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

190 Multivariate Analysis in Management, Engineering and the Sciences

FTIR characterization of complex biological systems.

then confirmed by standard biochemical assays.

approaches in the field of spectroscopy.

groups or classes.

differentiation, are studied. Indeed, in this case it is fundamental to be able to extract from

In Figure 1 we schematized the procedure that should be followed to successfully tackle the

**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

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

In this chapter, we will firstly introduce the most widely used multivariate analysis

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

the spectral data the relevant information of the process you are investigating [18-21].

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

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 carbohydrates [31, 33, 34].

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 of several biomolecules.

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 process under investigation [36].

Multivariate Analysis for Fourier Transform Infrared Spectra of Complex Biological Systems and Processes 193

original band half-width, this procedure introduces an enhancement of sharp lines, as those due to vapour and noise. For this reason, this analysis requires spectral data free of vapour

Furthermore, due to the intrinsic complexity of biological systems, their spectral analysis requires the support of appropriate multivariate analysis approaches able to tackle the study of high-dimensional data, to verify firstly the reproducibility of the results and then to

FTIR microspectroscopy is realized coupling to a FTIR spectrometer an infrared microscope characterized by an all reflecting optics, since typical lenses and condensers of visible microscopy - being made of glass, not transparent to the IR radiation - cannot be employed. The main advantage of FTIR microspectroscopy is that it offers the possibility to study selected areas of the sample under investigation, resulting particularly useful in the case of

Two main types of IR microscopy exist, depending on the detector employed, and both equipped with an IR thermal source (globar), whose spatial resolution is diffraction-limited. The first, conventional, generally equipped with a nitrogen cooled mercury cadmium telluride (MCT) detector, makes it possible to measure IR absorption spectra from a microvolume within the sample, selected by a variable aperture of the microscope, whose

The second type of IR microscope, more advanced, is equipped with a focal plane array (FPA), consisting of an array of infrared detector elements, that enables not only to collect the IR absorption spectrum of the sample, but also an IR chemical imaging, where the image contrast is given by the response of selected sample regions to particular IR wavenumbers. Depending mainly on the detection array, the spatial resolution in this kind of microscopy is approximately between 20 and 5 microns, making it possible to reach, therefore, a resolution

We should, however, add that the use of a synchrotron IR light source, with a brightness of at least two orders of magnitude higher than that of a conventional thermal source, makes it possible to achieve diffraction-limited spatial resolution with enhanced signal-to-noise ratio. In this way, synchrotron light could allow to explore the IR spectra at the subcellular level.

A final remark should be done concerning the spectral acquisition mode. Indeed, infrared measurements can be mainly performed in transmission, reflectance or attenuated total reflection (ATR) mode. Typically, measurements on complex biological systems are performed in transmission mode, using appropriate IR transparent supports for the deposition of the sample, such as BaF2, CaF2, ZnSe. In this case, the IR beam goes through the sample, that - depending mainly on its molar extinction coefficient - should have a

extract the most significant spectral information [18-21] (see for details paragraph 4).

systems characterized by an intrinsic heterogeneity, such as biological systems.

**3. FTIR microspectroscopy: Technical considerations**

absorption and with excellent signal to noise ratio.

side can be adjusted down to a few tens of microns.

uniform thickness, not exceeding 15-20 microns.

near to the diffraction limit.

**Figure 2.** FTIR absorption spectrum of a single intact murine oocyte. The measured absorption spectrum of a single intact murine oocyte (surrounded nucleolus, MI 10 H) is reported without any corrections. The oocyte - deposited on a BaF2 window - was measured in transmission by the IR microscope UMA 500, coupled to the FTIR spectrometer FTS 40A (both from Digilab), at a resolution of 2 cm-1. The absorption regions of the main biomolecules are indicated.

We should add that to obtain reliable results on the studied process it is crucial to standardize firstly the sample preparation, since - for instance - metabolic changes due to cell aging could result in significant spectral changes that could, in turn, hide the IR response specifically due to the process of interest, as it has been recently reported in the literature [37]. For these reasons, it is fundamental to check accurately the stage of cell growth in culture before performing spectroscopic measurements.

We should also briefly mention that, before spectral analyses, the measured IR spectra could require some corrections due to artifacts that can interfere with the spectroscopic response. For instance, single cells, or subcellular compartments, or particles of the size of the same order of that of the incident infrared light (�3-10 microns) could give rise to Mie scattering, that significantly distorts the measured spectrum, causing misinterpretation of the results. For this reason, before further analyses, it is strongly recommended to correct the measured spectra with opportune algorithms specifically developed to this aim [38].

Since the IR spectra of complex biological systems are due to the overlapping spectral features of multiple components, their analysis requires often the employment of resolution enhancement procedures to better resolve their absorption bands, an essential prerequisite for the identification of peak positions and their assignment to the vibrational modes of the different molecules. Among these, second derivative analysis is widely applied, as described in [39]. Since second derivative band intensity is inversely proportional to the square of the original band half-width, this procedure introduces an enhancement of sharp lines, as those due to vapour and noise. For this reason, this analysis requires spectral data free of vapour absorption and with excellent signal to noise ratio.

Furthermore, due to the intrinsic complexity of biological systems, their spectral analysis requires the support of appropriate multivariate analysis approaches able to tackle the study of high-dimensional data, to verify firstly the reproducibility of the results and then to extract the most significant spectral information [18-21] (see for details paragraph 4).
