Analysis of highly dynamic sub­cellular mechanisms by Lattice Light Sheet Microscopy

通过晶格光片显微镜分析高度动态的亚细胞机制

• 批准号: 413831413
• 金额: --
• 依托单位: Universität Hamburg
• 依托单位国家: 德国
• 项目类别: Major Instrumentation Initiatives
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/413831413?language=en
• 关键词: Analysis; highly; dynamic; sub; cellular

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).

目前生物医学研究中使用的大多数光学显微镜技术都是基于宽视场或共焦落射荧光照明。在这些模态中，激发轴和检测轴重叠，导致未聚焦的细胞区域的不必要曝光。当整个活细胞以高帧率或长时间成像时，这成为一个问题。结果是明显的光毒性和漂白。因此，在延长的时间段内对单个细胞进行成像以在亚细胞分辨率下监测随机事件提出了重大挑战。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。分离的照明和检测轴所做的光片显微镜提供了一个解决方案，光毒性和漂白问题。为此，两个物镜彼此垂直地布置，并且照明物镜将薄光片投射到样品中，仅照明实际成像的区域。薄片厚度理想地与检测物镜的z分辨率匹配，以最小化光损伤，从而增加用于后续生物过程的时空窗口。具有细胞分辨率的几种光片设计目前是市售的，通常适合于分析例如整个果蝇胚胎或植物根发育，而且也适合于使三维移动的细胞可视化（Fritz-Laylin等人，2017年）。最近，出现了能够研究亚细胞事件的第一个光片系统（Aguet等人，2016年，Cai et al.，2017年）。特别是诺贝尔奖获得者Erik Betzig的实验室已经开发了一种使用无衍射贝塞尔光束的相应方法（Gao，Shao，Chen，& Betzig，2014; T. Liu等人，2018）以产生薄的片材，同时提供更大的视场。这种技术的最新实现被称为晶格光片（LLS）显微术，因为它使用抖动贝塞尔光束（Chen等人，2014）用于片材均匀性。它允许产生理想尺寸的薄片（0.4-0.8 μm或更薄），以实现亚细胞分辨率，同时保持大视野以成像整个贴壁细胞。网格片在较长距离上较薄。由于片材厚度与检测物镜焦平面相匹配，因此仅聚焦的细胞结构被照射。这样，细胞被照射得少得多，并且Z轴分辨率被提高到超过共焦显微镜的能力（Aguet et al.，2016年; Chen等人，2014; Legant等人，2016年12月。Liu等人，2014年）。