Cryofixation

Muiier M and Moor H 1984 Cryofixation of thick specimens by high pressure freezing The Science of Bioiogicai Specimen Preparation ed J-P Revei, T Barnard and G H Haggis (O Hare, iL SEM, AMF 60666) pp 131-8... 

Figure 5.14 Freeze-etch electron micrograph of glycolipid nanotubes from 24 1 nonhydroxy galactocerebrosides (21) cryofixed from room temperature in water. Bar = 250 nm. Reprinted from Ref. 64 with permission of the Biophysical Society.

Richter K. Aspects of cryofixation and cryosectioning for the observation of hulk biological samples in the hydrated state by cryoelectron. Scanning Microsc Suppl 1996 10 375-386. 

Ding B, Turgeon R, Parthasarathy MV. Routine cryofixation of plant tissue by propane jet freezing for freeze substitution. J Electron Microsc Technol 1991 19 107-117. 

Hodson MJ, Sangster AG. Techniques for the microanalysis of higher plants with particular reference to silicon in cryofixed wheat tissues. Scanning Microsc 1990 4 407-408. 

Saga K (2005) Application of cryofixation and cryoultramicrotomy for biological electron micros copy. Med Mol Morphol 38 155 160... 

Hanyu, Y., Ichikawa, M., and Matsumoto, G. 1992. An improved cryofixation method Cryoquenching of small tissue blocks during microwave irradiation. J. Microsc. 165 255-271. Harnish, D. C., Scicchitano, M. S., Steven, J., Adelman, C., Lyttle, R., and Karathanasis, S. K. 2000. The role of CBP in estrogen receptor cross-talk with nuclear factor-kB in hepG2 cells. 

Freudenrich CC, Hockett D, Ingram P, LeFurgey A. In situ cryofixation of kidney for electron probe X-ray microanalysis. J. Struct. Biol. 1994 112 173-182. 

Cryofixation is done by the immersion of tissues in ice-cold isopentane for 3 min followed by freezing at -80°C. Fixed, frozen tumors are mounted in Tissue-Tek OCT 4583 compound (Sakura Finetek, Torrance, CA) and sectioned on a Microtome Plus (TBS). 

In order to use electron microscopy to visualise the microemulsion structure, the problem of the fixation of the liquid mixtures has to be solved. The method of choice is to solidify the microemulsion structure via cryofixation. However, given that the phase behaviour as well as the curvature of the amphiphilic film (see Fig. 1.18) and with it the microstructure of most micro emulsions show a strong temperature-dependence it has to be ensured that the cooling rate should be as high (>104 K/s) and the reorganisation kinetics of the microstructure as slow as possible. 

J. R. Bellare, H. T. Davis, L. E. Scriven, and Y. Talmon, An improved controlled-environment vitrification system (CEVS) for cryofixation of hydrated TEM samples, Proc. Xlth Int. Cong, on Electron Microscopy, Kyoto, Japan, Aug. 31-Sept. 7, 1986 J. Electron Microsc. 35 (Suppl. 

M. Muller and H. Moore, Cryofixation of suspensions and tissues by propane-jet freezing and high-pressure freezing, Proc. 42nd Annual Meeting, Microscopy Society of America (G. W. Bailey, ed.), San Francisco Press, San Francisco, 1984. 

Bachmann, L. and Schmitt. W. W. (1971). Improved cryofixation applicable to freeze-etching. Proc. Natl. Acad. Sci. U.S.A. 68,2149-2152. 

Switkes, M., Ruberti, J.W. Rapid cryofixation/freeze fracture for the study of nanobubbles at solid-liquid interfaces. Appl. Phys. Lett. 84, 4759 (2004)... 

Figure 2 The effects of specimen preparation on structure and element content of biological tissues. (A) Conventionally prepared section of heart muscle. Individual cells, their nuclei, the mitochondria, the Z-lines, and the myofilaments are clearly seen. (B) A heart muscle cell prepared by cryofixation and cryosectioning. Although the cellular structures are the same as in (A), the details are not as distinct. (C) A spectrum derived from material as in (A). The elements that can be Identified are Cu from the grid and Os, Pb, and U from the stain. The only element that can be detected from the specimen itself is S. (D) A spectrum derived from cryoprepared heart tissue. Here the major inorganic elements - Na, Mg, P, S, Cl, and K - are detected.

When investigating diffusible ions it is necessary to consider that treatment of the specimen before cryo-fixation may affect the results. For example, dicing tissue into small pieces before cryofixation is not suitable for highly metabolically active tissues such as the heart. It has been shown that in this tissue redistribution of ions can occur in times as short as 10 s. Similarly, in other tissues any delay between harvesting and fixation gives the possibility of element redistribution. In addition it has been shown that treatments such as the use of local anesthetics when taking skin biopsy samples can also affect elemental content. 

Apart from the minor changes to the above procedure to compensate for different types of tissue, a significant change to fixation will normally be necessary to accommodate immunohistochemical techniques, since antigens may be destroyed by the fixation. This may be a change to 0.5% glutaralde-hyde in paraformaldehyde and no postfixation in osmium, to removal of any conventional fixation chemical. In this case cryofixation is the solution, but subsequent sectioning is somewhat more involved. 

What type of sample collection method are you planning on using, i.e. distillation, direct purge-and-cryofixation ... 

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