Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.
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 x (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 microm error.


CONCLUSIONS
SFM is a valuable method for the evaluation of fluorescently labeled cells.
Varga
Viktor Sebestyén
VS
Hungarian Academy of Sciences, Clinical Gastroenterology Research Unit, Budapest, Hungary. vargasv@bel2.sote.hu
Bocsi
József
J
Sipos
Ferenc
F
Csendes
Gábor
G
Tulassay
Zsolt
Z
Molnár
Béla
B
D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D047569:Laser Scanning Cytometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D008214:Lymphocytes / Q000166:cytology; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012984:Software
D005456:Fluorescent Dyes