High-throughput single-cell sorting by stimulated Raman-activated cell ejection

Abstract

Single-cell sorting is essential to explore cellular heterogeneity in biology and medicine. Recently developed Raman-activated cell sorting (RACS) circumvents the limitations of fluorescence-activated cell sorting, such as the cytotoxicity of labels. However, the sorting throughputs of all forms of RACS are limited by the intrinsically small cross-section of spontaneous Raman scattering. Here, we report a stimulated Raman-activated cell ejection (S-RACE) platform that enables high-throughput single-cell sorting based on high-resolution multi-channel stimulated Raman chemical imaging, in situ image decomposition, and laser-induced cell ejection. The performance of this platform was illustrated by sorting a mixture of 1 m polymer beads, where 95% yield, 98% purity, and 14 events per second throughput were achieved. Notably, our platform allows live cell ejection, allowing for the growth of single colonies of bacteria and fungi after sorting. To further illustrate the chemical selectivity, lipid-rich Rhodotorula glutinis cells were successfully sorted from a mixture with Saccharomyces cerevisiae, confirmed by downstream quantitative PCR. Furthermore, by integrating a closed-loop feedback control circuit into the system, we realized real-time single-cell imaging and sorting, and applied this method to precisely eject regions of interest from a rat brain tissue section. The reported S-RACE platform opens exciting opportunities for a wide range of single-cell applications in biology and medicine. Significance statementImage-guided single-cell sorting is a potent tool in diverse biological applications. Current microfluidic cell sorting methods encounter challenges in handling smaller cells and are not applicable to tissue sections. To address these challenges, we have developed a stimulated Raman-activated cell ejection (S-RACE) platform, which is the first demonstration of single-cell ejection coupled with coherent Raman scattering. S-RACE allows label-free chemical imaging guided cell sorting through multispectral stimulated Raman scattering (SRS) imaging, on-the-fly image analysis, and laser-induced cell ejection. Versatile applications of S-RACE to a wide range of samples, such as polymer particles, single-live bacteria, single-live fungus, and tissue sections, are demonstrated.