K E Y WORDS. X-ray microanalysis, cryosectioning, X-ray imaging. S U M M A R YApplication of quantitative X-ray imaging to frozen hydrated tissue sections has presented a number of major problems including lack of a suitable algorithm which could deal effectively with mass loss due to radiation damage, problems of low characteristic X-ray signal to background ratios, and provide a means of analysis of the same location in both hydrated and dried states. This paper presents details of the application of our algorithm for analysis of frozen hydrated, then dried cryosections applied to quantitative X-ray imaging, which provides relatively high precision quantitative measurement of elemental content (related to both wet and dry weight) and water content of each pixel. This algorithm largely circumvents many of the problems of analysis of frozen hydrated tissue sections. Our algorithm for X-ray imaging obtains reasonably precise quantitative measurements coupled with morphological information by trading speed and image resolution.

A series of computer programs have been written for use in a multi-user resource center for biological microanalysis. They are tied together by a "Main Menu" program which acts as a traflic director and guides the investigator into whatever option is desired. These programs have been written with very clear instructions and interaction points. As a result, a complex handbook of options and responses is not required. Consquently, almost no training is required to use these programs as they are essentially self-explanatory. Extensive error checking has been included so that most errors are identified and corrections are requested without causing a program halt. Options available to the user provide for selected "region" analyses, elementally quantitative image analysis, and sorting of the resulting data files. A complete computer code printout is included in this report.

Biological samples pose problems for analysis with electron probe microanalysis (EPMA), since they have considerable differences in element composition depending on compartment and physiological state. Further problems exist because the compartment may be difficult to identify with certainty when cryo-preparation methods are used. It is therefore often necessary to collect many analysis points to provide a suitable sampling of a particular compartment from many specimens. Rejection of spectra because the values appear to be outliers may possibly introduce bias or risk rejection of significant data. Application of quantitative digital x-ray mapping overcomes many of these problems. Digital computers can be used to divide a sample region into many small units, collect x-ray counts at each "point," store these results, and reassemble this data into a compositional map at a later time. When enough analysis time is allowed for each point, and appropriate standards are used, the resulting data becomes quantitative.

Until quite recently, electron microprobe analysis techniques were limited to samples of “infinite” thickness, that is, to samples thick enough such that the entire excitation volume was contained within the material of interest. Thin film analysis was not possible with available matrix correction programs, which were based on the assumption of samples of “infinite” thickness. Now however, algorithms are available that permit analysis of thin samples.We have obtained one of the more versatile and sophisticated of these programs. In order to investigate the accuracy of this routine we have analyzed several BiSrCaCuO thin films at 15 kV and repeated the analysis at 30 kV. These films were thick enough such that at 15 kV conventional ZAF data reduction yielded acceptable totals (98-101 %) with minimal substrate x rays observed. At 30 kV, however abundant substrate x rays were observed and ZAF yielded very low totals. X-ray intensity ratios from 30 kV runs were used to estimate film thickness and matrix corrections were applied using the Waldo algorithm.

Application of quantitative x-ray imaging to frozen hydrated (FH) tissue sections has presented a number of major problems including lack of a suitable algorithm which could deal effectively with hydrated specimen mass loss from radiation damage, low characteristic x-ray signal to background signal, and lack of a suitable method which would permit analysis and comparison of the same cryosection in both hydrated and dried state. Our algorithm, using hydrated then dried cryosections, eliminates the major problems associated with analysis of FH tissue sections. This algorithm is suitable for application to quantitative x-ray imaging and will provide quantitative elemental images in both wet and dry weight units as well as quantitative images of water fractions.