光片显微镜的技术实现
Technical implementations of light sheet microscopy.
作者信息
Elisa Zagato, Toon Brans, De Smedt Stefaan C, Katrien Remaut, Kristiaan Neyts, Kevin Braeckmans
机构信息
Laboratory of General Biochemistry and Physical Pharmacy, Center for Nano- and Biophotonics, Ghent University, Belgium.
Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Belgium.
出版信息
Microsc Res Tech. 2018 Sep;81(9):941-958. doi: 10.1002/jemt.22981. Epub 2018 Jan 11.
Fluorescence-based microscopy is among the most successful methods in biological studies. It played a critical role in the visualization of subcellular structures and in the analysis of complex cellular processes, and it is nowadays commonly employed in genetic and drug screenings. Among the fluorescence-based microscopy techniques, light sheet fluorescence microscopy (LSFM) has shown a quite interesting set of benefits. The technique combines the speed of epi-fluorescence acquisition with the optical sectioning capability typical of confocal microscopes. Its unique configuration allows the excitation of only a thin plane of the sample, thus fast, high resolution imaging deep inside tissues is nowadays achievable. The low peak intensity with which the sample is illuminated diminishes phototoxic effects and decreases photobleaching of fluorophores, ensuring data collection for days with minimal adverse consequences on the sample. It is no surprise that LSFM applications have raised in just few years and the technique has been applied to study a wide variety of samples, from whole organism, to tissues, to cell clusters, and single cells. As a consequence, in recent years numerous set-ups have been developed, each one optimized for the type of sample in use and the requirements of the question at hand. Hereby, we aim to review the most advanced LSFM implementations to assist new LSFM users in the choice of the LSFM set-up that suits their needs best. We also focus on new commercial microscopes and "do-it-yourself" strategies; likewise we review recent designs that allow a swift integration of LSFM on existing microscopes.
基于荧光的显微镜技术是生物学研究中最成功的方法之一。它在亚细胞结构的可视化以及复杂细胞过程的分析中发挥了关键作用,如今常用于基因和药物筛选。在基于荧光的显微镜技术中,光片荧光显微镜(LSFM)展现出了一系列颇为有趣的优势。该技术将落射荧光采集的速度与共聚焦显微镜典型的光学切片能力相结合。其独特的配置仅允许激发样品的一个薄平面,因此如今能够实现对组织深处进行快速、高分辨率成像。样品被照射时的低峰值强度减少了光毒性效应,并降低了荧光团的光漂白现象,确保能够在数天内收集数据,且对样品的不良影响最小。毫不奇怪,LSFM的应用在短短几年内就有所增加,并且该技术已被应用于研究各种各样的样品,从整个生物体到组织、细胞簇和单细胞。因此,近年来已经开发出了许多装置,每一种都针对所使用的样品类型和手头问题的要求进行了优化。在此,我们旨在回顾最先进的LSFM实施方案,以帮助新的LSFM用户选择最适合其需求的LSFM装置。我们还关注新的商业显微镜和“自己动手做”策略;同样地,我们回顾了最近能够将LSFM快速集成到现有显微镜上的设计。