A new multiparameter flow cytometer: Optical and electrical cell analysis in combination with video microscopy in flow

Background: Flow cytometers, which are commercially available, do not neces sarily meet all demands of actual biomedical research. This is the case for the investigation of mechanisms involved in cell volume regulation, which requires electrical volume measurement and ratiometric multichannel fluores cence analysis for the simultaneous assessment of different physiologic par ameters (intracellular pH and the intracellular concentration of calcium io ns, etc). Methods and Results: We describe the construction of a new nonsorting flow cytometer designed for the simultaneous acquisition of seven parameters inc luding fluorescence signals, forward and perpendicular light scatter, cell volume according to the electrical Coulter principle, and flow cytometric i maging. The instrument is equipped with three different light sources. A tu nable argon-ion laser generates efficient excitation of the most standard f luorescent probes in the visible spectral range, and an are lamp provides t he means for ultraviolet excitation at low cost. Because of the spatial fil tering by the excitation and detection optics, two independent sets of dual fluorescence measurements can be performed, a prerequisite for flexible ra tiometric fluorescence analysis. A flow video microscope integrated into th e optical system optionally generates either brightfield or phase images of selected flowing particles. Only particles whose individual datasets meet predefined gating conditions are imaged in real time. To avoid smear effect s, the motion of the object to be imaged (speed approximate to 8 m/s) is fr ozen on the target of a CCD camera by flash illumination. For this purpose, a high radiance gas discharge lamp with 25-mJ electric pulse energy provid es an illumination time of 18 ns (full width half maximum). Test results ob tained from latex spheres and cells are shown. Conclusions: Test results indicate that our instrument can perform Coulter measurements in combination with flexible optical analysis. Moreover, integ ration of an adapted video microscope into a flow cytometer is an approach to overcome the gap between flow and image cytometry. (C) 1999 Wiley-Liss, Inc.