Automated Laser-Assisted Single-Cell Sorting for Cell Functional and RNA Sequencing

Accurate and efficient sorting of single target cells is crucial for downstream single-cell analysis, such as RNA sequencing, to uncover cellular heterogeneity and functional characteristics. However, conventional single-cell sorting techniques, such as manual micromanipulation or fluorescence-activated cell sorting, do not match current demands and are limited by low throughput, low sorting efficiency and precision, or limited cell viability. Here, we report an automated, highly efficient single-cell sorter, integrating laser-induced forward transfer (LIFT) with a high-throughput picoliter micropore array. The micropore array was surface-functionalized to manipulate liquid surface tension, facilitating the formation of single-cell picoliter droplets in the micropores to realize automated and highly efficient (>80%) single-cell isolation. Using an in-house built microscopic system, rare target cells were identified and automatically retrieved by LIFT with precise sorting efficiency (about 100%) for downstream single-cell analysis while maintaining high cell viability (about 80%). As a case demonstration, we demonstrated the accurate sorting of rare transfected PC-9 cells and post-transfection cell culture, minimizing cell loss and the risk of contamination. Furthermore, we performed single-cell RNA sequencing and showed that high-quality single-cell transcriptome information was efficiently and reliably obtained during cell sorting, preventing additional costs due to low sorting accuracy. The single-cell sorter will become invaluable for single-cell analysis, laying the foundation for multiomics analysis and precision medicine research. © 2025 American Chemical Society.