Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 147 (2015) 245–256

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Complementary analysis of tissue homogenates composition obtained by Vis and NIR laser excitations and Raman spectroscopy Emilia Staniszewska-Slezak, Kamilla Malek ⇑, Malgorzata Baranska Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The homogenized rat tissues are

studied by Raman spectroscopy with the use of various laser excitations.  Spectral profiles of heart, brain, liver, lung, intestine, and kidney are analyzed.  Dependence of the Raman signature on laser excitation is discussed for each tissue.  Similarities and differences in Raman spectra of tissues are described in detail.

a r t i c l e

i n f o

Article history: Received 13 November 2014 Received in revised form 5 March 2015 Accepted 11 March 2015 Available online 28 March 2015 Keywords: Tissue homogenates Raman spectroscopy Excitation laser lines Comparative analysis

a b s t r a c t Raman spectroscopy and four excitation lines in the visible (Vis: 488, 532, 633 nm) and near infrared (NIR: 785 nm) were used for biochemical analysis of rat tissue homogenates, i.e. myocardium, brain, liver, lung, intestine, and kidney. The Vis Raman spectra are very similar for some organs (brain/intestines and kidney/liver) and dominated by heme signals when tissues of lung and myocardium were investigated (especially with 532 nm excitation). On the other hand, the NIR Raman spectra are specific for each tissue and more informative than the corresponding ones collected with the Vis excitations. The spectra analyzed without any special pre-processing clearly illustrate different chemical composition of each tissue and give information about main components e.g. lipids or proteins, but also about the content of some specific compounds such as amino acid residues, nucleotides and nucleobases. However, in order to obtain the whole spectral information about tissues complex composition the spectra of Vis and NIR excitations should be collected and analyzed together. A good agreement of data gathered from Raman spectra of the homogenates and those obtained previously from Raman imaging of the tissue cross-sections indicates that the presented here approach can be a method of choice for an investigation of biochemical variation in animal tissues. Moreover, the Raman spectral profile of tissue homogenates is specific enough to be used for an investigation of potential pathological changes the organism undergoes, in particular when supported by the complementary FTIR spectroscopy. Ó 2015 Elsevier B.V. All rights reserved.

Introduction ⇑ Corresponding author at: Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland. Tel.: +48 12 663 2064; fax: +48 12 634 0515. E-mail address: [email protected] (K. Malek). http://dx.doi.org/10.1016/j.saa.2015.03.086 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.

The coupling of a Raman spectrometer to a microscope along with highly sensitive charge coupled device (CCD) detectors enabled collection of spectral information even from sub-cellular

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level of a biological sample. This has opened a wide range of application of Raman techniques for ex vivo, in vitro and in vivo investigations [1,2]. In the medical field, Raman biospectroscopy can complement histopathology and provide chemical information which is available from time-consuming and costly immunochemical and biochemical arrays. The main focus is placed on Raman imaging that clarifies changes in distribution of cellular components in tissues due to development of several pathologies like tumors, atherosclerosis, diabetes and others. There are several reports demonstrating a potential of Raman spectroscopy in chemical characterization of tissues including spatial distribution of biomacromolecules [3–31]. This work is a continuation of our ATR FTIR studies of tissue homogenates [32]. It should be stressed here that homogenization is a minimal method of tissue preparation and is an alternative for cutting tissue sections and then choosing a proper staining. The aim of those studies was to evaluate to what extent chemical information gathered from FTIR profile of homogenized tissue agrees with spectral characteristics recorded with the use of FTIR imaging technique. Based on second derivative IR spectra, it was possible to assign bands e.g. to triglycerides and cholesterol esters, proteins, phosphate macromolecules (DNA, RNA, phospholipids, phosphorylated proteins) and others (glycogen, lactate) while peaks related to amide I mode revealed the secondary structure of proteins. The comparison of spectral information gathered from FTIR spectra of the homogenates and those obtained previously from FTIR imaging of the tissue sections implicated that biochemistrybased changes are consistent with the expected composition of each tissue with results of FTIR imaging. Here, we present Raman spectra recorded for the same samples like in [32] and the goal of this work is to provide a comparative analysis of Raman spectra of six animal tissues, i.e. brain, lung, heart, liver, intestines and kidney to describe their spectral profiles. Additionally, since Raman features depend on the wavelength of the laser excitation as well as the contribution of resonance Raman (RR) effect of chromophoric components, we recorded Raman spectra of the tissues with the use of the most common laser lines in the radiation range from 488 to 785 nm. Although most Raman studies on animal and human tissues have been mainly carried out using the NIR laser excitation (785 nm) due to the RR effect of heme-based metalloproteins and fluorescence background [3,4,6,11,12,18], some investigations have been also performed with an excitation in the Vis region of radiation [13,15,21,26–29]. It is possible since recent technological developments allow for tight laser focusing (creating a small voxel), and consequently eliminating the contribution of fluorescent components to Raman spectrum registered from a larger volume of the laser spot [8,13,15,21]. However, laser excitation in the visible region of radiation can cause photochemical and thermal degradation of a biological sample resulting usually from high laser power. In addition, we put our attention on a detailed comparison of spectral information gathered from each Raman spectrum of a given tissue to provide a guide what biochemical composition of a tissue is accessible from such spectra.

Experimental Sample preparation Homogenates were obtained from 3 adult male Wistar rats (N = 3), Krf:(WI)WV (Charles River Laboratory, Germany), 13– 15 weeks of age and weighing between 200 g and 220 g. The rats were anesthetized with thiopental (60 mg/kg). All animal experiments were performed in accordance with institutional guidelines and were approved by the Animal Care and Ethics Committee of the Jagiellonian University.

