Application of phi (rho z) curves and a windowless detector to the quantitative x-ray microanalysis of frozen-hydrated bulk biological specimens.

Preliminary experiments aimed at establishing the validity of applying phi (rho z) curves to the analysis of elements from carbon to calcium in frozen-hydrated bulk biological specimens are described. Computer generated phi (rho z) curves permit simultaneous absorption and atomic number corrections to be made for quantitative analysis. The results so far obtained, whilst not conclusive, indicate that the method is valid for frozen-hydrated samples. The method allows non-ideal standards of inorganic salts to be used for quantitative light element analysis. Simultaneous oxygen analysis at 15kV with a windowless detector is possible using ice as a standard. With further refinements in spectrum processing, carbon analysis may also be possible using pure carbon as a standard. phi (rho z) curves are used to predict that absorption of x-rays from elements P to Ca will be low and that topographic changes in the specimen surface will have little effect on analyses, particularly with detectors using high take-off angles. Line-scan analyses support this prediction and in addition show that analyses of carbon and oxygen are strongly influenced by topography. The depth resolution of analysis is also predicted by phi (rho z) curves. For elements Na to Ca resolution at 15kV is approximately 2.0 micron and 1.0 micron at 10kV. These predictions are supported by experiments on frozen-hydrated sections. phi (rho z) curve analysis indicates that Na x-rays will be strongly absorbed in a frozen-hydrated specimen. It is shown experimentally that with a windowless detector the practical limit of Na detection in a frozen-hydrated bulk sample is between 10 and 25mM per kg wet wt at 15kV.