Under the lens: using Raman spectroscopy as a unique system in biofilm analyses

Biofilms are microbial communities that adhere to surfaces and each other, encapsulated in a protective extracellular matrix. These structures enhance resistance to antimicrobials, contributing to 65-80% of human infections. The transition from free-living cells to structured biofilms involves a myriad of molecular and structural adaptations. Raman spectroscopy is an analytical technique that has recently been adapted for biofilm analysis. The ability to operate without interference from water makes Raman spectroscopy a valuable tool for in situ characterization of biofilms, including direct analysis from clinical samples. The technique also offers the advantage of imaging speed and the capacity to generate extensive chemical and molecular data from samples, whilst also being non-destructive. However, Raman spectroscopy is often limited by its low sensitivity, particularly when applied to microbial analysis. This limitation has been addressed with the advent of surface-enhanced Raman spectroscopy and stimulated Raman scattering microscopy. When used in combination with traditional methods, these Raman technologies can be incredibly useful for understanding the mechanisms underlying biofilm development, antimicrobial susceptibility testing, and detection and discrimination of microorganisms. In this critical review, the application of Raman spectroscopy and its derivatives as a tool for biofilm characterization is discussed along with its associated advantages and challenges.