Ultra High-Throughput Multiparametric Imaging Flow Cytometry: Towards Diffraction-Limited Sub-Cellular Detection

Flow cytometry is widely recognized as the gold-standard technique for the analysis and enumeration of heterogeneous cellular populations and has become an indispensable tool in diagnostics,1 rare-cell detection2 and single-cell proteomics.3 Although contemporary flow cytometers are able to analyse many thousands of cells per second, with classification based on scattering or fluorescence criteria, the vast majority require unacceptably large sample volumes, and do not allow the acquisition of spatial information. Herein, we report a sheathless, microfluidic imaging flow cytometer that incorporates stroboscopic illumination for blur-free fluorescence and brightfield detection at analytical throughputs in excess of 60,000 cells/s and 400,000 cells per second respectively. Our imaging platform is capable of multi-parametric fluorescence quantification and subcellular (co-)localization analysis of cellular structures down to 500 nm with microscopy image quality. We demonstrate the efficacy of our approach by performing challenging high-throughput localization analysis of cytoplasmic RNA granules in yeast and human cells. Results suggest significant utility of the imaging flow cytometer in the screening of rare events at the subcellular level for diagnostic applications.