An Integrated Micromirror System for High‐Throughput Multi‐Parametric Cell Imaging

Imaging flow cytometry combines the high-throughput capabilities of conventional flow cytometry with single-cell morphological analysis, enabling the extraction of high-content information from large and heterogeneous cell populations. Whilst powerful, current imaging flow cytometers rely on complex optical assemblies to achieve high detection sensitivities and spatial resolution. Optofluidic systems that monolithically integrate microfluidics and optical components offer a route to both simplifying and miniaturizing imaging flow cytometers, whilst enhancing imaging performance. Herein, we present a sheathless optofluidic imaging flow cytometry platform (optoIFC) that incorporates microfabricated mirrors for simultaneous two-color fluorescence and brightfield imaging. The use of two-photon stereolithography enables the single-step fabrication of micromirrors directly within microfluidic channels, substantially simplifying device production. The use of such systems with stroboscopic fluorescence illumination provides for imaging throughputs exceeding 3,000 cells per second. To showcase the efficacy of the optoIFC, TNF-α-induced NF-κB nuclear translocation in HL-60 cells is investigated through dual-colour fluorescence imaging, confirming that the optoIFC enables precise cell sizing and high-content mapping of intracellular heterogeneity across large populations at high-throughput.