The water-mapping technique that we developed based on electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been further enhanced to map the subcellular distribution of water in frozen-hydrated biological cryosections. The new approach makes use of spectrum imaging, whereby EELS data are collected in parallel at each pixel. Several operations are required to process the spectra, including: subtraction of the detector dark current, deconvolution by the detector point-spread function, removal of plural inelastic scattering, and correction for the support film. The resulting single scattering distributions are fitted to standard reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the dark-field or bright-field image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variations in water content. Cryo-EELS has also been applied to determine the thickness of thin, hydrated tissue sections, thus providing information about the limits of the cryosectioning process itself.
