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Measurement accuracy in confocal microscopy
Author(s) -
Richard Delorme,
Mehdi Benchaïb,
Bryon Pa,
Catherine Souchier
Publication year - 1998
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1046/j.1365-2818.1998.00423.x
Subject(s) - confocal , colocalization , confocal microscopy , microscopy , light sheet fluorescence microscopy , scanning confocal electron microscopy , microscope , materials science , optics , fluorescence microscope , biological system , computer science , biophysics , fluorescence , biology , microbiology and biotechnology , physics
Confocal laser scanning microscopy provides optical serial sections through thick biological samples, making it possible to perform both three‐dimensional visualization and three‐dimensional quantitative analysis. On human lymphocytes, we measured geometrical features, cell contents in DNA and in cyclin A and CDK1 proteins, localization and colocalization of these two proteins. Cells were acquired at a vertical sampling step of 0.5 μm, which gives sufficient information about cell labelling. For the purpose of obtaining fast and reliable data at a reduced time cost, we examined various possibilities to simplify acquisition. For example, it might be possible to increase the vertical sampling step to 2.0 μm while preserving an acceptable accuracy of measurements. Further limiting the acquisition to the central sections appeared to give only rough estimations about the whole cells. Finally, we compared confocal microscopy to conventional two‐dimensional epifluorescence microscopy. Confocal microscopy appeared slightly less accurate as regards content estimation, but was an invaluable tool when investigating three‐dimensional structures and, more especially, localization of proteins.