500
Subject: [Microscopy] RE: TEM: Alternative to Uranyl Acetate Stain

Contents Retrieved from Microscopy Listserver Archives
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Sorry - just back from leave! Recently we have used it on 20yr old
epoxy blocks of unknown provenance and TAAB embedding resin. No
experience on LR White.

Will try Pal's bleach as we seem to have some non-specific staining.

It was in use here in 1989, when I started here, due to a safety scare
(yes even back in the good old days!). Hayat (1989) noted precipitates
above pH 6.8. We have never investigated the maintainance of pH. We
stain for 5-10min with KMnO4.

Dave



-----Original Message-----
X-from: Mike Reedy [mailto:mike.reedy-at-cellbio.duke.edu]
Sent: 19 April 2007 15:20
To: David Patton
Cc: cervantes-at-bendres.com


--
-mike reedy-

************************
Michael K. Reedy, M.D.
Duke Univ. Med. Center
Dept. Cell Biology, Box 3011 (for U.S. Mail)
458 Alex Sands Bldg, Research Dr (courier)
Durham, NC 27710

Office 919-668-2534
Lab 919-684-5674
Fax 919-681-9929
mike.reedy-at-cellbio.duke.edu
http://note.cellbio.duke.edu/Faculty/Research/Reedy.html

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From: paul_hazelton-at-umanitoba.ca
Date: Thu, 19 Apr 2007 12:40:16 -0500
Subject: [Microscopy] nova operator's manual

Contents Retrieved from Microscopy Listserver Archives
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Wim -

Here's the list of references. I haven't checked if all are available
or still in print.

I'm replying to the whole list in case anyone else is interested (can't
tell if you sent your message just to me).

Jessica Cervantes
Bend Research Inc
Bend, OR


The Cell, by Don W. Fawcett, MD. W. B. Saunders Company (out of
print).

Biomedical Electron Microscopy - Illustrated Methods and
Interpretations, by Arvid B. Maunsbach and Bjorn A. Afzelius. Academic
Press; 1st edition (January 15, 1999).

Cell and tissue ultrastructure: a functional perspective, by P. C.
Cross, and K. L. Mercer. W. H. Freeman & Co, 1993. 420 pp.

Histology: A text and Atlas, by Michael H. Ross, and Wojciech Pawlina.
Lippincott Williams & Wilkins; 1st edition (December 1, 2005). Was
updated 2006 (paperback) with CD-ROM and is available from Barnes &
Noble.

Color Atlas of Cytology, Histology and Microscopic Anatomy, by Wolfgang
Kuehnel. Thieme Medical Publishers; 4th edition (July 2003).

An Atlas of Ultrastructure, by Johannes Rhodin. W.B. Saunders Co.

Bloom and Fawcett: Concise Histology, by Don W. Fawcett, Ronald P.
Jensh. A Hodder Arnold Publication; 2nd edition (June 15, 2002).

For plant cells:
Introduction to the Fine Structure of Plant Cells, Myron Ledbetter and
Keith Porter. Springer-Verlag.



-----Original Message-----
X-from: Prof. dr. Wim Van den Broeck [mailto:wim.vandenbroeck-at-UGent.be]
Sent: Thursday, April 19, 2007 12:29 AM
To: Cervantes, Jessica

It seems that I've just been gifted with an LKB Nova ultratome, complete
with hydrolic table. However, there are no operator's manuals with the
microtome. Set it up and it is working fine, but would really like to
have an operator's manual so that I can check and make sure we know all
the little ins and outs of the machine.

Can any one help?????

Paul



Paul R. Hazelton, PhD
Electron Microscope Unit
University of Manitoba
Department of Medical Microbiology
531 Basic Medical Sciences Building
730 William Avenue
Winnipeg, Manitoba, Canada, R3E 0W3
e-mail: paul_hazelton-at-umanitoba.ca
Phone:204-789-3313
Pager:204-931-9354
Cell:204-781-1502
Fax:204-789-3926









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From: scott.streiker-at-udri.udayton.edu
Date: Thu, 19 Apr 2007 14:41:43 -0500
Subject: [Microscopy] viaWWW: Uptake of nanoparticles in cells using TEM

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Email: scott.streiker-at-udri.udayton.edu
Name: Scott Streiker

Organization: University of Dayton Research Institute

Title-Subject: [Filtered] Uptake of nanoparticles in cells using TEM

Question: Hello! I am trying to visualize the uptake of nanomaterials in cells using TEM. I have a cell pellet that I would like to embed in resin and then section using the ultramicrotome. One kit that I have the option to use is the Epofix Cold-Setting Resin from EMS. Has anyone used this kit before on biological samples? Or is there a better resin to try?

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From: bfoster-at-mme1.com
Date: Thu, 19 Apr 2007 16:44:57 -0500
Subject: [Microscopy] Re: viaWWW: Uptake of nanoparticles in cells

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html


For plant cells there are also books by Brian Gunning

Brian E.S Gunning and Martin W. Steer 'Plant Cell Biology; Structure and
Function' Jones and Bartlett Publishers (1996)

Brian E.S Gunning and Martin W. Steer 'Ultrastructure and the Biology of
Plant Cells' Edward Arnold (older version from mid 1970's

There is also a new DVD due out mid 2007 (I have seen an early version
which looks very good). You can find information
at:www.plantcellbiologyondvd.com/default.cfm

Ian

Ian Hallett
Sensory and Consumer Science - Microscopy
HortResearch, Mt Albert Research Centre
Private Bag 92 169, Auckland Mail Centre
Auckland 1142, New Zealand
+64-9-815 4200 ext 7002

-----Original Message-----
X-from: cervantes-at-bendres.com [mailto:cervantes-at-bendres.com]
Sent: Friday, 20 April 2007 3:35 a.m.
To: Ian Hallett

Wim -

Here's the list of references. I haven't checked if all are available
or still in print.

I'm replying to the whole list in case anyone else is interested (can't
tell if you sent your message just to me).

Jessica Cervantes
Bend Research Inc
Bend, OR


The Cell, by Don W. Fawcett, MD. W. B. Saunders Company (out of
print).

Biomedical Electron Microscopy - Illustrated Methods and
Interpretations, by Arvid B. Maunsbach and Bjorn A. Afzelius. Academic
Press; 1st edition (January 15, 1999).

Cell and tissue ultrastructure: a functional perspective, by P. C.
Cross, and K. L. Mercer. W. H. Freeman & Co, 1993. 420 pp.

Histology: A text and Atlas, by Michael H. Ross, and Wojciech Pawlina.
Lippincott Williams & Wilkins; 1st edition (December 1, 2005). Was
updated 2006 (paperback) with CD-ROM and is available from Barnes &
Noble.

Color Atlas of Cytology, Histology and Microscopic Anatomy, by Wolfgang
Kuehnel. Thieme Medical Publishers; 4th edition (July 2003).

An Atlas of Ultrastructure, by Johannes Rhodin. W.B. Saunders Co.

Bloom and Fawcett: Concise Histology, by Don W. Fawcett, Ronald P.
Jensh. A Hodder Arnold Publication; 2nd edition (June 15, 2002).

For plant cells:
Introduction to the Fine Structure of Plant Cells, Myron Ledbetter and
Keith Porter. Springer-Verlag.



-----Original Message-----
X-from: Prof. dr. Wim Van den Broeck [mailto:wim.vandenbroeck-at-UGent.be]
Sent: Thursday, April 19, 2007 12:29 AM
To: Cervantes, Jessica

Hi, Scott

This would definitely be another of those interesting applications to
try with the new Tomo AFM/Ultramicrotome from NT-MDT. The AFM is
superb at imaging nanoparticles and TOMO uses the Leica
ultramicrotome for sectioning.

While I don't have any pictures of nanoparticles, I do have a really
neat movie I can share with you showing the serial sectioning of
nanotubes in epoxy, followed by the dynamic 3D reconstruction from
Dr. DeWith at the Dutch Polymer Institute at the
TU/Eindhoven. Contact me off-line if you are interested. I can
send it via YouSendIt so that you can download it easily. Also, if
you are interested in seeing if this is a good solution for your
application, I encourage you to correspond directly with Dr. Efimov
at NTMDT (see CC above). He may be able to run a sample for you.

Hope this is helpful.

Best regards,
Barbara Foster

Microscopy/Microscopy Education
313 S Jupiter Rd, Suite 100
Allen, TX 75002
P: 972-954-8011
W: www.MicroscopyEducation.com

Caveat: MME is working with NTMDT in support of the TOMO.

MME is now scheduling customized, on-site courses through July. Call
us today for details.

P. S.
Need a good general reference or light microscopy text for next
semester? Call us today to learn more about "Optimizing LIght
Microscopy". Copies still available through MME... even for
class-room lots ... and we give quantity discounts. Call Ken Piel at
(972)954-8011.

At 04:16 PM 4/19/2007, scott.streiker-at-udri.udayton.edu wrote:



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From: eknoppel-at-cc.usu.edu
Date: Thu, 19 Apr 2007 16:57:51 -0500
Subject: [Microscopy] viaWWW: Lung tissue

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Email: eknoppel-at-cc.usu.edu
Name: Edward L. Knoppel

Organization: USDA-ARS-PPRL

Title-Subject: [Filtered] Lung tissue

Question: Without using a microwave, freezing or perfusion of the tissue; is there a "really" good procedure for fixing pieces of animal lung tissue that someone would be gracious enough to share? Thanks in advance, Ed

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From: ewing.hr-at-bruker-axs.com
Date: Thu, 19 Apr 2007 16:58:33 -0500
Subject: [Microscopy] viaWWW: Job Opening: Senior Salesperson - Microanalysis

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Email: ewing.hr-at-bruker-axs.com
Name: Doug Skinner

Organization: Bruker AXS Microanalysis

Title-Subject: [Filtered] Job Opening: Senior Salesperson - Microanalysis

Question: Senior Salesperson - Microanalysis

Bruker AXS, Inc., a leading manufacturer of Analytical Instrumentation, has a job opening for a Senior Salesperson, working out of the Bruker Canada office in Milton, Ontario.

