Comparison of intracellular water content measurements by dark-field imaging and EELS in medium voltage TEM

Knowledge of the water content at the subcellular level is important to evaluate the intracellular concentration of either diffusible or non-diffusible elements in the physiological state measured by the electron microprobe methods. Water content variations in subcellular compartments are directly related to secretion phenomena and to transmembrane exchange processes, which could be attributed to pathophysiological states. In this paper we will describe in details and compare two local water measurement methods using analytical electron microscopy. The first one is based on darkfield imaging. It is applied on freeze-dried biological cryosections; it allows indirect measurement of the water content at the subcellular level from recorded maps of darkfield intensity. The second method uses electron energy loss spectroscopy. It is applied to hydrated biological cryosections. It is based on the differences that appear in the electron energy loss spectra of macromolecular assemblies and vitrified ice in the 0-30 eV range. By a multiple least squares (MLS) fit between an experimental energy loss spectrum and reference spectra of both frozen-hydrated ice and macromolecular assemblies we can deduce directly the local water concentration in biological cryosections at the subcellular level. These two methods are applied to two test specimens: human erythrocytes in plasma, and baker's yeast (Saccharomyces Cerevisiae) cryosections. We compare the water content measurements obtained by these two methods and discuss their advantages and drawbacks.