The X-ray microanalysis of frozen-hydrated sections in scanning electron microscopy: An evaluation |
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Authors: | Brij L. Gupta Theodore A. Hall |
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Affiliation: | Department of Zoology, Biological Microprobe Laboratory, Cambridge University, Downing Street, Cambridge CB2 3EJ, UK |
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Abstract: | The present status of the technique to measure concentrations of electrolyte elements and dry mass in 1 μm thick frozen-hydrated sections of soft biological tissues with electron probe X-ray microanalysis in a scanning electron microscope is critically reviewed. The technique is to quench-freeze fresh specimens to < − 180°C, cut 1 μm thick hydrated cryosections −70°C), transfer on to a cold stage (< −170°C) of a suitable microanalytical arrangement, obtain scanning transmission images to identify the cell and tissue compartments, locate an electron probe (several μm2 to 100 nm) on the areas of interest and collect X-ray quanta. The X-ray quanta are collected with suitable spectrometers (WDS and EDS) and processed with a computer using a comprehensive programme based on continuum normalization procedures (‘Hall’ programme). The cryosections are analysed first in a hydrated state and second after dehydration within the microanalyser column to obtain directly elemental concentrations in mM kg−1 wet wt and mM kg−1 dry wt of the compartments identified under the beam. The local water-fractions are estimated and the elemental concentrations converted into mM 1−1 water. In the past 7 years the technique has been applied to obtain fully quantitative information on Na, K, Cl, P, S, Ca and H2O in more than ten types of tissue. |
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Keywords: | Address for correspondence: Dr Brij L. Gupta Zoology Department Downing Street Cambridge CB2 3EJ UK. |
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