Label-free single-cell analysis in microdroplets using a light-scattering-based optofluidic chip

Droplet-based single-cell analysis is a very powerful tool for studying phenotypic and genomic heterogeneity at single-cell resolution for a variety of biological problems. In conventional two-phase droplet microfluidics, due to the mismatch in optical properties between oil and aqueous phases, light scattering mainly happens at the oil/water interface that disables light-scattering-based cell analysis confined in microdroplets. Detection and analysis of cells in microdroplets thus mostly rely on the fluorescence labeling of cell samples, which may suffer from complex operation, cytotoxicity, and low fluorescence stability. In this work, we propose a novel light-scattering-based droplet screening (LSDS) that can effectively detect and characterize single cells confined in droplets by adjusting the optical properties of droplets in a multiangle optofluidic chip. Theoretical and simulated calculations suggest that refractive index (RI) matching in droplet two-phase materials can reduce or eliminate droplets' scattered signals (background signal), enabling the differentiation of scattered signals from single cells and particles within droplets. Furthermore, by using a set of multiangle (from −145° to 140°) optical fibers integrated into the optofluidic chip, the scattered light properties of droplets with the RI ranging from 1.334 to 1.429 are measured. We find that the smaller the RI and size of microparticles inside droplets are, the smaller the RI difference between two-phase materials Δn is required. Especially, when Δn is smaller than 0.02, single cells in droplets can be detected and analyzed solely based on light scattering. This capability allows to accurately detect droplets containing one single cell and one single gel bead, a typical droplet encapsulation for single-cell sequencing. Altogether, this work provides a powerful platform for high-throughput label-free single-cell analysis in microdroplets for diverse single-cell related biological assays.