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The conventional microbial cell analyses are mostly population-averaged methods that conceal the characteristics of single-cell in the community. Single-cell analysis can provide information on the functional and structural variation of each cell, resulting in the elimination of long and tedious microbial cultivation techniques. Raman spectroscopy is a label-free, noninvasive, and  in-vivo  method ideal for single-cell measurement to obtain spatially resolved chemical information. In the current review, recent developments in Raman spectroscopic techniques for microbial characterization at the single-cell level are presented, focusing on Raman imaging of single cells to study the intracellular distribution of different components. The review also discusses the limitation and challenges of each technique and put forward some future outlook for improving Raman spectroscopy-based techniques for single-cell analysis. Raman spectroscopic methods at the single-cell level have potential in precision measurements, metabolic analysis, antibiotic susceptibility testing, resuscitation capability, and correlating phenotypic information to genomics for cells, the integration of Raman spectroscopy with other techniques such as microfluidics, stable isotope probing (SIP), and atomic force microscope can further improve the resolution and provide extensive information. Future focuses should be given to advance algorithms for data analysis, standardized reference libraries, and automated cell isolation techniques in future.

Recent developments in Raman spectral analysis of microbial single cells: Techniques and applications