Lees Robert M, Bianco Isaac H, Campbell Robert A A, Orlova Natalia, Peterka Darcy S, Pichler Bruno, Smith Spencer LaVere, Yatsenko Dimitri, Yu Che-Hang, Packer Adam M
Science and Technology Facilities Council, Octopus imaging facility, Research Complex at Harwell, Harwell Campus, Oxfordshire, UK.
Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK.
Nat Protoc. 2025 Mar 17. doi: 10.1038/s41596-024-01120-w.
The goal of this protocol is to improve the characterization and performance standardization of multiphoton microscopy hardware across a large user base. We purposefully focus on hardware and only briefly touch on software and data analysis routines where relevant. Here we cover the measurement and quantification of laser power, pulse width optimization, field of view, resolution and photomultiplier tube performance. The intended audience is scientists with little expertise in optics who either build or use multiphoton microscopes in their laboratories. They can use our procedures to test whether their multiphoton microscope performs well and produces consistent data over the lifetime of their system. Individual procedures are designed to take 1-2 h to complete without the use of expensive equipment. The procedures listed here help standardize the microscopes and facilitate the reproducibility of data across setups.
本协议的目标是改善跨大量用户群体的多光子显微镜硬件的特性描述和性能标准化。我们特意专注于硬件，仅在相关情况下简要提及软件和数据分析程序。在这里，我们涵盖了激光功率的测量和量化、脉冲宽度优化、视野、分辨率以及光电倍增管性能。目标受众是在光学方面专业知识较少、在实验室中构建或使用多光子显微镜的科学家。他们可以使用我们的程序来测试其多光子显微镜在系统使用寿命内是否性能良好并产生一致的数据。各个程序设计为在不使用昂贵设备的情况下1 - 2小时内完成。此处列出的程序有助于使显微镜标准化，并促进不同设置下数据的可重复性。
