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Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis
Author(s) -
Varga Viktor Sebestyén,
Bocsi József,
Sipos Ferenc,
Csendes Gábor,
Tulassay Zsolt,
Molnár Béla
Publication year - 2004
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20027
Subject(s) - fluorescence , microscopy , fluorescence microscope , scanning electron microscope , calibration , cytometry , biomedical engineering , microscope , laser scanning , rhodamine , materials science , optics , flow cytometry , biology , medicine , laser , mathematics , physics , microbiology and biotechnology , statistics , composite material
Background Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. Methods We applied a commercial motorized fluorescent microscope system. The scanning was done at 20× (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno‐Brite beads were used. Results With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after ±5 μm error. Conclusions SFM is a valuable method for the evaluation of fluorescently labeled cells. © 2004 Wiley‐Liss, Inc.