Tissue samples were thawed before use and washed out a few times with phosphate buffer (pH 7.4). Homogenization of tissues was performed immediately after collection of organs from animals. Aliquots of 200 mg of each tissue (myocardium, brain, liver, lung, intestine, and kidney) were weighed and placed in a glass mortar and pestle tissue grinder. The tissues were homogenized on ice with 1 ml of phosphate buffer (pH 7.4). No chemicals or protease inhibitors were added to homogenized tissues. All homogenized tissue samples prepared according this procedure were stored at 80 °C until analysis. Next 20 ll of each samples were placed on a slide glass (cleaned with ethanol and dried before measurements). Thin smears of whole blood were prepared on a glass slide. Tail blood samples were obtained from rats (N = 3, three thin smears per animal) by pinprick where the first drop of blood was wiped away and the next drop of blood was put on a window. Blood samples were collected before Raman measurements. All samples were dried in the desiccator at room temperature before Raman measurements. Raman spectroscopy Raman spectra of rat tissues were recorded using a WITec confocal CRM alpha 300 Raman microscope equipped with air cooled lasers operating at 488, 532, 632.8 and 785 nm and a CCD detector cooled to 82 °C. The lasers were coupled to the microscope via a single mode optical fiber with a diameter of 50 lm. A dry Olympus MPLAN (100/0.90NA) objective was used. The scattered radiation was directed to a spectrometer using a multi-mode fibers (50 and 100 lm core diameter for 488, 532 nm and 632.8 and 785 nm, respectively). Two Raman spectrographs were used and optimized for excitations in the Vis and NIR regions. Both spectrographs are equipped with the back-illuminated CCD cameras, but a deep-depletion camera is optimized for the 785 nm laser excitation. Gratings 600 and 300 g mm 1 were used for Vis and NIR excitation lines, respectively. Registered spectral regions were 0–4300, 0– 3700, 0–2600 and 0–3200 cm 1 for 466, 532, 632.8, and 785 nm lasers, respectively. Spectral resolution is ca. 3 cm 1 for 488 and 532 nm laser excitation and ca. 6 cm 1 for lines 632.8 and 785 nm. The monochromator of the spectrometer was calibrated using a radiation spectrum from a calibrated xenon lamp (Witec UV-light source). Additionally, standard alignment procedure (single point calibration) was performed before measurement with the use of the Raman scattering line produced by a silicon plate (520.7 cm 1). For all measurements, integration time was 0.5 s with 120 accumulations, then averaged. Laser power on a sample position was between 10 and 44 mW and a sample was illuminated through a 100 air objective (Olympus, MPLAN, FL N, NA = 0.90). Firstly, Raman features of the used glass substrate do not contribute to spectra of tissues as excluded by the comparison of Raman spectra of a clean glass slide and samples. Before further analysis all spectra were baseline corrected (a rubber method, 10 iterations), smoothed (9 points in a Savitzky–Golay algorithm), and then vector normalized in the fingerprint region. WITec Project 2.06 software was used to process data. Each Raman spectrum presented here was averaged from 90 spectra for each tissue collected from 3 animals. Results and discussion Even though, organs used in our experiment were washed out a few times with buffer, they can still contain blood whose resonance Raman signal of the heme group in hemoglobin of erythrocytes strongly contributes to spectra of tissues. Strong highly resolved bands have been reported in red blood cells when using

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a laser excitation from Vis to NIR region [33,34]. Therefore, we collected Raman spectra of rat blood using the same protocol like for tissues to identify unambiguously RR bands of RBCs signal. Fig. 1 depicts the comparison of Raman spectra of blood collected with the use of laser lines exciting at wavelengths of 488, 532, 633 and 785 nm. For assignments and discussions on band origins the reader is referred to previously published studies [8,33–35]. Several bands are observed in the fingerprint region of these spectra and the most pronounced bands were chosen as marker bands for RBCs (labeled with asterisks in Fig. 1). If their presence was observed in spectra of tissues, they were excluded from our analysis. In turn Fig. 2 as an example show all spectra of the liver tissue collected from three animals and recorded with the use of 532 nm from 30 points of the sample deposit that then were averaged. An examination of these spectra indicates good reproducibility of spectral information between animals and measurement points. A similar reproducibility was obtained for spectra of the other tissues presented below. Below, we discuss bands observed for each tissue in Raman spectra collected with the use of each laser line (Figs. 3–8) and finally we summarize the overall biochemical information gathered from Raman spectroscopy studies. Tables 1–3 present sets of Raman bands observed for all tissues in spectra collected for the given excitation line. To provide a useful comparison of our results and those gathered from Raman imaging of tissue crosssection, we collect Raman bands positions and their assignment from several Raman spectroscopy studies in Table 4 [3–5,7,11,12, 16,18,20,24,30]. One can also notice from Figs. 1 and 3–8 that

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Fig. 2. Raw spectra of the liver tissue from three animals (blue, red and green spectra for each animal) recorded using a 532 nm laser excitation that demonstrate the intra- and inter-sample variability. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Raman spectra of the rat brain tissue recorded using four excitation lines in the 300–1750 and 2800–3100 cm 1 region.

Fig. 1. Raman spectra of rat blood recorded using four excitation lines in the 300– 1750 and 2800–3100 cm 1 region. Asterisks denote selected marker bands of blood.

