A small excitation window allows long-duration single-molecule imaging, with reduced background autofluorescence, in C. elegans neurons

• DOI: 10.1016/j.optcom.2023.129700
• Publication Date: 2023-06-23T00:15:22Z
• Authors: Aniruddha Mitra; Elizaveta Loseva; Guus H. Haasnoot; Erwin J.G. Peterman
• Keywords: Photobleaching; Autofluorescence; Fluorescence-lifetime imaging microscopy; Live cell imaging

Single-particle imaging using laser-illuminated widefield epi-fluorescence microscopy is a powerful tool to investigate molecular processes in vivo. Performing high-quality single-molecule such biological systems, however, remains challenge due difficulties controlling the number of fluorescing molecules, photobleaching, and autofluorescence background. Here, we show that by exciting only small, 5-15 μm wide region chemosensory neurons live C. elegans, can significantly improve duration quality imaging. Small-window illumination (SWIM) allows long-duration single-particle since fluorescently labelled proteins are excited upon entering small area, limiting their photobleaching. Remarkably, also find excitation window improves signal-to-background ratio individual particles. With help theoretical calculations, explain improved reduced background, mostly caused out-of-focus autofluorescence. We demonstrate potential this approach studying dendritic transport ciliary calcium channel protein, OCR-2, elegans. reveal OCR-2-associated vesicles continuously transported back forth along length dendrite switch between directed diffusive states. Furthermore, perform tracking quantitatively characterize dynamics. SWIM be readily applied other vivo systems where intracellular or cytoskeletal dynamics occur elongated protrusions, as axons, dendrites, cilia, microvilli extensions fibroblasts.