A wide variety of cellular processes—including cell division, growth regulation, neural transmission, and ionic regulation—are regulated via transient shifts and/or steady-state shifts in the levels of molecules acting as signal transducers (for example, calcium flux, pH shift, and receptor binding). In order to investigate the mechanics of these complex multicomponent processes, bioscientists need to pinpoint and quantitate these event(s) in living cells and tissues.

A variety of probes have been developed that allow the scientist to visualize and quantitate physiological events using fluorescence microscopy. Specific probes for calcium, for example, can distinguish changes in free versus sequestered calcium via shifts in the ratio of the probe’s fluorescence intensity at two different wavelengths. Ratio imaging microscopy allows the researcher to pinpoint the cellular location of the chemical shift utilizing low-light level imaging techniques, while ratio fluorescence microscopy allows one to quantitate the event with sensitive photo-detectors. However, numerous technical problems complicate...

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