SpringerProtocols: Abstract: Recent Advances in High-Pressure Freezing: Equipment- and Specimen-Loading Methods

Recent Advances in High-Pressure Freezing: Equipment- and Specimen-Loading Methods

By: Kent L. McDonald², Mary Morphew³, Paul Verkade⁴, Thomas Müller-Reichert⁴

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This chapter is an update of material first published by McDonald in the first volume of this book. Here, we discuss the improvements in the technology and the methodology of high-pressure freezing (HPF) since that article was published. First, we cover the latest innovation in HPF, the Leica EM PACT2. This machine differs significantly from the BAL-TEC HPM 010 high-pressure freezer, which was the main subject of the former chapter. The EM PACT2 is a smaller, portable machine and has an optional attachment, the Rapid Transfer System (RTS). This RTS permits easy and reproducible loading of the sample and allows one to do correlative light and electron microscopy with high time resolution. We also place more emphasis in this article on the details of specimen loading for HPF, which is considered the most critical phase of the whole process. Detailed procedures are described for how to high-pressure freeze cells in suspension, cells attached to substrates, tissue samples, or whole organisms smaller than 300 μm, and tissues or organisms greater than 300 μm in size. We finish the article with a brief discussion of freeze substitution and recommend some sample protocols for this procedure.

Affiliation(s): (2) Electron Microscope Laboratory, University of California, Berkeley, CA
(3) The Boulder Laboratory For 3-D Electron Microscopy of Cells, University of Colorado, Boulder, CO
(4) Electron Miscroscope Facility, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

Book Title: Electron Microscopy: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 369 | Pub. Date: Feb-27-2007 | Page Range: 143-173 | DOI: 10.1007/978-1-59745-294-6_8

Subject: Biochemistry

Key Words: High-pressure freezing - high-pressure freezer - freeze substitution - cryofixation - EM specimen preparation - cryoEM - electron tomography - vitreous cryosections - correlative light - electron microscopy

References

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1.	Moor, H. (1987) Theory and practice of high-pressure freezing, in Cryotechniques in Biological Electron Microscopy (Steinbrecht, R. A. and Zierold, K., eds.), Springer-Verlag, Berlin, pp. 175–191.

2.	McDonald, K. L. (1999) High pressure freezing for preservation of high resolution fine structure and antigenicity for immunolabeling, in Electron Microscopy Methods and Protocols, 1st ed (Hajibagheri, M. A. N., ed.), Humana Press, Totowa, NJ, pp. 77–97.

3.	Studer, D., Graber, W., Al-Amoudi, A., and Eggli, P. (2001) A new approach for cryofixation by high-pressure freezing. J. Microsc. (Oxford) 203, 285–294.

4.	Moor, H. and Riehle, U. (1968) Snap-freezing under high pressure: a new fixation technique for freeze-etching. Proc. 4th Eur. Elect. Microsc. 2, 33–34.

5.	Steinbrecht, R. A. and Müller, M. (1987) Freeze substitution and freeze drying, in CryoTechniques in Biological Electron Microscopy (Steinbrecht, R. A. and Zierold, K., eds.), Springer-Verlag, Berlin, pp. 149–172.

6.	Kiss, J. Z. and Staehelin, L. A. (1995) High pressure freezing, in Techniques in Modern Biomedical Microscopy; Rapid freezing, freeze fracture, and deep etching (Severs, N. J. and Shotton, D. M., eds.), Wiley-Liss, New York, pp. 89–104.

7.	McDonald, K. (1994) Electron microscopy and EM immunocytochemistry, in Drosophila melanogaster: Practical Uses in Cell and Molecular Biology (Goldstein, L. S. B. and Fyrberg, E., eds.), Methods in Cell Biology 44, Academic Press, San Diego, pp. 411–444.

8.	McDonald, K. L., Sharp, D. J., and Rickoll, W. (2000) Preparing thin sections of Drosophila for examination in the transmission electron microscope, in Drosophila: A Laboratory Manual, 2nd ed. (Sullivan, W., Ashburner, M., and Hawley, S., eds.), CSHL Press, Cold Spring Harbor, NY, pp. 245–271.

9.	Kaneko, Y. and Walther, P. (1995) Comparison of ultrastructure of germinating pea leaves prepared by high-pressure freezing-freeze substitution and conventional chemical fixation. J. Electron Microsc. 44, 104–109.

10.	Murk, J. L., Posthuma, G., Koster, A. J., et al. (2003) Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography. J. Microsc. (Oxford) 212, 81–90.

11.	Small, J. V. (1981) Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks. J. Cell Biol. 91, 695–205.

12.	Plattner, H. and Bachmann, L. (1982) Cryofixation: A tool in biological ultrastructural research. Int. Rev. Cytol. 79, 237–304.

13.	Gilkey, J. C. and Staehelin, L. A. (1986) Advances in ultrarapid freezing for the preservation of cellular ultrastructure. J. Electron Microsc. Tech. 3, 177–210.

14.	Studer, D., Michel, M., Wohlwend, M., Hunziker, E. B., and Buschmann, M. D. (1995) Vitrification of articular cartilage by high-pressure freezing. J. Microsc. (Oxford) 179, 321–332.

15.	Manninen, A., Verkade, P., LeLay, S., et al. (2005) Caveolin-1 is not essential for biosynthetic apical membrane transport. Molec. Cell. Biol. 25, 10087–10096.

16.	Walther, P. (2003) Recent progress in freeze-fracturing of high-pressure frozen samples. J. Microsc. (Oxford) 212, 34–43.

