PDF(Pubmed)
Abstract：
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.
摘要：
膜电位(MP)变化可以提供细菌功能和代谢状态或应激水平的简单读出。虽然存在几种光学方法来测量可兴奋细胞中MP的快速变化，缺乏这种方法来绝对和精确测量细菌细胞中的稳态膜电位（MPs）。用于测量MP的常规基于电极的方法不适用于在小细菌细胞中校准光学方法。虽然基于Nernstian指标的光学测量已成功使用，它们不能提供MP或其变化的绝对或精确定量。我们提出了一个小说,校准MP记录方法来解决这个差距。我们的方法基于（i）独特的VoltageFluor（VF）光学传感器，其荧光寿命通过光诱导电子转移（PeT）作为MP的函数而变化，并且（ii）用于高通量读出的定量相量-FLIM分析。这种方法可以轻松记录MP更改，量化和可视化。使用我们的初步枯草芽孢杆菌特异性MP与VF寿命校准，我们估计未扰动的枯草芽孢杆菌细胞的MP为-65mV，化学去极化细胞的MP为-14mV。我们的工作为深入了解细菌电生理学和生物电研究铺平了道路。
