QUANTITATIVE WATER MAPPING OF CRYOSECTIONED CELLS BY ELECTRON-ENERGY-LOSS SPECTROSCOPY

A direct technique based on electron energy-loss spectroscopy (EELS) i n the scanning transmission electron microscope (STEM) has been develo ped to map subcellular distributions of water in frozen-hydrated biolo gical cryosections. Previously, methods for water determination have b een indirect in that they have required the cryosections to be dehydra ted first. The new approach makes use of spectrum-imaging, where EELS data are collected in parallel at each pixel. Several operations are r equired to process the spectra including: subtraction of the detector dark current, deconvolution by the detector point-spread function, rem oval of plural inelastic scattering and correction for the support fil m, The resulting single scattering distributions are fitted to standar d reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the darkfield or brightfield image from a hydrated cryosection shows minimal structure, the process ed EELS image reveals strong contrast due to variations in water conte nt. Reference spectra have been recorded from the major biomolecules ( protein, lipid, carbohydrate, nucleic acid) as well as from vitrified water and crystalline ice. It has been found that quantitative results can be obtained for the majority of subcellular compartments by fitti ng only water and protein reference spectra, and the accuracy of the m ethod for these compartments has been estimated as +/- 3.5%. With the present instrumentation the maximum allowed dose of 2 x 10(3) e/nm(2) limits the useful spatial resolution to around 80 nm for +/- 5% precis ion at a single pixel. By averaging pixel intensities a value of 56.8% with a precision of +/- 2 0% has been determined for the water conten t of liver mitochondria. The water mapping technique may prove useful for applications to cell physiology and pathophysiology.