Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control

• Published in: Nature protocols Vol. 6; no. 12; pp. 1929 - 1941
• Main Authors: Cole, Richard W, Jinadasa, Tushare, Brown, Claire M
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.12.2011; Nature Publishing Group
• Subjects: 631/1647/328/1978; Analysis; Analytical Chemistry; Biological Techniques; Biomedical and Life Sciences; Computational Biology/Bioinformatics; Drying; Image processing; Imaging, Three-Dimensional - methods; Lasers; Life Sciences; Light; Microarrays; Microscope and microscopy; Microscopy; Microscopy, Confocal - methods; Microscopy, Confocal - standards; Microspheres; Optics; Organic Chemistry; Protocol; Quality Control; Sample preparation; Weights and Measures; Canada; United States; New York; Montreal Quebec Canada; United States > US; Quebec Canada
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2011.407

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.