Raman and infra-red microspectroscopy: towards quantitative evaluation for clinical research by ratiometric analysis

Abstract

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.

Detection and classification of Bacteria using Raman Spectroscopy Combined with Multivariate Analysis

“…Another characteristic peak for biological samples is the distinct Amide I peak which appears at 1668 cm -1 . These spectral features were found to be consistent with the earlier reported literature and have been compiled inTable 1 12,[34][35][36][37][38] . The Raman shifts for all the four strains closely resembles each other and therefore, it is quite challenging to identify strains based on merely comparing the spectral frequencies or amplitude.…”

“…The Raman shifts for all the four strains closely resembles each other and therefore, it is quite challenging to identify strains based on merely comparing the spectral frequencies or amplitude. Therefore, [12,[34][35][36][37][38] . in order to discriminate the bacteria unequivocally, one has to resort to multivariate analyses tools.…”

“…However, biological samples are very complex as even a single cell or a bacterium is composed of a number of biomolecules such as proteins, lipids, carbohydrates and nucleic acids. Different types of analysis techniques are used to extract information from biological samples 12,16 . Since the molecular structure of the biomolecules are unique, distinctive spectral fingerprints can be obtained through Raman spectroscopic analysis.…”

“…Raman spectroscopy is based upon the principle of inelastic scattering of photons incident on the sample. It has found applications in the area of materials science as well as in biology [11][12][13][14][15] . In the realm of biolchemical analysis it is essential that a technique is non-invasive, preferably labelfree and non-destructive.…”

Analysis of Demineralized Mammalian Bone Tissue by Fourier Transform Infrared Microscopy

“…In particular, microscopic infrared spectroscopy, which allows measurement of the infrared absorption spectrum of microscopic areas, has recently attracted attention [16][17][18][19][20] . Generally, the histology of lesion sample slices are observed using a microscope; however, the infrared absorption spectrum of microscopic areas observed can be obtained without staining tissue slices when the procedure is conducted using microscopic infrared spectroscopy [21][22][23] .…”