2/17/98

One of my facility users has some freeze-dried sperm which she now needs

to embed and section for TEM. May I ask anyone with some experience

in this for advice?! I don't know specifically what she needs to see,

yet.



* Tina (Weatherby) Carvalho * tina@pbrc.hawaii.edu *

* Biological Electron Microscope Facility * (808) 956-6251 *

* University of Hawaii at Manoa * http://www.pbrc.hawaii.edu/bemf*

Aloha Tina,



The main problem as I see it will be some kind of fixation and contrasting

without rehydrating the specimens. In my checkered past we had a freeze-dryer

/ molecular distillation dryer / whatever that had a side access port and a

small heated chamber on it. We placed small amounts of OsO4 crystals and

paraformaldehyde powder in the chamber (one at a time), gently heated the

chamber, opened the valve and did vapor fixation on the dried specimens.

After each fixation step we re-pumped the chamber to remove as much of the

excess vapors as possible.



You could probably do something similar with a bell jar or desiccator, a few

feet of Tygon tubing, a test tube and a vacuum stopcock. There are a few

things to watch out for:



1. Do the osmium vapor fixation first! We found that if we hit the specimens

with aldehyde first it interfered with the penetration of the osmium vapors.

The aldehyde probably formed some kind of "crust" or barrier at the surface of

the specimens.



2. Be careful (of course) pumping out the osmium vapors. We equipped the

vacuum pump inlet with a liquid nitrogen cold trap for this purpose. We also

changed the pump oil very frequently. It tended to turn black quickly,

indicating that the pump oil was a very good secondary osmium trap!



3. extend the infiltration time with lots of changes of resin. The osmium

tended to diffuse out of the tissue and we ended up with specimens embedded in

a black cloud of osmium in the final blocks. Too much of this, and it

interferes with the polymerization of the resin (we used Spurr's).



Hope this helps!



All the best,



Bob

*********************************

Robert (Bob) Chiovetti

E. Licht Company / 1-800-865-4248

rchiovetti@aol.com

Tina, I have taken tissue that I prepared for and viewed in the SEM

several times and just placed them into absolute ethanol, then

embedded them normally. I have done this with very delicate

specimens. Of course they were fixed and postfixed previously for

SEM. If these tissue have not and you need the contrast, you probably

could run the samples down a descending series of alcohols to water

and then fix, etc. Another option would be to expose the sample to

osmium tetroxide vapors first before going into the 100%. That maybe

the only fixation you need before embedding. I would try both and

compare.

Good luck

Hank Adams

Electron Microscopy Lab

New Mexico State University

Las Cruces,NM 88003

phone: 505-6463600

fax: 505-6465665

hadams@NMSU.Edu

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