Lazzari-Dean Julia R, Miller Evan W
Department of Chemistry, University of California, Berkeley, Berkeley, California, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
Bioelectricity. 2021 Sep 1;3(3):197-203. doi: 10.1089/bioe.2021.0007. Epub 2021 Sep 9.
Membrane potential ( ) exerts physiological influence across a wide range of time and space scales. To study in these diverse contexts, it is essential to accurately record absolute values of , rather than solely relative measurements. We use fluorescence lifetime imaging of a small molecule voltage sensitive dye (VF2.1.Cl) to estimate mV values of absolute membrane potential. We test the consistency of VF2.1.Cl lifetime measurements performed on different single-photon counting instruments and find that they are in striking agreement (differences of <0.5 ps/mV in the slope and <50 ps in the -intercept). We also demonstrate that VF2.1.Cl lifetime reports absolute under two-photon (2P) illumination with better than 20 mV of resolution, a nearly 10-fold improvement over other lifetime-based methods. We demonstrate that VF-FLIM is a robust and portable metric for across imaging platforms and under both one-photon and 2P illumination. This work is a critical foundation for application of VF-FLIM to record absolute membrane potential signals in thick tissue.
膜电位（ ）在广泛的时间和空间尺度上发挥生理影响。为了在这些不同的背景下研究膜电位，准确记录膜电位的绝对值而非仅仅是相对测量值至关重要。我们使用一种小分子电压敏感染料（VF2.1.Cl）的荧光寿命成像来估计绝对膜电位的毫伏值。我们测试了在不同单光子计数仪器上进行的VF2.1.Cl寿命测量的一致性，发现它们惊人地一致（斜率差异<0.5 ps/mV，截距差异<50 ps）。我们还证明，在双光子（2P）照明下，VF2.1.Cl寿命报告的绝对膜电位分辨率优于20 mV，比其他基于寿命的方法提高了近10倍。我们证明，VF - FLIM是一种用于跨成像平台以及在单光子和2P照明下测量膜电位的强大且便携的指标。这项工作是将VF - FLIM应用于记录厚组织中绝对膜电位信号的关键基础。