Raman spectra of investigated biosamples and recorded by 632.8 nm laser excitation exhibit low S/N ratio. Since a similar Raman spectra of blood and lung were recorded by Ingle and coworkers [27] by using a different Raman instrumentation, a primary reason for this observation is probably a low quantum efficiency of the detector and grating that are usually optimized for the 488 and 532 nm excitations. Thus, a good quality 633 nm Raman bio-spectrum is recorded when Raman scattering is

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high-intensity bands in the region above 2800 cm 1 which are assigned to the symmetric stretching modes of the CH2 and CH3 of lipids (ca. 2890 and 2855 cm 1) including olefinic lipids (3012 cm 1) [10,37]. Another band in this spectral region (at 2931 cm 1) is usually attributed to the m(CH3) mode of lipids and proteins together. Its high intensity is observed for each biosample if Raman spectrum collected with the 488 and 532 nm lasers is not dominated by the heme bands. In turn, this spectral region shows the presence of low-intensity bands when Raman spectrum is recorded using the NIR laser excitation. A high-content of lipids is also confirmed by the presence of bands at 1447 and 1309 cm 1 originating from the deformation and torsional vibrations of the CH2 groups [6,37]. The former is present in the spectra recorded by using 488, 532 and 785 nm on contrary to the 1306 cm 1 band that is found for the 488 nm excitation only. The other prominent bands assigned to lipids are observed in the 785 nm spectrum at 1298, 1264, 1128 and 1064 cm 1 [3,6], see the bands assignment in Table 3. This spectrum also shows the presence of cholesterol and phospholipids at 608 and 716 cm 1, respectively [6,11]. Raman spectra exhibit the presence of proteins by amide I and III bands as well as by the specific vibrations of aromatic amino acid residues and disulphide bridges. The position of the amide I in the regions of 1658–1650, 1680–1655 and 1665–1660 cm 1 shows the contribution of proteins with a-helical, b-sheet and random coils/turns conformations, respectively. In all Raman spectra of the brain tissue, except the 633 nm spectrum, an intense band at 1663 cm 1 is observed in the amide I region whereas this band shifts to 1670 cm 1 in the 633 nm spectrum and is accompanied by a 1454 cm 1 band typical for proteins [the d(CH3) mode], see Fig. 2.

Fig. 4. Raman spectra of the rat lung tissue recorded using four excitation lines in the 300–1750 and 2800–3100 cm 1 region.

enhanced by molecular resonance like for hemoglobin or by interaction with the metal surface like in SERS spectra [36]. Changes in overall intensity of raw Raman spectra due to switching laser excitation are illustrated in Fig. 9 for lung tissue. The lowest intensity is observed upon excitation by the red laser line whereas the other Vis spectra and NIR spectrum are comparable in the fingerprint region. A decrease in intensity of the high-wavenumber region is specific for NIR Raman spectrum resulting from recording this spectrum by using a deep-depletion CCD camera. From that reason, most Raman bio-analysis reported so far has been performed in the spectral region below 1800 cm 1 [4,5,7,8,11,12,14,16–18,20,23–25,30,31].

Spectral features of brain tissue Raman spectra of the brain tissue collected by the use of all laser lines are depicted in Fig. 3 while the positions of bands together with the assignment are listed in Tables 1–3. At first glance, Raman spectra collected using 488 and 532 nm laser excitations are similar, and then the laser excitation at 633 nm provides a poor Raman spectrum. Shifting laser excitation to the NIR region of radiation provides Raman spectrum of a different profile than the other spectra. Very weak-intensity bands of heme structures are only observed in the 532 nm spectrum at 754 and 1585 cm 1 indicating that this sample was almost RBCs-free. As it is expected from the known chemical composition of the white and gray matter of the brain, the Raman spectra of this tissue should reflect high lipid content. This was clearly observed in the FTIR spectrum recorded for the same tissue in our previous studies [32]. The use of the blue and green laser lines results in appearing

Fig. 5. Raman spectra of the rat heart tissue recorded using four excitation lines in the 300–1750 and 2800–3100 cm 1 region.

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488 nm excitation line but not in liver tissue imaged by using the 532 nm laser [29]. This band is easily recognized in NIR Raman spectra of brain tissue, see Table 4. Therefore, the discussed here bands of the homogenized brain tissue originate from tryptophan and phospholipids, respectively. Surprisingly, none of spectral features attributed to nucleic acids is found in the 785 nm spectrum on contrary to Raman imaging of cross-sections of the brain tissue [18,24,30,31]. To our best knowledge, there is no report showing Raman features of brain tissue recorded by using 488 and 532 nm, thus we cannot compare information gathered here from the homogenate with other methods of tissue preparation. On the other hand, the comparison of the NIR Raman spectra clearly show that chemical information identified in the spectrum of the homogenized tissue is very similar to those reported for tissue cross-sections, see Tables 3 and 4 [16,18,24,30]. Spectral features of lung tissue The mean Raman spectra of the lung tissue are presented in Fig. 4 while the positions of bands are collected in Tables 1–3. The comparison of these spectra with Raman spectra of rat blood in Fig. 1 clearly shows that spectral profile of the lung tissue recorded with the use of 532 and 633 nm laser excitations is dominated by resonance Raman signals of erythrocytes (green and red traces in Figs. 1 and 4). This problem was also noticed in Raman studies on unfixed (air-dried) cross sections of lung tissues collected by using these laser lines [26,27]. Despite this fact bands present in all spectra provide some information about lipids and proteins. Our 532 and 633 nm Raman spectra and their counterparts reported in the literature are almost identical in the Fig. 6. Raman spectra of the rat liver tissue recorded using four excitation lines in the 300–1750 and 2800–3100 cm 1 region.

