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Sample Preparation: fixation

Here are some thoughts on fixation methods:

While chemical fixation (e.g., 4% formaldehyde, 0.25% glutaraldehyde) usually preserves the ultrastructure of the specimen quite well, it may not preserve its elemental content. In particular, as membranes get damaged by the fixation, they can become permeable for ions. Also, to reduce radiation damage and diffusion artifacts during the long scan times that are typical for highresolution X-ray fluorescence scans we usually use dried specimen. Then the time that is needed to dry the specimen also is an issue (during the drying process the osomotic pressure changes and cell membranes can break).

Nevertheless, some of our users did have success using chemical fixation as described above in investigating trace metals in cells. Sometimes a a brief rinse in ethanol after air drying can help to remove salt residues.

Fixation in cold (-20C) acetone/methanol has the advantage that the drying process is much more gentle on the cell structures, as surface tension of methanol/acetone is much less than that of water. In contrast to form- or glutaraldehyde, however, there is no crosslinking of proteins, just precipitation. Also, membranes (lipids) tend to be removed.

It would seem that chemical fixation methods can preserve the content of elements that are typically tightly bound to proteins (e.g., Zn) well, but one probably should be very cautious in the interpretation of maps of highly diffusible ions.

Probably the best fixation method would be cryofixation, where the specimen is cooled to liquid nitrogen temperatures rapidly enough to avoid ice crystal formation. This can be achieved, for example, by plungeing the specimen rapidly into liquid ethane, though this particular method limits the thickness where adequate fixation to something on the order of 10 microns. Another way to achive vitrification of the specimen is by using a high pressure freezing device, then regions up to 300 microns deep into the specimen can be adequately preserved.



last update 26 July, 2009