Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control
Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control
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DOI：
10.1038/nprot.2011.407
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发表时间：
2011-12-01
期刊：
影响因子：
14.8
通讯作者：
Brown, Claire M.
中科院分区：
文献类型：
--
作者：
Cole, Richard W.;Jinadasa, Tushare;Brown, Claire M.
该协议概述了收集和分析点扩散函数 (PSF) 的程序。它描述了如何制备荧光微球样品、设置共焦显微镜以正确收集微球的 3D 共焦图像数据并执行 PSF 测量。 PSF 分析用于确定显微镜的分辨率并识别显微镜图像质量的任何问题。 PSF 几何形状用作识别物镜、共焦激光扫描组件和其他中继光学器件问题的指标。提供了 PSF 异常的可能原因的识别以及提高显微镜性能的解决方案。微球样品制备需要 2-3 小时加上过夜干燥时间。显微镜设置需要 2 小时（激光预热 1 小时），而收集和分析 PSF 图像需要额外的 2-3 小时。
This protocol outlines a procedure for collecting and analyzing point spread functions (PSFs). It describes how to prepare fluorescent microsphere samples, set up a confocal microscope to properly collect 3D confocal image data of the microspheres and perform PSF measurements. The analysis of the PSF is used to determine the resolution of the microscope and to identify any problems with the quality of the microscope's images. The PSF geometry is used as an indicator to identify problems with the objective lens, confocal laser scanning components and other relay optics. Identification of possible causes of PSF abnormalities and solutions to improve microscope performance are provided. The microsphere sample preparation requires 2-3 h plus an overnight drying period. The microscope setup requires 2 h (1 h for laser warm up), whereas collecting and analyzing the PSF images require an additional 2-3 h.