Scanning Electron Microscopy


Lumbricals from the hind feet of young rats are dissected free, stretched to approximately 125% of resting length, and mounted on individual, simple plastic forms. After recovery in physiological saline, the isolated muscles are incubated for periods of 40 to 60 min. in one of a series of hypertonic bathing solutions. The composition of each bathing solution is identical, except for osmolality which is increased with lactose. At least one muscle from each animal is incubated in a control solution to serve as a control muscle for that particular set of 5 to 8 muscles. Mounted muscles are removed from the bathing solutions and quickly plunged into chilled liquid propane. Tissue is freeze-dried at low temperature, fixed with osmium tetroxide vapor, and embedded in brominated EPON 826. Dilution of the embedding medium by tissue solids, assessed by reduction of the embedding plastic Br Lα signal, is used to establish intracellular hydration; or conversely, intracellular solids fractions. The Br Lα signal is monitored along with S Kα, K Kα, Cl Kα, Na Kα, and the continuum region from 4.2 to 7.2 keV using a simultaneous ED-WD spectrometer electron probe microanalyzer. This provides sufficient information to present intracellular concentrations in units of mmol/kg wet weight, mmol/kg water, or mmol/kg dry weight. Cell volume change relative to a suitable control is determined from the ratio of sulfur signals from the experimental and control cells. The response of cells to hypertonic challenge is assessed by comparing actual cell volume change with the change expected of a simple osmotic bag. Simultaneously, the intracellular electrolyte data provides information on the mechanism of cellular response to osmotic shifts.

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