Analysis of highly dynamic sub­cellular mechanisms by Lattice Light Sheet Microscopy

目前在生物医学研究中使用的大多数光学显微镜技术都是基于广角或共聚焦的会聚荧光照明。在这些模式中，激发轴和检测轴重叠，导致不必要的暴露不在焦点上的细胞区域。当整个活细胞以高帧率或长时间成像时，这就成为一个问题。结果就是明显的光毒性和漂白。因此，对单个细胞进行长时间成像以监测亚细胞分辨率下的随机事件是一项重大挑战。然而，这种能力对于在分子机制水平上理解细胞和致病过程的动力学至关重要。在光片显微镜中，照明和检测轴的分离为光毒性和漂白问题提供了解决方案。为此，两个物镜彼此垂直排列，照明物镜将一薄层光投射到样品中，只照亮实际成像的区域。薄片厚度与检测物镜的z分辨率理想匹配，以最大限度地减少光损伤，从而增加后续生物过程的时空窗口。目前市面上有几种具有细胞分辨率的薄片设计，通常适用于分析整个果蝇胚胎或植物根系发育，但也适用于三维可视化细胞运动（Fritz-Laylin等人，2017）。最近，第一个能够研究亚细胞事件的光片系统出现了（Aguet et al., 2016; Cai et al., 2017）。特别是诺贝尔奖得主Erik Betzig的实验室开发了一种使用非衍射贝塞尔光束的方法(Gao, Shao, Chen, & Betzig, 2014；Liu et al., 2018)生成薄的薄片，同时提供更大的视野。这项技术的最新实现被称为晶格光片（LLS）显微镜，因为它使用抖动贝塞尔光束（Chen et al., 2014）来实现片的均匀性。它允许生成薄片（0.4-0.8 μm及更薄），其理想尺寸适合亚细胞分辨率，同时保持大视野以成像整个贴壁细胞。晶格片在较长的距离上越薄。由于薄片的厚度与检测物镜的焦平面相匹配，因此只照亮焦内的细胞结构。通过这种方式，细胞受到的照射要少得多，并且z分辨率提高到超过共聚焦显微镜的能力（Aguet et al., 2016; Chen et al., 2014; Legant et al., 2016； Z. Liu et al., 2014）。

Most light microscope techniques currently employed in biomedical research are based on widefield or confocal epifluorescence illumination. In these modalities, the excitation and detection axis overlap, leading to unnecessary exposure of cellular regions that are not in focus. This becomes a problem when whole, living cells are imaged at high frame rates or for long time. Pronounced phototoxicity and bleaching are the consequence. Hence, imaging of individual cells for extended time periods to monitor stochastic events at sub-cellular resolution presents a major challenge. Such capabilities, however, are critically essential to understand the kinetics of cellular and pathogenic processes at the level of molecular mechanisms. Separation of the illumination and detection axis as done in light sheet microscopy offers a solution to photo-toxicity and bleaching problems. For this, two objectives are arranged perpendicular to each other, and the illumination objective projects a thin sheet of light into the sample only illuminating the region that is actually imaged. The sheet thickness is ideally matched to the detection objective’s z-resolution, to minimize photo-damage, thus increasing the spatio-temporal window for following biological processes. Several light sheet designs with cellular resolution are currently commercially available, typically suited to analyse e.g. whole drosophila embryos or plant root development, but also to visualize cells moving in three dimensions (Fritz-Laylin et al., 2017). Recently, first light sheet systems that are capable of studying sub-cellular events emerged (Aguet et al., 2016; Cai et al., 2017). Especially the lab of Nobel laureate Erik Betzig has developed a respective method using non­diffracting Bessel beams (Gao, Shao, Chen, & Betzig, 2014; T.­L. Liu et al., 2018) to generate sheets that are thin and at the same time provide a larger field of view. The latest implementation of this technique is called lattice light sheet (LLS) microscopy since it uses dithered Bessel beams (Chen et al., 2014) for sheet homogeneity. It allows the generation of sheets (0.4-0.8 μm and thinner) that are ideally sized for sub-cellular resolution while keeping a large field of view to image whole, adherent cells. The lattice sheet is thinner over a longer distance. Since the sheet thickness is matched to the detection objectives focal plane, only cellular structures in focus are illuminated. This way, cells are irradiated much less and, z­resolution is improved beyond capabilities of confocal microscopes (Aguet et al., 2016; Chen et al., 2014; Legant et al., 2016; Z. Liu et al., 2014).