Local illumination-enhanced live-cell single-molecule fluorescence and single-channel patch-clamp coupling technology

Channel proteins act as precise molecular regulators of transmembrane transport, which is a fundamental process essential for maintaining cellular homeostasis. These proteins dynamically modulate their functional states through conformational changes, thereby 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 in single-molecule imaging in cellular environments. To address this limitation, we develop a spatially selective optical excitation system capable of localized illumination. By integrating tunable optical modules, we generate 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 achieves submicron-scale alignment between the excitation zone and the micropipette contact area, enabling simultaneous electrophysiological recording and background-suppressed fluorescence detection within the clamped membrane domain. Experimental validation demonstrates that the systemcan perform single-molecule fluorescence imaging and trajectory analysis within a specified observation areas, with imaging resolution inversely related to the size of the illuminated region. Optimized optical design allows for precise excitation targeting while minimizing background illumination, thereby achieving high signal-to-noise ratio single-molecule imaging and significantly reducing photodamage. Integration with cell-attached patch-clamp configurations establishes a dual-modality platform for synchronized acquisition of single-molecule fluorescence images and single-channel recordings. The validation using mechanosensitive mPiezo1 channels confirms the system’s compatibility with single-channel recording, indicating that optical imaging induces no detectable interference to electrophysiological signal acquisition. This method overcomes longstanding challenges in the simultaneous application of single-molecule imaging and electrophysiological techniques in live-cell environments. It establishes a novel experimental framework for investigating the structure-function relationships of channel proteins and membrane-related molecular machines through spatially coordinated optoelectronic measurements on live-cell membranes, which has broad applicability in molecular biophysics and transmembrane transport mechanism research.