17.	Shimoni, E. and Müller, M. (1998) On optimizing high-pressure freezing: from heat transfer theory to a new microbiopsy device. J. Microsc. (Oxford) 192, 236–247.

18.	Al-Amoudi, A., Chang, J. J., Leforestier, A., et al. (2004) Cryoelectron microscopy of vitreous sections. EMBO J. 23, 3583–3588.

19.	Zhang, P., Bos, E., Heymann, J., Gnaegi, H., Kessel, M., Peters, P. J., and Subramaniam, S. (2004) Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr. J. Microsc. (Oxford) 216, 76–83.

20.	Craig, S., Gilkey, F. C., and Stähelin, L. A. (1987) Improved specimen support cups and auxiliary devices for the Balzers high pressure freezing apparatus. J. Microsc. (Oxford) 148, 103–106.

21.	Hess, M. W., Müller, M., Debbage, P. L., Vetterlein, M., and Pavelka, M. (2000) Cryopreparation provides new insight into the effects of Brefeldin A on the structure of the HepG2 golgi apparatus. J. Struct. Biol. 130, 63–72.

22.	Sawaguchi, A., Yao, X., Forte, J. G., and McDonald, K. L. (2003) Direct attachment of cell suspensions to high-pressure freezing specimen planchettes. J. Microsc. (Oxford) 212, 13–20.

23.	Dahl, R. and Staehelin, L. A. (1989) High-pressure freezing for the preservation of biological structure: theory and practice. J. Electron Microsc. Tech. 13, 165–174.

24.	Robards, A. W. and Sleytr, U. B. (1985) Low temperature methods in biological electron microscopy, in Practical Methods in Electron Microscopy, vol. 10 (Glauert, A. M., ed.), Elsevier, Amsterdam.

25.	Hohenberg, H., Mannweiler, K., and Müller, M. (1994) High-pressure freezing of cell suspensions in cellulose capillary tubes. J. Microsc. (Oxford) 175, 34–43.

26.	O’Toole, E. T., McDonald, K. L., Mäntler, J., et al. (2003) Morphologically distinct microtubule ends in the mitotic centrosome of Caenorhabditis elegans. J. Cell Biol. 163, 451–456.

27.	McDonald, K. and Müller-Reichert, T. (2002) Cryomethods for thin section electron microscopy, in Guide to Yeast Genetics and Molecular and Cell Biology, Parts B and C (Guthrie, G. and Fink, G. R., eds.), Meth. Enzymol. 351, Academic Press, San Diego, pp. 96–123. <Occurrence Type="DOI"><Handle>10.1016/S0076-6879(02)51843-7</Handle></Occurrence>

28.	Lonsdale, J. E., McDonald, K. L., and Jones, R. L. (1999) High pressure freezing and freeze substitution reveal new aspects of fine structure and maintain protein antigenicity in barley aleurone cells. Plant J. 17, 221–229.

29.	Samuels, A. L., Giddings, T. H. Jr., and Staehelin, L. A. (1995) Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants. J. Cell Biol. 130, 1345–1357.

30.	Reipert, S., Fischer, I., and Wiche, G. (2004) High-pressure freezing of epithelial cells on sapphire coverslips. J. Microsc. (Oxford) 213, 81–85.

31.	Morphew, M. and McIntosh, J. R. (2003) The use of filter membranes for high-pressure freezing of cell monolayers. J. Microsc. (Oxford) 212, 21–25.

32.	Schlegel, A., Giddings, T. H., Ladinsky, M. S., and Kirkegaard, K. (1996) Cellular origin and ultrastructure of membranes induced during poliovirus infection. J. Virol. 70, 6576–6588.

33.	Müller-Reichert, T., O’Toole, E. T., Hohenberg, H., and McDonald, K. L. (2003) Cryoimmobiliztion and three-dimensional visualization of C. elegans ultrastructure. J. Microsc. (Oxford) 212, 71–80.

34.	Oegema, K., Desai, A., Rybina, S., Kirkham, M., and Hyman, A. A. (2001) Functional analysis of kinetochore assembly in Caenorhabditis elegans. J. Cell Biol. 153, 1209–1226.

35.	Hohenberg, H., Tobler, M., and Müller, M. (1996) High pressure freezing of tissue obtained by fine needle biopsy. J. Microsc. (Oxford) 183, 133–139.

36.	Monaghan, P., Perusinghe, N., and Müller, M. (1998) High-pressure freezing for immunocytochemistry. J. Microsc. (Oxford) 192, 248–258.

37.	Giddings, T. H. (2003) Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples. J. Microsc. (Oxford) 212, 53–61.

38.	Hess, M. W. (2003) On plants and other pets: practical aspects of freeze substitution and resin embedding. J. Microsc. (Oxford) 212, 44–52.

39.	Rensing, K. H. and Samuels, A. L. (2004) Cellular changes associated with rest and quiescence in winter-dormant vascular cambium of Pinus contorta. Trees 18, 373–380.

40.	Segui-Simmaro, J. M., Austin II, J. R., White, E. A., and Staehelin, L. A. (2004) Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by high-pressure freezing. Plant Cell 16, 836–856.

41.	Sabatini, D. D., Bensch, K., and Barnett, R. J. (1963) Cytochemistry and electron microscopy. The preservation of cellular structures and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19–58.

42.	Koster, A. J. and Klumperman, J. (2003) Electron microscopy in cell biology: integrating structure and function. Nat. Rev. Mol. Cell Biol. Suppl, SS6–SS10.

43.	McIntosh, J. R., Nicastro, D., and Mastronarde, D. (2005) New views of cells in 3-D: an introduction to electron tomography. Trends Cell Biol. 15, 43–51.

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