摘要：SummaryAccessing enormous uncultivated microorganisms (microbial dark matter) in various Earth environments requires accurate, nondestructive classification, and molecular understanding of the microorganisms inin situand at the single-cell level. Here we demonstrate a combined approach of random forest (RF) machine learning and single-cell Raman microspectroscopy for accurate classification of phylogenetically diverse prokaryotes (three bacterial and three archaeal species from different phyla). Our RF classifier achieved a 98.8 ± 1.9% classification accuracy among the six species in pure populations and 98.4% for three species in an artificially mixed population. Feature importance scores against each wavenumber reveal that the presence of carotenoids and structure of membrane lipids play key roles in distinguishing the prokaryotic species. We also find unique Raman markers for an ammonia-oxidizing archaeon. Our approach with moderate data pretreatment and intuitive visualization of feature importance is easy to use for non-spectroscopists, and thus offers microbiologists a new single-cell tool for shedding light on microbial dark matter.Graphical abstractDisplay OmittedHighlights•Random forest models classify prokaryotic species with high accuracy of >98%•Both bacteria and archaea are classified using minimally preprocessed Raman data•Feature importance reveals what biomolecules contribute to species classification•Raman marker bands for some archaeal species are discoveredMolecular spectroscopy techniques; Microbiology; Machine learning