One would simply assign this band to a-helical and turn structures of the brain proteins, however a contribution of the stretching mode of the olefinic C@C bonds cannot be excluded since this tissue is lipid-rich. This is found in agreement with our FTIR studies for this homogenate [32] as well as with Raman mapping of tissue cross sections of human brain [6]. On the other hand, the secondary structure of proteins is expressed in Raman spectra by positions of amide III bands. For brain, all Raman spectra show only a band at ca. 1265 cm 1 assigned to a-helical structures [6,12,16,38,39]. This is congruent with FTIR spectrum of this sample that exhibited the largest contribution of a-helices in the overall composition of proteins [32]. In addition, the changes in tertiary structure of proteins can be monitored by changes in position and intensity of bands attributed to vibrations of amino acid residues which are sensitive to local environment through hydro- and hydrophilic interactions. In Raman spectra of the brain recorded with the use of 488 and 532 nm lasers, we found only features at 1006 and ca. 560 cm 1 typical for phenylalanine and tryptophan, respectively [6]. The NIR Raman spectrum exhibits in turn the presence of weak bands assigned to phenylalanine (1001 cm 1), tryptophan (883 cm 1) [11] and tyrosine (648 cm 1) [5] as well as a medium intensity band characteristic for cysteine (541 cm 1) [9]. The most pronounced bands of nucleic acids are observed at 1093 and 565 cm 1 in the Raman spectra recorded with the use of excitation lines at 488 and 532 cm 1 only. They are usually attributed to the symmetric stretches of the PO2 group of DNA and cytosine/guanine, respectively [16]. However, one can expect the presence of the marker band of DNA at ca. 785 cm 1 confirming the assignment of the 565 and 1093 cm 1 bands to nucleic acid. The 785 cm 1 band of weak intensity was found in Raman maps of the nucleus of endothelial cells irradiated by a

Fig. 7. Raman spectra of the rat intestinal tissue recorded using four excitation lines in the 300–1750 and 2800–3100 cm 1 region.

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Fig. 8. Raman spectra of the rat renal tissue recorded using four excitation lines in the 300–1750 and 2800–3100 cm 1 region.

fingerprint region (the high-wavenumber region is not shown in the cited publications) [26,27]. The differences found in the 532 nm spectra regard the contribution of resonance effect of

Table 1 Positions of Raman bands (in cm Brain

Lung

RBCs [26]. The high-wavenumber region in the spectra of the homogenized lung tissue exhibits the presence of a typical for a biosample band at 2933 cm 1 originating from lipids and proteins. The latter is also represented by amide I and III bands but they are found in the 488, 532 and 785 nm Raman spectra only. Interestingly, an amide I band is found at 1641 and 1654 cm 1 in the spectra with excitation at 532 and 785 nm, respectively, showing a contribution of different protein conformations. Both wavenumbers of the amide I band are found in agreement with other Raman studies on human, pig and rat lung tissues by using 514 and 785 nm laser line [5,12,13]. The 1654 cm 1 band is typical for such a collagen-type tissue however, we did not found other bands characteristic for this protein as suggested in [12], most likely, due to contribution of Raman bands of erythrocytes to spectral features of lung tissue. The comparison of bands positions of the homogenized and cross-sectioning lung tissue for the laser line at 785 nm (Tables 3 and 4) shows that these Raman spectra are considerably distinct [3,5,12,14]. The main differences are found in the 1000–1700 cm 1 region in that the opposite changes in intensity are observed. Because these tissue specimens were snap-frozen without fixing and embedding medium, the preparation method cannot fully explain the observed difference. Likely, the contribution of the RR effect of erythrocytes in the homogenized sample strongly disturbs Raman profile collected for this material by using the NIR excitation. Other bands observed in the spectral region below 1450 cm 1 in the 488, 532 and 785 nm spectra are assigned to different biomolecules, see Tables 1–3. All of them exhibit the presence of a band at ca. 1450 cm 1 attributed to proteins and lipids whereas the aromatic amino acid residues such as Phe and Trp give their specific bands in the visible range of excitation only. In turn a 1082 cm 1 band of the phosphate group is found in the 532 nm spectrum only while the conformation of the SAS bond in proteins can be monitored by a laser excitation in the NIR region, c.f. Tables 1–3, Fig. 4. Spectral features of heart tissue Similarly to Raman spectra of lung, spectra of myocardium collected with the visible laser excitations are obscured by signals of

1

) with their assignment observed in spectra of tissues for 488 nm excitation line [3–7,10,12–14,16,37,38,39–41]. Heart

3051

Liver

Intestines

Kidney

Assignment

3055

3054

3051

2934 2886 2859 1663

2962 2934 2896 2856 1660

m(CH) aromatic amino acid residues m(@CH) olefinic lipids mas(CH3) lipids and proteins ms(CH3) lipids and proteins ms(CH3) lipids ms(CH2) lipids Amide I: a-helix/random coil; m(C@C) lipids m(C@C) tryptophan

3012 2932 2893 2857 1663

2933 2892

2934 2891

2931 2890 1661

1447

1451

1654 1551 1445 1398 1362

1365 1318

1316

1453 1401 1364 1314

1449

1345 1313

1451 1400 1364 1310

1306 1269 1246 1220 1127 1093 1074 1006 563

1130

1004

1242 1226 1155 1128

1000

1128 1100

1006 565

d(CH2/CH3) proteins; d(CH2) lipids , cholesterol, phospholipids

ms(COO ); CH2 deformation Guanine/tryptophan Adenine, guanine, CH deformation of proteins d(CH2) lipids Torsional (CH2) lipids Amide III: a-helix; collagen Amide III: unhydrated b-sheet Amide III: hydrated b-sheet m(CC/CN) proteins ms(COC) glycogen; ms(PO2) DNA

