This paper reviews various facets of Raman spectroscopy. This encompasses biomolecule fingerprinting and conformational analysis, discrimination of healthy vs. diseased states, depth-specific information of materials and 3D Raman imaging.

Biomolecular structure elucidation is one of the major techniques for studying the basic processes of life. These processes get modulated, hindered or altered due to various causes like diseases, which is why biomolecular analysis and imaging play an important role in diagnosis, treatment prognosis and monitoring. Vibrational spectroscopy (IR and Raman), which is a molecular bond specific technique, can assist the researcher in chemical structure interpretation. Based on the combination with microscopy, vibrational microspectroscopy is currently emerging as an important tool for biomedical research, with a spatial resolution at the cellular and sub-cellular level. These techniques offer various advantages, enabling label-free, biomolecular fingerprinting in the native state. However, the complexity involved in deciphering the required information from a spectrum hampered their entry into the clinic. Today with the advent of automated algorithms, vibrational microspectroscopy excels in the field of spectropathology. However, researchers should be aware of how quantification based on absolute band intensities may be affected by instrumental parameters, sample thickness, water content, substrate backgrounds and other possible artefacts. In this review these practical issues and their effects on the quantification of biomolecules will be discussed in detail. In many cases ratiometric analysis can help to circumvent these problems and enable the quantitative study of biological samples, including ratiometric imaging in 1D, 2D and 3D. We provide an extensive overview from the recent scientific literature on IR and Raman band ratios used for studying biological systems and for disease diagnosis and treatment prognosis.

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Rapid detection and discrimination of pathogenic bacteria for food safety, environmental pollution, medical diagnoses, and chemical and biological threats remains a considerable challenge. In the present work, we demonstrate positively charged Ag/Au bimetallic nanoparticles (Ag/Au bmNPs) as a potential surface-enhanced Raman scattering (SERS) substrate for label-free detection and discrimination of three bacteria, viz., Escherichia coli, Salmonella typhimurium, and Bacillus subtilis with excellent reproducibility. The approach relies on a priori synthesis of Ag/Au bmNPs and subsequent SERS studies on bacteria. The positive surface charge on Ag/Au bmNPs offers significant advantages of short acquisition time at very low power, high sensitivity, and a simple operating procedure without the need of very specific procedures or protocols used to capture the bacteria. The reproducible and specific intrinsic fingerprint of the cell wall and intracellular components of three bacteria obtained by label-free SERS enables precise discrimination and classification of three bacteria using multivariate analyses such as principal component analysis and canonical discriminant analysis.

The cellular proteolytic machinery orchestrates protein turnover and regulates several key biological processes. This study addresses the roles of Lon, a major ATP-dependent protease, in modulating the responses of Escherichia coli strain MG1655 to low and high amounts of sodium salicyclate (NaSal), a widely used clinically relevant analgesic. NaSal affects several bacterial responses, including growth and resistance to multiple antibiotics. The loss of lon reduces growth in response to high, but not low, amounts of NaSal. From amongst a panel of Lon substrates, MarA was identified to be the downstream target of Lon. Thus, stabilization of MarA in the absence of lon lowers growth of the strain in the presence of higher amounts of NaSal. The steady-state transcript levels of marA and its target genes, acrA, acrB and tolC, are higher in the Dlon strain compared with the WT strain. Consequently, the resistance to antibiotics, e.g. tetracycline and nalidixic acid, is enhanced in Dlon in a marA-dependent manner. Furthermore, the target genes of MarA, i.e. acrB and tolC, are responsible for NaSal-mediated antibiotic resistance. Studies using atomic force microscopy demonstrated that ciprofloxacin led to greater cell filamentation, which is lower in the Dlon strain due to higher levels of MarA. Overall, this study delineates the roles of Lon protease, its substrate MarA and downstream targets of MarA, e.g. acrB and tolC, during NaSal-mediated growth reduction and antibiotic resistance. The implications of these observations in the adaptation of E. coli under different environmental conditions are discussed.