Chan Zuckerberg Biohub, San Francisco, CA, USA.
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, USA.
Nat Protoc. 2018 Nov;13(11):2462-2500. doi: 10.1038/s41596-018-0043-4.
We describe the implementation and use of an adaptive imaging framework for optimizing spatial resolution and signal strength in a light-sheet microscope. The framework, termed AutoPilot, comprises hardware and software modules for automatically measuring and compensating for mismatches between light-sheet and detection focal planes in living specimens. Our protocol enables researchers to introduce adaptive imaging capabilities in an existing light-sheet microscope or use our SiMView microscope blueprint to set up a new adaptive multiview light-sheet microscope. The protocol describes (i) the mechano-optical implementation of the adaptive imaging hardware, including technical drawings for all custom microscope components; (ii) the algorithms and software library for automated adaptive imaging, including the pseudocode and annotated source code for all software modules; and (iii) the execution of adaptive imaging experiments, as well as the configuration and practical use of the AutoPilot framework. Setup of the adaptive imaging hardware and software takes 1-2 weeks each. Previous experience with light-sheet microscopy and some familiarity with software engineering and building of optical instruments are recommended. Successful implementation of the protocol recovers near diffraction-limited performance in many parts of typical multicellular organisms studied with light-sheet microscopy, such as fruit fly and zebrafish embryos, for which resolution and signal strength are improved two- to fivefold.
我们描述了一种自适应成像框架的实现和使用，该框架用于优化光片显微镜中的空间分辨率和信号强度。该框架称为 AutoPilot，包括用于自动测量和补偿活标本中光片和检测焦平面之间不匹配的硬件和软件模块。我们的方案使研究人员能够在现有的光片显微镜中引入自适应成像功能，或使用我们的 SiMView 显微镜蓝图来设置新的自适应多视角光片显微镜。该方案描述了：（i）自适应成像硬件的机械光学实现，包括所有定制显微镜组件的技术图纸；（ii）用于自动自适应成像的算法和软件库，包括所有软件模块的伪代码和注释源代码；（iii）自适应成像实验的执行，以及 AutoPilot 框架的配置和实际使用。自适应成像硬件和软件的设置每个需要 1-2 周。建议具有光片显微镜经验，并且对软件工程和光学仪器构建有一定了解。该方案的成功实施可恢复许多典型多细胞生物中光片显微镜研究的近衍射极限性能，例如果蝇和斑马鱼胚胎，其分辨率和信号强度提高了两到五倍。
