Sequential quantitative X-ray elemental imaging of frozen-hydrated and freeze-dried biological bulk samples in the SEM.

ABSTRACT

Planed frozen-hydrated (FH) bulk biological samples of chicken retina were analysed by X-ray elemental imaging in a scanning electron microscope and reanalysed after freeze-drying in the microscope column. Sequential elemental imaging of the same bulk sample in this way provides improved information on element distributions. There was no evidence of element redistribution during the freeze-drying process. Quantitative elemental images were obtained and interpreted to deduce relative and absolute element concentrations in different regions of the retina. Water concentrations were determined from the difference in oxygen concentrations at 15 kV and 5 kV in FH and freeze-dried (FD) samples, respectively. Two accelerating voltages were used to maintain similar X-ray excitation volumes. Water concentrations were also estimated by relating measured oxygen concentration in FH samples to the concentration of oxygen in solutions of a generalized protein in water and by comparing concentrations of phosphorous or sulphur in the FH and FD states.