High-throughput single cell analysis using multi-focus Raman spectroscopy under randomly interleaved scattering projection.

Raman microspectroscopy is a powerful tool for label-free monitoring of single-cell dynamics. However, the traditional single-point acquisition mode is extremely inefficient because it only analyzes one cell at a time. We propose a method that combines multi-focus Raman excitation with random interleaving of scattering projections. The approach uses a time-sharing multi-focus array to simultaneously excite multiple biological cells and synchronously projects their Raman spectra into a single spectral channel with varying shifts. The spectral shifts of the cells are randomly interleaved during data acquisition, resulting in a sequence of mixed spectra from which a compressive sensing method is utilized to reconstruct the time-lapse Raman spectra of the individual cells. The method's feasibility and performance are validated by numerical modeling and experimental investigations into biological spore germination. The results indicated that the throughput of single cell analysis can be increased by up to a factor of 15. The reconstructed spectra of the individual cells demonstrated exceptional fidelity, faithfully capturing the cellular changes in the individual cells. The developed technology paves the way towards high-throughput, label-free monitoring of living single cells, which has promising applications in cell biology.