Ratio-based indicators for cytosolic Ca with visible light excitation.

Calcium ions (Ca) play central roles in cellular physiology. Fluorescent indicators for Ca ions revolutionized our ability to make rapid, accurate, and highly parallel measurement of Ca 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 Ca 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 Ca 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[]phosphole-based fluorescent Ca indicators for ratio-based imaging of Ca in living cells that can be used with modern light emitting diode (LED)-equipped fluorescence microscopes. We report isoCaRed-1Me, a Ca indicator that enables 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 Ca imaging using modern microscopes.