Properties of frozen sections relevant to quantitative microanalysis

Abstract

SUMMARY 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 μm frozen‐hydrated sections by the continuum method, with spatial resolution of the order of 1 μm, 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.

X‐ray microanalysis of freeze‐dried and frozen‐hydrated cryosections

“…Similar observations were reported by Wendt-Gallitelli and Wolburg (1984). Hall (1986b) ascribes this effect to inappropriate correction for extraneous background. Nevertheless, the size of the ice crystal segregation zones has to be considered as a possible source of error in the evaluation of X-ray microanalytical data.…”

Radiation damage in dry and frozen hydrated organic material

“…For example, the carbon in carrageenan was lost with a rate given by 0, = 50e/A' (5 x lo3 e/nm') at 300K but 0, = 670e/A2 (6.7 x lo4 elm') at 100 K. For the nitrogen in nitrocellulose, 0, was 14.4 e/A2 (140 e/nm') at 300K and 17e/A2 (1700e/nm2) at 100K. Similar effects were seen by X-ray spectroscopy (Cantino et al, 1986;Hall, 1986). Other laboratories (Isaacson et al, 1978;Leapman, 1982;Vesely, 1984) have shown a reduction in fluorine concentrations due to electron irradiation, but more recent studies have discovered some more stable fluorine compounds (Ciliax et al, 1990).…”

Quantitative X‐ray microanalysis of biological specimens

“…Although much of the quantitative techniques in biological X-ray microanalysis were established during the seventies, the field still is in an active state of development. Areas in which progress is being made or expected are mainly a) more accurate determination of local mass either with the use of the corrected continuum signal or by alternative techniques (Hall, 1986), b) quantitative analysis of light (Z < 11) elements, and c) determination of the local water content. In addition, increased computer power has opened possibilities for fully quantitative mapping, where quantitative analysis is carried out on each pixel of the X-ray map (see, e.g., Fiori, 1986; Heyman and Saubermann, 1987; Saubermann and Heyman, 1987;Somlyo et al, 1986).…”