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High‐resolution low‐temperature scanning electron microscopy for observing intracellular structures of quick frozen biological specimens
Author(s) -
Inoué Takao,
Koike Hirotami
Publication year - 1989
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1989.tb02913.x
Subject(s) - liquid nitrogen , endoplasmic reticulum , electron microscope , scanning electron microscope , cryofixation , intracellular , sublimation (psychology) , biophysics , chemistry , biological specimen , materials science , ultrastructure , anatomy , biology , biochemistry , composite material , optics , psychology , ecology , physics , organic chemistry , psychotherapist
SUMMARY Intracellular structures of rapidly frozen biological tissues were observed in 3‐D under a low‐temperature scanning electron microscope using a newly developed side‐entry type cryo‐holder. The present low‐temperature SEM is simple, easy to operate and effective for observing biological materials at high magnification. Biological tissues (the pancreas, small intestine, brown adipose tissue and Harderian gland) freshly removed from the mouse were immediately frozen in liquid propane cooled with liquid nitrogen, and their surfaces were manually fractured using a precooled razor blade in liquid nitrogen before introducing the cryo‐holder into the SEM. When intracellular structures were revealed after appropriate sublimation, the specimens were coated with gold using a metal evaporator fitted to the side of the microscope column at one of the specimen chamber ports. The cryo‐holder was connected to a copper braid coming from a liquid nitrogen reservoir to maintain a low temperature. Using this method, intracellular structures such as the mitochondria and endoplasmic reticulum were demonstrated at high magnifications. Ribosomal granules were discerned on the rough endoplasmic reticulum of the pancreatic acinar cells. Granular substances, presumably elementary particles, were also recognized on the mitochondrial cristae of the brown adipose tissue. The method was particularly effective for studying the 3‐D configuration of lipid droplets which had been difficult to preserve by chemical fixation.