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
M. Hardt et H. Plattner, 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, J STRUCT B, 128(2), 1999, pp. 187-199
We analyzed preparative and analytical aspects of the dynamic localization
of Ca2+ during cell stimulation, using a combination of quenched how and en
ergy-dispersive X-ray microanalysis (EDX). Calcium (or Sr, as a substitute)
was retained as fluorides during freeze-substitution, followed by epoxide
embedding. The quenched-how used allowed analyses, during stimulation, in t
he subsecond time range. Sections of 500 nm were analyzed and no artificial
Ca or Sr leakage was recognizable, We calculated a primary beam spread fro
m 63 to 72 nm that roughly indicated the resolution of EDX/structure correl
ation, These values are quite compatible with the size of potential structu
res of interest, e.g., Ca stores (similar to 100-nm thickness) or cilia (si
milar to 250-nm diameter). We used widely different standards to calibrate
the ratio of CaKalpha net counts in relation to actual [Ca]. Calibration cu
rves showed a linear relationship and a detection limit of [Ca] = 2 mM, whi
le [Ca] in cytosol was 3 mM and in stores was 43 mM, both in nonactivated c
ells. Eventually Sr2+ can rapidly be substituted for Ca2+ in the medium bef
ore and during stimulation, thus allowing one to determine Me2+ fluxes. Wit
h our "model" cell, Paramecium, we showed that, upon stimulation (causing r
apid Ca2+ mobilization from subplasmalemmal stores), Ca was immediately exc
hanged for Sr in stores, (C) 1999 Academic Press.