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Flow cytometers (FCM) are built for particle measurements. In principle, concentration
measurement of a homogeneous solution is not possible with FCM due to the lack of a trigger
signal. In contrast to FCM slide based cytometry systems could act as tools for the measurement
of concentrations using volume defined cell counting chambers. These chambers enable to
analyze a well defined volume. Sensovation AG (Stockach, Germany) introduced an automated
imaging system that combines imaging with cytometric features analysis. Aim of this study was
to apply this imaging system to quantify the fluorescent molecule concentrations.
The Lumisens (Sensovation AG) slide-based technology based on fluorescence digital imaging
microscopy was used. The instrument is equipped with an inverted microscope, blue and red
LEDs, double band-pass filters and a high-resolution cooled 16-bit digital camera. The
instrument was focussed on the bottom of 400μm deep 6 chamber slides (IBIDI GmbH,
Martinsried, Germany) or flat bottom 96 well plates (Greiner Bio One GmbH, Frickenhausen,
Germany). Fluorescent solutions were imaged under 90% pixel saturation in a broad
concentration range (FITC: 0.0002-250 μg/ml, methylene blue (MethB): 0.0002-250 μg/ml).
Exposition times were recorded. Images were analysed by the iCys (CompuCyte Corp.,
Cambridge, MA, USA) image analysis software with the phantom contour function. Relative
fluorescence intensities were calculated from mean fluorescence intensities per phantom contours
divided by the exposition time.
Solution concentrations could be distinguished over a broad dynamic range of 3.5 to 5.5 decades
log (range FITC: 0.0002-31.25μg/ml, MethB: 0.0076-31.25μg/ml) with a good linear relationship
between dye concentration and relative fluorescence intensity. The minimal number of
fluorescent molecules per pixel as determined by the mean fluorescence intensity and the
molecular weight of the fluorochrome were about 800 molecules FITC and ~2.000 MethB.
The novel slide-based imaging system is suitable for detection of fluorescence differences over a
broad range of concentrations. This approach may lead to novel assays for measuring
concentration differences in cell free solutions and cell cultures e.g. in secretion assays.
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