High Pressure Freezing/Freeze Substitution of Some Key
Organisms Used in M6lecular Genetic Studies.
Kent McDonald
Electron Microscope Laboratory, 26 Giannini Hall, University of California, Berkeley, CA 94720-3330
It has long been recognized that rapid freezing followed by freeze substitution [l]
improves preservation of cytological detail. The reason ultrarapid freezing is not more
commonly used is due to the small size of sample that can be adequately frozen. By most
common freezing methods cytoplasm is only well-preserved to a depth of about 10 pm
[2]. Beyond that, heat can not be removed fast enough to prevent ice crystal damage.
However, with the development and commercial availability of high pressure freezing
technology [3], it became possible to get good preservation in non-cryoprotected
samples
to a depth of 200 pm[4.5], and in theory to a depth of 600 p [3]. When ultrapid freezing
is followed by freeze substitution [6,7] and resin embedding, it is possible to use
conventional thin section techniques with transmission EM to evaluate cell fine structure.
The power of genetics has never been more evident than in today’s biology, and
the most effective genetic systems are probably Saccharomyces cerevisiae (budding
yeast), Drosophila melanogaster (a fruit fly), and Caenorhabditis elegans (a nematode
worm). Interestingly, these organisms are difficult to prepare for electron microscopy,
probably because they are surrounded by extracellular layers such as cell wall, vitelline
envelope, or cuticle which act as diffusion barriers and make the rapid exchange of
fixatives, solvents and resins difficult. We believe that it is important to work out
optimum methods of preserving ultrastructure in these organisms in order to characterize
mutant phenotypes and to improve immunolabelling at the EM level. We will present
evidence that high pressure freezing is probably the fixation method of choice for these
organisms (Figs. 1 & 2).
References
1. Simpson, W.L. Anat. Rec. 80 (1941) 205.
2. Robards, A. W., 8z Sleytr, U. B. Practical Methods in Electron Microscopy”
(Ed. A. M. Glauert), Vol.
10, Elsevier, Amsterdam (1985).
3. Moor, H. “Cryotechniques in Biological Electron Microscopy” (edited by R. A. Steinbrecht and K.
Zierold), p. 175. Springer-Verlag, Berlin (1987).
4. Studer, D. et al., Scanning Microscopy,
S3 (1989) 253.
5. McDonald, K. Meth. Cell Biol. 44 (1994) 441.
6. Steinbrecht, R.A., & Miiller, M. “Cryotechniques in Biological Electron Microscopy. ” (Eds. R.A.
Steinbrecht, and K. Zierold). p. 149. Springer-Verlag, Berlin (1987).
7. Hippe-SanwaId, S. Microsc. Res. Tech. 24 (1993) 400.
FIG. 1
S. cerevisiae cell prepared by high pressure freezing and freeze substitution.
FIG. 2 - Microvilli in the gut of C. e&am
substitution.
prepared by high pressure freezing and freeze