Microwave-Assisted Freeze Substitution of Biological and Biomedical Samples (See Video)
Cryo Microwave Sample Processor with the TotalTemp Thermal Platform System specially engineered to operate inside of the Ted Pella BioWave Microwave Oven
Freeze substitution fixation (FS) of hydrated samples frozen in vitreous ice provides exceptional preservation of structure for light and electron microscopy, and enables immunological detection of thermo-labile antigens that otherwise are damaged/destroyed by processing at ambient or elevated temperatures. Its use as a research tool or in clinical pathology has, however, been limited by the relatively lengthy periods required for passive diffusion of fixatives and organic solvents into the frozen hydrated material.
The invention utilizes controlled microwave (MW) irradiation to accelerate the FS process; and comprises systems, devices and methods for microwave-assisted processing of samples under cryo-conditions, typically -80°C. The entire MWFS procedure is now accomplished in less than 4 hours as compared to the approximately 4-5 days previously required for FS process.
The Cryo Microwave Sample Processor System combines two specific technologies, controlled microwave irradiation and temperature cycling. The controlled temperature cycling device, a Thermal Platform (a.k.a. Hot/Cold Plate) was specially engineered by TotalTemp using some alternative nonmetallic materials to allow it to operate inside of a microwave environment. The Thermal Platform is cooled with Liquid Nitrogen (L-N2) giving it a precisely controlled temperature range of -100°C to +200°C.
The microwave oven was provided to TotalTemp by the customer to be modified for the installation of a bulkhead interface plate to allow the input of AC power, L-N2 and a nitrogen gas purge to prevent excessive condensation inside the oven while running the platform at cryo temperatures. The nitrogen gas purge is directed inside of a clear polycarbonate cover that rests over the platform surface. TotalTemp machined a custom designed adapter plate with recessed pockets to support vials and petri dishes for thermal conduction during the FS process. The Microwave Oven is a Ted Pella Biowave Model 3451 which has special Magnetron for continuous power from 80-750W, not pulsing power like a standard kitchen oven.
TotalTemp Thermal Platform inside of oven showing custom Adapter Plate designed to support specific vials and Petri dishes for tissue sample preparation
Time Comparison Chart of sample preparation while under microwave irradiation (red line) vs. sample preparation without microwave irradiation (purple line). Note the tremendous time difference on the “Minutes” scale.
Images of Bacillus Subtilis, using ambient and cryo-preparative methods
Mid-log phase cultures were prepared by conventional chemical fixation (A), MW-assisted chemical fixation (B), traditional FS (C), or by rapid FS without (D) or with MW irradiation (E-F). Inserts show detailed cell wall structure at a magnification equivalent to that in panel D. Samples processed rapidly by FS without MW irradiation exhibited poor preservation (D). Samples processed by MWFS exhibited structures such as membranes, ribosomes, and cell wall material that were obscured or poorly discernible by other techniques. Bars: 50 nm.