Optical Determination of Absolute Membrane Potential in Cellular Physiology

Membrane potential (Vmem) is a voltage across the plasma membrane of all cells, arising from differences in ionic concentration. Changes in Vmem over milliseconds transmit electrical impulses in neurons, and Vmem dynamics over hours to days can modulate differentiation and body patterning. A complete understanding of Vmem requires techniques that can measure its absolute value (in millivolts) across a broad range of time and length scales.In this dissertation, we develop fluorescence lifetime imaging (FLIM) of small molecule voltage sensitive dyes (VoltageFluors, VFs) as a platform for optical recording of absolute Vmem. We review strategies for absolute Vmem detection, highlighting a need for optical techniques complementary to traditional electrode-based recording. Using the VoltageFluor VF2.1.Cl, we demonstrate that its fluorescence lifetime (τfl) can report on absolute Vmem with biologically relevant Vmem resolution (5 mV RMSD for Vmem changes, 19 mV RMSD for Vmem in single trials). Using FLIM, we studied the slow Vmem response following EGF stimulation of A431 carcinoma cells. Because FLIM is robust to motion and photobleaching, we were able to report a 10-15 mV hyperpolarizing response over the course of 15 minutes. Through a combination of pharmacology and absolute Vmem recordings, we demonstrate that the Ca2+ activated K+ channel KCa3.1 mediates the observed hyperpolarization. We then investigated the downstream effects of this Vmem change on EGF-induced Ca2+ signaling and protein phosphorylation. To broaden the reach of FLIM-based absolute Vmem imaging, we characterized the τfl of 11 additional VoltageFluors. First, we explored the fluorescence lifetime of a library of conformationally restricted VF dyes, revealing that synthetic modifications to the dimethylaniline electron donor can modulate the photoinduced electron transfer Vmem sensing trigger of VFs across a wide range. By relating photophysical characterization to VF performance in detection of fast Vmem events, we establish mechanism-based guidelines for VF design. We then developed a red-shifted absolute Vmem imaging platform with FLIM of a carborhodamine VF, which displays the highest absolute Vmem sensitivity of all VFs tested (~4 ps/mV), as well as excellent photostability and low phototoxicity. With red-shifted FLIM, we can make hundreds of sequential Vmem recordings on the same field of view and report Vmem during cardiomyocyte action potentials at acquisition speeds of 20 Hz. Towards absolute Vmem imaging in thick tissue, we characterize Vmem-dependent changes in τfl across a spectrum of VFs under two photon illumination. We observe Vmem sensitivity in all cases, and we identify two additional VFs (carboVF and RhoVR(Me)) with the capability to report absolute Vmem with high fidelity. Finally, we test and discuss various models to describe fluorescence lifetimes of VFs. Together, this work demonstrates that FLIM of VF dyes is a broadly useful strategy for absolute Vmem measurement, expanding the possible scope of Vmem recordings and enabling new biological inquiry into Vmem signaling