The position is to secure sales for BAXS Microanalysis Products in Canada and act as the technical sales representative to the existing customer base and prospective customers. Significant travel throughout Canada will be required.

Job responsibilities:

ï Prospecting for new customers
ï Following up sales leads provided by the company
ï Presenting and demonstrating company products
ï Supplying quotations and technical information, formulating sales strategies, as well as negotiating and securing sales orders
ï Maintain contact with existing customers to assure their satisfaction, develop good working relationships with SEM salespeople, collect and report market information and provide routine sales forecasts.

Bachelor's degree (B.S. or B.A.) from four-year College; or five years experience and/or training; or equivalent combination of education and experience. Three years experience in scientific equipment sales is desired. This position requires excellent verbal communication and interaction skills. Fluency in French and English is preferred.

Candidates meeting these requirements should send a resume to:

Doug Skinner
Bruker AXS Microanalysis
ewing.hr-at-bruker-axs.com



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From: cervantes-at-bendres.com
Date: Thu, 19 Apr 2007 17:57:57 -0500
Subject: [Microscopy] stabilization ofmembranes

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Did I miss the replies to this query somehow? Is there a technical
problem, or were the replies private? Would it be possible to post
them?

Let's not be shy (if that's it) about posting to the whole list. I've
gotten so much out of this resource (especially lately). I've had to
re-post replies to my query for others who have had the same question
and hadn't gotten the messages.

Thanks for keeping the information flowing; it's invaluable.
Jessica Cervantes
Bend Research Inc
Bend, OR

-----Original Message-----
X-from: drk-at-SHCC.org [mailto:drk-at-SHCC.org]
Sent: Wednesday, April 18, 2007 10:49 AM
To: Cervantes, Jessica

Fellow Microscopists,


A few days ago I posted a query for suggestions on how to stabilize
lysosomal membranes in cultured cells prior to immunocytochemistry. We
have
tried several of the tips but without success. Thanks to those who
replied!
We have been following the condition of the lysosomes using a GFP tag
that
we know to be specifically compartmentalized. We note that following
fixation and a rinse in TRIS-Glycine the GFP is still compartmentalized.
However, perhaps in 90% EtOH and definitely in 1:1 LR White:90% EtOH we
see
the GFP moves from the lysosomes to the cytoplasm and nucleus. It seems
that
ethanol and certainly LR White contribute to the deterioration of
membranes.
Can anyone suggest an embedding media that might be a little gentler on
membranes and still possibly work for surface label immunocytochemistry?
We
are thinking of trying Nanoplast, a water soluble media but any
suggestions
are welcome.


Thanks! Doug

Douglas R. Keene
Assistant Investigator
Micro-Imaging Center
Shriners Hospital for Children
3102 S.W. Sam Jackson Park Road
Portland, Oregon 97239
503-221-3434
drk-at-shcc.org


==============================Original
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7, 22 -- Date: Wed, 18 Apr 2007 10:43:58 -0700
7, 22 -- From: Doug Keene {drk-at-SHCC.org}
7, 22 -- Subject: stabilization ofmembranes
7, 22 -- Sender: drk-at-SHCC.org
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13, 16 -- MIME-Version: 1.0
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13, 16 -- Subject: RE: [Microscopy] stabilization of membranes
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From: tom-at-tomkaye.com
Date: Thu, 19 Apr 2007 18:47:51 -0500
Subject: [Microscopy] stabilization ofmembranes

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html


I would like to 2nd the idea that replying to the list is a good thing. This
is my only resource for such information so I would rather delete than miss
an opportunity to learn something.

Thanks!

Tom Kaye

Not affiliated with anything.

-----Original Message-----
X-from: cervantes-at-bendres.com [mailto:cervantes-at-bendres.com]
Sent: Thursday, April 19, 2007 6:02 PM
To: tom-at-tomkaye.com

Did I miss the replies to this query somehow? Is there a technical
problem, or were the replies private? Would it be possible to post
them?

Let's not be shy (if that's it) about posting to the whole list. I've
gotten so much out of this resource (especially lately). I've had to
re-post replies to my query for others who have had the same question
and hadn't gotten the messages.

Thanks for keeping the information flowing; it's invaluable.
Jessica Cervantes
Bend Research Inc
Bend, OR

-----Original Message-----
X-from: drk-at-SHCC.org [mailto:drk-at-SHCC.org]
Sent: Wednesday, April 18, 2007 10:49 AM
To: Cervantes, Jessica

Fellow Microscopists,


A few days ago I posted a query for suggestions on how to stabilize
lysosomal membranes in cultured cells prior to immunocytochemistry. We
have
tried several of the tips but without success. Thanks to those who
replied!
We have been following the condition of the lysosomes using a GFP tag
that
we know to be specifically compartmentalized. We note that following
fixation and a rinse in TRIS-Glycine the GFP is still compartmentalized.
However, perhaps in 90% EtOH and definitely in 1:1 LR White:90% EtOH we
see
the GFP moves from the lysosomes to the cytoplasm and nucleus. It seems
that
ethanol and certainly LR White contribute to the deterioration of
membranes.
Can anyone suggest an embedding media that might be a little gentler on
membranes and still possibly work for surface label immunocytochemistry?
We
are thinking of trying Nanoplast, a water soluble media but any
suggestions
are welcome.


Thanks! Doug

Douglas R. Keene
Assistant Investigator
Micro-Imaging Center
Shriners Hospital for Children
3102 S.W. Sam Jackson Park Road
Portland, Oregon 97239
503-221-3434
drk-at-shcc.org


==============================Original
Headers==============================
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7, 22 -- Date: Wed, 18 Apr 2007 10:43:58 -0700
7, 22 -- From: Doug Keene {drk-at-SHCC.org}
7, 22 -- Subject: stabilization ofmembranes
7, 22 -- Sender: drk-at-SHCC.org
7, 22 -- To: Microscopy-at-Microscopy.Com
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[216.228.161.112])
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==============================Original Headers==============================
25, 20 -- From tom-at-tomkaye.com Thu Apr 19 18:47:51 2007
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25, 20 -- From: "Tom Kaye" {tom-at-tomkaye.com}
25, 20 -- To: {microscopy-at-microscopy.com}
25, 20 -- Subject: 2nd for posting to the list
25, 20 -- Date: Thu, 19 Apr 2007 18:48:03 -0500
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From: tom-at-tomkaye.com
Date: Thu, 19 Apr 2007 19:07:06 -0500
Subject: [Microscopy] Need help with ID

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Hello All,

Do any of you know what this thing is? It looks like a "bug" but it's
attached to a probable piece of fungus. Any ideas welcome, this is a head
scratcher for everyone that has seen it.

Thanks!

Tom Kaye

Tomkaye.com/images/fungus_lrg.jpg Long shot, the bug thing is lower right


Tomkaye.com/images/fungus2_lrg.jpg Close up.


==============================Original Headers==============================
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8, 20 -- From: "Tom Kaye" {tom-at-tomkaye.com}
8, 20 -- To: {microscopy-at-microscopy.com}
8, 20 -- Subject: Need help with ID
8, 20 -- Date: Thu, 19 Apr 2007 19:07:18 -0500
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From: Rosey.VanDriel-at-csiro.au
Date: Thu, 19 Apr 2007 23:19:42 -0500
Subject: [Microscopy] viaWWW: Lung tissue

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html


Hello Ed,
There is a method which works really well in fetal mouse lung. (Cole TJ,
Solomon NM, van Driel R, Monk JA, Bird D, Richardson SJ, Dilley RJ,
Hooper SB. Am J Respir Cell Mol Biol 2004 May; 30(5):613-9 "Altered
epithelial proportions in the fetal lung of glucocorticoid receptor null
mice")

We used the method as published by Williams, M. C. 1977. "Conversion of
lamellar body membranes into tubular myelin in alveoli of fetal rat
lungs". J. Cell Biol. 72:260-277.

It involves use of veronal (barbitone) and maleate buffers, but is
really simple to do.

However, I do not know how well it works with inflated lungs.

Briefly:
Dissect lungs from embryo, place in drop of fixative and slice gently
into mm cubes. Fix in 4% Paraformaldehyde + 2% Glutaraldehyde + 4%
Sucrose in HEPES buffered saline pH 7.4 for 3 to 4 hours at room temp.
Rinse 2 min in cold veronal acetate pH 7.4
Postfix in 1.5% OsO4 in Veronal Acetate -at- 4degrees C overnight
Rinse 3 x 10 min -at-4 deg C in Tris Maleate pH 5.2
En bloc stain with 1.5% UrAc in Tris Maleate pH5.2, 90 min on ice, in
dark
Cold dehydration, 5 minute changes in graded acetones on ice (10, 20,
30, 40, 50, 60, 70, 80, 90, 95%)Then Absolute dry acetone 4 x 10
minutes, the last 2 changes at room temp.
Absolute acetone:Epon Mix, 50:50, 30 minutes, rotating
Epon, 3 x 60 minute changes, can leave one change overnight, rotating
Embed in fresh Epon, polymerize overnight at 60 to 65 degrees C.

If you would like the buffer recipes, let me know.

Good luck!

