Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis

Viktor Sebestyén Varga, József Bocsi, Ferenc Sipos, Gábor Csendes, Zsolt Tulassay, Béla Mohaár

Research output: Article

30 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)53-62
Number of pages10
JournalCytometry Part A
Volume60
Issue number1
DOIs
Publication statusPublished - júl. 2004

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ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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