A clogging-free microfluidic impedance and imaging flow cytometry based on three-dimensional hydrodynamic focusing

Abstract Microfluidic impedance and imaging flow cytometry have been widely used for in quantifying and classifying single cells due to their label-free nature and cost-effectiveness. However, state-of-the-art microfluidic impedance and imaging flow cytometry suffers from key limitations, including poor signal quality and susceptibility to cell clogging. This study presents a microfluidic impedance and imaging flow cytometry based on three-dimensional hydrodynamic focusing, in which single microbeads were confined within the focused sample flow by transparent and insulating sheath streams in the lateral and vertical directions, thereby enabling the simultaneous capture of high-quality single-microbead impedance profiles and brightfield images without risking channel blockage. The key geometrical parameters of the microfluidic channel and the key experimental parameters of the sample-to-sheath flow ratio were designed and optimized using numerical simulations. As for platform demonstration, four-frequency impedance amplitude profiles and bright-field images of 10 μ m and 15 μ m microbeads were quantified and subsequently used for classification. This study presented a microfluidic impedance and imaging flow cytometry that integrates high-quality signals with capability to operate without clogging, thereby overcoming the limitations of conventional counterparts and providing a reliable tool for single-cell analysis.