Abstract
Observation of highly dynamic processes inside living cells at the single molecule level is key for a better understanding of biological systems. However, imaging of single molecules in living cells is usually 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, for example, in a reflected geometry with a high numerical aperture imaging objective, have been developed. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-colour acquisition of -stacks. We demonstrate fast volume scanning by imaging a two-colour giant unilamellar vesicle (GUV) hemisphere. In addition, the high contrast enabled the imaging and tracking of single lipids in the GUV cap. The enhanced reflected scanning light-sheet microscope enables fast 3D scanning of artificial membrane systems and potentially live cells with single-molecule sensitivity and thereby could provide quantitative and molecular insight into the operation ofcells.
Highlights
The ability to image and track single molecules in living cells in real time and three-dimensions (3D) represents one of the big challenges in microscopy
The light-sheet setup enables fast 3D imaging of giant unilamellar vesicle (GUV) and single cells using a thin light sheet reflected by a goldcoated atomic-force-microscopy cantilever placed next to the sample of interest
We presented an reflected light-sheet microscope (RLSM) optimized for fast 3D two-colour imaging
Summary
The ability to image and track single molecules in living cells in real time and three-dimensions (3D) represents one of the big challenges in microscopy. A powerful approach to improve the precision of single molecule localization, is to maximize the collection of emitted photons and reduce the background signal from out-of-focus fluorophores. Total internal reflection fluorescence microscopy (TIRFM), widely used for single molecule studies, illuminates only a thin plane near the glass–sample interface. This reduction in illumination volume results in a low background and high contrast of single fluorophores. Due to the intrinsically restricted geometry of illumination, TIRFM is not suitable for 3D imaging of whole cells.
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