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Microwave-Assisted Freeze Substitution

Microwave with cryo-platform
Microwave with cryo-platform. Credit: NIAID

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.

 
Transmission electron microscope images of Bacillus subtilis
Transmission electron microscope images of Bacillus subtilis, using ambient and cryo-preparative methods. Mid-log phase cultures were prepared by conventional chemical fixation (A), microwave-assisted chemical fixation (B), traditional freeze substitution (C), or by rapid freeze substitution without (D) or with microwave irradiation (E-F). Inserts show detailed cell-wall structure at a magnification equivalent to that in panel D. Samples processed rapidly by freeze substitution without microwave irradiation exhibited poor preservation (D). Samples processed by microwave-assisted freeze substitution exhibited structures such as membranes, ribosomes, and cell-wall material that were obscured or poorly discernible by other techniques. Bars: 50 nm. Credit: NIAID
 

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.

References

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.

Last Updated August 16, 2013

Last Reviewed August 16, 2013