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.

Detection and identification of surface molecular contamination is important for improving process and product yields in a wide range of industrial applications. In particular, molecular compounds can easily deposit on the surface of hardware, optical components, semiconductor devices, medical devices, etc. with the risk of impairing their functionality. In this work, qualitative and quantitative metrological methodologies for surface molecular contamination detection based on Fourier transform infrared spectroscopy and micro-Raman spectroscopy are presented. The specificity of detection of two ubiquitous industrial contaminants i.e. poly(methylphenylsiloxane) and paraffin oil by infrared and Raman fingerprints is first demonstrated. Moreover, in order to obtain homogeneously contaminated surfaces that can be used as standard materials for Raman calibration, films of different thicknesses of contaminants were prepared on calcium fluoride windows, within a contamination range of 70–900 ng cm⁻², by a spin coating method. The amount of contaminants spread on the surface was quantified by applying a pre-set FT-IR calibration curve in accordance with the European Cooperation for Space Standardization procedure (ECSS-Q-ST-70-05C) and it was subsequently used to calibrate the Raman equipment which demonstrated a sensitivity up to 10⁻⁸ g cm⁻². A real case study of industrial contaminated surfaces i.e. a glass lens for a laser cutting machine is also presented to assess the applicability of both techniques in molecular contamination monitoring. In the discussed case Raman analysis turned out to be particularly useful for punctual investigation of the surface, especially when the sample is not transparent to the infrared radiation.