Use of standard fluorescence microscopy to assess modifications in the plasma membrane potential and in the intracellular concentration of inorganic ions in cultured cells

As an alternative to electrophysiological procedures, fluorescent probes have been widely used to assess modifications in the plasma membrane potential (PMP) of living cells, mostly via spectrophotometric or spectrofluorimetric analysis of samples of suspended cells. Another suggested procedure for the quantitative study of PMP changes using fluorescent probes utilizes confocal microscopy and image analysis of cells loaded with potentiometric probes. A modification of this latter procedure employs standard fluorescent microscopy and image analysis (FM-IA). We have used this methodology to study modifications in the PMP and intracellular ionic concentrations for the case of cultured epithelial cells. In this chapter we describe the FM-IA method, illustrate its utilization with examples taken from our published work and discuss possible applications and shortcomings.