Center for Plant Molecular Biology (ZMBP), University of Tübingen, Auf der Morgenstelle 32, Tübingen, Germany.
Max Planck Institute for Solid State Physics, Heisenbergstrasse 1, Stuttgart, Germany.
J Microsc. 2022 Jan;285(1):40-51. doi: 10.1111/jmi.13070. Epub 2021 Dec 8.
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 of cells.
在单细胞水平上观察活细胞内的高度动态过程对于更好地理解生物系统至关重要。然而，在活细胞中对单个分子进行成像通常受到空间和时间分辨率、光漂白和信号背景比的限制。为了克服这些限制，已经开发出了具有薄选择性平面照明的光片显微镜，例如，在具有高数值孔径成像物镜的反射几何形状中。在这里，我们开发了一种具有主动光学的反射式光片显微镜，用于快速、高对比度的双色采集 - 堆叠。我们通过对双色巨单层囊泡 (GUV) 半球进行成像来演示快速体积扫描。此外，高对比度还能够对 GUV 帽中的单个脂质进行成像和跟踪。增强的反射扫描光片显微镜能够以单细胞灵敏度快速扫描人工膜系统和潜在的活细胞，从而为细胞的运作提供定量和分子见解。
