Preparation and transfer of ultrathin frozen-hydrated and freeze-dried cryosections for microanalysis in scanning transmission electron microscopy.
A cooling chain preparation technique is described which enables electron microscopy and X-ray microanalysis of biological cells and tissues on an ultrastructural level. Following this method the biological object is quickly frozen without any chemical pretreatment. Sections of about 100 nm thickness are cut at a temperature below 173 K in a cryoultramicrotome by means of a dry glass knife and then they are transferred under cold nitrogen gas atmosphere at 120 K in a transfer chamber to the electron microscope. After locking the specimen under vacuum into the cold stage of a scanning transmission electron microscope, analysis of structure and elemental distribution is done at 165 K. In frozen-hydrated sections morphological details are obscured by ice and characteristic X-ray peaks are overlapped by a high continuum radiation. After freeze-drying ultrastructure can be well imaged without any staining, and the peak-to-background ratio of the characteristic X-rays is much enhanced. The technique is illustrated by results of yeast cells, rat liver, guinea pig heart muscle and cultured mouse fibroblast cells. The results are discussed with respect to its present limitations and possibilities.
D000818:Animals; D002467:Cell Nucleus / Q000032:analysis; D002478:Cells, Cultured; D002713:Chlorine / Q000032:analysis; D003593:Cytoplasm / Q000032:analysis; D004577:Electron Probe Microanalysis / Q000379:methods*; D005612:Freeze Drying; D005629:Frozen Sections; D006168:Guinea Pigs; D008099:Liver / Q000648:ultrastructure; D051379:Mice; D008855:Microscopy, Electron, Scanning; D008867:Microtomy; D008928:Mitochondria / Q000032:analysis; D009206:Myocardium / Q000648:ultrastructure; D011188:Potassium / Q000032:analysis; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure; D013048:Specimen Handling / Q000379:methods*
D002713:Chlorine; D011188:Potassium