Freeze substitution

Steinbrecht R A and Muiier M 1987 Freeze-substitution and freeze-drying Cryotechniques in Bioiogicai Eiectron Microscopy ed R A Steinbrecht and K Zieroid (Beriin Springer) pp 149-72... 

Lauchli, A., Spurr, A.R. Wittkop, R.W. (1970). Electron probe analysis of freeze substituted, epoxy resin embedded tissue for ion transport studies in plants. Planta, 95, 341-50. 

Van Zyl, J., Forrest, Q.G., Hocking, C. Pallaghy, C.K. (1976). Freeze-substitution of plant and animal tissue for the localisation of water-soluble compounds by electron probe microanalysis. Micron, 7, 213-24. 

Roos N. Freeze-substitution and other low temperature embedding methods, in Electron Microscopy in Biology—A Practical Approach (Harris JR, ed.), IRL Press, Oxford, UK, 1991, pp. 39-58. 

Deparaffinize with two xylene changes, 5 min each Use freeze-dried or freeze-substituted tissue can also use chemically fixed... 

Unfixed, freeze dried, freeze substituted, or chemically fixed Chemically fixed, use 15-25 im sections... 

Czymmek KJ, Bourett TM, Howard RJ. Immunolocalisation of tubulin and actin in thick-sectioned fungal hyphae after freeze-substitution fixation and methacrylate de-embedment. JMicrosc 1996 181 153-161. 

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. 

In a previous review of this topic (5), it was suggested that molecular distillation drying followed by resin embedding needed more work before it could be evaluated as a preparative technique for microanaly-tical work as there had only been one investigation that had used it (59). Unfortunately, as far as this author is aware, there has only been one more study (12) that has used the technique to prepare material for micro-analysis. It is possible that workers have been put off using it by the apparent fall from grace of the related freeze substitution technique or, more likely, by the expense of molecular distillation drying apparatus. 

Hajibagheri MA, Flowers TJ. Use of freeze-substitution and molecular distillation drying in the preparation of Dunaliella parva for ion localization studies by x-ray microanalysis. Microsc Res Technol 1993 24 395-399. 

Fixation by rapid freezing followed by either freeze substitution or cryosectioning can also overcome some of the problems of standard immersion fixation and resin embedding. These are more specialized techniques and will not be dealt with here. Discussion of the methods can be found in Polak and Varndell (6), Hayat (7), and Verkleij and Leunissen (8). 

Monaghan, P and Robertson, D (1990) Freeze-substitution without aldehyde or osmium fixatives ultrastructural and implication for immunocytochemistry. J Microsc 158, 355—363... 

Standard chemical fixation fails to preserve extracellular materials. In contrast, Os04-microwave heating or the high-pressure freezing-freeze-substitution technique is able to preserve such materials (Eggli and Graber, 1994). The former technique is simpler... 

Eggli, P. S., and Graber, W. 1994. Improved ultrastructural preservation of rat ciliary body after high pressure freezing and freeze substitution A perspective view based upon comparison with tissue processed according to a conventional protocol or by osmium tetroxide microwave fixation. Microsc. Res. Technol. 29 11-22. 

Craig, R., Padron, R., and Alamo, L. (1991). Direct determination of myosin filament symmetry in scallop striated adductor muscle by rapid freezing and freeze substitution./. Mol. Biol. 220, 125-132. 

B. A. I. van den Bergh, M. A. Salomons-de Vries and J. A. Bouwstra, Interactions between liposomes and stratum corneum studied by freeze substitution electron microscopy, Int. J. Pharm. 167 51-61 (1998). 

Y3. Yu, Y., Leng, C. G., Kato, Y., and Ohno, S., Ultrastructural study of glomerular capillary loops at different perfusion pressures as revealed by quick-freezing, freeze-substitution and conventional fixation methods. Nephron 76, 452-459 (1997). 

Menco B. P. Cunningham A. M., Qasba P., Levy N. and Reed R. R. (1997) Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse a freeze-substitution ultrastructural. J. Neuxocytol. 26, 691-706. 

Rather than being lyophilised cells may be freeze substituted (Pearse, 1953). After treating cells with isopentane at liquid nitrogen temperature they are flooded in several changes of absolute methanol at the temperature of solid C02 for 2-3 h. 

Kaeser, W., Freeze-substitution of plant tissues with a new medium containing dimethoxypropane, J. Microscopy, 154, 273-278, 1988. 

Figure 12.2. The structure of ice cream mix and ice cream. (A). Fat globules (F) in mix with crystalline fat within the globule and adsorbed casein micelles (C), as viewed by thin section transmission electron microscopy. (B). Close-up of an air bubble (A) with adsorbed fat, as viewed by low temperature scanning electron microscopy. (C). Air bubble (A) with adsorbed fat cluster (FC) that extends into the unfrozen phase, as viewed by thin section transmission electron microscopy with freeze substitution and low temperature embedding.

Fig. 3 shows SFM image of one of the sections (thickness of - 100 nm) of K562 cell embedded in epoxy resin. It should be noticed that topographical contrast and the identification of the K562 internal ultrastructure critically depend on the procedure of cell preparation before embedding (chemical fixation or high-pressure freezing and freeze-substitution). 

Fig. 7. The effect of adsorbed protein on structure of ice-cream mix, ice cream, and melted ice cream. A-B, ice-cream mix with no surfactant and with added surfactant, respectively, as viewed by thin-section transmission electron microscopy. f= fat globule, c = casein micelle, arrow = crystalline fat, bar = 0.5 pm. See Reference 24 for methodology. C-D, ice cream with no surfactant and with added surfactant, respectively, as viewed by low-temperature scanning electron microscopy, a = air bubble, f = fat globule, bar = 4 pm. See Reference 34 for methodology. E-F, ice cream with no surfactant and with added surfactant respectively, as viewed by thin-section transmission electron microscopy with freeze substitution and low-temperature embedding. a = air bubble, f= fat globule, c = casein micelle, fc = fat cluster, bar = 1 pm. See Reference 13 for methodology. G-H, melted ice cream with no surfactant and with added surfactant respectively, as viewed by thin-section transmission electron microscopy. f= fat globule, c = casein micelle, fn = fat network, bar = 1 pm in G and 5 pm in H. See Reference 24 for methodology.

Menco BP. 1984. Ciliated and microvillous structures of rat olfactory and nasal respiratory epithelia. A study using ultra-rapid cryo-fixation foUowed by freeze-substitution or freeze-etching. CeU Tissue Res 235 225-241. 

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