FLUORESCENCE IMAGING OF INTRACELLULAR CA2+

The measurement of intracellular Ca2+ concentrations ([Ca2+]i) is of critical importance, because many cellular functions are tightly regulated by [Ca2+]i. The fluorescent indicator, fura-2, has been used frequently to measure [Ca2+]i because of its sensitivity and specificity, and because it can be loaded into living cells with little disruption of function. Most importantly, the peak excitation wavelength of fura-2 changes when it binds Ca2+. As a consequence, measurements of fluorescence at two excitation wavelengths can be used to obtain an estimate of [Ca2+]i that is independent of dye concentration and cell thickness. Fura-2 acetoxymethyl ester (AM) is a lipid-soluble derivative that is often used because of its ability to pass through cell membranes. There are, however, several problems with the use of fura-2 AM such as intracellular compartmentation and incomplete deesterification. The availability of low-light-level cameras and computer hardware for the digitization of fluorescent images has made quantitative fluorescence microscopy possible. This technique has shown a striking spatial heterogeneity of [Ca2+]i in a variety of cell types, and has revealed substantial new information on dynamic intracellular biochemistry and signal transduction. However, the current imaging technology is not fully developed because of dye and instrumentation limitations. Further development of techniques and new probes are required to improve temporal and spatial resolution.