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

项目摘要 光片显微镜已经成为研究各种生物学问题的必要工具，从 从发育问题到神经回路的映射。薄片显微镜因其优异的性能而备受关注 厚样品的光学切片，低光毒性，可进行多天高帧成像 使捕获正在放电的神经元成为可能的速率。许多不同类型的光片显微镜已经被 设计和建造，其中许多都具有令人印象深刻的能力。高速同步多视点(hs- SiMView)系统和扫描共焦对准平面激发(SCAPE)显微镜 体积成像速度为每秒1到10个体积，分辨率为0.5到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纤维层。晶格薄片显微镜(LLSM)可以实现令人印象深刻的亚 200 nm分辨率，但该系统是各向异性的，仅限于较小的样品。 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，它将是 能够在所有维度上以320 nm的分辨率进行快速各向同性成像，分辨率可低至180 nm x 180 nm x 320 nm，使用全结构照明方法。这台显微镜将采用一种简单的立式。 样例安装几何结构，并将进一步自然地纳入多方向灯片，这将有助于 消除阴影伪影。这个系统将有大约30倍的体积分辨率的状态- 用于斑马鱼幼虫或果蝇成像的最先进的光片显微镜系统。通过允许生物学家 具有研究亚细胞结构或追踪神经过程的分辨率的图像模型生物，这 该项目将帮助破译神经回路，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.