Abstract
While great progress has been achieved in developing optical methods for measuring fast changes in membrane potential (like action potentials) in excitable cells, less progress has been made in precise (and calibrated) measurements of steady state resting membrane potentials (RMPs) and small changes in RMPs (in excitable or non-excitable cells). In excitable cells, small changes in RMPs are associated with multiple physiological processes such as sub-threshold events in neuronal signaling and in synaptic plasticity. They also play an important role in cell differentiation and proliferation of cardiomyocytes. In non-excitable cells, such as bacterial colonies, RMP changes play important roles in intercellular communication, coordination, metabolism, and stress response. Accurate and precise recordings of minute RMP changes require noise-immune optical tools. We have been developing an RMP (calibrated) recording approach that is capable of measuring small changes RMPs. Our approach relies on 3 components: (i) a novel optical transducer (voltage sensing dye) that utilizes intrinsic photoinduced electron transfer (PeT) mechanism to sense RMP by fluorescence lifetime changes; (ii) a novel time-gated SPAD array imager that allows widefield fluorescence lifetime imaging (FLIM); (iii) a quantitative phasor analysis of the recoded FLIM data. This package provides high-throughput, quantification of pixel-wise lifetime information. Two aspects of our approach - the novel VSD and the data analysis - have been tested in excitable cardiac muscle cells (NVRM) and in non-excitable bacterial cells (B. subtilis). B. subtilis RMPs have been estimated for normal cultures and for cultures under perturbing chemical conditions. Drug induced changes of NVRM RMPs have been also estimated. Implementation of our 3-pronged approach towards studying electrical signaling/coupling networks in bacterial biofilms and in cardiomyocytes will be discussed.
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