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D. Lesciute et al. / Medical Physics in the Baltic States 7 (2009) 16 - 19 from the FPA detector. Firstly, the interferograms has to be recalculated to the infrared spectra by using fast Fourier transformation (FFT) and secondly, all spectra has to be baseline corrected. The main feature of spectral data obtained from an array detector is large amount of data. Mathematical treatment of the data with aim to reduce its amount has to be performed. Common procedure is integration of spectra over certain spectral regions. The chemical images representing distribution of different chemical components in the sample can be rationed to each other. This procedure is very efficient in determination of cancerous areas of a biological sample. Usually cancer cells are assumed to have only very small differences from healthy cells in respect to relative concentration of different chemical component presenting in the cells. Such differences can be visualized only in so called rationed images and finally used for recognition of cancerous cells [4]. 2. Infrared spectral features of biological molecules The biological cells are classified according to their primary physiological functions. In such a way the cells can be assigned to the different groups. Epithelia, nerve, sex and blood cells are examples of such grouping. From molecular point of view all biological cells consist from molecules of four types: proteins, lipids, nucleonic acids and hydrocarbons [5]. Spectral bands arising from all these molecules are found in infrared absorption spectra of a biological tissue. Relative intensity of the bands is changing in process of development of cancer. All the amino acids are constituted from four major parts according to general structure where central or α – atom of carbon is attached to the carbonyl group, amino group and hydrogen atom (Fig. 1). Every amino acid has distinctive structure in the secondary chain, which is denoted by letter R in figure 1. Primary structure of the protein or polypeptide is formed by combining separate segments (blocks) of amino acid to the linear chain via condensation reaction between amino and carboxyl groups adjacent in the linear acids chain. The bond between the blocks of amino acid is called peptide bond. The structural unit of the main polypeptide chain is constituted from several functional groups: C-N group, C-H group, NH2 group and C=H group. All the groups determine IR absorption spectrum of protein. [6] The main absorption bands are as follows: amide I band at 1650 cm -1 caused by C=O bond vibration (80%) and C-N bond vibration (20%); amide II band at 1545 cm -1 caused by N-H vibration (60%) and C-N vibration (40%); amide III band at 1236 cm -1 caused by C-N vibration; amide A – at 3290 cm -1 caused by N-H vibration. Most of the proteins have non linear structures forming complex secondary structures which locate in more than one plane. Regular secondary structures of proteins are α-helix and β-pleated sheet. Both of these periodic secondary structures have hydrogen bond 17 between oxygen atoms in main carboxyl group and hydrogen atoms in main NH- group. These structural changes are followed by shifting of corresponding spectral bands. The shift depends on the vibrations of the main chain. Analysis of these shifts gives empirical rules: IR absorption band at 1660 – 1650 cm -1 is ascribed to αhelix, 1640 – 1620 cm -1 - to β-pleated sheet conformation, 1695 – 1660 cm -1 – to β-pleated sheet and β-strand conformations with absorption band at 1650 – 1640 cm -1 is ascribed to unorganized structure [7]. These empirical rules are useful for studies of biological tissues analyzing the data from the vibrational spectra. Amino group + H3N H Cα R Radical Carboxylic O ion C O - Fig. 1. The main structural components of amino acids Proteins are the second most abundant compounds in human body after water (70%). Human DNA (deoxyribonucleic acid), which contains all the genetic information, is constituted from proteins. Also, molecules of proteins are present in many fundamental parts of the cells. Therefore, proteins are very significant in biological organisms. They have functions like ferment catalysis, transportation and accumulation of nutrients, immune protection, exciting and transmitting nerve impulses, governing the processes of reproduction and development of cells. Primary structure of all