The use of frozen-hydrated bulk specimens for x-ray microanalysis

The recent development of versatile instrumentation has removed a serious obstacle for the use of frozen-hydrated bulk specimens in studies of biological problems. Methods for freezing, fracturing, mounting, etching, and coating are now known and offer a wide spectrum of technical possibilities to choose from for a particular application. However, progress in biological studies is still limited by the problems encountered during the analysis. In order to keep the X-ray source volume commensurate with the size of cells, low accelerating voltages have to be used. Poor peak to background ratios are, thereby, obtained and the sensitivity of the method is deteriorated. Superficial freeze-drying improves the peak to background ratios, but it is no expedient because of the difficulty in determining the degree of dehydration. The most serious problem is specimen charging which occurs even after coating. The degree of charging is not uniform, neither within the sample nor between specimen and standard. Therefore, the determination of relative concentrations becomes uncertain.