Membrane potential (Vm) has been implicated in embryonic development, yet its quantitative role remains poorly understood. Developmental bioelectricity spans fast voltage transients in excitable tissues and slower shifts in resting Vm alongside morphogenesis. While fast activity can be monitored with existing intensity-based approaches, calibrated comparison of resting Vm across cell types and developmental stages in vivo has remained out of reach, as live embryos introduce photon-limited imaging, optical heterogeneity, and variable indicator expression that confound intensity readouts.
This dissertation addresses this gap by establishing an integrated in vivo voltage imaging framework in developing zebrafish embryos. As a foundation, a genetic toolkit for the eFRET-based indicator Ace2N-mNeon is developed to achieve cell-type-specific, membrane-targeted expression across embryonic tissues. This platform enables high-speed intensity recordings of rapid voltage activity in nascent neurons and cardiomyocytes, revealing synchronous neuronal firing and the progressive maturation of cardiac conduction between 1 and 4 days post-fertilization. To move beyond relative intensity readouts, a cell-resolved FLIM workflow is introduced that pairs membrane-focused segmentation with electrophysiological calibration, converting donor lifetime into absolute Vm estimates and resolving systematic differences on the order of 10 mV in vivo. Finally, to improve upon the sensitivity limits of current indicators, alternative eFRET constructs are screened and a theoretical spectral-perturbation model is developed, together defining design constraints for next-generation lifetime-readable voltage sensors.
Together, this work provides the first quantitative, cell-resolved framework for mapping absolute resting Vm in intact vertebrate embryos, opening new routes to test whether spatial voltage patterns play an instructive role in embryonic patterning alongside biochemical and mechanical cues. ... Read more