**1. Introduction**

188 Multivariate Analysis in Management, Engineering and the Sciences

2008;62(8) 906-915.

[48] Oxley J., Smith J., Brady J., Dubnikova F., Kosloff R., Zeiri L., Zeiri Y. Raman and Infrared Fingerprint Spectroscopy of Peroxide-Based Explosives. Applied Spectroscopy

> Fourier transform infrared (FTIR) spectroscopy is a label-free and non invasive technique that exerts an enormous attraction in biology and medicine, since it allows to obtain in a rapid way a biochemical fingerprint of the sample under investigation, giving information on its main biomolecule content. This spectroscopic tool is successfully applied not only to the study of the structural properties of isolated biomolecules, such as proteins, nucleic acids, lipids, and carbohydrates, but also to the characterization of complex biological systems, for instance intact cells, tissues, and whole model organisms.

> In particular, FTIR microspectroscopy, obtained by the coupling of an infrared microscope to a FTIR spectrometer, makes it possible to collect the IR spectrum from a selected sample area down to ~ 20 microns x 20 microns when conventional IR source and detector are employed, and down to of a few micrometers when more specialized and sensitive detectors and the highly brilliant synchrotron light source are used. In this way, FTIR microspectroscopy provides detailed information on several biological processes in situ, among which stem cell differentiation [1-5], somatic cell reprogramming [6], cell maturation [7, 8], amyloid aggregation [9-12] and cancer onset and progression [13-15], making it possible to disclose the infrared response not only from single cells, but also from subcellular compartments [8, 16, 17].

> The FTIR spectra of biological systems are very complex since they consist of the overlapping absorption of the main biomolecules; for this reason, to pull out the significant and non-redundant information contained in the spectra it is necessary to apply an appropriate multivariate analysis, able to process very high-dimensional data. This is even more crucial when time-dependent biological processes, such as cell maturation or

© 2012 Ami et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Ami et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

differentiation, are studied. Indeed, in this case it is fundamental to be able to extract from the spectral data the relevant information of the process you are investigating [18-21].

Multivariate Analysis for Fourier Transform Infrared Spectra of Complex Biological Systems and Processes 191

occurred, but also to disclose the most significant cellular processes responsible for the different oocyte destiny, thus validating the visual inspection of the infrared spectra [7].

Fourier transform infrared (FTIR) microspectroscopy is a powerful technique that allows to obtain a molecular fingerprint of the sample under investigation in a rapid and non-invasive way. In the case of complex biological systems it provides simultaneously, in a single measurement, information on the main biomolecules, such as lipids, proteins, nucleic acids, and carbohydrates, requiring also a very limited amount of sample. For these reasons, it became recently a very attracting tool for biomedical research [20, 22-24], being successfully employed for the study of several biological systems, from intact cells [6, 7, 25] to tissues [11,

As an example, in Figure 2 it is reported the FTIR absorption spectrum of a single intact murine oocyte. As shown, its IR response is very complex, being due to the absorption of the main biomolecules. In particular, between 3050 - 2800 cm-1 and 1500 - 1350 cm-1 the absorption of the lipid acyl chains occurs, while around 1740 cm-1 the ester carbonyl absorbs [29]. Moreover, the amide I and amide II bands - mainly due to the C=O stretching and the NH bending of the peptide bond respectively - give information on the protein secondary structure [30], while the spectral range between 1000 and 800 cm-1 is very informative on nucleic acid absorption, since it is due in particular to sugar vibrations sensitive to their conformation and to backbone vibrational modes [31, 32]. Finally, we should also mention the very complex spectral range between 1250 - 1000 cm-1, mainly due to phosphodiester groups of nucleic acids and phospholipids and to the C-O absorption of glycogen and other

Making it possible to obtain a sample biochemical fingerprint in a rapid and non destructive way, FTIR microspectroscopy is widely applied to the in situ characterization of cellular processes, such as cell maturation, differentiation, and reprogramming [3, 5-7, 25, 35], and to the detection of several diseases, as, for instance, cancer [13-15] and neurodegenerative disorders [10, 11], whose onset is accompanied by changes in the composition and structure

Since water has a strong absorption in the mid-infrared spectral range, samples have to be dried rapidly before IR measurements, in particular when working in transmission mode (see for details the following paragraph). The suitability of such "dry-fixing" has been proved by Raman spectroscopy, a vibrational tool complementary to FTIR, whose response is not affected by water. In particular, Raman measurements performed on differentiating human embryonic stem cells, hydrated and dry-fixed, demonstrated that the rapid desiccation didn't affect the spectroscopic response of the main biomolecules. Indeed, in both cases the same temporal pattern of the differentiation marker bands - due to tryptophan, nucleic acid backbone and base vibrations - was observed during the biological

26, 27] and whole model organisms (i.e. the nematode *Caenorhabditis elegans*) [9, 28].

**2. FTIR microspectroscopy of complex biological systems**

carbohydrates [31, 33, 34].

of several biomolecules.

process under investigation [36].

In Figure 1 we schematized the procedure that should be followed to successfully tackle the FTIR characterization of complex biological systems.

**Figure 1.** Scheme of the FTIR approach to study complex biological systems. The IR absorption spectra are analysed by resolution enhancement approaches (e.g. second derivatives) to resolve the overlapped absorption components and to monitor their variations during the process under investigation. The spectroscopic results are validated by an appropriate multivariate analysis approach, to identify firstly specific marker bands of the studied process. The interpretation of the spectroscopic data should be then confirmed by standard biochemical assays.

Several multivariate analysis approaches exist and for the scope of this book they can be divided into two main categories: regression and classification techniques. In the first category fall all methods that allow to derive a model describing the relationship between two sets of variables. The second category includes techniques to split observations into groups or classes.

In this chapter, we will firstly introduce the most widely used multivariate analysis approaches in the field of spectroscopy.

We will then illustrate the basic principles and experimental details for the application of principal component - linear discriminant analysis (PCA-LDA) to the analysis of FTIR spectral data of complex biological systems. The potential of these combined tools will be described on illustrative examples of cell biological process studies. In particular, we will discuss in details its application on our FTIR study of murine oocytes characterized by two different types of chromatin organisation around the nucleolus, strongly affecting their development after fertilization. In this case, PCA-LDA analysis made it possible to identify not only the maturation stage in which the fate separation between the two kinds of oocytes occurred, but also to disclose the most significant cellular processes responsible for the different oocyte destiny, thus validating the visual inspection of the infrared spectra [7].
