Fluorescence and bioluminescence measurement of cytoplasmic free calcium.

The first measurements of cytosolic free Ca2", [Ca2"]1, in living cells were made with aequorin in giant muscle cells (Ridgway & Ashley, 1967) in the late 1960s. During the 1970s two further methods were introduced, bis-azo absorbance dyes (mainly arsenazo III) and calciumselective microelectrodes (Ashley & Campbell, 1979). However, even as late as 1981, [Ca'1] had been reliably measured in very few cell types other than giant cells of invertebrates and the techniques were largely confined to the laboratories of those specialized in excitable cell physiology and familiar with microelectrodes or microinjection and advanced optical or electronic technologies. In 1982 a new generation of fluorescent dyes was introduced, with quin2 (Tsien et al., 1982a) and a chemical trick for loading it non-disruptively into populations of cells of any size (Tsien, 1981). This technique in its basic form is simple enough, and needs only basic laboratory instrumentation, so that it is now routinely used in hundreds oflaboratories. The technique has been rapidly developed with the invention of the superior dyes, fura-2 and indo1, and technologies for monitoring [Ca2"], in single cells, whole fields ofidentified cells, and even localized areas within cells, and also improvements in time resolution down to the millisecond range. An outline of these developments forms part of this review. Clearly, with hundreds of papers already published we can only point out the major features, advantageous and problematic, of the technique and hope to provide a critical guide to the literature that details the important points. Another development has been the refinement of the use of the calcium sensitive photoproteins aequorin and obelin. Important novel data on microinjected small single cells are now coming from specialist laboratories and the techniques are not as fearsomely difficult as commonly thought. Part of this review is therefore devoted to an explanation of this method, its advantages and pitfalls, and its successes. We will not consider bisazo dyes or calcium-selective microelectrodes. There are several detailed accounts of these techniques and their place in relation to other approaches (e.g. Thomas, 1982; Blinks et al., 1982; Rink, 1983; Tsien & Rink, 1983). Nor will we review the fluorine derivatives of the fluorescent dyes that have provided n.m.r. probes (Metcalf et al., 1985). This review covers the technical considerations of using fluorescent and bioluminescent probes for measuring [Ca2+]1; we make no attempt to cover the extensive and proliferating data obtained from such measurements.