Freeze substitution is a preparative process that preserves frozen specimens for electron or light microscopy. It involves replacing vitreous ice in samples with one or more fixatives dissolved in an organic solvent to chemically cross-link reactive molecules as they are immobilized at the instant of freezing. Specimens processed by freeze substitution are considered significantly closer to native state when examined than those preserved in other forms.
Our unit has designed, developed, and deployed a cryo-thermal device for greatly accelerating freeze substitution processing of biomaterials for light and electron microscopy (figure above). The device, fabricated by Total Temp Technologies in San Diego, California, enables complete and highly reproducible freeze substitution with tightly regulated temperature control within our Pelco 3451 laboratory microwave processing oven. By balancing flow of liquid nitrogen coolant and internal electric heaters, the platform and sample temperature are set, maintained, and transitioned within a range of -100 and 100 degrees C, while simultaneously accelerating replacement of ice with organic medium and reactions of fixatives with the samples using microwave excitation. Using this system, protocols that require 48 to more than 100 hours to complete by passive diffusion methods are routinely performed in less than 90 minutes, with comparable or superior structural preservation (shown below). This enables freezing, fixing, embedding, sectioning, staining, and examining cryo-samples in one or two days, which previously would have required a week or more to complete.
Dorward DW, Nair V, Hansen BT, Fischer ER. Device and method for microwave assisted cryo-sample processing. Patent application publications: US 2011/0229928 A1, EP 2331930 A1
Rayavara K, Rajapandi T, Wollenberg K, Kabat J, Fischer ER, Desai SA. A complex of three related membrane proteins is conserved on malarial merozoites. Mol Biochem Parasitol. 167:135-43. 2009.