We have examined the ability of real-time simultaneous measurement of bright field/fluorescent images of cells in an on-chip bright field/fluorescent multi-imaging flow cytometer system. The system consists of (1) a disposable microfluidic hydrofocusing flow cytometry chip, (2) an optical microscopy module with splittable bright field/fluorescent multi-imaging optics, and (3) a real-time image-processing module with a 200 images/s high-speed digital camera. In the double "Y" shape three-way-inlet microfluidic pathways fabricated in the poly(dimethylsiloxane) (PDMS) microchip, we applied fluorescent polystyrene standard beads and HeLa cells stained with fluorescent dye, Hoechst 33258, and measured the z-axis (depth) dependence of the morphological index; the intensity profile of cells and nuclei. Then, we measured the tendency of the blur of bright field/fluorescent images in the simultaneous measurement of bright field/fluorescent images on a single light-receiving surface, and found that their blurs were similar within the same range of the depth of the microfluidic pathway for small cell cluster measurement, 25μm. Hence, the fluorescent images were applied as supporting information of the bright field images of cell clusters at the focal plane for the cell number counting. The result indicates the potential of precise identification of various types of cells by simultaneous morphological analysis of bright field and fluorescent images distributed with a single camera in a wider depth of microfluidic chip as a substitute for conventional biomarker detection.
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