Towards optical measurements of membrane potential values in Bacillus subtilis using fluorescence lifetime.

Membrane potential (MP) changes can provide a simple readout of bacterial functional and metabolic state or stress levels. While several optical methods exist for measuring fast changes in MP in excitable cells, there is a dearth of such methods for absolute and precise measurements of steady-state membrane potentials (MPs) in bacterial cells. Conventional electrode-based methods for the measurement of MP are not suitable for calibrating optical methods in small bacterial cells. While optical measurement based on Nernstian indicators have been successfully used, they do not provide absolute or precise quantification of MP or its changes. We present a novel, calibrated MP recording approach to address this gap. Our method is based on (i) a unique VoltageFluor (VF) optical transducer, whose fluorescence lifetime varies as a function of MP via photoinduced electron transfer (PeT) and (ii) a quantitative phasor-FLIM analysis for high-throughput readout. This method allows MP changes to be easily recorded, quantified and visualized. Using our preliminary Bacillus subtilis -specific MP versus VF lifetime calibration, we estimated the MP for unperturbed B. subtilis cells to be -65 mV and that for chemically depolarized cells as -14 mV. Our work paves the way for deeper insights into bacterial electrophysiology and bioelectricity research.