Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

Abstract

Observation of highly dynamic processes inside living cells at the single molecule level is key for a quantitative understanding of biological systems. However, imaging of single molecules in living cells usually is limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination have recently been developed. For example, a reflected light-sheet design combines the illumination by a thin light-sheet with a high numerical aperture objective for single-molecule detection. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-color acquisition of z-stacks. We demonstrate fast volume scanning by imaging a two-color giant unilamellar vesicle (GUV) hemisphere. In addition, the high signal-to-noise ratio enabled the imaging and tracking of single lipids in the cap of a GUV. In the long term, the enhanced reflected scanning light sheet microscope enables fast 3D scanning of artificial membrane systems and cells with single-molecule sensitivity and thereby will provide quantitative and molecular insight into the operation of cells.