Ratiometric Ca2+ Measurements Using the FlexStation Scanning Fluorometer

Many commercial organizations currently use the Fluorometric Imaging Plate Reader (FLIPR�: Molecular Devices, Sunnyvale, CA) to conduct highthroughput measurements of intracellular Ca²⁺ concentration (see Chapter 7 ), taking advantage of its rapid kinetics, reliability, and compatibility for automation. For the majority of industrial applications, the primary limitation of FLIPR (i.e., its requirement for single wavelength fluorescent probes using visible light excitation) is not a significant issue. Indeed, visible light probes offer certain benefits over their ultraviolet (UV)-excited ratiometric counterparts, such as reduced sample autofluorescence and higher absorbance, thereby allowing relatively low concentrations of dye to be used. However, under certain circumstances researchers may prefer to conduct high-throughput experiments with ratiometric dyes, particularly when issues of dye leakage, photobleaching, or signal-to-noise ratio become a concern.