A Quantitative Microfluidic Flow Cytometer Based on Spaced Uniform Optical Field

Rapid, quantitative analysis of single-cell proteins with arbitrary spatial distributions carries pivotal significance across fundamental biological research and clinical applications. However, as the golden method, flow cytometry can only quantify cell surface proteins due to non-uniform excitation illumination and the lack of calibration microbeads with known numbers of internal fluorescent molecules. This study reported a quantitative microfluidic flow cytometer utilizing aligned microfabricated metal apertures to selectively extract the central portions of Gaussian beams, forming a spaced uniform optical field. Based on this spaced uniform optical field, multicolor fluorescence signals originating from antibody-binding events with arbitrary distributions on single microbeads were converted into the number of proteins. The geometries of the microfluidic cuvette and the uniformity of fluorescence intensities across the excitation fields were quantitatively validated. Using this methodology, calibration curves with high linearities for all nine fluorescence channels were established, and a mixture of rainbow calibration microbeads was analyzed, where five types of fluorescent probes were quantitatively measured, enabling the classification of the rainbow mixture into five distinct subpopulations. The presented flow cytometer enables quantitative analysis of single-cell proteins with arbitrary spatial distributions and thus demonstrates strong potential for fundamental biological research and clinical applications at the single-cell level.