Robust Spontaneous Raman Flow Cytometry for Single-Cell Metabolic Phenome Profiling via pDEP-DLD-RFC

A full-spectrum spontaneous single-cell Raman spectrum (fs-SCRS) captures the metabolic phenome for a given cellular state of the cell in a label-free, landscape-like manner. Herein a positive dielectrophoresis induced deterministic lateral displacement-based Raman flow cytometry (pDEP-DLD-RFC) is established. This robust flow cytometry platform utilizes a periodical positive dielectrophoresis induced deterministic lateral displacement (pDEP-DLD) force that is exerted to focus and trap fast-moving single cells in a wide channel, which enables efficient fs-SCRS acquisition and extended stable running time. It automatically produces deeply sampled, heterogeneity-resolved, and highly reproducible ramanomes for isogenic cell populations of yeast, microalgae, bacteria, and human cancers, which support biosynthetic process dissection, antimicrobial susceptibility profiling, and cell-type classification. Moreover, when coupled with intra-ramanome correlation analysis, it reveals state- and cell-type-specific metabolic heterogeneity and metabolite-conversion networks. The throughput of ≈30–2700 events min⁻¹ for profiling both nonresonance and resonance marker bands in a fs-SCRS, plus the >5 h stable running time, represent the highest performance among reported spontaneous Raman flow cytometry (RFC) systems. Therefore, pDEP-DLD-RFC is a valuable new tool for label-free, noninvasive, and high-throughput profiling of single-cell metabolic phenomes.