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Channel proteins act as precise molecular regulators of transmembrane transport, a fundamental process essential for maintaining cellular homeostasis. These proteins dynamically modulate their functional states through conformational changes, forming the structural basis for complex physiological processes such as signal transduction and energy metabolism. Single-molecule fluorescence spectroscopy and single-channel patch-clamp electrophysiology represent two cornerstone techniques in modern biophysics: the former enables molecular-resolution analysis of structural dynamics, while the latter provides direct functional characterization of ion channel activity. Despite their complementary capabilities, integrating these techniques to simultaneously monitor protein conformational dynamics and functional states remains technically challenging, primarily due to the strong autofluorescence background inherent to single-molecule imaging in cellular environments. To address this limitation, we developed a spatially selective optical excitation system capable of localized illumination. By integrating tunable optical modules, we generated a dynamically adjustable excitation field on living cell membranes, achieving precise spatial registration between the excitation volume and the patch-clamp recording site. This system achieved submicron-scale alignment between the excitation zone and the micropipette contact area, enabling simultaneous electrophysiological recording and background-suppressed fluorescence detection within the patched membrane domain. Experimental validation demonstrated the system’s ability to perform single-molecule fluorescence imaging and trajectory analysis within designated observation areas, with imaging resolution inversely correlated with the size of the illuminated region. Optimized optical design allowed for precise excitation targeting while minimizing background illumination, resulting in high signal-to-noise ratio single-molecule imaging with significantly reduced photodamage. Integration with cell-attached patch-clamp configurations established a dual-modality platform for synchronized acquisition of single-molecule fluorescence images and single-channel recordings. Validation using mechanosensitive mPiezo1 channels confirmed the system’s compatibility with single-channel recordings, demonstrating that optical imaging induces no detectable interference with electrophysiological signal acquisition. This methodology overcomes longstanding challenges in the concurrent application of single-molecule imaging and electrophysiological techniques in live-cell environments. It establishes a novel experimental framework for investigating structure–function relationships in channel proteins and membrane-associated molecular machines through spatially coordinated optoelectronic measurements on live-cell membranes, with broad applicability in molecular biophysics and studies of transmembrane transport mechanisms.
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Keywords:
- Channel Proteins /
- Live-Cell Dynamic Imaging /
- Single-Molecule Fluorescence Imaging /
- Cell-Attached Recording /
- Single-Molecule Tracking
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