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Summary A direct technique based on electron energy‐loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been developed to map subcellular distributions of water in frozen‐hydrated biological cryosections. Previously, methods for water determination have been indirect in that they have required the cryosections to be dehydrated first. The new approach makes use of spectrum‐imaging, where 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 darkfield or brightfield image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variations in water content. 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 fitting only water and protein reference spectra, and the accuracy of the method for these compartments has been estimated as ± 3·5%. With the present instrumentation the maximum allowed dose of 2 × 103 e/nm2 limits the useful spatial resolution to around 80 nm for ± 5% precision 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 content of liver mitochondria. The water mapping technique may prove useful for applications to cell physiology and pathophysiology.

Abstract. 2014 Simultaneous measurements have been made in the scanning transmission electron microscope to determine relative microanalytical sensitivities for electron energy loss (EELS) and energy-dispersive x-ray spectroscopies (EDXS). A photodiode-array parallel detector was used for EELS, and an ultrathin window Si(Li) detector subtending a solid angle of 0.18 sterad was used for EDXS. Energy loss spectra were acquired in the first or second difference mode to reduce channelto-channel gain variations and to detect weak signals on large backgrounds. Both EELS and EDXS data were analyzed using multiple least squares procedures to fit reference spectra; this gave not only the fitting coefficients but also their standard deviations. Results showed that EELS is the method of choice for analysis of elements with Z 10. For higher atomic numbers (10 Z 25) EDXS is preferable to EELS if the K edge is used. However, EELS has the higher sensitivity for most elements in this atomic number range if the L23 edge is analyzed. For sodium detection limits are approximately a factor of 10 better with EELS. As the atomic number increases this factor becomes smaller although EELS still has a significant advantage for phosphorus. The occurence of the "white-line" resonance at the L23 edge for Z > 20 boosts the advantage of EELS for calcium but at iron EELS and EDXS have about the same sensitivity. Calculated estimates of the relative sensitivities for EELS and EDXS are in reasonable agreement with the experimental data.

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