Ratio-based indicators for cytosolic Ca2+ with visible light excitation

Calcium ions (Ca2+) play central roles in cellular physiology. Fluorescent indicators for Ca2+ ions revolutionized our ability to make rapid, accurate, and highly parallel measurement of Ca2+ concentrations in living cells. The use of ratio-based imaging with one particular indicator, fura-2, allowed practitioners to correct for a number of experimental confounds, including dye bleaching, variations in sample thickness, and fluctuations in illumination intensity. Ratio-based imaging with fura-2 was the most accurate and reliable method for measuring Ca2+ concentrations. Two drawbacks to fura-2 exist. First, it requires ultraviolet (UV) excitation, which is more toxic to living cells than visible light. Second, our ability to use fura-2 for accurate, stable, ratio-based determinations of Ca2+ concentration in living cells is fast becoming a method of the past. This is due, in part, because modern microscopes are phasing out the use of mercury arc lamps that provide the UV excitation needed for fura-2 imaging. To address this problem, we describe the design, synthesis, and cellular application of benzo[b]phosphole-based fluorescent Ca2+ indicators for ratio-based imaging of Ca2+ in living cells that can be used with modern light emitting diode (LED)-equipped fluorescence microscopes. We show that isoCaRed-1Me has absorbance spectra in the visible range, shows Ca2+ selectivity over Mg2+, and displays a Ca2+-dependent excitation spectra. These unique properties enable ratio-based imaging in immortalized cell lines, primary mammalian hippocampal neurons, and human induced pluripotent stem cell-derived cardiomyocytes. These data show that isoCaRed-1Me will be useful for ratio-based Ca2+ imaging using modern microscopes.