Single cell and subcellular measurements of intracellular Ca²⁺ concentration.

Increases in bulk average cytoplasmic Ca(2+) concentration (Ca(2+)) are derived from the combined activities of many Ca(2+) channels. Near (<100 nm) the mouth of each of these channels the local Ca(2+) rises and falls more quickly and reaches much greater values than occurs in the bulk cytoplasm. Even during apparently uniform, steady-state [Ca(2+)] increases large local inhomogeneities exist near channels. These local increases modulate processes that are sensitive to rapid and large changes in [Ca(2+)] but they cannot easily be visualized with conventional imaging approaches. The [Ca(2+)] changes near channels can be examined using total internal reflection fluorescence microscopy (TIRF) to excite fluorophores that lie within 100 nm of the plasma membrane. TIRF is particularly powerful when combined with electrophysiology so that ion channel activity can be related simultaneously to the local subplasma membrane and bulk average Ca(2+). Together these techniques provide a better understanding of the local modulation and control of Ca(2+) signals.