the proteins is made of linear amino acids chain, which can form various secondary and tertiary structures. Eukaryotic proteins are mostly consisted from 20 different α-amino acids complex. The acids in that complex have unique side chains. Lipids are other important compounds of the cell. They are constructional material in the cells, responsible for supply of energy and water, also they have protective function. Mostly, long chains of fatty acids make lipids hydrophobic. Lipids have a lot of absorption bands in the so called ‘fingerprint’ spectral region caused by vibrations of C-H group. Four absorption bands at 3000 – 2800 cm -1 are common to most of the lipids: absorption bands at 2962 cm -1 are caused by antisymmetric vibration of methyl group (νasCH3), the bands at 2872 cm -1 – by symmetric vibration of metyl group (νsCH3), absorption bands between 2936 - 2916 cm -1 are caused by antisymmetric vibration of CH2 group (νasCH2) and the bands between 2863 – 2843 cm -1 – by symmetric vibration of CH2 group (νsCH2) [8]. In most standard tissue preparation methods non polar solvents like ethanol are used to eliminate lipids from the tissue. FTIR spectroscopy does not require such separation methods. Nucleic acids are responsible for transferring genetic information and proteins biosynthesis in living organism. IR absorption of nucleic acids is caused by vibrations of several functional groups. Functional groups are located on the main periodic structure of nucleic acid. Spectral
D. Lesciute et al. / Medical Physics in the Baltic States 7 (2009) 16 - 19 band at 1080 cm -1 is caused by symmetric vibration of PO2 and at 1240 cm -1 – by antisymmetric vibration of PO2 [7]. 3. Experimental Prostate samples were obtained from Vilnius University hospital Santariskiu Klinikos by means of cancerous tissue surgery. The prostate slices were taken from inner tissue and outer tissue. Such 10 microns thick slices were prepared by microtome from frozen samples. Similar slices from neighbouring layers of the tissue were prepared for histological observation. a b Fig. 2. Optical images of two prostate tissue slices: a) taken from inner tissue; b) taken from outer tissue. Spectra were obtained using FTIR spectrometer Vertex 70 combined with microscope “Hyperion 3000” from “Bruker” and with FPA MCT 64 × 64 detector. The resolution of the spectrometer was set to 8 cm -1 . Falsecolour images representing the distribution of cancerous cells in the tissue were obtained by integrating the area under the spectral band of specific amide I and amide II spectral bands. The OPUS software was used for this purpose. 4. The results and discussions The samples - 20 microns slices of cancerous prostate tissue - were investigated in this work by using two methods: infrared absorption spectroscopy at selected points of the sample and infrared spectroscopical imaging of whole area of the sample. Firstly, the samples were analyzed visually and only areas of very different density (e.g. transparent or completely dark) have been chosen for the single point infrared measurements. Optical images of selected areas are presented in fig. 2. The optical images were captured with optical microscope set to ×15 magnification. 18 Optical density 1.2 1.0 0.8 0.6 0.4 0.2 0.0 3500 3000 2500 2000 1500 Wavenumber, cm -1 Fig. 3. FTIR absorption spectra of slice taken from outer prostate tissue: red – dark spots; blue – light spots. 1000 The IR spectra were recorded in 4000 – 900 cm -1 spectral region e.g. in the region where CaF2 support plate for the tissue slices is transparent. The spectra typical for transparent and dark areas of the slices taken from outer prostate tissue are presented in fig. 3. Close look to the spectra reveals that they correspond to the same type of the cells, just thickness of the slice corresponding to blue spectrum is larger than that corresponding to the red spectrum. Identification of the spectral bands is presented in table 1. The assignment was made using data obtained for other biological tissues [3]. Table 1. Assignment of the spectral bands to normal vibrations Wave number (cm -1 ) Assignment 3295 Amide A (N-H bond) 3050 Amide B 2960 νas CH3 2930 νas CH2 2874 νs CH3 2852 νs CH2 1655 Amide I (νC=O, δC-N, δN-H) 1545 Amide II (δ N-H, ν C-N) 1476 δ CH2 1380 δ CH3 1240 νas PO - 2 1170 νas CO-O-C 1078 νs PO - 2 1050 νs CO-O-C 1030 νs C-O
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