Properties of frozen sections relevant to quantitative microanalysis

Difficulties in the quantitative X-ray microanalysis of frozen sections may conceivably arise from ice-crystal damage and from electron-beam damage. X-ray peak-to-continuum ratios are commonly taken as a quantitative index of elemental concentrations. But recent reports suggest that in dehydrated frozen sections such ratios vary greatly with the scale of ice-crystal formation existing prior to sublimation. The experiments in these reports are re-interpreted here; it is argued that peak intensities may be affected by ice-crystal scale but that ratios of peak to continuum should not be affected after corrections for exogenous continuum. The accuracy of the peak-to-continuum method is affected by beam-induced loss of mass from microvolumes during analysis. Mass loss can be reduced or slowed by a cold-stage. For example, the radiation sensitivity for loss of chlorine from PVC is reduced by a factor of 1000 or more with reduction of temperature from 300 to 100 K. For sections of soft tissue the effectiveness of cooling is not nearly so striking but at 100 K, analyses of 1 micron frozen-hydrated sections by the continuum method, with spatial resolution of the order of 1 micron, can be completed before substantial mass loss occurs. However, analysis of frozen-hydrated sections by the continuum method at much higher resolution, say 100 nm resolution in 100 nm sections, is precluded by mass loss. Measurements of local mass can be achieved with much lower dose by observation and calibration of the electron transmission or backscattering. But even with these methods, several problems remain in achieving quantitative X-ray analysis at very high resolution.