Superresolution Light Sheet Microscopy for imaging model organisms with 200nm resolution

项目摘要 光片显微镜已成为研究各种生物学问题的重要工具， 发展问题映射神经回路。光片显微镜是有吸引力的，由于其出色的 厚样品中的光学切片，低光毒性，可允许多天成像和高帧 这使得捕捉放电神经元成为可能。许多不同类型的光片显微镜已被 设计和建造，其中许多都有令人印象深刻的能力。高速同步多视图（hs- SiMView）系统和扫描共焦对准平面激发（SCAPE）显微镜可以执行 体积成像，每秒1至10个体积，分辨率为0.5至2微米。虽然这些系统 产生奇妙的结果，他们没有分辨率来研究亚细胞细节或追踪轴突， 树突穿过致密的神经鞘。点阵光片显微镜（LLSM）可以实现令人印象深刻的亚显微镜， 200 nm的分辨率，但该系统是各向异性的，并限于较小的样品。 在这里，我们建议开发一种新型的单物镜结构照明光片显微镜， 能够在所有维度上以320 nm的分辨率快速各向同性成像，并且分辨率低至180 nm x 180 nm x 320 nm，使用全结构化照明方法。这台显微镜将采用一个简单的直立 样品安装几何形状，并将进一步自然地结合多方向光片，这将有助于 消除阴影伪影。该系统将具有大约30倍的状态体积分辨率- 最先进的光片显微镜系统，用于斑马鱼幼虫或果蝇成像。通过让生物学家 图像模型生物的分辨率，以研究亚细胞结构或跟踪神经过程，这 该项目将有助于解码神经电路，破译发育途径，并阐明 疾病

Project Summary Light Sheet Microscopy has become an essential tool for investigating a wide variety of biological problems from developmental questions to mapping neural circuits. Light Sheet Microscopy is attractive due to its excellent optical sectioning in thick samples, low phototoxicity which can allow imaging over multiple days and high frame rates which make it possible to capture firing neurons. Many different types of Light Sheet Microscope have been designed and built, many of which have impressive capabilities. The high-speed Simultaneous Multi-View (hs- SiMView) system and the Swept Confocally Aligned Planar Excitation (SCAPE) microscope can perform volumetric imaging at 1 to 10 volumes per second with resolutions of 0.5 to 2 microns. While these systems produce wonderful results, they do not have the resolution to investigate sub-cellular details or trace axons and dendrites through dense neuropil. The Lattice Light Sheet Microscope (LLSM) can achieve an impressive sub- 200 nm resolution but the system is anisotropic and limited to smaller samples. Here we propose to develop a novel single-objective structured-illumination light-sheet microscope that will be capable of fast isotropic imaging at a resolution of 320 nm in all dimensions and resolutions down to 180 nm x 180 nm x 320 nm using a full structured illumination approach. This microscope will employ an easy upright sample mounting geometry and will further naturally incorporate a multi-direction light sheet which will help to eliminate shadowing artifacts. This system will have approximately 30 times the volumetric resolution of state- of-the-art Light Sheet Microscope systems for imaging zebrafish larvae or fruit flies. By allowing biologists to image model organisms with the resolution to investigate subcellular structures or trace neural processes, this project will help decode neural circuitry, decipher developmental pathways, and elucidate the mechanisms of disease.