Continuous microfluidic 3D focusing enabling microflow cytometry for single-cell analysis.

Single-cell analysis is developed with an aim to understand the cellular heterogeneity, including protein content, DNA, RNA, as well as the cellular metabolism. Microflow cytometry is an emerging technology to study cells at a single-cell level in a high-throughput manner and offers rich biochemical information of cells by taking advantage of microfluidic focusing. With the advancements in microfabrication technologies, microfluidics has been advanced to focus the cells from "2D" to "3D", which improves sensing capabilities of flow cytometry and brings new types of flow cytometry to the world. In this review, we first present a comprehensive review of recent developments of microfluidic technologies for 3D focusing according to the underlying physics. We then discuss the applications of microfluidic 3D focusing on advanced microflow cytometry, such as impedance flow cytometry, optical signal-based flow cytometry, imaging flow cytometry, and deformability flow cytometry. Finally, we discuss the challenges and perspectives of microfluidic 3D focusing in flow cytometry for single-cell analysis. Due to the superior capabilities of microflow cytometry employing 3D focusing, this technology will be highly promising in the near future, with further applications in biology and medicine.