1095

1106

ms(PO2): DNA, phospholipids m(CC) lipids, phospholipids

1006 785 567

1006

Phenylalanine Nucleic acids Tryptophan/cytosine/guanine

565

E. Staniszewska-Slezak et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 147 (2015) 245–256 Table 2 Positions of Raman bands (in cm Brain 3012 2931 2885 2853 1661 1447 1340 1309 1267 1091 1066 1006 603 569 549

Brain

) with their assignment observed in spectra of tissues for 532 nm excitation line [3–7,10,12–14,16,17,37,38,40–44]. Heart

2931

2929

Liver

Intestines

Kidney

Assignment

3062

3060 3012 2931 2889 2856 1661

3062

m(CH) aromatic amino acid residues m(@CH) olefinic lipids m(CH3) lipids and proteins ms(CH3) lipids ms(CH2) lipids Amide I: a-helix/random coil; m(C@C) lipids

1449 1344 1309

1453

2931

1661 1641 1438

1453

1082

1082

1002

1082 1006

1236 1093 1068 1006

Lung

1654

1440 1394 1374 1351

1335 1298 1264

1086 1006

) with their assignment observed in spectra of tissues for 785 nm excitation line [3–7,10,12–14,16,37,38,40–45]. Heart

Liver

Intestines

Kidney

Assignment

2926

2927

2921 2882

2929

1656 1600 1543 1445 1393

1656 1602 1540 1443 1391

1656 1607 1541 1445 1402

1656 1604 1550 1440 1402

m(CH3) lipids and proteins ms(CH3) lipids ms(CH2) lipids Amide I: a-helix/random coil; m(C@C) lipids Amide I: a-helix

1342

1353 1338

1345 1327 1303

1310

1314 1262

1128 1085 1064

1119

1001 963 930 883

1000 966

1117 1088 1044 999 966 923 893 850 823

1120 1090 1070 1002 970 926 885

1236 1125 1090 1060 1001 966 929 884 858 781

714 660

Amide I: b-turn d(CH2/CH3) proteins; d(CH2) lipids, cholesterol, phospholipids Adenine, guanine, CH deformation of proteins Torsional (CH2) lipids Amide III: a-helix; collagen Amide III: hydrated b-sheet ms(PO2 ): DNA, phospholipids m(CC) lipids, phospholipids Phenylalanine Cholesterol Tryptophan/cytosine/guanine Cholesterol

1

1255

756 740 716

2933 2880 2859 1661

558

3930 2883 2855 1660

1442

1

Lung

Table 3 Positions of Raman bands (in cm

251

718

716

716

1257 1227 1123 1095 1077 1040 999 962 925 886 854 826 780 752 713 663

648 608 576 541 534

530

409

411

420

541 522 424 411

534 407

RBCs, in particular those recorded with the use of 532 and 633 nm, see Figs. 1 and 5. Excluding the heme bands, the comparison of spectra of lung and heart, as the heme-rich tissues, clearly shows that both samples present different Raman features expressing differences in chemical composition, details in Tables 1 and 2. For instance, the 488 nm excitation leads to appearing bands at 678 and 1551 cm 1 assigned to guanine and tryptophan, respectively, in the spectrum of heart only whereas vibrations of DNA and

530 431 405

Bilirubin, phenylalanine

m(C@C) tryptophan d(CH2/CH3) proteins; d(CH2) lipids, cholesterol, phospholipids

ms(COO ); CH2 deformation Nucleic acids Adenine, guanine, CH deformation of proteins Collagen d(CH2) lipids; amide III Amide III: a-helix; collagen Amid III: b-sheet; cytosine Amide III: a-helix m(CN) proteins; m(CC) lipids ms(PO-2): DNA, phospholipids m(CC) lipids, phospholipids Proline Phenylalanine CH3 deformation: lipids, proteins [24], m(CACa) peptide backbone Tryptophan Tyrosine, glycogen, proline Tyrosine Cytosine/nucleic acids Tryptophan Adenine/nucleic acids Phospholipids Guanine/nucleic acids Tyrosine Cholesterol Tryptophan/cytosine, guanine Cholesterol m(SS) cysteine ggt conformation Cholesterol Undefined

phenylalanine are present in the 532 nm spectrum of lung [40]. The cardiac extracellular matrix mainly contains fibrillar collagens but FTIR spectrum of the heart homogenate studied by us in [32] did not show a typical set of bands for collagen. Here, only Raman spectrum collected with the NIR laser line can indicate the presence of collagen by the presence of bands at 1656, 1338 and 1251 cm 1 [6,12,39]. The same amide I band also appears in the blue-laser excited spectrum but this is a small shoulder and

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Table 4 Positions of Raman bands (in cm Positions [cm

1

]

Brain tissue 1740 vw 1658–1670 m-s 1618 w, sh 1604–1607 w 1584 w, sh 1450 vs 1436 vs 1340 s 1340 w 1301 s 1296 vs 1274 w 1263–1266 w 1250 s 1227–1234 w 1207 w 1171–1175 w 1156 s 1130 w 1099 m 1081 m 1062 m 1032 w 1002 s 957 m 926–934 vw 853 m 828 vw 782 vw 758 vw 725 w 718 vw-w 700 w-m 663–666 w 642 vw-m 621 vw-m 550 524 m, br Lung tissue 1738 vw 1655–1660 s-vs 1605 m, sh 1585 w, sh 1438–1449 vs 1322 m 1297 m 1270 m 1252–1259 s 1123–1127 w 1064 w 1000–1004 m-s 970 vw 963 vw 941–949 w 874 w 852 w 829 w 778 w 717 w 700 vw 668 vw 644 vw 621 vw Liver tissuea,b 3010–3013b w 2855–2962b vs 1658–1663b m 1658a vs 1640–1680b m 1605a vw 1595–1597b s 1449a,b s-vs

