A pre-cryogenic holder (cryo-holder) facilitating cryo-specimen observation under a conventional scanning electron microscope (SEM) is described. This cryo-holder includes a specimen-holding unit (the stub) and a cryogenic energy-storing unit (a composite of three cylinders assembled with a screw). After cooling, the cryo-holder can continue supplying cryogenic energy to extend the observation time for the specimen in a conventional SEM. Moreover, the cryogenic energy-storing unit could retain appropriate liquid nitrogen that can evaporate to prevent frost deposition on the surface of the specimen. This device is proved feasible for various tissues and cells, and can be applied to the fields of both biology and material science. We have employed this novel cryo-holder for observation of yeast cells, trichome, and epidermal cells in the leaf of Arabidopsis thaliana, compound eyes of insects, red blood cells, filiform papillae on the surface of rat tongue, agar medium, water molecules, penicillium, etc. All results suggested that the newly designed cryo-holder is applicable for cryo-specimen observation under a conventional SEM without cooling system. Most importantly, the design of this cryo-holder is simple and easy to operate and could adapt a conventional SEM to a plain type cryo-SEM affordable for most laboratories.
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