Quantitative x-ray microanalysis of frozen-hydrated bulk biological specimens.

Fully frozen-hydrated bulk specimens can be fractured at < -170 decrees C to reveal a surface with sufficient topographical detail in a secondary electron image to identify subcellular structures. Metal coating prevents charging and reduces thermal damage. Quantitative analyses can be carried out at -165 degrees C using standards of either simple aqueous salt solutions or matching standards. The latter can be chemically analysed body fluids or fixed and leached tissue equilibrated with salt solutions. Analytical precision is limited by surface topography of the specimen and difficulties with standard preparation. Sodium is currently difficult to detect at concentrations below 50 mMl-1 whereas other elements can be detected in the 2-5 mMl-1 range. Preliminary analyses of fluid compartments in cicada filter chamber are consistent with physiological measurements, and analyses of ion gradients in insect Malpighian tubules are consistent with analyses which have been made on frozen-hydrated sections and reported in the literature. Cellular concentrations of ions in suspected ion regulating cells in brine fly larvae have been shown to respond to changes in environmental ion concentrations.