Abstract
The contractile vacuole (CV) regulates internal osmolality of free-living protozoa such as amoebae. The CV membrane is known to contain a V-H+ ATPase but the other ion transporters involved in the water pumping mechanism are not yet established. Here we continue our earlier investigation by analytical electron microscopy to measure vacuolar and cytoplasmic ion concentrations inAcanthamoeba castellanii. The present aim is to determine concentrations of ions transported across the CV membrane of amoebae grown under different osmotic conditions.Subcellular ion concentrations in freeze-dried cryosections can generally be determined from x-ray microanalysis using the Hall method.4However, this approach is not suitable for measuring ion concentrations in the CV, which contains mainly inorganic salts and little, if any, organic compounds. A new method based on electron scattering measurements was therefore devised to deal with this highly aqueous compartment. This method involves: (i) measuring the volume of water in the hydrated cryosectioned vacuole with parallel electron energy loss spectroscopy (PEELS); (ii) measuring the mass of vacuolar salt in the freeze-dried cryosection by dark-field scanning transmission electron microscopy (STEM); and (iii) measuring the ratios of ions by energy-dispersive x-ray spectroscopy (EDXS).
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