Rosey

Rosemary van Driel
Electron Microscopy
New and Emerging Zoonotic Diseases
Australian Animal Health Laboratory
CSIRO Livestock Industries
Private Bag 24
Geelong Vic 3220
Australia

International Phone: +61 3 5227 5209
International Fax: +61 3 5227 5555
email: Rosey.VanDriel-at-csiro.au






-----Original Message-----
X-from: eknoppel-at-cc.usu.edu [mailto:eknoppel-at-cc.usu.edu]
Sent: Friday, 20 April 2007 08:00
To: Van Driel, Rosey (LI, Geelong)

This Question/Comment was submitted to the Microscopy Listserver
using the WWW based Form at
http://microscopy.com/MicroscopyListserver/MLFormMail.html
------------------------------------------------------------------------
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Remember this posting is most likely not from a Subscriber, so when
replying
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Email: eknoppel-at-cc.usu.edu
Name: Edward L. Knoppel

Organization: USDA-ARS-PPRL

Title-Subject: [Filtered] Lung tissue

Question: Without using a microwave, freezing or perfusion of the
tissue; is there a "really" good procedure for fixing pieces of animal
lung tissue that someone would be gracious enough to share? Thanks in
advance, Ed

------------------------------------------------------------------------
---

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From: David.Patton-at-uwe.ac.uk
Date: Fri, 20 Apr 2007 03:49:14 -0500
Subject: [Microscopy] Need help with ID

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Just a guess - looks like the moulted exoskeleton of a caterpillar-like
insect larva. The nice wavy tubes look like the air circulation tubes
that connect to spiracles.

Dave

-----Original Message-----
X-from: tom-at-tomkaye.com [mailto:tom-at-tomkaye.com]
Sent: 20 April 2007 01:11
To: David Patton

Hello All,

Do any of you know what this thing is? It looks like a "bug" but it's
attached to a probable piece of fungus. Any ideas welcome, this is a
head
scratcher for everyone that has seen it.

Thanks!

Tom Kaye

Tomkaye.com/images/fungus_lrg.jpg Long shot, the bug thing is lower
right


Tomkaye.com/images/fungus2_lrg.jpg Close up.


==============================Original
Headers==============================
8, 20 -- From tom-at-tomkaye.com Thu Apr 19 19:07:05 2007
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-0500
8, 20 -- From: "Tom Kaye" {tom-at-tomkaye.com}
8, 20 -- To: {microscopy-at-microscopy.com}
8, 20 -- Subject: Need help with ID
8, 20 -- Date: Thu, 19 Apr 2007 19:07:18 -0500
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This incoming email to UWE has been independently scanned for viruses by
McAfee anti-virus software and none were detected


This email was independently scanned for viruses by McAfee anti-virus software and none were found


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From: niko.hellsten-at-stratum.fi
Date: Fri, 20 Apr 2007 04:50:21 -0500
Subject: [Microscopy] cross section sample preparation

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Hello everyone

I'm doing basic cross section preparation for light microscopy work. My
samples are stainless steel plated with 0-50 micrometers thick layers of
copper and chromium.

I would like to make the interfaces clearer than they are after
grinding/polishing. I have thought about etching/corroding the surface with
some acid or other chemical.

I would like to hear from the experts what kind of chemicals they might
recommend. Or if etching my samples chemically would do me any good at all.

Thanks in advance

Niko Hellstén
Product Engineer
Stratum Oy
mail: niko.hellsten-at-stratum.fi
GSM: +358-(0)440-955301


==============================Original Headers==============================
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7, 22 -- From: "Niko Hellsten" {niko.hellsten-at-stratum.fi}
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From: Joseph_Oparowski-at-bose.com
Date: Fri, 20 Apr 2007 06:31:22 -0500
Subject: [Microscopy] cross section sample preparation

Contents Retrieved from Microscopy Listserver Archives
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Hi

I do not know if it would be appropriate with your material but I always
suggest people fracture samples for cross section. The usual method is to
use liquid nitrogen to embrittle the specimen.

This method is mainly used for the SEM but many clients use LM as well. The
SEM is very good at detecting poor cross sections through cutting or
polishing but the fracture route has never let me down.

Steve Chapman
Senior Consultant Protrain
For electron microscopy consultancy and training world wide
Tel +44 1280 816512 Fax +44 1280 814007
Mobile +44 7711 606967 Web www.emcourses.com


----- Original Message -----
X-from: {niko.hellsten-at-stratum.fi}
To: {protrain-at-emcourses.com}
Sent: Friday, April 20, 2007 10:51 AM

Hello Niko,

If you are just trying to delineate the interfaces, I would etch the copper layer. This is assuming that your configuration is the SS base metal / a Cu under layer / a Cr outer layer. Of more importance is the polishing of the sample before etching. You must ensure that the polishing method does not introduce smearing of the polished surface, especially at the copper plating.

A flat etch to remove copper and reveal the copper grain boundaries consists of;

20 ml ammonium hydroxide
20 ml of water
5 ml of 3% hydrogen peroxide

The hydrogen peroxide is added just prior to etching. Etching is performed by swabbing the polished surface for approximately 10-30 sec.

For more on polishing methods see;

"ASM Handbook, Vol. 9 - Metallography and Microstructures", ASM International, 2004, Materials Park, OH 44073

For etchants and precautions for safely using them see;

"Metallographic Etching, 2nd Ed.", Petzow, G., ASM International, 1999, Materials Park, OH 44073


Good luck.

Joseph

Joseph M. Oparowski
Center for Materials Science
Bose Corporation Joseph_Oparowski-at-bose.com
The Mountain, M/S 415 Phone: (508) 766-1371
Framingham, MA 01701-9168 Fax: (508) 766-1313


-----Original Message-----
X-from: niko.hellsten-at-stratum.fi [mailto:niko.hellsten-at-stratum.fi]
Sent: Friday, April 20, 2007 5:55 AM
To: Oparowski, Joseph

Hello everyone

I'm doing basic cross section preparation for light microscopy work. My
samples are stainless steel plated with 0-50 micrometers thick layers of
copper and chromium.

I would like to make the interfaces clearer than they are after
grinding/polishing. I have thought about etching/corroding the surface with
some acid or other chemical.

I would like to hear from the experts what kind of chemicals they might
recommend. Or if etching my samples chemically would do me any good at all.

Thanks in advance

Niko Hellstén
Product Engineer
Stratum Oy
mail: niko.hellsten-at-stratum.fi
GSM: +358-(0)440-955301


==============================Original Headers==============================
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28, 24 -- From Joseph_Oparowski-at-bose.com Fri Apr 20 06:31:21 2007
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From: klemari_cz-at-yahoo.com
Date: Fri, 20 Apr 2007 07:03:59 -0500
Subject: [Microscopy] ultramicrotome

Contents Retrieved from Microscopy Listserver Archives
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Hello everyone,

our institution has acquired money to buy an
ultramicrotome for TEM sample preparation of material
samples, such as layered structures (clay minerals or
thin layers on Si support).

To my knowledge, there are currently two
ultramicrotomes on the market - EM UC6 from Leica and
Power-Tome XL from RMC. I have talked to sales people
from both companies, and of course they both claim
that their cutting technique is the best for cutting
hard materials because it does not introduce internal
vibrations. RMC is motor driven while Leica boasts
with "the Gravity Stroke". I can imagine both
techniques having some troubles vibrationwise so I am
torn and confused. So here comes the question. Has
anybody ever tried to section hard materials on these
machines and compared the results?

Thanks a lot for your time.

Have a nice weekend,

Mariana

¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤

Mariana Klementova
Institute of Inorganic Chemistry of the ASCR, v.v.i.
250 68 Husinec-Rez 1001
Czech Republic

phone: ++420-266 17 31 44
mobile: ++420-723 52 66 19
fax: ++420-220 94 15 02
e-mail: klemari-at-iic.cas.cz
http://www.iic.cas.cz/~jem3010/english/

¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤ ¤

__________________________________________________
Do You Yahoo!?
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From: Walter.Bobrowski-at-pfizer.com
Date: Fri, 20 Apr 2007 07:16:29 -0500
Subject: [Microscopy] stabilization ofmembranes

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Jessica, have you attempted ICC following routine aldehyde fixation and
osmium post-fixation followed by standard epoxy embedding? The osmium
will preserve the membranes and you *may* still get successful ICC. Give
a holler and I'll send you a (work in progress) protocol that appears to
yield successful IHC on LM thick sections using RT reagents (no
microwaves, no heated steam baths).

Best regards,

Walter F. Bobrowski
Sr. Scientist
Pfizer Global R&D
2800 Plymouth Rd.
Ann Arbor, MI 48105

TEL: 734-622-7814
FAX: 734-622-5718

"The ultimate human freedom is the ability to choose one's attitude in a
given set of circumstances." -Viktor Frankl



-----Original Message-----
X-from: cervantes-at-bendres.com [mailto:cervantes-at-bendres.com]
Sent: Thursday, April 19, 2007 7:03 PM
To: Bobrowski, Walter

Did I miss the replies to this query somehow? Is there a technical
problem, or were the replies private? Would it be possible to post
them?

Let's not be shy (if that's it) about posting to the whole list. I've
gotten so much out of this resource (especially lately). I've had to
re-post replies to my query for others who have had the same question
and hadn't gotten the messages.

Thanks for keeping the information flowing; it's invaluable.
Jessica Cervantes
Bend Research Inc
Bend, OR

-----Original Message-----
X-from: drk-at-SHCC.org [mailto:drk-at-SHCC.org]
Sent: Wednesday, April 18, 2007 10:49 AM
To: Cervantes, Jessica

Fellow Microscopists,


A few days ago I posted a query for suggestions on how to stabilize
lysosomal membranes in cultured cells prior to immunocytochemistry. We
have
tried several of the tips but without success. Thanks to those who
replied!
We have been following the condition of the lysosomes using a GFP tag
that
we know to be specifically compartmentalized. We note that following
fixation and a rinse in TRIS-Glycine the GFP is still compartmentalized.
However, perhaps in 90% EtOH and definitely in 1:1 LR White:90% EtOH we
see
the GFP moves from the lysosomes to the cytoplasm and nucleus. It seems
that
ethanol and certainly LR White contribute to the deterioration of
membranes.
Can anyone suggest an embedding media that might be a little gentler on
membranes and still possibly work for surface label immunocytochemistry?
We
are thinking of trying Nanoplast, a water soluble media but any
suggestions
are welcome.


