INTRACELLULAR FLUORESCENT-PROBE CONCENTRATIONS BY CONFOCAL MICROSCOPY

A general method is described that; takes advantage of the optical sec tioning properties of a confocal microscope to enable measurement of b oth absolute and relative concentrations of fluorescent molecules insi de cells. For compartments within cells that are substantially larger than the point spread function, the fluorescence intensity is simply p roportional to the concentration of the fluorophore. For small compart ments, the fluorescence intensity is diluted by contributions from reg ions outside the compartment. Corrections for this dilution can be est imated via calibrations that are based on the intensity distribution f ound in a computationally synthesized model for a cell or organelle th at has been blurred by convolution with the microscope point spread fu nction. The method is illustrated with four test cases: estimation of intracellular concentration of a fluorescent calcium indicator; estima tion of the relative distribution between the neurite and soma of a ne uronal cell of the InsP(3) receptor on the endoplasmic reticulum; esti mation of the distribution of the bradykinin receptor along the surfac e of a neuronal cell; and relative distribution of a potentiometric dy e between the mitochondria and cytosol as a means of assaying mitochon drial membrane potential.