Quantitative elemental X-ray imaging of frozen-hydrated biological samples.

It is shown that quantitative X-ray imaging of planed, frozen-hydrated, biological bulk samples that have not been etched is possible. X-ray imaging represents a better alternative to static beam (selected area) analysis of fractured frozen-hydrated samples. This procedure avoids the undesirable necessity of etching planed frozen-hydrated samples to provide an interpretable electron image. Qualitative oxygen and carbon X-ray images, which can be acquired in a short time, can be used for distinguishing morphological features and remove the requirement for electron images. In test samples of frozen-hydrated albumin, containing salts, analyses by X-ray images compared well with static beam (selected area) analyses from the same samples. An example of an analysis of frozen-hydrated insect Malpighian tubules is given in which the response to ouabain treatment was analysed. In this example X-ray imaging showed that ouabain resulted in a significant increase in cytoplasmic and luminal Na and a significant decrease in cytoplasmic and luminal K. X-ray imaging also showed that there was a significant increase in cellular water content. The presence of a potassium gradient in soybean root nodules was also demonstrated. The use of standard deviation images for processing low count images increases analytical precision but results in underestimates of the true concentrations.