UC Berkeley

Membrane potential, Vmem, is the voltage across a cellular plasma membrane, produced by differences in ion concentration across the semipermeable membrane. Changes in Vmem over millisecond timescales transmit the electrical signals of action potentials in neurons and cardiomyocytes. Slower changes in Vmem over minutes, hours, or days affect processes such as differentiation, and set the resting membrane potential, which can affect neuronal and cardiac excitability. Vmem may also vary across substructures within cells, like organelles or dendritic spines, to enable localized electrical signaling. Forming a complete picture of the role of Vmem across these varied time and length scales requires techniques that can measure the value of Vmem in millivolts and reliably report both the dynamic changes in voltage and the absolute millivolt value of Vmem at rest.In this dissertation, we develop fluorescence lifetime imaging microscopy (FLIM) of the VoltageFluor (VF) small-molecule voltage-sensitive dyes as a method for optically reporting absolute Vmem, and then expand the method to additional dye scaffolds and cellular targets. We review existing tools and methods for recording Vmem, highlighting the need for additional optical techniques alongside electrophysiology. Initially using the VoltageFluor VF2.1.Cl, we demonstrate that its fluorescence lifetime, τfl, can report Vmem in multiple cell lines with voltage resolution at a biologically relevant level (5 mV RMSD voltage changes, and 19 mV RMSD for Vmem in a single trial in HEK293T cells). We optically read out the Vmem of hundreds of cells and report voltage distributions consistent with the values measured electrophysiologically, with 100-fold improvement in throughput over electrophysiology. To expand this FLIM technique to enable optical determination of Vmem in more complex cultures, such as neurons, we investigate two chemical-genetic targeting strategies. The first, VoltageSpy, has a 7-fold improvement in voltage resolution over the existing genetically encoded voltage indicator (GEVI) systems for reporting Vmem using FLIM. The second, VF-HaloTag, has a 10-fold improvement in resolution over the same GEVI FLIM indicator. VF-HaloTag also enables FLIM imaging of individual neurons and even individual dendrites with good spatial and lifetime resolution. We also develop a red-shifted, carborhodamine-based voltage indicator with comparable sensitivity to VF2.1.Cl. This red-shifted indicator is robust to photoxicity and photobleaching, while exhibiting a monoexponential fluorescence decay, requiring fewer photons for a reliable determination of τfl. This allows both rapid and extended FLIM imaging, allowing us to resolve cardiac action potentials in lifetime, and image sensitive cells for minutes at a time. Finally, we utilize FLIM as a tool for examining structural modifications to the VF scaffold. We report a consistent trend across three dye series (six dyes), demonstrating the effect of altering the position of the molecular wire in the VF scaffold on τfl. This overall work showcases the utility of FLIM to optically read out voltage, and shows how the technique can be expanded to measure voltage at various spatial and time scales, broadening the toolbox for optical readout of Vmem.