Thanks! Doug

Douglas R. Keene
Assistant Investigator
Micro-Imaging Center
Shriners Hospital for Children
3102 S.W. Sam Jackson Park Road
Portland, Oregon 97239
503-221-3434
drk-at-shcc.org


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27, 31 -- From Walter.Bobrowski-at-pfizer.com Fri Apr 20 07:16:23 2007
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From: as-at-astonmet.com
Date: Fri, 20 Apr 2007 07:42:48 -0500
Subject: [Microscopy] Re: cross section sample preparation

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html


Niko,

To avoid smearing, especially in the final stages
of polishing, try using Buehler's MasterMet and
MasterPrep on a spongey pad such as their
Chemocloth. This is good for flatness and
preserving delicate features even with layers of very different materials.

Alan Stone
ASTON


Note: Buehler is not the only supplier of these
products. Other metallographic suppliers may have
similar products, but I am not familiar with them.




At 04:51 AM 4/20/2007, you wrote:



} ----------------------------------------------------------------------------
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From: p.veys-at-cra.wallonie.be
Date: Fri, 20 Apr 2007 07:46:45 -0500
Subject: [Microscopy] viaWWW: Stereology : grids and methods

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Email: p.veys-at-cra.wallonie.be
Name: Pascal Veys

Title-Subject: [Filtered] Stereology : grids and methods

Question: To anyone who could be give me a tip...

We are investigating grid counting for particles counting in feed, but also taking into account their relative size : a particle being 5 times bigger than all others should be counted as 5 instead of 1). Basically we intend to use eyepiece square grids reticles. Does anyone has other proposals (eg type of grid) ? We are also looking for a good reference method description including a discussion on biases (such as that of counting only two adjacent lengths of the square...), is there an ultimate good and recent book of reference available ?

Thanks for any help.

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From: fab-at-tariffenet.it
Date: Fri, 20 Apr 2007 07:47:13 -0500
Subject: [Microscopy] viaWWW: need help fo digital camera

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Email: fab-at-tariffenet.it
Name: fabio

Organization: University of Catania

Title-Subject: [Filtered] need help fo digital camera

Question: Dear All,
I would like to mount a digital camera (such as a Canon Powershot A-540 or A-560)on my Reichert Microstar IV light microscope (equipped with a trinocular viewing body).
I know I need an adaper for the camera (LADC52F) and an adapter for the microscope trinocular body.
Which kind of adapter I need?
Any suggestions?

Thank You

Fabio
University of Catania, Italy.
fab-at-tariffenet.it






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From: oshel1pe-at-cmich.edu
Date: Fri, 20 Apr 2007 08:02:39 -0500
Subject: [Microscopy] Re: Need help with ID

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Tom,

Looks like 2 badly collapsed mite larvae. Given the scale bar on the
lower-mag shot, these are probably recently hatched. The long, fuzzy
things sticking out of the circles are sensory setae. These are
dorsal views, and the legs are the long, straight fuzzy things that
some out from underneath them. The confusing bunch of things sticking
out of the end of the one larva (whose posterior end is under the 2nd
larva) are the pedipalps and chelicerae (soft parts at this stage).
The 2 larger structures sticking "north" out of the mess are the
forelegs, and joints are just visible. No clue about the taxon, but
judging by size, I'd guess Astigmata, or whatever that group is these
days.

Mind, this could all be empty arm-waving, but I'd be willing to bet a
pitcher of good beer on it.

Phil

} Hello All,
}
} Do any of you know what this thing is? It looks like a "bug" but it's
} attached to a probable piece of fungus. Any ideas welcome, this is a head
} scratcher for everyone that has seen it.
}
} Thanks!
}
} Tom Kaye
}
} Tomkaye.com/images/fungus_lrg.jpg Long shot, the bug thing is lower right
}
}
} Tomkaye.com/images/fungus2_lrg.jpg Close up.
--
Philip Oshel
Microscopy Facility Supervisor
Biology Department
024C Brooks Hall
Central Michigan University
Mt. Pleasant, MI 48859
voice: (989) 774-3576
dept. fax: (989) 774-3462

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From: j.janssen-at-nki.nl
Date: Fri, 20 Apr 2007 08:20:59 -0500
Subject: [Microscopy] viaWWW: grids and methods

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Email: j.janssen-at-nki.nl
Name: J.W.R.M. Janssen

Organization: Dutch Cancer Institute

Title-Subject: [Filtered] grids and methods

Question: Dear Pascal,

Here is a good reference:
Recent developments for quantifying immunogold label on transmission EM thin sections Terry M Mayhew Centre for Integrated Systems Biology & Medicine, School of Biomedical Sciences, University of Nottingham, UK
Succes, Hans.

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From: JBABBITT-at-fresco.com
Date: Fri, 20 Apr 2007 10:44:44 -0500
Subject: [Microscopy] RE: Cross-sectioning packaging film

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I am a relative newcomer to the microscopy/histology field and need some advice on sample preparation. I am trying to section a multilayer packaging film (e.g. polyester, aluminum foil, polyethylene lamination) and am having trouble keeping the sample flat, so I can get a good reading on individual layer thicknesses. I am using a cryostat microtome for sample preparation. Is there a simple technique I can employ, or a type of embedding compound that works better for industrial applications? Thanks to any who can give me a hand, I appreciate it.

R. Jefferson Babbitt, Ph.D.
Analytical Services Manager
Fres-co System USA
3005 State Rd.
Telford, PA 18969-1021
voice - 215-721-4600 x2149
cell - 267-236-4027
fax - 215-799-8017
email - jbabbitt-at-fresco.com





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From: cervantes-at-bendres.com
Date: Fri, 20 Apr 2007 10:47:39 -0500
Subject: [Microscopy] stabilization ofmembranes

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Thanks Walter. I'm definitely interested in your protocol.

Jessica Cervantes
Bend Research Inc
Bend, OR

-----Original Message-----
X-from: Walter.Bobrowski-at-pfizer.com [mailto:Walter.Bobrowski-at-pfizer.com]
Sent: Friday, April 20, 2007 5:22 AM
To: Cervantes, Jessica

Jessica, have you attempted ICC following routine aldehyde fixation and
osmium post-fixation followed by standard epoxy embedding? The osmium
will preserve the membranes and you *may* still get successful ICC. Give
a holler and I'll send you a (work in progress) protocol that appears to
yield successful IHC on LM thick sections using RT reagents (no
microwaves, no heated steam baths).

Best regards,

Walter F. Bobrowski
Sr. Scientist
Pfizer Global R&D
2800 Plymouth Rd.
Ann Arbor, MI 48105

TEL: 734-622-7814
FAX: 734-622-5718

"The ultimate human freedom is the ability to choose one's attitude in a
given set of circumstances." -Viktor Frankl



-----Original Message-----
X-from: cervantes-at-bendres.com [mailto:cervantes-at-bendres.com]
Sent: Thursday, April 19, 2007 7:03 PM
To: Bobrowski, Walter

Did I miss the replies to this query somehow? Is there a technical
problem, or were the replies private? Would it be possible to post
them?

Let's not be shy (if that's it) about posting to the whole list. I've
gotten so much out of this resource (especially lately). I've had to
re-post replies to my query for others who have had the same question
and hadn't gotten the messages.

Thanks for keeping the information flowing; it's invaluable.
Jessica Cervantes
Bend Research Inc
Bend, OR

-----Original Message-----
X-from: drk-at-SHCC.org [mailto:drk-at-SHCC.org]
Sent: Wednesday, April 18, 2007 10:49 AM
To: Cervantes, Jessica

Fellow Microscopists,


A few days ago I posted a query for suggestions on how to stabilize
lysosomal membranes in cultured cells prior to immunocytochemistry. We
have
tried several of the tips but without success. Thanks to those who
replied!
We have been following the condition of the lysosomes using a GFP tag
that
we know to be specifically compartmentalized. We note that following
fixation and a rinse in TRIS-Glycine the GFP is still compartmentalized.
However, perhaps in 90% EtOH and definitely in 1:1 LR White:90% EtOH we
see
the GFP moves from the lysosomes to the cytoplasm and nucleus. It seems
that
ethanol and certainly LR White contribute to the deterioration of
membranes.
Can anyone suggest an embedding media that might be a little gentler on
membranes and still possibly work for surface label immunocytochemistry?
We
are thinking of trying Nanoplast, a water soluble media but any
suggestions
are welcome.


Thanks! Doug

Douglas R. Keene
Assistant Investigator
Micro-Imaging Center
Shriners Hospital for Children
3102 S.W. Sam Jackson Park Road
Portland, Oregon 97239
503-221-3434
drk-at-shcc.org


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32, 17 -- Date: Fri, 20 Apr 2007 08:47:36 -0700
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From: walck-at-southbaytech.com
Date: Fri, 20 Apr 2007 11:18:39 -0500
Subject: [Microscopy] re: cross section sample preparation

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

I can see your problem. You have a soft material that smears with relatively hard materials and they have much different chemistries. I would like to suggest an alternative to bringing out your structure. After polishing, instead of using a chemical etch, you can use ion etching. What I would do is to ion polish at a low angle for about 10 minutes and then follow that with a high angle etch at about 35 degrees for a short time. With copper samples for light microscopy, we found that 2.5 min gave a good grain appearance at 500X and could see the interfaces with elelctroless copper layers. Since the ion polishing and etching is a physical process, it is less sensitive to the chemical nature of the materials in your cross section. We make and sell the IBS/e that can be used for this purpose and have gotten very nice results with electroplated copper samples with light microscopy. Gatan has a nicely prepared booklet that shows light microscopy results as well.

Please contact me offline and we could arrange to run some samples for you. I would only ask that we could put the results on our web site as an application note.

Disclaimer: South Bay Techonology, Inc. makes and sells the IBS/e ion beams sputter and etch system as well as metallurgical polishing equipment.

