Ratio-based indicators for cytosolic Ca ²⁺ 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[<i>b</i>]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 report isoCaRed-1Me, a Ca²⁺ 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 Ca²⁺ imaging using modern microscopes.