Fluorescence Imaging Applied to the Measurement of Ca²⁺ in Mammalian Neurons

Abstract

Interest in high resolution photometry in conjunction with cytoplasmic indicator molecules has increased markedly in the past few years. The pioneering experiments in the field date back to the late 1960’s and early 1970’s (c.f. Reynolds, 1972; and for more recent reviews, Ashley and Campbell, 1979; DeWeer and Salzberg, 1986). For the most part these early studies employed the luminescent, Ca binding protein, aequorin, together with intensified target (SIT) TV cameras. Three factors, however, have always limited the use of aequorin: 1) short supply, 2) difficulty in introducing it into cells, 3) kinetics that make calibrated measurements of Ca all but impossible. Synthetic azo dyes with somewhat lower affinities for Ca²⁺ have also been around for a number of years, in use primarily for molluscan neurons and striated muscle (c.f. Brown, et al.,1976; Ahmed and Connor, 1979; Miledi, et al.,1977; Baylor, et al., 1986). More recently such studies have been extended to mammalian neurons and tumor-derived cell lines (Bolsover and Spector, 1986), using arsenazo III, the highest affinity dye of this class. Its large change in extinction coefficient at 660 nm, where tissue absorbance is low, has made it possible to track Ca²⁺ changes during physiological activity in buried processes of molluscan neurons (Graubard and Ross, 1986; Connor, Kretz, and Shapiro, 1986) and in Purkinje cell dendrites in tissue slices (Ross and Werman, 1987). No membrane permeable forms of the azo dyes exist at present, so their use is limited to cells where injections can be made. Both aequorin and arsenazo III continue to be valuable indicators in spite of the inevitable hyperbole that has accompanied the introduction of newer dyes.