-Scott

Scott D. Walck, Ph.D.
Technical Director
South Bay Technology, Inc.
1120 Via Callejon
San Clemente, CA 92673

TEL: +1-949-492-2600
FAX: +1-949-492-1499
walck-at-southbaytech.com

-------- Original Message --------
} From: niko.hellsten-at-stratum.fi
} Sent: Friday, April 20, 2007 5:54 AM
} To: Walck-at-SouthBayTech.com
} Subject: [Microscopy] cross section sample preparation
}
} ----------------------------------------------------------------------------
} The Microscopy ListServer -- CoSponsor: The Microscopy Society of America
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} Hello everyone
}
} I'm doing basic cross section preparation for light microscopy work. My
} samples are stainless steel plated with 0-50 micrometers thick layers of
} copper and chromium.
}
} I would like to make the interfaces clearer than they are after
} grinding/polishing. I have thought about etching/corroding the surface with
} some acid or other chemical.
}
} I would like to hear from the experts what kind of chemicals they might
} recommend. Or if etching my samples chemically would do me any good at all.
}
} Thanks in advance
}
} Niko Hellstén
} Product Engineer
} Stratum Oy
} mail: niko.hellsten-at-stratum.fi
} GSM: +358-(0)440-955301
}
}
} ==============================Original Headers==============================
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From: cgarber-at-2spi.com
Date: Fri, 20 Apr 2007 12:20:50 -0500
Subject: [Microscopy] Preparation of multilayer packaging films

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Hi

I would try freezing the sample and using a single bend fracture procedure
as set out below. We have used this technique on polythene freezer bags and
many other materials for SEM but the technique is equally applicable to LM.

"Over the many years that clients and I have been investigating the cross
sections of materials by for the best method is to fracture the material.
The SEM is very clever in that it sees a cut surface and tells us "this is a
cross section cut with a sharp scalpel blade" or "this is a cross section
cut with a blunt scalpel blade" etc etc.
Preparation method A

1. Cut down the material to 1cm by 3cms place it into liquid nitrogen
until it stops bubbling.

2. Remove the material and crack it using either heavy duty tweezers or
fine pliers. If you are unable to crack the material and are forced to flex
it in order for it to crack this is not good enough! In the latter case
reduce or neck the material as shown, even a 0.5mm long crack could provide
a great deal of detail in the SEM?

3. When the pieces have dried out (condensation) they may both be
observed by LM and SEM

Fibres that will not fracture by the above method could be fractured by one
of two other methods.

Method B

1. Insert the material in a small diameter tube (thin drinking straws are
ideal). Cut the straw down to about 3cms tall. Block one end with wax,
modelling clay or similar material.

2. Using a syringe force water into the straw and block the end as above.

3. Drop the straw into liquid nitrogen then follow method A part 2 above.

4. When the pieces have dried out (condensation) they may be observed by
both LM and SEM

Method C

1. Drill 2mm to 3mm holes in a pair of stubs as shown in diagram 2.

2. Infiltrate the holes with a water soluble carbon solution and push a
bundle of fibres through the carbon solution.

3. When dry follow method A part 2 except use a blade to initiate the
crack

4. When the pieces have dried out (condensation) they may be observed by
both LM and SEM

Method C has been used with materials like freezer bags that were failing.
In this case the material was spun into a small spiral and then infiltrated
with the carbon solution."


The above data was taken from our "hints and tips" web page.

Steve Chapman
Senior Consultant Protrain
For electron microscopy consultancy and training world wide
Tel +44 1280 816512 Fax +44 1280 814007
Mobile +44 7711 606967 Web www.emcourses.com

----- Original Message -----
X-from: {JBABBITT-at-fresco.com}
To: {protrain-at-emcourses.com}
Sent: Friday, April 20, 2007 4:45 PM

R. Jefferson Babbitt, Ph.D wrote the following:
================================================================
I am a relative newcomer to the microscopy/histology field and need some
advice on sample preparation. I am trying to section a multilayer packaging
film (e.g. polyester, aluminum foil, polyethylene lamination) and am having
trouble keeping the sample flat, so I can get a good reading on individual
layer thicknesses. I am using a cryostat microtome for sample preparation.
Is there a simple technique I can employ, or a type of embedding compound
that works better for industrial applications? Thanks to any who can give
me a hand, I appreciate it.
========================================================================
We have done this same kind of system in our own laboratory. The
quick-and-dirty way to get a cross-section is liquid nitrogen fracturing
but as you point out, getting a good edge-on view is not necessarily easy.
Another danger: If you don't know in advance exactly how many layers there
are, one or more outer layers can split off further down from the fracture
surface and what you think is the fracture surface indeed might not be the
facture surface.

Hence, for SEM work, we always gold coat the two sides so that so long as we
can see in the fracture surface the two gold layers, we know we have the
entire cross-section, and in order to keep it "straight", we mount is using
an angled SEM mount (available from SPI Supplies as well as all of our major
competitors), which is then tilted 45 deg. for the head on view.

But we have pretty consistently found that a TEM view, while the sample work
up is more tedious, is far more rewarding. Most of the time one wants to
look at such films, is because there is an adhesion failure between one or
more of the layers and the TEM view is able to resolve features (such as
contaminants) between the layers or the dispersion of inorganic fillers in
one or more of the layers. We still gold coat such films (with passivation
layers) before embedding to ensure that there is not interaction of the
embedding resin with the polymer film layers. Our preferred embedding resin
is our own SPI-Pon 812 epoxy- based embedding resin (but we believe
equivalent results will be obtained with the equivalent sold by others).

One other thing: The cross-sectioning, irrespective of what method you are
using, must be done cryo, with an ultramicrotome using a diamond knife. A
glass knife will not last very long against the aluminum foil layer.

Since you are a "neighbor" to SPI Supplies, we would be happy to have you
visit and we would show you how we do these kinds of films.

Disclaimers: Mentioned in this posting are some of our own products and we
have the vested interest in promoting their use.

Chuck

===================================================
Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400
President
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: drk-at-SHCC.org
Date: Fri, 20 Apr 2007 12:36:52 -0500
Subject: [Microscopy] responses to membrane stabilization querry (long)

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Fellow Microscopists:

Many thanks to those who responded to my request for suggestions for
stabilizing membranes (lysosomes in particular). My specific aim in this
experiment is to stabilize cultured cells containing GFP tagged lysosomes
for surface-label immunocytochemistry with the hope of co-localizing GFP
expressing protein (using confocal images overlaid onto TEM images cut from
the next serial section) and another potentially interactive protein
localized with immunogold on the TEM section. We are able to follow GFP
emission through the protocol using our confocal microscope. In cultures
fixed in media buffered 4% paraformaldehyde/1% glutaraldehyde, we see that
GFP remains compartmentalized up to 90% ethanol but after the introduction
of 1:1 90% EtOH:LRWhite, GFP is no longer compartmentalized and instead is
distributed throughout the cytoplasm and nucleus. I am now in the process
of trying some of the ideas. A preliminary result suggests that the
lysosomes are stabilized somewhat more by the inclusion of 4% picric acid;
also by progressively lowering temperature during graded ethanol dehydration
to minus 20C, embedding in Lowicryl HM20 at -20C, and polymerization at
-20C. We have yet to section the HM20, but we hope that confocal microscopy
on 0.5um sectioned HM20 will reveal compartmentalized GFP. Then we'll see
what mess we've made of the additional antibody-binding epitopes. We love a
challenge!

To address the request that the responses be shared, here is a summary.
Since some of these responses were made privately, I have not included the
author's names.

Response #1:

what may help: high-pressure immobilization (cryo-fixation; expensive but
really good),
followed by freeze-substitution at -90 (8 to 48 hrs)/-60 (8hrs)/-30 (6 to 8
hrs) as described in several papers.

It is worth trying:
Aceton + 0.1/0.2% OsO4, +0.5% Uac +/-GA (0.1 to 1%) +/-FA (0.25 to 2%)
(MANY variants possible, I know)
or
EtOH, no OsO4, but add some or all other chemicals/fixatives
MeOH, no OsO4, but add some or all ...
Aceton + 2%GA (ready available as it is)
Aceton + 0.2%GA + UAc 0.5% ...
and so on.

The advantage of cryo-substitution at very low temperatures:
The chemicals are not reactive at low temperature initially (only slowly at
-60, and then at higher temperatures). The chemicals become evenly
distributed in the cell/tissue, and then react at the same time upon
warming, everywhere, similarly. Result: tissue, cells and membranes are
often better preserved. in addition, you may add some water (1 - 5%?) to the
freeze-substitution medium, (YES!!), as suggested by Paul Walther a few
years ago.
We have done this, others as well, with success.

Response #2:

Have you tried tannic acid! It is supposed to stabilize plasma membranes,
don't know about Lysosomal membranes,

Response #3"

While it would involve doing freeze-substitution, Lowicryl HM 20
is great for membranes. You would need to do low temperature dehydration
after room temperature or 4 degree C aldehyde fixation. I used to use a
small amount of glutaraldehyde with the methanol free buffered formalin. I
also used uranyl acetate to help preserve membranes and dehydrated cold with
methanol. You can look at my paper for details:

The Journal of Histochemistry and Cytochemistry 40(10):1491-1500, 1992
"Immunocytochemical Localization of Lysozyme and Surfactant Protein A in Rat
Type II Cells and Extracellular Surfactant"

Response #4:

I have a couple of thoughts on the problem. I don't think you are getting
any buffering capacity from the culture medium. And if there is any serum
in the medium, you have titrated all the aldehydes with the serum and have
no functional fixative left when you are trying to fix the cells.

Summary of additional responses:

These included longer fixation times, fixation at ambient temperature not
4C, and the importance of including Calcium in the fixation buffer.

Again, thank you to those who responded and to all interested microscopists!

Doug Keene
Douglas R. Keene
Assistant Investigator
Micro-Imaging Center
Shriners Hospital for Children
3102 S.W. Sam Jackson Park Road
Portland, Oregon 97239
503-221-3434
drk-at-shcc.org


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From: gerard.cox-at-goodrich.com
Date: Fri, 20 Apr 2007 12:48:07 -0500
Subject: [Microscopy] New SEM Purchase

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Our group wants to buy a new SEM to replace an old AMRAY machine for failure analysis and quality control investigations. We also need EDS capabilities, and do not anticipate the need for any other analysis tools. It seems like we will need to go to 10 000X only or thereabouts, and we would like to have low vacuum capabilities for imaging nonmetallic components / painted parts. We want a large chamber, and a large range of stage motion (100 mm X 80 mm X 80 mm min.) We need the new machine to last a really long time, be easily maintained, and spare parts should be available for the indefinite future, as I do not anticipate funding for anything like this again for over a decade. I think that analog imaging is preferred to digital acquisition because of alleged "software fudging" in the latter (or something.)

