Preparation of cultured and isolated cells for X-ray microanalysis.
Various electron microscopical preparation techniques are compared with regard to the preservation of the intracellular element distribution as determined by X-ray microanalysis in scanning and scanning transmission electron microscopy. By use of chemical agents for fixation and dehydration ions are redistributed and washed out. This is also true for freeze-substitution. Whole cells are prepared by cryofixation followed by freeze-drying. Interference of intracellular measurements by extracellular elements can be avoided by appropriate washing the cells before cryofixation. The washing medium has to be carefully selected in order to avoid distortions of the original intracellular element content. These problems are circumvented by the preparation of freeze-dried cryosections from cryofixed cells. This is demonstrated by data of the intracellular elemental composition in cultured cells (fibroblasts, Staphylococcus aureus bacteria) and in cells isolated from rat tissue (kidney papillary collecting duct and liver). Cryofixation of a single cell in a defined functional state is illustrated by results obtained from streaming Amoeba proteus cells, cryofixed under light microscopical control. The main conclusion is that X-ray microanalysis of cells in functional states requires cryofixation and cryopreparation techniques which have to be adapted to the particular cell biological problem to be investigated.
Zierold
K
K
Max-Planck-Institut für Systemphysiologie, Dortmund, FRG.
Schäfer
D
D
D000656:Amoeba / Q000032:analysis*; D000818:Animals; D002478:Cells, Cultured; D004577:Electron Probe Microanalysis / Q000379:methods*; D005347:Fibroblasts / Q000032:analysis*; D005612:Freeze Drying; D005615:Freezing; D007668:Kidney / Q000032:analysis / Q000166:cytology; D008099:Liver / Q000032:analysis / Q000166:cytology; D008855:Microscopy, Electron, Scanning; D011309:Preservation, Biological; D051381:Rats; D013211:Staphylococcus aureus / Q000032:analysis*