Dec 3, 2024
4:30pm - 4:45pm
Hynes, Level 2, Room 202
Andrea Pianetti1,Pietro Bertolotti1,Cosimo D'Andrea2,Giuseppe Maria Paternò1,2,Guglielmo Lanzani1,2
Istituto Italiano di Tecnologia1,Politecnico di Milano2
Andrea Pianetti1,Pietro Bertolotti1,Cosimo D'Andrea2,Giuseppe Maria Paternò1,2,Guglielmo Lanzani1,2
Istituto Italiano di Tecnologia1,Politecnico di Milano2
Understanding and controlling bacterial motility and response to external stimuli is indeed pivotal for leveraging bacteria in the creation of smart and living materials. Recent studies have unveiled the dynamic nature of bacterial membrane potential: it is now evident that membrane potential regulates a wide array of bacterial physiological processes and behaviors, including membrane transport, motility, antibiotic resistance, communication, and environmental sensing. Despite these advancements, numerous questions remain. These include the information encoding capacity of membrane potential dynamics, the origins of excitability and electrical signaling, the feasibility of artificial control of membrane potential, and many others <sup>[1]</sup>.<br/>Traditional patch-clamp techniques, while effective for certain applications, are inadequate for studying dynamic changes in bacterial membrane potential due to the requirement for cell immobilization and the diminutive size of bacterial cells. Consequently, there is an urgent need for developing novel, minimally invasive methods to measure membrane potential dynamics <sup>[2]</sup>.<br/>Fluorescent molecular probes with voltage-dependent optical properties have emerged as a promising solution for noninvasive studies of membrane voltage. Recent advancements that optical electrophysiology measurements will soon become routine in human cell electrophysiology <sup>[3]</sup>. This approach is now being extended to non-eukaryotic cells, such as bacteria. In this context, Nernstian dyes have been widely employed due to their high efficency and low toxicity. However, estimating membrane potential based solely on fluorescence intensity is fraught with challenges due to various factors such as spatial uniformity of excitation and fluctuations in light source power, which can lead to significant experimental errors. In contrast, fluorescence lifetime is an intrinsic property that depends solely on the local environment of the dye and is independent of many external experimental parameters. Thus, measuring both fluorescence intensity and lifetime can provide more accurate and reliable data.<br/>In this study, we investigated the dependency of the fluorescence lifetime of the TMRM dye on the progressive depolarization of membrane potential in both gram-positive and gram-negative bacteria. Our findings demonstrate that this phenomenon is ubiquitous across different bacterial types, underscoring its potential as a robust tool for electrophysiological studies of motile and free-living bacteria in various environments. Furthermore, we employed Fluorescence Lifetime Imaging Microscopy (FLIM) to precisely correlate bacterial motility behavior with membrane potential modulation, using both intensity and lifetime as observables. Additionally, we demonstrated the internalization of DIANEP dyes in bacterial membranes, which have primarily been used in eukaryotic cells, to evaluate membrane potential based on variations in spectral characteristics beyond photoluminescence dynamics.<br/>These advancements enable the use of optical methods in bacterial electrophysiology, providing new insights into the dynamic processes of bacterial behavior and physiology. This approach could also be extended to study the detailed electrophysiology of other small motile cells, such as algae and viruses, which have not been extensively explored before but might serve as a new foundation for developing living materials.<br/>[1] Cohen AE, Venkatachalam V. Bringing bioelectricity to light. Annu Rev Biophys. 2014;43:211-32. doi: 10.1146/annurev-biophys-051013-022717.<br/>[2] Benarroch JM, Asally M. The Microbiologist's Guide to Membrane Potential Dynamics. Trends Microbiol. 2020 Apr;28(4):304-314. doi: 10.1016/j.tim.2019.12.008.<br/>[3] Yuecheng Zhou, Erica Liu, Holger Müller, and Bianxiao Cui. Optical Electrophysiology: Toward the Goal of Label-Free Voltage Imaging, Journal of the American Chemical Society 2021 143 (28), 10482-10499. DOI: 10.1021/jacs.1c02960.