Quantitative energy-dispersive X-ray microanalysis of calcium dynamics in cell suspensions during stimulation on a subsecond time scale: preparative and analytical aspects as exemplified with paramecium cells.
We analyzed preparative and analytical aspects of the dynamic localization of Ca(2+) during cell stimulation, using a combination of quenched flow and energy-dispersive X-ray microanalysis (EDX). Calcium (or Sr, as a substitute) was retained as fluorides during freeze-substitution, followed by epoxide embedding. The quenched-flow used allowed analyses, during stimulation, in the subsecond time range. Sections of 500 nm were analyzed and no artificial Ca or Sr leakage was recognizable. We calculated a primary beam spread from 63 to 72 nm that roughly indicated the resolution of EDX/structure correlation. These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (approximately 100-nm thickness) or cilia (approximately 250-nm diameter). We used widely different standards to calibrate the ratio of CaK(alpha) net counts in relation to actual ¿Ca. Calibration curves showed a linear relationship and a detection limit of ¿Ca = 2 mM, while ¿Ca in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells. Eventually Sr(2+) can rapidly be substituted for Ca(2+) in the medium before and during stimulation, thus allowing one to determine Me(2+) fluxes. With our "model" cell, Paramecium, we showed that, upon stimulation (causing rapid Ca(2+) mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores.
Hardt
M
M
Faculty of Biology, University of Konstanz, Konstanz, D-78434, Germany.
Plattner
H
H
D000818:Animals; D002118:Calcium / Q000032:analysis*; D002923:Cilia / Q000648:ultrastructure; D015925:Cryopreservation; D003911:Dextrans / Q000494:pharmacology; D004577:Electron Probe Microanalysis; D005089:Exocytosis; D017348:Microscopy, Electron, Scanning Transmission; D016805:Paramecium tetraurelia / Q000378:metabolism* / Q000648:ultrastructure; D011137:Polystyrenes; D011145:Polyvinyls; D012680:Sensitivity and Specificity
D003911:Dextrans; D011137:Polystyrenes; D011145:Polyvinyls; C006960:chelex; C039691:aminoethyldextran; D002118:Calcium