American Chemical Society Acta Medica Okayama 2470-1343 8 2023 Detection of Membrane Potential-Dependent Rhodopsin Fluorescence Using Low-Intensity Light Emitting Diode for Long-Term Imaging 4826 4834 EN Shiho Kawanishi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Atsushi Shibukawa Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayuki Sakamoto Department of Optical Neural and Molecular Physiology, Graduate School of Biostudies Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and Faculty of Medicine, Dentistry and Pharmaceutical Sciences Microbial rhodopsin is a family of photoreceptive membrane proteins that commonly consist of a seven-transmembrane domain and a derivative of vitamin-A, retinal, as a chromophore. In 2011, archaeorhodopsin-3 (AR3) was shown to exhibit voltage-dependent fluorescence changes in mammalian cells. Since then, AR3 and its variants have been used as genetically encoded voltage indicators, in which mostly intense laser stimulation (1-1000 W/cm(2)) is used for the detection of dim fluorescence of rhodopsin, leading to high spatiotemporal resolution. However, intense laser stimulation potentially causes serious cell damage, particularly during long-term imaging over minutes. In this study, we present the successful detection of voltage-sensitive fluorescence of AR3 and its high fluorescence mutant Archon1 in a variety of mammalian cell lines using low-intensity light emitting diode stimulation (0.15 W/cm2) with long exposure time (500 ms). The detection system enables real-time imaging of drug-induced slow changes in voltage within the cells for minutes harmlessly and without fluorescence bleaching. Therefore, we demonstrate a method to quantitatively understand the dynamics of slow changes in membrane voltage on long time scales. No potential conflict of interest relevant to this article was reported.