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

• 成果类型: Article
• 署名作者: Zhou, Xinqi; Belavek, Kayla J.; Navarro, Marisol X.; Martinez, Kayli N.; Hinojosa, Abigail; Miller, Evan W.
• 署名单位: University of California System; University of California Berkeley; University of California System; University of California Berkeley; University of California System; University of California Berkeley
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13853
• DOI: 10.1073/pnas.2410436122
• 发表日期: 2025-02-18
• 关键词: n-h bonds; calcium indicators; fluorescent-probe; red; wavelength; rhodamine; scaffold; fura-2; DESIGN; oxide

摘要

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 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.