We are looking at a JEOL JSM-6490LV and a Hitachi S-3400N specifically, (my manager's choices) and I have seen a CamScan and a few FEI models. My initial impressions are that the CamScan unit is a bit specialized and that the service/support won't be as effective (we are in southern Ontario) and that the FEI machines will be WAY too expensive and more tailored to bio applications / really high-end research. As to the JEOL and Hitachi, I was told the JEOL is slightly better and the company is less likely to disappear (ie, better support and spare parts in the long-term) while the service and support for the Hitachi people is superior, both initially and long-term.

I realize I am asking a great deal, and would appreciate any input into this matter. Has anyone bought or used either of the models listed above? Does anyone have any comments on what we do or do not want? Is Peltier cooling instead of LN2 really a good idea? Any input as to EDS machines and software to buy or to avoid? Thanks for your help.

Gerard Cox


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From: kraftpiano-at-gmail.com
Date: Fri, 20 Apr 2007 15:10:08 -0500
Subject: [Microscopy] SEM: Replacing diffusion pump water cycle with coolant.

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Just a brief question to the list: I've been working on the cooling
system to my JSM-840 when an idea struck me. I am having problems
because the water pump that I have to circulate the chilled water is
not powerful enough to produce the pressure needed to circulate water
through the SEMs diffusion pumps. The pump is actually rated for a
coolant, though (Antifreeze/DI Water mixture) and I was wondering
what you guys think about instead of using water to chill the
diffusion pumps and amplifier electronics, using some form of coolant
with a lower viscosity than water, which would alleviate my need for
such high pressures and let me use the pump that fits the chiller I
have.

--Justin A. Kraft

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From: TJJ-at-stowers-institute.org
Date: Fri, 20 Apr 2007 16:04:09 -0500
Subject: [Microscopy] Recommended humidity level

Contents Retrieved from Microscopy Listserver Archives
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Can anybody give me an optimal % relative humidity that is recommended
for EM rooms? We recently had some failures with formvar grids (we
think) due to excessive room humidity when they were being made. I can
bring in a de-humidifier, but what would be considered to be optimal?

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110



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From: gary-at-gaugler.com
Date: Fri, 20 Apr 2007 16:36:17 -0500
Subject: [Microscopy] Re: SEM: Replacing diffusion pump water cycle

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

You did not say what brand chiller you have.
What flow rate and pressure do you need? For
just a diffusion pump (no electronics), I don't think a huge
rate is needed. I think you need to set the temperature
and have the correct flow rate rather than
concentrate on pressure. If you can't get the
rate, then that is what you should focus on,
IMO.

Haskris and probably others specifically say
to NOT use anti-freeze. Some folks survive
very well just using distilled water. That
has not been my experience. I find that 10%
mix of 100% Ethylene Glycol and DI works great. Most
chillers use really cheap brass fittings rather
than Imperial brass. Consequently, a bad mix
of coolant will cause corrosion and also lead to
algae growth. The other gotcha are the hoses.
Use opaque ones rather than transparent ones.
Keeping the light out inhibits algae growth too.

In my Haskris R50 chiller with 5 gallon reservoir,
water or the mix results in the same pressure
at same flow rate. What will cause pressure to
change is a dirty tank filter, or especially the
external toilet paper filter (50u fiber mesh).

gary g.



At 12:12 PM 4/20/2007, you wrote:

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From: drk-at-SHCC.org
Date: Fri, 20 Apr 2007 16:36:45 -0500
Subject: [Microscopy] Recommended humidity level

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Hello Teri,

If it is for formvar that you wish to keep humidity low, you might consider
casting your formvar films within a glove bag flushed with dry nitrogen gas.
We use such a system here in our Oregon lab with good success. The bags we
use are from I2R and the web address is:
http://www.i-2-r.com/glove_bag/l_glove_bags/p_x_r/x_r.htm. I have no
financial interest.

Doug

Douglas R. Keene
Assistant Investigator
Micro-Imaging Center
Shriners Hospital for Children
3102 S.W. Sam Jackson Park Road
Portland, Oregon 97239
503-221-3434
drk-at-shcc.org

-----Original Message-----
X-from: TJJ-at-stowers-institute.org [mailto:TJJ-at-stowers-institute.org]
Sent: Friday, April 20, 2007 2:08 PM
To: drk-at-SHCC.org

Can anybody give me an optimal % relative humidity that is recommended
for EM rooms? We recently had some failures with formvar grids (we
think) due to excessive room humidity when they were being made. I can
bring in a de-humidifier, but what would be considered to be optimal?

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110



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From: ejb1176-at-gmail.com
Date: Fri, 20 Apr 2007 16:44:11 -0500
Subject: [Microscopy] Re: SEM: Replacing diffusion pump water cycle with

Contents Retrieved from Microscopy Listserver Archives
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Hi Justin,
Does JEOL list a spec, for the flow rate? Too much may give you
excess vibration.

Could the lines/pump have some blockage?

Restricting the outlet, a little, will increase the backpressure, it
might be enough to trip the interlock, if that's shutting things off.

I've added up to 5% ETHYLENE GLYCOL to recirculating cooling systems
(EBEAM and SEM and TEM) with no long-term deleterious effects. Not
sure how much that will change the viscosity.

I've always been advised against running DI water alone as it is ion
hungry and may leach metal pipes. I've always used grocery store
distilled water.

Best of Luck.

Ed Basgall, PhD
Irvine, CA 92612

email: ejb1176-at-gmail.com


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From: tivol-at-caltech.edu
Date: Fri, 20 Apr 2007 17:19:30 -0500
Subject: [Microscopy] Re: Recommended humidity level

Contents Retrieved from Microscopy Listserver Archives
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On Apr 20, 2007, at 2:04 PM, TJJ-at-stowers-institute.org wrote:

} Can anybody give me an optimal % relative humidity that is recommended
} for EM rooms? We recently had some failures with formvar grids (we
} think) due to excessive room humidity when they were being made. I can
} bring in a de-humidifier, but what would be considered to be optimal?
}
Dear Teri,
You need not only to be concerned about the formvar, but you also do
not want condensation on the column or electronics. Too low a humidity
will also allow the generation of static electricity. About 40% RH is
a very good value, and the maximum permissible depends on the
difference in temperature between the room and the scope and is usually
about 60%.
Yours,
Bill Tivol, PhD
EM Scientist
Electron Cryo-Microscopy Facility
Broad Center, Mail Code 114-96
California Institute of Technology
Pasadena CA 91125
(626) 395-8833
tivol-at-caltech.edu


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From: kraftpiano-at-gmail.com
Date: Fri, 20 Apr 2007 17:43:12 -0500
Subject: [Microscopy] Re: SEM: Replacing diffusion pump water cycle with coolant.

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Sorry, I wasn't very clear about the chiller before. It's not a name
brand chiller- it's actually a part from a larger system, but I don't
know what that larger system is- I bought it locally as industrial
surplus. Here's what I know about the recirculator system:

Pump: Iwaki Magnetic drive pump. 12 L/min. Max. head 2.1m.

There is a heat exchanger unit and all of the condenser piping
necessary to chill the water, and it can divide the output a maximum
of three ways, so I can have the electronics set on a different water
circuit than the diffusion pumps. When I say that the pressure is not
enough to push the water through, I mean that the pump pumps, but very
little water is pushed through, and it is shutting down because of
thermal overload. I know that the JEOL manual specifies 5 L/min. at
12-36 PSI, but I'm not sure how that translates from "Max head" to
PSI. The pump's flow rate is more than enough to handle it, but it
isn't making it through. That's why I was thinking about lower
viscosity fluids, which should flush through at a higher flow rate and
a lower PSI.

--Justin.

On 4/20/07, kraftpiano-at-gmail.com {kraftpiano-at-gmail.com} wrote:
}
}
}
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} Just a brief question to the list: I've been working on the cooling
} system to my JSM-840 when an idea struck me. I am having problems
} because the water pump that I have to circulate the chilled water is
} not powerful enough to produce the pressure needed to circulate water
} through the SEMs diffusion pumps. The pump is actually rated for a
} coolant, though (Antifreeze/DI Water mixture) and I was wondering
} what you guys think about instead of using water to chill the
} diffusion pumps and amplifier electronics, using some form of coolant
} with a lower viscosity than water, which would alleviate my need for
} such high pressures and let me use the pump that fits the chiller I
} have.
}
} --Justin A. Kraft
}
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From: vitalylazar-at-att.net
Date: Fri, 20 Apr 2007 18:05:05 -0500
Subject: [Microscopy] Re: SEM: Replacing diffusion pump water cycle

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

ditto that- 30 psi is enough, unless cooling system is clogged. If it is
clogged, then concentrate your efforts on unclogging the cooling system,
rather than increasing pressure. The highest water pressure that I can
imagine being required for any SEM/TEM is in mid-40 psi. I typically set
much lesser pressure, between 25 and 35 psi. Compensating poor flow in a
clogged system with higher pressure is a bad habit. Plus, high pressure
erodes rubber and plastic components of the cooling system, and that alone
accelerates formation of clogs. Especially with old instrument.

I suggest to have at least 1 flowmeter with flow control (it is cheap). And
pressure reducer with bypass line for the water pump - every decent chiller
has that anyway. Pump itself shall be capable of at least 100 psi. Not for
everyday operation, but for troubleshooting, especially if you have old
instrument. You can easily detect presence of a clog (if not location...),
by adjusting and monitoring pressure and flow rate. More than one flowmeter
is good to have if cooling system has parallel branches.

