Low-temperature specimen preparation apparatus and techniques for low-temperature scanning electron microscopy (LTSEM) and synchrotron soft x-ray microscopy

Abstract

We have developed equipment and techniques for low-temperature scanning electron microscope (LTSEM) and x-ray microscope examinations of animal tissue samples. A cryoprobe for rapidly freezing tissue in vivo, a low-temperature saw for cutting large blocks of frozen tissue without warming, cardboard containers for sample storage in liquid nitrogen (LN2), tools for holding and manipulating frozen specimens, and an under-LN2 specimen viewer are described here. An LTSEM specimen holder for small blocks of tissue and a low-temperature trim saw for precisely cutting such blocks are described elsewhere. We have found such equipment, along with various commercial and specially fabricated dewars, to greatly facilitate our LTSEM experiments.To minimize ice crystal size by freezing as rapidly as possible and to maintain tissue in as close to normal physiologic state as possible during freezing, we use a cryoprobe for freezing 1 cm2 surfaces of tissue in vivo. The cryoprobe (Fig. 1) is a highly-polished 1 mm thick, 10 mm diam.