Hyperspectral Chemical Imaging of Single Bacterial Cell Structure by Raman Spectroscopy and Machine Learning

In this work, biomolecules, such as membrane proteins, lipids, and DNA, were identified and their spatial distribution was mapped within a single Escherichia coli cell by Raman hyperspectral imaging. Raman spectroscopy allows direct, nondestructive, rapid, and cost-effective analysis of biological samples, minimizing the sample preparation and without the need of chemical label or immunological staining. Firstly, a comparison between an air-dried and a freeze-dried cell was made, and the principal vibrational modes associated to the membrane and nucleic acids were identified by the bacterium’s Raman chemical fingerprint. Then, analyzing the Raman hyperspectral images by multivariate statistical analysis, the bacterium biological status was investigated at a subcellular level. Principal components analysis (PCA) was applied for dimensionality reduction of the spectral data, then spectral unmixing was performed by multivariate curve resolution–alternating least squares (MCR-ALS). Thanks to multivariate data analysis, the DNA segregation and the Z-ring formation of a replicating bacterial cell were detected at a sub-micrometer level, opening the way to real-time molecular analysis that could be easily applied on in vivo or ex vivo biological samples, avoiding long preparation and analysis process.