Since the second half of the 20th century laser-excited Raman spectroscopy as fast non-invasive and slightly damaging method has been widely used in investigation of different biological objects. During this time, in general, two main problems can be formulated for the use of Raman spectroscopy to study biological samples: (1) very low signal intensity of the Raman spectrum bands that requires enhanced laser power and a well-developed mathematical apparatus for treatment of spectra; and (2) rapid damage of radiated samples that requires a very careful selection of the radiation power and duration for each biological object. Unfortunately, due to these peculiarities, not all types of Raman spectroscopy are effective when working with biological objects. The solution of these problems is critical for the successful use of Raman spectroscopy. However, the aforementioned problems do not prevent the use of different methods of Raman spectroscopy for various biological and environmental studies.

Table 3 shows some examples of substances and objects that were detected using different methods of Raman spectroscopy.

As we have already mentioned, the advantages of IR spectroscopy include noninvasiveness, non-damaging or slightly damaging effects (even compared to the Raman spectroscopy), relatively rapid obtaining of results, and (unlike the Raman spectroscopy) the possibility to evaluate polar molecules (alcohols, phenolic compounds, etc.) [4]. As a result, different types of IR spectroscopy are quite actively used for the monitoring of environmental pollution as well as for solving different biomedical and other tasks, such as tumor diagnostics (reviewed in [4,5,92,93]). Moreover, IR spectroscopy is a cheap method compared to its analogues [5]. A study of biological samples usually requires the use of near-infrared (1000–2500 nm, 10,000–4000 cm⁻¹) and mid-infrared (2500–25,000 nm, 4000–400 cm⁻¹) spectroscopy [4]. Unfortunately, the low sensitivity of near-infrared (NIR) spectroscopy and the limitations of mid-IR spectroscopy in the case of dealing with aqueous solutions, as well as the lower resolution of the method compared to Raman spectroscopy [3], result in significant problems with microscopic studies [4] that limit the potential of the method in relation to living objects and environmental studies.

The result of measuring biological samples is an IR spectrum, which, like in the case of Raman spectroscopy, represents a superposition of numerous overlapping peaks, whose presence and intensity depend on the chemical bonds in the studied compounds and their conformation. Based on the presence of typical chemical groups, it is possible to determine the presence of proteins, amino acids, fatty acids, polysaccharides, phenolic compounds, etc. However, such a large number of peaks significantly complicates their interpretation and requires the use of special mathematical apparatus, which (in contrast to Raman spectroscopy) has already been developed. The obtained spectral data are treated using various multiparametric analysis methods, such as principal component analysis (PCA), soft independent modeling of class analogy (SIMCA), hierarchical clustering analysis (HCA), canonical variation analysis (CVA), factor discriminant analysis (FDA), partial least squares discriminant analysis (PLSDA), and various regression models, including multiple linear regression (MLR), principal component regression (PCR), partial least squares regression (PLSR), etc. [33]. When applying these methods, there is no need to use the values of individual peaks; in these cases, whole (or selected sections of) IR spectra can be used without isolating individual peaks.

Table 4 shows some examples of using different methods of IR spectroscopy for environmental studies.

All the aforesaid advantages of IR (especially NIR) spectroscopy, as well as the availability of relatively inexpensive and cheap equipment, make it possible to perform environmental studies using IR spectroscopy (reviewed in [6]). Since IR and especially NIR spectroscopy provide the possibility of working with aqueous solutions, this method allows for the in vivo qualitative and quantitative assessment of the composition of animals and plants and the evaluation of changes occurring in them. The objects of IR spectroscopy may include single-celled fungi, lichens, microalgae, plant and animal tissues (membranes, bones, plant organs), multicellular plants and animals, and products of their vital activities (consumed food, excretions, feces, etc.) [6].