FLUORESCENCE LIFETIME IMAGING OF INTRACELLULAR CALCIUM IN COS CELLS USING QUIN-2

We describe the first fluorescence lifetime images of cells. To demonstrate this new capability we measured intracellular Images of Ca2+ in COS cells based on the Ca2+-dependent fluorescence lifetime of Quin-2. Apparent fluorescence lifetimes were measured by the phase-modulation method using a gain-modulated image intensifier and a slow-scan CCD camera. We describe methods to correct the images for photobleaching during acquisition of the data, and to correct for the position-dependent response of the image intensifier. The phase angle Quin-2 images were found to yield lower than expected Ca2+ concentrations, which appears to be the result of the formation of fluorescent photoproducts by Quin-2. Fluorescence lifetime imaging (FLIM) does not require wavelength-ratiometric probes and appears to provide new opportunities for chemical imaging of cells.