Improved Cryofixation Applicable to Freeze Etching

Abstract

Freeze etching of solute model systems (e.g., glycerol or ferritin solutions) demonstrates that cryofixation can introduce serious artifacts due to the segregation of the dissolved or dispersed material from the solvent. Since, in ARTIFACT PROBLEMS IN CRYOFIXATIONSince the introduction of freeze etching (2, 3), methods of adequate cryofixation for electronmicroscopic specimens have become of particular concern. In addition to offering an interesting alternative to chemical fixation for the study of cellular material, cryofixation provides practically the only method for the study of solute systems by electron microscopy.Investigations of frozen-fluid specimens most clearly demonstrate the limits of cryofixation. The commonly available cooling rates are too low to prevent the rearrangement of solutes or particles during freezing. The well-known appearance of freeze-etched glycerol solutions is just one example. When frozen by the standard technique (by dropping a small metal specimen holder with about 1 mm3 of solution into a liquid cryomedium) the glycerol and water separate into two different phases, i.e., compartments of water surrounded by a glycerol network (Fig. 3). For a given cooling rate, the size of the water compartments depends on the glycerol concentration and on the presence of other solute materials. Similar pictures are obtained when freeze-etching aqueous solutions of salts or sugar (4), organic solutions (e.g., benzoic acid in benzene) (5) Freeze-etching techniques for biological specimens use cryoprotectants that are mainly capable of preventing one very-gross form of segregation, namely, the formation of large ice crystals that cause cell damage. On the other hand, cryoprotectants might introduce artifacts of their own, either be segregation from the aqueous solution during freezing or by their toxic effects on the biological system. In investigations of solutions or colloidal systems, cryoprotectants are of no use at all. Therefore, several attempts have been made to increase the speed of freezing, which is known to improve the quality of a cryofixed electron-microscopic specimen (3,(6)(7)(8)(9) To overcome this difficulty, a spray-freezing method was developed, which can be applied to freeze-etching.Since this "spray-freeze-etch" method has previously been described only in a short note (11), the details of the procedure are given in this paper. THE TECHNIQUE OF SPRAY-FREEZE-ETCHING PrinciplesThe approach of the spray-freeze-etch technique is to increase the cooling rate by injection of very small droplets of the * It is recommended that different techniques should be evaluated by the use of simple model systems, such as glycerol solutions. Segregation patterns of solutions react very sensitively to changes in freezing conditions and are much easier to reproduce and interpret than freeze-etched cells.

A study of rapid mitochondrial structural changes in vitro by spray-freeze-etching

“…Samples were withdrawn from the incubation cell and spray-frozen (Bachmann and Schmitt [1], Bachmann and Schmitt-Fumian [2]) using apparatus constructed in this laboratory (Lang et al [16]). …”

“…The development of spray-freeze-etching by Bachmann and Schmitt (1) in 1971 suggested that it might be possible to examine changes in mitochondrial structure by spray-freezing without the use of fixatives or cryoprotectants. This new tech-nique also made it potentially possible to follow the time course of rapid changes in mitochondrial structure after an alteration in incubation conditions.…”

Action of Polymyxin B on Bacterial Membranes: Morphological Changes in the Cytoplasm and in the Outer Membrane of Salmonella typhimurium and Escherichia coli B

“…An additional structural change, the formation of blebs in the outer membrane of gram-negative bacteria (9,13,31,40), was reinvestigated by the application of spray-freezing and freeze-etching (1,2,24). Freeze-etching has been described for Escherichia coli B to give a relatively close approximation to the living state (5).…”

Microdomain Arrangement of the SERCA-type Ca2+ Pump (Ca2+-ATPase) in Subplasmalemmal Calcium Stores of Paramecium Cells

“…(a) Cells were either sandwiched between two thin copper sheets and vigorously injected into melting propane ( ‫ف‬ 123K) according to Gulik-Krzywicki and Costello (1978), or (b) cells were sprayed into melting propane according to Bachmann and Schmitt (1971) and Plattner et al (1972) using the quenched-flow set-up and the follow-up methodology described by Knoll et al (1991). Cells were then processed by freeze-fracturing and platinum/carbon replication in a Balzers BAF 300 unit.…”