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 eva luate the intracellular concentration of either diffusible or non-diffusibl e elements in the physiological state measured by the electron microprobe m ethods. Water content variations in subcellular compartments are directly r elated to secretion phenomena and to transmembrane exchange processes, whic h could be attributed to pathophysiological states. In this paper we will d escribe in details and compare two local water measurement methods using an alytical electron microscopy. The first one is based on darkfield imaging. It is applied on freeze-dried biological cryosections; it allows indirect m easurement of the water content at the subcellular level from recorded maps of darkfield intensity. The second method uses electron energy loss spectr oscopy. It is applied to hydrated biological cryosections. It is based on t he differences that appear in the electron energy loss spectra of macromole cular assemblies and vitrified ice in the 0-30 eV range. By a multiple leas t squares (MLS) fit between an experimental energy loss spectrum and refere nce spectra of both frozen-hydrated ice and macromolecular assemblies we ca n deduce directly the local water concentration in biological cryosections at the subcellular level. These two methods are applied to two test specime ns: human erythrocytes in plasma, and baker's yeast (Saccharomyces Cerevisi ae) cryosections. We compare the water content measurements obtained by the se two methods and discuss their advantages and drawbacks.