With the deepening of microbial research, the traditional "Culture-First, Screen-Second" strategy has shown pronounced limitations when confronted with complex environmental samples and in the quest for functionally significant microbes. Taking gut and natural-environment samples as examples, more than 99% of prokaryotes remain uncultivable under standard laboratory conditions, leaving myriad microorganisms with crucial functions undiscovered and uncharacterised. To address this challenge, a "Screen-First, Culture-Second" workflow based on single-cell Raman spectroscopy (SCRS) combined with heavy-water (D₂O) metabolic labelling has been proposed. This approach employs in situ, label-free SCRS to rapidly pinpoint individual cells exhibiting specific metabolic activities, followed by precise cultivation and taxonomic identification of those cells. By measuring the assimilation rate of D₂O in single cells, SCRS enables the efficient isolation of microbes endowed with targeted functions—for instance, phosphate solubilisation or pollutant degradation.This review summarises the core techniques underpinning this strategy and showcases representative applications. Then it analyses in detail its advantages for functional-microbe discovery, highlighting its considerable potential to enhance screening throughput, sensitivity and activity retention. Meanwhile, the review also discusses the trends, including automation, AI-assisted spectral recognition, multi-omics integration and genome-informed medium design. Finally, the review envisions broad prospects for this strategy in building microbial resource libraries, monitoring antimicrobial resistance and other emerging fields. Overall, the "Screen-First, Culture-Second" platform offers a systematic reference framework for high-throughput functional screening and sorting and is aimed to accelerate the widespread adoption of single-cell Raman technologies in microbiological research and application.
