Lattice Light Sheet Microscope (LLSM)

晶格光片显微镜 (LLSM)

• 批准号: 413891558
• 金额: --
• 依托单位: Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
• 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 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/413891558?language=en
• 关键词: Lattice; Light; Sheet; Microscope; LLSM

One major goals of light microscopy is to analyze the dynamic behavior of molecular and cellular networks under physiological conditions. The technical obstacles in achieving this goal has always been the simultaneous pursuit of imaging depth, speed, spatial resolution and specimen viability. The development of light sheet microscopy, which couples a separate excitation lens in perpendicular axis to the detection lens, has made it possible to confine the excitation of the specimen only to the volume being observed. This reduced phototoxicity significantly and simultaneously increased the image acquisition speed through its wide-field light collection mode.However, the diffracting Gaussian beam used for the light sheet in common systems imposed limits on the optical resolution that could be achieved. This limitation was overcome by introducing a non-diffracting Bessel beam in light sheet microscopy (1). The further improved ‘lattice light sheet microscope’ (LLSM) generated an ultrathin light sheet (~ 400 nm) improving the axial resolution and allowing for very fast volumetric imaging (2). Importantly, the LLSM caused significantly less photodamage compared to commonly used methods for 3D high-resolution live-specimen imaging (i.e. spinning disk confocal). The LLSM is thus a novel tool to analyze fast 3D sub-cellular dynamics in cells or small organisms with high SNR and low photodamage. Recent applications of the LLSM have successfully visualized and quantified sub-cellular processes with unprecedented details and imaging volumes (i.e. (2–6)). The optional LLS-SIM (structured illumination microscopy) mode or the combination of LLSM with single molecule localization microscopy offer super-resolution applications with spatial resolutions beyond the diffraction limit (2, 7).With this proposal we aim at establishing the first commercially available LLSM in Germany within the centrally located Advanced Medical Bioimaging core facility of the Charité – Universitätsmedizin Berlin (AMBIO). This way we ensure that the LLSM will be accessible to all academic researchers, maintained by experts, and that the researchers receive professional training and assistance to pursue their biological applications. The high-profile research projects within the Berlin research community (listed in 2.5) all require fast, 3D live-specimen imaging with low phototoxicity and would greatly benefit from the LLSM. Three of these groups have already used an early version of the LLSM with very promising results from cultured cells and living brain. Beyond these projects, about 30% of the usage time will be available for any other research group with suitable LLSM projects within the German research community. We will publish an annual report about the performance (and limitations) of the LLSM to help the research community in evaluating whether this novel method would be suitable for their project.

光学显微镜的一个主要目标是分析生理条件下分子和细胞网络的动态行为。实现这一目标的技术障碍一直是同时追求成像深度，速度，空间分辨率和标本的生存能力。光片显微镜的发展，它耦合一个独立的激励透镜在垂直轴的检测透镜，使其有可能限制激发的标本只被观察到的体积。这大大降低了光毒性，同时通过其宽视场光收集模式提高了图像采集速度。然而，普通系统中用于光片的衍射高斯光束对可以实现的光学分辨率施加了限制。通过在光片显微镜中引入无衍射贝塞尔光束克服了这一限制（1）。进一步改进的“晶格光片显微镜”（LLSM）产生了一个平行光片（~ 400 nm），提高了轴向分辨率，并允许非常快速的体积成像（2）。重要的是，LLSM引起的光损伤显着减少相比，常用的方法为3D高分辨率活体标本成像（即旋转盘共焦）。因此，LLSM是一种新的工具，用于分析细胞或小生物体中的快速3D亚细胞动力学，具有高SNR和低光损伤。LLSM的最新应用已经成功地可视化和量化了具有前所未有的细节和成像体积的亚细胞过程（即（2-6））。可选的LLS-SIM（结构照明显微镜）模式或LLSM与单分子定位显微镜的组合提供了空间分辨率超过衍射极限的超分辨率应用（2，7）。通过该提案，我们的目标是在位于中心的Charité - Universitätsmedizin柏林（AMBIO）先进医学生物成像核心设施内建立德国第一个商业化的LLSM。通过这种方式，我们确保LLSM将可供所有学术研究人员使用，由专家维护，并且研究人员接受专业培训和协助，以追求其生物学应用。柏林研究界的高调研究项目（列于2.5）都需要快速、低光毒性的3D活体标本成像，并将大大受益于LLSM。其中三个小组已经使用了LLSM的早期版本，从培养的细胞和活体大脑中获得了非常有希望的结果。除了这些项目之外，大约30%的使用时间将可用于德国研究界内任何其他具有合适LLSM项目的研究小组。我们将发布一份关于LLSM性能（和局限性）的年度报告，以帮助研究界评估这种新方法是否适合他们的项目。