1

) with their assignment observed in Raman images of tissues (mainly 785 nm excitation line). Assignment

Refs.

m(C@O) lipids Amide I, m(C@C) lipids

[16] [16,18,30] [24,30] [24,30] [18,24,30] [16,24,30] [18,24,30] [30] [16] [16] [18,24] [16] [18,30] [30] [16,30] [30] [24,30] [30] [16,18,24,30] [24,30] [30] [16,18,24,30] [16,24,30] [16,18,24,30] [30] [18,30] [24,30] [24,30] [18,24,30] [24,30] [30] [16,24] [18,24] [24,30] [24,30] [24,30] [30] [30]

Tyrosine, tryptophan Phenylalanine, tyrosine Pyridine ring (nucleic acids), phenylalanine d(CH2/CH3) proteins (collagen); d(CH2) lipids, cholesterol, phospholipids CH2 bending, paraffin wax CH3/CH2 wagging of collagen, nucleic acids Amide III Amide III Aliphatic side chains, lipids Amide III Amide III (a-helix) Amide III (b-sheet) Cholesterol, phospholipids Hydroxyproline, tyrosine, tryptophan, phenylalanine Cholesterol, phospholipids, paraffin wax Carotenoids, CAN stretching of proteins Aliphatic side chains, phenylalanine, lipids, paraffin wax Nucleic acids, phospholipids Nucleic acids Aliphatic side chains, lipids, paraffin wax Phenylalanine Phenylalanine Hydroxyapatite, carotenoid/cholesterol m(CACa) peptide backbone, glycogen, proline, valine Tyrosine, proline Tyrosine, DNA, cytosine, uracil, thymine DNA, nucleic acids Tryptophan Adenine Phospholipids Cholesterol, lipids Nucleic acids, cysteine Tyrosine Phenylalanine Tryptophan, cytosine, guanine S-S in proteins

m(C@O) lipids Amide I Bilirubin, phenylalanine Phenylalanine CH2 proteins, lipids CH2 proteins Lipids Lipids Amide iii CAC lipids, CAN protein Lipids Phenylalanine CH3 deformation: lipids, proteins CH3 deformation: lipids, proteins Proteins, collagen phospholipids, collagen Tyrosine Tyrosine Nucleic acids Phospholipids Cholesterol, lipids Nucleic acids Tyrosine Phenylalanine

mas(@CH) mas(CH2/CH3), ms(CH2/CH3) m(C@C) lipids Amide I Bilirubin, phenylalanine Vitamin A CH2 of proteins and lipids

[5] [3,5,12] [5] [3] [3,5,12] [5] [5] [5] [3,5] [3,5,12] [3] [3,5,12] [3,5] [12] [3,5] [5] [3,5] [3] [3] [3,5] [3,5] [3,5] [3] [3] [15] [15] [15] [7] [15] [7] [15] [7,15]

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1

a

1340 m 1303–1313b 1264–1277b 1256a s 1164–1166b 1125a w 1089–1099b 1003a vs 970a vw 936a w 853a w 829a w 782a w 746a w 719a w 668a vw 644a vw 622a w

w w w w

Intestinal tissue 1657–1665 vs 1449–1451 vs 1340 w 1297 m 1266 m 1244 m 1209 w 1126 w 1060 w 1100 m 1004 vs 936 vs 854 vs 827 w 815 m 783 vw 759 w 721 vw 700 vw 643 w 621 w Renal tissuea,b 1728a vw 1657a vs 1633b w 1618a w 1545b vs 1460b s 1448a s 1406a s 1259a m 1123a w 1097a m 1003a s 938a s 854a m-s 743a w 673a w

]

Assignment

Refs.

Adenine, guanine, CH deformation of proteins CH2 deformation of lipids @CH deformation of lipids Amide III Vitamin A CAC lipids, CAN protein DNA Phenylalanine CH3 deformation: lipids, proteins Protein backbone Tyrosine Tyrosine Nucleic acids Nucleic acids Lipids Nucleic acids Tyrosine Phenylalanine

[7] [15] [15] [7] [15] [7] [15] [7] [7] [7] [7] [7] [7] [7] [7] [7] [7] [7]

Amide I CH3 deformation proteins, lipids Collagen Lipids Amide iii Amide iii, collagen Phenylalanine m(CN) proteins; m(CC) lipids m(CC) lipids, phospholipids Nucleic acids Phenylalanine m(CACa) peptide backbone, collagen Hydroxyproline, proline, tyrosine, collagen Tyrosine Hydroxyproline, proline Nucleic acids Tryptophan Lipids Cholesterol Tyrosine Phenylalanine

[4] [4] [4] [4] [4] [4] [4] [4] [4] [4] [4] [4] [4] [4] [4] [4] [4] [4] [4] [4] [4]

Phospholipids Amide I

[20] [20] [13] [20] [13] [13] [20] [20] [20] [20] [11] [20] [11] [11,20] [11,20] [11,20]

Tyrosine, tryptophan Amide II CH2 proteins, lipids

ms(COO ); CH2 deformation Amide III CAC lipids, CAN protein Nucleic acid Phenylalanine Proline Proline, tyrosine Thymine Cysteine

vs – very strong, s – strong, m – medium, w – weak, vw – very weak. a 785 nm excitation line. b 532 nm excitation line.

