Abstract
Optical methods have recently become available for continuously imaging the free concentrations of important ions and second messengers such as calcium, sodium and hydrogen inside living cells. These ion levels are found to undergo remarkable changes upon stimulation of quiescent cells with growth factors known to stimulate phosphoinositide breakdown. In serum-starved REF-52 fibroblasts, growth factors such as serum, vasopressin, or PDGF (platelet-derived growth factor) cause intracellular [Na+] to increase from about 4 m M to 8 m M . If mitogen treatment is combined with pharmacological depolarization of the membrane potential, repetitive [Ca2+]j spikes result in these rat fibroblasts. The mechanism of this oscillation has been investigated by light-flash release of intracellular messengers such as inositol 1,4,5-trisphosphate (Ins(l,4,5)P3), Ca2+, and diacylglycerol, as well as more traditional biochemical techniques. The key feedback pathway appears to be Ca2+-stimulation of phospholipase C production of Ins(l,4,5)P3.
Introduction
Recent technical advances now enable cytosolic free Ca2+ concentrations ([Ca2+] i), Na+ concentrations ([Na+]i), and pH to be continuously imaged inside individual living cells with micrometer spatial resolution and subsecond time resolution (for recent reviews see Tsien, 1988 Tsien, , 1989 . This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to those ions (Grynkiewicz et al. 1985; Minta and Tsien, 1989; Rink et al. 1982 ). Binding of these ions shifts the fluorescence spectrum of the corresponding indicator. The ratio of excitation or emission amplitudes at two wavelengths measures the free ion concentration while canceling out intensity variations due to non-uniform cell thickness or dye content. A fluorescence microscope equipped to acquire images at two wavelengths and ratio them can thus produce dynamic images of intracellular messenger levels. 
Results and discussion


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