利用干涉式散射光學顯微術進行大腸桿菌膜電位的無標記成像

Abstract

在可興奮性細胞中，包括神經元、肌肉細胞以及某些內分泌細胞，動作電位是由電壓閘控離子通道的開啟與關閉所產生，這些通道允許在電化學梯度驅動下的離子通過，導致膜電位變化並傳遞電信號。目前常用的膜電位量測技術包括全細胞貼片鉗與外源性電壓敏感螢光探針。然而，膜片鉗技術可能造成細胞損傷，而電壓敏感螢光探針容易受到光毒性與光漂白的影響，限制了訊號獲取的時間與穩定性。近年來，基於散射的干涉顯微術有了重大進展，能以高靈敏度與高速度進行無標記的奈米尺度結構檢測，為觀測快速電生理事件（例如細菌的電生理現象）開啟了新的契機。部分研究已經利用基於干涉的光學方法來成像神經科學中離子通道的活性，顯示此技術具有偵測膜電位的潛力。然而，有關細菌膜電位的研究仍相對有限，目前仍缺乏能有效探測此類快速過程的無標記方法。 在本研究中，我們利用基於散射的干涉顯微術來量測細菌的動作電位。我們以大腸桿菌作為模型系統，在其細胞中基因表現電壓感測螢光指示蛋白 ViBac1，以作為動作電位（Vm）的訊號。同時，我們使用干涉散射顯微術（iSCAT），這是一種基於散射干涉的顯微技術，用以量測與動作電位相關的散射訊號。我們同時記錄了 1 分鐘長度的螢光與 iSCAT 影像，以捕捉發生在數秒時間尺度上的自發膜電位動態。對螢光與 iSCAT 影像資料分別進行空間與時間濾波後，我們探討了兩種信號之間的相關性。結果顯示螢光膜電位訊號與 iSCAT 訊號之間存在約 0.3 的負相關，且不同細胞之間有顯著差異。此外，我們設計了兩種控制實驗條件：其一為在無 L-阿拉伯糖培養的細胞（缺乏螢光反應），其二為經 CCCP 處理的細胞（喪失膜電位）。這些對照實驗確認了所觀測到的散射訊號確實與膜電位變化相關。我們的結果顯示，細菌自發的膜電位動態會產生可由 iSCAT 顯微術量測的訊號。然而，膜電位變化同時伴隨非特異性散射背景，可能來自細胞分子層級的波動。 本研究證實了以 iSCAT 顯微術進行無標記、直接觀察單一細菌膜電位動態的可行性，為以非侵入且長時間量測方式研究膜電位在細胞代謝與能量調控中的角色奠定了基礎。

In excitable cells, including neurons, muscle cells, and certain endocrine cells, membrane potentials are generated through the opening and closing of voltage-gated ion channels, allowing ion fluxes driven by electrochemical gradients, resulting in changes in membrane potential that propagate electrical signals. Current techniques, such as whole cell patch clamp and exogenous voltage-sensitive fluorescent probes, are commonly used to measure membrane potential. However, the patch clamp technique may damage cells, and voltage-sensitive fluorescent probes are susceptible to phototoxicity and photobleaching, thereby limiting signal acquisition. Recent advancements in scattering based interference microscopy have enabled label-free detection of nanoscale structures with high sensitivity and speed, opening new opportunities for monitoring rapid electrical events such as bacterial electrophysiology. Some studies have already employed interference-based optical methods to image ion channel activity in neuroscience, demonstrating their applicability for membrane potential detection. However, research on bacterial membrane potential remains limited, and effective label-free methods for probing these fast processes are still lacking. In this study, we demonstrate the measurement of bacterial membrane potentials using scattering-based interference microscopy. We used E. coli as our model system, in which fluorescence voltage indicator ViBac1 was genetically encoded, providing a membrane potential (Vm) readout. Meanwhile, we employed interferometric scattering microscopy (iSCAT), a scattering-based interference microscopy technique, to measure the scattering signal associated with membrane potentials. Simultaneous fluorescence and iSCAT videos were recorded for 1 minute to capture spontaneous membrane potential dynamics in the imescales of a few seconds. Spatial and temporal filtering were applied to both the fluorescence and iSCAT image data, aiming to explore correlations between the two signals. A modest anticorrelation of -0.3 was observed between the time-varying fluorescence Vm signal and the iSCAT signals, with a considerable cell-to-cell heterogeneity. Control experiments were performed under two conditions: cells grown without L-arabinose (lacking fluorescence response) and cells treated with CCCP (lacking membrane potential). These controls confirmed that the observed scattering signals are related to membrane potential changes. Our results show that the spontaneous bacterial membrane potential dynamics create a signal that can be measured by iSCAT microscopy. However, the membrane potential is accompanied by a nonspecific scattering background, presumably due to the molecular fluctuations of the cell. This study demonstrates the feasibility of label-free, direct visualization of membrane potential dynamics at single bacterium resolution, paving the way for investigating its roles in cellular metabolism and bioenergetic regulation through noninvasive, long-term measurements.