Likely places for clogs in JEOL-840 are: quick connections, lower (hottest)
water line coils on both ODPs, and of course filters if any are present.
Less likely places are electronics and obj. lens. But this is never certain.

To remove clog, reverse the direction of water flow, or use small amount
water with compressed air in reversed direction. But first disconnect
various parts of cooling circuit, and determine where the clog is located.
If you reverse the whole thing at once, clog might get worse and harder to
remove. You will need a variety of hoses and fittings to do this work
efficiently.

Flow rates at room T~20C and water T~17C: 1gpm is great, 0.5gpm is OK, 0.2
gpm is critically low, below that expect thermal protection to trip at any
moment.

Vitaly Feingold
SIA
2773 Heath Lane
Duluth GA 30096
Ph. 770-232-7785
Fax 770-232-1791
www.sia-cam.com
----- Original Message -----
X-from: {gary-at-gaugler.com}
To: {vitalylazar-at-att.net}
Sent: Friday, April 20, 2007 5:37 PM


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From: hanke-at-mee-inc.com
Date: Fri, 20 Apr 2007 18:42:58 -0500
Subject: [Microscopy] Re: cross section sample preparation

Contents Retrieved from Microscopy Listserver Archives
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Fracture method will not work on this sample. Stainless steel and copper
are very ductile and will bend and neck down before breaking. These
metals are not made brittle by cold temperatures.

Sample preparation, i.e. good polishing methods is the key to this
evaluation. This combination can be polished mechanically, but a good
procedure and technique will be required.

--
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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From: gary-at-gaugler.com
Date: Fri, 20 Apr 2007 20:49:45 -0500
Subject: [Microscopy] Re: 2nd for posting to the list

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

The reason NOT to post to the list is that you get
volumes of "Out Of Office" messages. This is the
same as when you make a new posting. Most of the
defunct messages are trapped by Eudora but quite
a few still get though....sigh.

Some do not have this subject but say that they
are gone from xx to yy.

gary g.



At 03:50 PM 4/19/2007, you wrote:
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From: W.Muss-at-salk.at
Date: Sat, 21 Apr 2007 06:14:22 -0500
Subject: [Microscopy] Re:AskAMicroscopist: negative staining whith a small lipo protein

Contents Retrieved from Microscopy Listserver Archives
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Dear Alvaro,
interesting question....
Normally (e.g. as I use it sometimes for negative staining of virus particles)
you will have at least two negative staining solutions:
i) hydrous PTA (1, 2 or other percentage)
ii) hydrous uranylacetate 1-4%.......
for (small) lipoproteins (esp. for HDL-like) I am not quite sure wether simple
saying so or giving advice in that way is helpful.

As I understand negative staing, also (a) positive result(s)will depend on pH
(and therefore [with what ingredients] buffering the solution eventually to
which "special" pH), and (absorption/adhesion) technique of specimen
preparation.
I don't think your carbon-coated formvar-filmed copper grids are a
problem....but,. if I remember correctly, there are several (also older)
papers/articles on negative staining techniques for e.g. liposomes (for an
imagination on such try search phrase } } "negative staining","liporotein"
{ {) reporting some very specific caudeles for adhesion of lipoprotein
material....(for instance: see*) below)
Some prefer also prefixation of mixed particles with hydrous Osmium tetroxide
before negative staining with the heavy metal solution, sometimes only hydrous,
somtetimes with an additive (like sucrose).....
so it may depend also on the type of material you'd like to have demonstrated
and wether you are able to get the (macro-)molecule adhering in a stabilized
form to the formvar-carbon-surface of the grid or not (cf. *)

Once determined [by trial&error (perhaps, unluckily)] how to proceed with the
grids' properties = hydrophilization and immediate use or storage for
months...see *), adhesion time/technique, eventually prestabilization of the
specimen by additional fixation, the right negative staining solution (also in
terms of i) element [PTA, UO2, AmmMolybdat,Uranylformate, etc.],
ii)concentration, iii) pH of the solution, iv) specific additives necessary?)
the technique itself yields reliable and rapid rsults, even within 5-10 minutes
(as I have read in some papers) ==} that's what I would like to wish you in
overcoming your problem.


Best regards
Wolfgang Muss, Salzburg, AUSTRIA

cf:
*) Original paper in:
J Lipid Res. 1980 Nov;21(8):981-92.
Unilamellar liposomes made with the French pressure cell: a simple preparative
and semiquantitative technique.
Hamilton RL Jr, Goerke J, Guo LS, Williams MC,Havel RJ.
Ex Material & Methods-section:
Negative staining of liposomes often causes artifactual images resulting from
disruption of small liposomes that subsequently form larger multilamellar
structures. Although the mechanism of this process is not understood, it may be
caused in part by the intense hydrophobicity of freshly evaporated carbon on
grid surfaces. Our attempts to modify the surface film of freshly prepared
carbon-coated grids (200-300 mesh copper grids, Fullum, Schenectady,NY) by glow
discharge, polylysine, or addition of albumin to sample or grid did not prove
satisfactory. However, we have learned empirically that parlodioncovered
grids with carbon films made with a Siemens evaporator (VI3 G 500) permit even
spreading of liposomes without apparent structural changes, provided that the
grids have been aged 6- 12 months on the shelf. Carbon rods for filming were
obtained from Ringsdorff-Herke (Spektral Kohlen, Bonn-Bad Godesberg, West
Germany). With such “matured” carbon-coated grids, liposomes were prepared for
electron microscopy with 2% potassium phosphotungstate at pH 6.45-6.5 by the
drop procedure described previously (1 1). We found that the lipid
concentration in the sample was also important for preparing uniform spreads.
Optimal results were more consistently obtained with samples containing
1- 10 mg/ml PC. Improved results can be obtained for more dilute samples by
increasing contact time of the sample on the grid to 2-3 min, and by using
a tighter 400 mesh grid.......



Zitat von alvarobq-at-fcien.edu.uy:

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} Below is the result of your feedback form (NJZFM-ultra-55). It was submitted
} by (alvarobq-at-fcien.edu.uy) from
} http://www.microscopy.com/Ask-A-Microscopist/Ask-A-Microscopist.html on
} Friday, September 15, 2006 at 08:40:57
} ---------------------------------------------------------------------------
}
} Email: alvarobq-at-fcien.edu.uy
} Name: Alvaro D. Olivera
}
} Organization: Science University
}
} Education: Undergraduate College
}
} Location: Montevideo - Uruguay
}
} Question:
}
} I'm TEM technician and advanced undergraduate in Biochemistry.
} I'm trying negative staining whith a small lipo protein HDL like. I'm
} using CARBON-FORMVAR COATED Cu GRIDS and PTA 2% and 3%. I can't see it.
} What do you suggest?
}
} Many thanks, Alvaro.
}
} ---------------------------------------------------------------------------
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} ==============================Original Headers==============================
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} protein
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From: randerson20-at-tampabay.rr.com
Date: Sat, 21 Apr 2007 06:36:33 -0500
Subject: [Microscopy] 2nd for posting to the list

Contents Retrieved from Microscopy Listserver Archives
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Hmmm. I get maybe 3 or 6 out of office msgs/post.

