Application of scanning electron microscopy to x-ray analysis of frozen-hydrated sections. II. Analysis of standard solutions and artificial electrolyte gradients.
New specimen handling and analytic techniques for the application of x-ray microanalysis to studies of cell and organ biology have been recently described (Saubermann et al., 1981, J. Cell Biol. 88:257-267). Based on these techniques, absolute quantitative standardization has been established through x-ray analysis of frozen-hydrated and then dried sections of independently measured standard solutions of elements. These experiments demonstrate that the specific techniques employed have a probable error of less than 10%. Artificial electrolyte gradients established in gelatin were subjected to analysis to determine whether there was elemental displacement under non-membrane-limited conditions at the temperatures employed for sectioning (-30 degrees to -40 degrees C). No significant difference was observed between such gradients in serial sections cut at -30 degrees and -80 degrees C. Similarly, no additional ice-crystal-damage artifact was found in sections cut at -30 degrees C when compared with sections cut at -80 degrees C. Thus, in terms of ice-crystal size, gradient maintenance, and compartmental differentiation, cryosectioning at -30 degrees to -40 degrees C was not associated with redistribution incompatible with 1- to 2-micrometers spatial resolution, and absolute measurements of elemental concentration were practical within regions of this size.
D002712:Chlorides / Q000032:analysis; D003460:Crystallization; D004573:Electrolytes / Q000032:analysis; D004577:Electron Probe Microanalysis / Q000379:methods*; D005629:Frozen Sections; D008855:Microscopy, Electron, Scanning; D008867:Microtomy; D011188:Potassium / Q000032:analysis; D012015:Reference Standards; D013048:Specimen Handling / Q000379:methods; D013455:Sulfur / Q000032:analysis
D002712:Chlorides; D004573:Electrolytes; D013455:Sulfur; D011188:Potassium