is accompanied by an amide III band at 1226 cm 1 originating from a b-sheet structure of proteins. Apart from that, a band at ca. 1623 cm 1 appears in the 633 nm spectrum suggesting the presence of proteins with b-sheet structures or tryptophan [11]. Since the same band is found in the 633 nm spectrum of lung, one cannot exclude that this band results from resonance Raman effect of red blood cells. Taking into account this effect on the Raman spectra, we suggest that the heart tissue should be examined using 488 and 785 nm excitation lines. The lipid bands in the high-wavenumber

range and at 1445 cm 1 are present in both spectra. Exciting Raman effect by the blue laser line, we can also observe a Raman signature of guanine at 1362 and 678 cm 1 which is absent in the NIR Raman spectrum. In turn, the latter exhibits a number of bands attributed to vibrations of proline (1044 cm 1), phenylalanine (999 cm 1) and cysteine (530 cm 1). Interestingly to our best knowledge, this tissue has not been extensively investigated by employing Raman spectroscopy imaging. A few reports have shown in situ investigations of cytochrome redox state in cardiomyocytes when Raman scattering was excited by a 532 nm

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Examining other spectral features of the liver tissue, we observed that vibrations of lipids appear at 1313 cm 1 in the 532 nm Raman spectrum and phospholipids at 716 cm 1 in the 785 nm Raman spectrum. Apart from the typical band for Phe at ca. 1000 cm 1 that is present in all spectra, only bands at ca. 530 cm 1 attributed to cysteine are found in the NIR spectrum. A typical component of the liver tissue is bilirubin found in Raman difference spectra between tissue compartments by Krafft et al. [7]. Here, we observe only one of bilirubin bands located at 1604 cm 1, however, we do not exclude the presence of other spectral features of this biomolecule (1264, 1226, 971 cm 1) since they can be overlapped by other bands, c.f. Fig. 6. Next, several bands exhibit the presence of nucleic acids, mainly purine and pyrimidine bases, but they are specific for the excitation line. In the 488 nm spectrum, we observe bands at 1364 and 679 cm 1 assigned to guanine and 565 cm 1 specific for cytosine and guanine whereas the green and NIR laser lines excite only a band at 1342 cm 1 specific for adenine and guanine. It is worth to stress that a band at 1080–1090 cm 1 of the symmetric stretches of the phosphate group are absent in all spectra of the liver tissue. The comparison of Raman peaks positions and overall profile for the homogenized tissue and cross-sections reported in [7, unfixed tissue, laser: 785 nm] and [15, tissue fixed with 4% formalin, laser: 532 nm] and collected in Tables 2–4 shows differences in bands intensity in the region 1700–1100 cm 1 in NIR and Vis Raman spectra but almost all bands are found in spectra of the homogenate and cross-sections. More pronounced differences are found in the NIR spectra and they are similar to those discussed for lung. Spectral features of intestinal tissue

Fig. 9. Averaged raw spectra of the lung tissue showing the overall variation of intensity due to changing excitation lines.

laser line [8,28]. Both works have reported the presence of three intensive bands at 751, 1130, and 1582 cm 1 assigned to heme proteins. An additional processing and analysis of Raman spectra have showed the presence of other peaks mainly contributed to changes in redox state of heme.

Spectral features of liver tissue Raman spectra of liver homogenate are depicted in Fig. 6. The strong signals of RBC are observed in spectrum excited using the green-laser line only (green trace in Fig. 6). Despite that the high-wavenumber region in this spectrum shows the presence of high-intensity band at 2931 cm 1 accompanied by a 1453 cm 1 band in the fingerprint region that are an evidence for biomacromolecules [15]. The other spectra also show characteristic peaks of heme but they do not dominate in the spectral profile. This is also confirmed by the lack of signal when the red laser line was used (red trace in Fig. 6). Interestingly, a position of an amide I band shifts from 1661 to 1656 cm 1 after changing excitation lines from Vis to NIR radiation. This is consistent with studies on Raman spectroscopy imaging of cross sections of liver by using 532 and 785 nm laser lines [7,15]. Kochan et al. assigned a 1661 cm 1 band to the stretching mode of the lipidic C@C bond since they analyzed lipid droplets in the liver tissue (excitation: 532 nm) [15] while Krafft and coworkers attributed a band at 1656 cm 1 to proteins and lipids (excitation: 785 nm) [7]. A similar observation was found in the corresponding spectra of kidney and intestines studied here, as described below.

Average Raman spectra of intestines are presented in Fig. 7. All of them show a weak Raman signal of red blood cells. Similarly to Vis Raman spectra of brain, the high-wavenumber region shows a high content of lipids including the presence of olefinic lipids, see blue and green traces in Figs. 3 and 7. This is consistent with our FTIR spectroscopy studies that showed that the highest lipid to protein ratio is specific for brain and intestines, ca. 0.92 and 0.66, respectively. Actually, spectral features observed in spectra recorded using 488 and 532 nm are almost identical for both tissues. In turn, the 633 nm spectrum of intestines is free of Raman signal on contrary to brain whereas the NIR spectra of both tissues differ significantly. Intestinal tissue is mainly composed of protein filaments and this composition is reflected in several Raman peaks assigned to protein moieties, see Table 3. For example, collagen vibrations appear at 1338 and 1262 cm 1 along with bands of Phe (1602 and 1001 cm 1), Trp (884 cm 1) and Cys (534 cm 1) [3–6,9,11]. Apart from that, bands of nucleic acids and phospholipids are observed in the NIR spectrum at 781 and 716 cm 1, respectively. When we compared the recorded here NIR spectrum of this tissues with those for frozen cross-sections of colon tissue reported in [4], we found first of all decreasing intensity of amide I and III bands and bands in the 1000–800 cm 1 due to the homogenization of the tissue. The spectral information gathered from the latter is very similar to those for the spatially resolved Raman spectra of the colon tissue, except bands observed in the region below 800 cm 1. Only a few bands are found in this region (at 534, 716 and 781 cm 1) whereas Raman mapping indicated the presence of lipids and amino acid residues, see Tables 3 and 4. Spectral features of renal tissue Raman spectra of the kidney homogenate displayed in Fig. 8 show that this sample exhibits a low content of blood. Similarly to the previous tissues, Raman spectrum collected with the use of a red laser line is Raman signal-free. The protein and lipid

