Quantitative electron probe microanalysis of biological thin sections: methods and validity.

Abstract A quantitative method of electron probe X-ray analysis of thin sections proposed by Hall [1] and based on the relationship between characteristic peak and X-ray continuum was evaluated in conjunction with a multiple least squares computer fitting program designed for the subtraction of peak from background counts and for the deconvolution of overlapping peaks. The theory of this quantitation is discussed including the effects of higher Z elements in normally low Z biological matrices. Estimates of the inherent statistical errors of the deconvolution program and the extent of systematic errors introduced by changes in detector resolution and energy calibration are given. The minimal detectable concentration of K in a typical biological thin section and using probe currents readily tolerated by the specimen over a 100 sec counting time were found to be approximately 10 mmol/kg. The minimal detectable mass in a 100 sec collection time using a thermionic gun was calculated to be approximately 10 −19 g in agreement with previous analysis of the iron core of single ferritin molecules [7]. The quantitation of Na and K in thin polymer sections is illustrated and the use of biological materials containing covalently bound elements as standards is described. Quantitation of P and Mg of such specimens gave results within 10% of chemical analysis. The effects of the transmission function of the detection system, ionization cross section and fluorescence yield on the X-ray counts generated by different elements are evaluated by analyzing binary standards and fitting the calculated transmission function to the X-ray continuum. There was excellent agreement between the experimentally determined continuum shape, the calculated transmission function and the results obtained with binary standards. The continuum followed the behavior expected of thin organic sections up to 1 ∥micron. Above this thickness the absorption of the low energy continuum within the specimen was observed. The effects of contamination and mass loss due to radiation damage were evaluated by measuring X-ray continuum and characteristic peaks as a function of electron dose. Contamination was proportional to probe current and inversely proportional to probe diameter. The mass loss due to radiation damage, as measured by X-ray continuum counts, was significantly different in serum albumin and in sucrose specimens. There was a 50% loss of sucrose mass at a dose of 0.03 C/cm 2 at room temperature. An Arrhenius plot of the process gave a thermal activation energy of 900 K. We interpret this finding to indicate that the mass loss is primarily due to vaporization or diffusion of small organic fragments. Serum albumin films showed 13% mass loss at doses up to 760 C/cm 2 . The sources of extraneous, other than the specimen, continuum were evaluated and characterized. It is shown that such contributions to the continuum can be recognized as originating from a thick target X-ray spectrum (specimen grid and holder, electron optical column) or by a low energy profile that is inconsistent with the X-ray transmission function of the beryllium window of the detector (scattered electron spectrum). Methods for correcting for such extraneous contributions to the continuum are given, and the optimum region (lowest error specimen continuum) for quantitation is evaluated. Examples of quantitative analysis of cryosections of human red blood cells, frog striated and rabbit smooth muscle are given and shown to be, within the limits of statistical error, in agreement with the results of chemical analyses. The total theoretically attainable improvement in minimal mass detection is estimated to be a factor of approximately 300, suggesting a detectable minimal mass of 10 −22 g. We conclude that quantitative analysis of ultrathin biological sections with a spatial resolution of at least 2000 A and an absolute accuracy of approximately 10% is feasible with the method described.