Toward measurements of absolute membrane potential 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...
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Elsevier
2025-03-01
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| Series: | Biophysical Reports |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2667074725000011 |
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| author | Debjit Roy Xavier Michalet Evan W. Miller Kiran Bharadwaj Shimon Weiss |
| author_facet | Debjit Roy Xavier Michalet Evan W. Miller Kiran Bharadwaj Shimon Weiss |
| author_sort | Debjit Roy |
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| description | 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 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. In this study, we used a fluorescence lifetime-based approach to obtain a single-cell-resolved distribution of the membrane potential and its changes upon extracellular chemical perturbation in a population of bacterial cells for the first time. Our method is based on 1) a unique VoltageFluor (VF) optical transducer, whose fluorescence lifetime varies as a function of MP via photoinduced electron transfer and 2) a quantitative phasor-FLIM analysis for high-throughput readout. This method allows MP changes to be easily visualized, recorded and quantified. By artificially modulating potassium concentration gradients across the membrane using an ionophore, we have obtained a Bacillus subtilis-specific MP versus VF lifetime calibration and estimated the MP for unperturbed B. subtilis cells to be −65 mV (in minimal salts glycerol glutamate [MSgg]), −127 mV (in M9), and that for chemically depolarized cells as −14 mV (in MSgg). We observed a population-level MP heterogeneity of ∼6-10 mV indicating a considerable degree of diversity of physiological and metabolic states among individual cells. Our work paves the way for deeper insights into bacterial electrophysiology and bioelectricity research. |
| format | Article |
| id | doaj-art-50b9833ca9b644cdba8aba764cd22245 |
| institution | Kabale University |
| issn | 2667-0747 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biophysical Reports |
| spelling | doaj-art-50b9833ca9b644cdba8aba764cd222452025-02-02T05:29:29ZengElsevierBiophysical Reports2667-07472025-03-0151100196Toward measurements of absolute membrane potential in Bacillus subtilis using fluorescence lifetimeDebjit Roy0Xavier Michalet1Evan W. Miller2Kiran Bharadwaj3Shimon Weiss4UCLA-DOE Institute for Genomics and Proteomics, Department of Biological Chemistry, University of California at Los Angeles, Los Angeles, California; Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, CaliforniaDepartment of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California; California Nano Systems Institute, University of California at Los Angeles, Los Angeles, California; Corresponding authorDepartments of Chemistry, Molecular and Cell Biology, Helen Wills Neuroscience Institute, University of California at Berkeley, Berkeley, CaliforniaUCLA-DOE Institute for Genomics and Proteomics, Department of Biological Chemistry, University of California at Los Angeles, Los Angeles, California; Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, CaliforniaUCLA-DOE Institute for Genomics and Proteomics, Department of Biological Chemistry, University of California at Los Angeles, Los Angeles, California; Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California; Department of Physiology, University of California at Los Angeles, Los Angeles, California; California Nano Systems Institute, University of California at Los Angeles, Los Angeles, California; Department of Physics, Institute for Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan, Israel; Corresponding authorMembrane 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 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. In this study, we used a fluorescence lifetime-based approach to obtain a single-cell-resolved distribution of the membrane potential and its changes upon extracellular chemical perturbation in a population of bacterial cells for the first time. Our method is based on 1) a unique VoltageFluor (VF) optical transducer, whose fluorescence lifetime varies as a function of MP via photoinduced electron transfer and 2) a quantitative phasor-FLIM analysis for high-throughput readout. This method allows MP changes to be easily visualized, recorded and quantified. By artificially modulating potassium concentration gradients across the membrane using an ionophore, we have obtained a Bacillus subtilis-specific MP versus VF lifetime calibration and estimated the MP for unperturbed B. subtilis cells to be −65 mV (in minimal salts glycerol glutamate [MSgg]), −127 mV (in M9), and that for chemically depolarized cells as −14 mV (in MSgg). We observed a population-level MP heterogeneity of ∼6-10 mV indicating a considerable degree of diversity of physiological and metabolic states among individual cells. Our work paves the way for deeper insights into bacterial electrophysiology and bioelectricity research.http://www.sciencedirect.com/science/article/pii/S2667074725000011 |
| spellingShingle | Debjit Roy Xavier Michalet Evan W. Miller Kiran Bharadwaj Shimon Weiss Toward measurements of absolute membrane potential in Bacillus subtilis using fluorescence lifetime Biophysical Reports |
| title | Toward measurements of absolute membrane potential in Bacillus subtilis using fluorescence lifetime |
| title_full | Toward measurements of absolute membrane potential in Bacillus subtilis using fluorescence lifetime |
| title_fullStr | Toward measurements of absolute membrane potential in Bacillus subtilis using fluorescence lifetime |
| title_full_unstemmed | Toward measurements of absolute membrane potential in Bacillus subtilis using fluorescence lifetime |
| title_short | Toward measurements of absolute membrane potential in Bacillus subtilis using fluorescence lifetime |
| title_sort | toward measurements of absolute membrane potential in bacillus subtilis using fluorescence lifetime |
| url | http://www.sciencedirect.com/science/article/pii/S2667074725000011 |
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