Detection of Membrane Potential-Dependent Rhodopsin Fluorescence Using Low-Intensity Light Emitting Diode for Long-Term Imaging.
Shiho KawanishiKeiichi KojimaAtsushi ShibukawaMasayuki SakamotoYuki SudoPublished in: ACS omega (2023)
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/cm 2 ) 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.
Keyphrases
- single molecule
- energy transfer
- light emitting
- drug induced
- high resolution
- loop mediated isothermal amplification
- real time pcr
- label free
- liver injury
- induced apoptosis
- mass spectrometry
- optical coherence tomography
- microbial community
- ms ms
- bone marrow
- single cell
- hydrogen peroxide
- dna methylation
- copy number
- cell cycle arrest
- mesenchymal stem cells
- photodynamic therapy
- cell death
- signaling pathway
- cell therapy
- high speed
- human health
- fluorescence imaging
- wild type