Evaluation and experimental monitoring of calcium in plasma membrane inner nanoscale cytoplasm regions

So far, measurements of free calcium in the nanoscale cytosolic region near the plasma membrane remain a challenge in cell biology. We present a convenient approach that combines membrane-bound dyes with cytosolic calcium probes. Manipulations with distinct laser excitations of these dyes allow monitoring of calcium in both the cytosol and the sub-membrane region. Whereas Förster resonance energy transfer induces secondary fluorescence of the calcium probe at distances under 10 nm from the membrane upon membrane dye excitation, direct excitation of the probe visualizes the bulk calcium. Experiments on HEK293 cells and neurons disclose that at rest sub-membrane calcium concentration is large exceeding 400 nM. Furthermore calcium entry through ion channels, exchangers, or ionophores, as well as spontaneous calcium neuronal oscillations, increase calcium concentration up to 1 µM and 800 nM in the pre-membrane domain and in the cytoplasm, respectively. This approach provides a new tool for calcium nanodomain studies.