Ron

gary-at-gaugler.com wrote:
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} gary g.
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} At 03:50 PM 4/19/2007, you wrote:
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} }
} } I would like to 2nd the idea that replying to the list is a good thing. This
} } is my only resource for such information so I would rather delete than miss
} } an opportunity to learn something.
} }
} } Thanks!
} }
} } Tom Kaye
} }
} } Not affiliated with anything.
} }
} } -----Original Message-----
} } X-from: cervantes-at-bendres.com [mailto:cervantes-at-bendres.com]
} } Sent: Thursday, April 19, 2007 6:02 PM
} } To: tom-at-tomkaye.com
} } Subject: [Microscopy] RE: stabilization of membranes
} }
} }
} }
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} }
} } Did I miss the replies to this query somehow? Is there a technical
} } problem, or were the replies private? Would it be possible to post
} } them?
} }
} } Let's not be shy (if that's it) about posting to the whole list. I've
} } gotten so much out of this resource (especially lately). I've had to
} } re-post replies to my query for others who have had the same question
} } and hadn't gotten the messages.
} }
} } Thanks for keeping the information flowing; it's invaluable.
} } Jessica Cervantes
} } Bend Research Inc
} } Bend, OR
} }
} } -----Original Message-----
} } X-from: drk-at-SHCC.org [mailto:drk-at-SHCC.org]
} } Sent: Wednesday, April 18, 2007 10:49 AM
} } To: Cervantes, Jessica
} } Subject: [Microscopy] stabilization ofmembranes
} }
} } ------------------------------------------------------------------------
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} } The Microscopy ListServer -- CoSponsor: The Microscopy Society of
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} } ----
} }
} } Fellow Microscopists,
} }
} }
} } A few days ago I posted a query for suggestions on how to stabilize
} } lysosomal membranes in cultured cells prior to immunocytochemistry. We
} } have
} } tried several of the tips but without success. Thanks to those who
} } replied!
} } We have been following the condition of the lysosomes using a GFP tag
} } that
} } we know to be specifically compartmentalized. We note that following
} } fixation and a rinse in TRIS-Glycine the GFP is still compartmentalized.
} } However, perhaps in 90% EtOH and definitely in 1:1 LR White:90% EtOH we
} } see
} } the GFP moves from the lysosomes to the cytoplasm and nucleus. It seems
} } that
} } ethanol and certainly LR White contribute to the deterioration of
} } membranes.
} } Can anyone suggest an embedding media that might be a little gentler on
} } membranes and still possibly work for surface label immunocytochemistry?
} } We
} } are thinking of trying Nanoplast, a water soluble media but any
} } suggestions
} } are welcome.
} }
} }
} } Thanks! Doug
} }
} } Douglas R. Keene
} } Assistant Investigator
} } Micro-Imaging Center
} } Shriners Hospital for Children
} } 3102 S.W. Sam Jackson Park Road
} } Portland, Oregon 97239
} } 503-221-3434
} } drk-at-shcc.org
} }
} }
} } ==============================Original
} } Headers==============================
} } 7, 22 -- From drk-at-SHCC.org Wed Apr 18 12:43:18 2007
} } 7, 22 -- Received: from mail.SHCC.org (mail.shcc.org [64.213.211.202])
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} } 12:43:18 -0500
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} } Microscopy-at-Microscopy.Com;
} } 7, 22 -- Wed, 18 Apr 2007 10:41:57 -0700 (PDT)
} } 7, 22 -- Date: Wed, 18 Apr 2007 10:43:58 -0700
} } 7, 22 -- From: Doug Keene {drk-at-SHCC.org}
} } 7, 22 -- Subject: stabilization ofmembranes
} } 7, 22 -- Sender: drk-at-SHCC.org
} } 7, 22 -- To: Microscopy-at-Microscopy.Com
} } 7, 22 -- Cc: drk-at-SHCC.org
} } 7, 22 -- Reply-to: drk-at-SHCC.org
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} } Headers==============================
} }
} }
} } ==============================Original Headers==============================
} } 13, 16 -- From cervantes-at-bendres.com Thu Apr 19 17:57:57 2007
} } 13, 16 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com
} } [216.228.161.112])
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} } l3JMvuoK002012
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} } 13, 16 -- MIME-Version: 1.0
} } 13, 16 -- Content-Type: text/plain;
} } 13, 16 -- charset="us-ascii"
} } 13, 16 -- Subject: RE: [Microscopy] stabilization of membranes
} } 13, 16 -- Date: Thu, 19 Apr 2007 15:57:55 -0700
} } 13, 16 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C47F-at-BRIEX04A}
} } 13, 16 -- In-Reply-To: {200704181748.l3IHmsrE012175-at-ns.microscopy.com}
} } 13, 16 -- References: {200704181748.l3IHmsrE012175-at-ns.microscopy.com}
} } 13, 16 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com}
} } 13, 16 -- To: {Microscopy-at-microscopy.com}
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} }
} }
} } ==============================Original Headers==============================
} } 25, 20 -- From tom-at-tomkaye.com Thu Apr 19 18:47:51 2007
} } 25, 20 -- Received: from tomkaye.com (mxgateway.techpro.com [64.4.207.8])
} } 25, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with
} } ESMTP id l3JNlodW014261
} } 25, 20 -- for {microscopy-at-microscopy.com} ; Thu, 19 Apr 2007
} } 18:47:51 -0500
} } 25, 20 -- Received: from tk7c797a275194 [24.15.58.103] by
} } tomkaye.com with ESMTP
} } 25, 20 -- (SMTPD32-8.14) id AFAD3312012A; Thu, 19 Apr 2007 18:47:57 -0500
} } 25, 20 -- From: "Tom Kaye" {tom-at-tomkaye.com}
} } 25, 20 -- To: {microscopy-at-microscopy.com}
} } 25, 20 -- Subject: 2nd for posting to the list
} } 25, 20 -- Date: Thu, 19 Apr 2007 18:48:03 -0500
} } 25, 20 -- Message-ID: {GOEAIJOJDGCJBPIFHOBGKEPBOOAA.tom-at-tomkaye.com}
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} } 25, 20 -- Importance: Normal
} } ==============================End of - Headers==============================
} }
}
}
} ==============================Original Headers==============================
} 7, 20 -- From gary-at-gaugler.com Fri Apr 20 20:49:45 2007
} 7, 20 -- Received: from qsmtp4.mc.surewest.net (qsmtp.mc.surewest.net [66.60.130.145])
} 7, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l3L1niqX031833
} 7, 20 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 20:49:45 -0500
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} 7, 20 -- X-Mailer: QUALCOMM Windows Eudora Version 7.1.0.9
} 7, 20 -- Date: Fri, 20 Apr 2007 18:49:48 -0800
} 7, 20 -- To: tom-at-tomkaye.com
} 7, 20 -- From: Gary Gaugler {gary-at-gaugler.com}
} 7, 20 -- Subject: Re: [Microscopy] 2nd for posting to the list
} 7, 20 -- Cc: MSA listserver {microscopy-at-microscopy.com}
} 7, 20 -- In-Reply-To: {200704192350.l3JNo7lp017141-at-ns.microscopy.com}
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}
}


==============================Original Headers==============================
4, 19 -- From randerson20-at-tampabay.rr.com Sat Apr 21 06:36:32 2007
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4, 19 -- Date: Sat, 21 Apr 2007 07:36:28 -0400
4, 19 -- From: Ronald Anderson {randerson20-at-tampabay.rr.com}
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4, 19 -- To: gary-at-gaugler.com, Listserver {Microscopy-at-Microscopy.Com}
4, 19 -- Subject: Re: [Microscopy] Re: 2nd for posting to the list
4, 19 -- References: {200704210149.l3L1ntal032017-at-ns.microscopy.com}
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From: zaluzec-at-microscopy.com
Date: Sat, 21 Apr 2007 08:25:22 -0500
Subject: [Microscopy] Out of Office messages & posting comments to the list

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Colleagues.....

Replying to the list is not only appropriate, but should be done in almost
all cases. The obvious exception I could see is when a private reply
is appropriate, or a vendor is discussing a reply to a query with detailed
product information about a commerical item to an individuals request.

Remember the primary purpose of this list is to share our communal
expertise, if you post messages privately then this knowledge distribution
is curtailed. Of course, some people post summaries of replies
to their questions. This is a good procedure, and is also suggested
in the FAQ, and I would encourage people who receive alot of private replies to do this
as a courtesy to the community. Consider it part of the way you
pay the astronomical annual fees charged to each of you for being
a member of this forum.

FYI, there is nothing I can do to stop "out of the office" type replies, since
those come from the individuals and never pass through the listserver
filters. If you read the Listserver FAQ, I request that if you are unavailable
then you should unsubscribe from the list and then resubscribe when
you return. There are alot of people that do this (THANK YOU), but
obviously some do not.

In principle you won't miss anything, if you unsubscribe for a short while,
as all the messages are archived, however, there are clearly people who
choose not to follow this request for various reasons.

An alternative should you be wary of missing something, or if
your company policy requires you to use Out of the Office messages, would
be to have 2 addresses. One for your official mail (to which you could
add the Out of the Office reply), while a second which is only used for
Listserver traffic. This would be the logical approach for those
subscribers who use the Out of the Office message frequently.

As Gary G. mentioned most Email programs today allow you
to filter mail by topic, and I certainly, also use this mechanism to
refine the distribution of all Emails I personally get into appropriate folders.
If you do this then the issue becomes relatively minor, considering
the proponderance of SPAM/JUNK mail.

If it is not obvious to you by now all Listserver Email has the
following text in the subject line
[Microscopy]
that was done in order to allow you to create a filter
to put all listserver Email into a folder.

Fortunately for all of you the primary filters on the subscription
lists now catch nearly all of the junk/spam mail which is sent
to "microscopy-at-". For the record, long ago spam exceeded 500
messages/day, which I filter out for you. So a few ocassional
out of the office replies is a small price to pay for the information you
get for subscribing to this forum.

Cheers,


Nestor
Your Friendly Neighborhood SysOp.

==============================Original Headers==============================
12, 13 -- From zaluzec-at-microscopy.com Sat Apr 21 08:25:22 2007
12, 13 -- Received: from [206.69.208.22] (mac22.zaluzec.com [206.69.208.22])
12, 13 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3LDPJrW012128
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12, 13 -- References: {200704211136.l3LBaXsH031128-at-ns.microscopy.com}
12, 13 -- Date: Sat, 21 Apr 2007 08:25:18 -0500
12, 13 -- To: microscopy-at-microscopy.com
12, 13 -- From: "Nestor J. Zaluzec" {zaluzec-at-microscopy.com}
12, 13 -- Subject: Out of Office messages & posting comments to the list
12, 13 -- Content-Type: text/plain; charset="us-ascii"
==============================End of - Headers==============================




From: keith.morris-at-ucl.ac.uk
Date: Sat, 21 Apr 2007 09:12:21 -0500
Subject: [Microscopy] AskAMicroscopist: count gold particles

Contents Retrieved from Microscopy Listserver Archives
http://www.microscopy.com/MicroscopyListserver/MicroscopyArchives.html

Dear Alida,

Software image analysis of TEM film or digital images isn't always easy as
it is often difficult for the software to distinguish the objects you wish
to count from the rest of the image. However our brains can generally do a
far better job in picking out (discriminating) what we want to count or
measure. But you have be careful with things like sampling procedure and
image quality to make sure you don't bias the results.

Generally, even with modern computers, it is often quicker to count by eye
than get software to do this for you. This is particularly so if there are
only a few objects (less than 50) in the image. However if you can easily
software threshold (detect) the objects you wish to count then the software
can count them automatically very quickly. But often the 'detected'
thresholded binary overlay will need editing to remove false objects, and
this may take far longer than simply counting by eye.

In most cases careful preparation of the specimen and optimising image
capture and microscope optics will be essential to produce images are most
suited to semi-automatic image analysis software. For example if you wish to
count cells under optical microscopy, plate them at lower densities so that
they will not overlay each or frequently touch and so can be easily
separated and counted by software algorithms. Things like blue DAPI
fluorescence staining of the nucleus will enable cell numbers to be counted
very quickly semi-automatically, although it will get into trouble if a lot
of cells are multi-nuclear or in different focus planes. Often though, even
if the software doesn't automatically count the objects as 'accurately' as
we can by eye, the biological variation across samples is far higher than
these counting errors. Indeed different users will invariably produce
different counts for the same samples.

If, as is often the case with TEM and optical phase contrast transmission
images, it is very difficult to only threshold the objects of interest, then
manual counting is generally the quickest option. Image analysis software
can still help here though. Biomedical image analysis programs like
MetaMorph (http://www.moleculardevices.com/pages/software/me