[3dem] TEMs and sample preferences

[reinhard rachel]

Dear Liz, 
wonderful situation ... congratulation.

> We are in the final stages of setting up our two new microscopes and I have

> a little question about samples. The 200 kV microscope will be dedicated to

> the imaging of cryo-specimens. However, we also have a new 120 kV
microscope. 
> What are the current preferences about specimens to image with an
instrument? 
> The three basic specimen types we image in our EM core lab are cryo,
standard 
> sections, and negatively stained samples. Does anyone have information on
the 
> types and amounts of contamination that are associated with each? Along with

> maintenance costs (perhaps)? We also have another TEM we use for basic 
> training and routine screening.

the question arises what the aim is, with each of your specimen, and what type
and contrast and so on. There is no simple answer; you will get 10 or 20 or
more replies, and most likely as many different answers. 
the negative staining can most likely be done on the 120 kV machine (at which
voltage, depends again on the molecular mass, the type of stain, etc)  - but if
you aim for high-resolution cryo-negative staining, the extra resolution of the
200 kV will help in the image processing (do you aim for this?). 
the cryo-work can be done on the 120 kV, at 80, 100, 120 kV (which I favor) -
in many cases, you may have sufficient contrast. Again, what type of instrument
is it? from which brand? some are really ideal for cryo - others are not
(vacuum, objective lense, imaging spectrometer, camera, ...). What type of
specimen? do you want to image liposomes? or single protein molecules at high
resolution? semi-crystalline samples? then, the 200 kV with image processing
will be nice (although you will - initially - may have less contrast). 
sections? what type of sample, of processing, of thickness? standard sections:
the 120 kV might be ok, and again, you may vary the voltage. (But, first
optimize your sample preparation). Thicker sections, and possibly tomo? 200 kV,
definitely, if it is sufficient. On the other hand, if the sections are weakly
stained only, go to one of the machines, get it tuned to 100, 80 or even 60 kV
(hopefully, you have an alignment not only for one, but for two or three  or
more voltages?), and have fun. 

No, there is no simple answer at all. It all depends, the specimen being the
most critical point(thickness, staining, quality of processing etc), objective
lense, apertures, operator, camera, availability of image processing, and your
experience. - 

kind regards,
Reinhard


-- 
PD Dr. Reinhard Rachel
Universitaet Regensburg
Centre for EM / Anatomy
Faculty for Biology&Preclin.Med.
Universitaetsstr. 31
D-93053 Regensburg - Germany
tel +49 941 943 2837, 1720
fax +49 941 943 2868
mail reinhard.rachel at biologie.uni-regensburg.de
office: VKL 3.1.29 
President of the German Society for Electron Microscopy - Deutsche
Gesellschaft für Elektronenmikroskopie - DGE
www.dge-homepage.de