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content of the sample is specified in the region above 2800 cm 1 in the Raman Vis spectra. However a 3012 cm 1 band of unsaturated lipids is absent, on contrary to brain and intestinal spectra, and despite the fact that our FTIR studies on tissue homogenates suggested a similar content of olefinic lipids for the three tissues [25]. Analyzing the fingerprint region of both Vis Raman spectra (488 and 532 nm), we observe that the spectral features of renal tissue are very similar to Vis spectra of liver tissue (see Figs. 6 and 8). Only the 488 nm spectra show differences in intensities and shapes of bands centered at ca. 1100 cm 1. This region is characteristic for the phosphate groups in DNA and phospholipids. Lyng et al. have also proposed to assign this broad spectroscopic feature to the COC stretching vibrations of glycogen [21]. Our FTIR studies of homogenates studied here clearly showed that a high content of glycogen is specific for the liver tissue while FTIR spectrum of the renal tissue exhibits a higher intensity of bands attributed to the phosphate groups of phospholipids and nucleic acids than liver. Thus, the observed spectral differences in Raman spectra simply result from different composition of phospholipids, nucleic acids and polysaccharides. Among Vis Raman spectra of the renal homogenate (blue and green traces in Fig. 8), the 488 nm spectrum is less obscured by RR signal of heme. Here, one can find a protein/ lipid band at 1660 cm 1, whereas the 1275–1230 cm 1 region exhibits amide III bands. In turn, an overall profile of the NIR Raman spectrum is similar to the corresponding spectrum of liver, c.f. black traces in Figs. 6 and 8. Comparison of NIR Raman spectra of air-dried and fresh normal kidney tissues investigated in [11] and [20], respectively, one can notice an increase in intensity of the region below 1200 cm 1 when a sample was freestyle frozen and then kept at room temperature before Raman measurements [11]. The opposite effect is found in our studies when we again observe low-intensity bands in the 1200–400 cm 1 range, see Tables 3 and 4.

Conclusion Our results demonstrate the potential of Raman spectroscopy to characterize selected tissue in the form of homogenized samples. Unlike FTIR spectra, which must be mathematically processed (by using e.g. second derivative) to reveal differences between tissues, Raman spectra of each tissue clearly illustrate differences in chemical composition. It is not the aim of this work to provide a detailed comparison between IR and Raman profiles of biological samples since this has been widely discussed in the literature. To summarize briefly our FTIR and Raman investigations on tissue homogenates, reported in [32] and in this work, the former results clearly characterized the secondary structure of proteins and properties of lipids along with a variation in composition of carbohydrates whereas an advantage of Raman spectra is information about amino acid residues, nucleotides and nucleobases. In the presented Raman studies, we noticed that the prosthetic group of hemoglobin is the main contributor to the Raman spectrum of most tissues under 532 nm-wavelength excitation. This indicates that the 532 nm laser lines cannot be successfully employed in such investigations if complete removing of blood from the homogenized sample is limited due to morphological structure of tissue. Next, the 633 nm wavelength excitation provides low S/N ratio or no Raman signals of the homogenized tissues, showing that this laser line cannot be universally applicable in Raman bio-analysis. In general if we compare corresponding spectra of all tissues we noticed that the Vis Raman spectra of brain/intestines and kidney/ liver are very similar whereas heme-rich tissues of lung and myocardium exhibit different spectral profile. In turn, the NIR Raman spectra are specific for each tissue and more informative than

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the corresponding spectra collected with the Vis excitations. Most bands in NIR Raman spectra appear at similar wavenumbers but the most pronounced differences correspond to relative intensities, especially in the region of 850–1500 cm 1. However, the high-wavenumber region in the Vis Raman spectra provides a clear evidence for a high content of lipids, including unsaturated fractions. For the latter, we noticed that the contribution of resonance Raman effect to a spectral profile of tissue can lead to the loss of a 3012 cm 1 band. This is clearly shown in our Vis Raman spectra of brain and myocardium. FTIR spectra of these homogenized tissues reported in [32] revealed a higher content of unsaturated lipids in myocardium than in brain whereas we observed Raman characteristics of lipids unsaturation in the Vis Raman spectra of brain only. Since most studies on tissues in means of Raman spectroscopy imaging have been performed for the 785 nm excitation, we compared our NIR Raman spectra with spectra from Raman imaging of tissue sections. For brain and colonic tissues, we found that clusteraverage Raman spectra of healthy tissue sections [4,18,23] are almost identical with the spectrum of the brain and intestinal homogenates recorded here except differences in bands intensity in the 700–1000 cm 1 region. These changes revealed by cluster analysis of the brain section mainly showed variation in composition of cholesterol, cholesterol esters and phospholipids [18] while for colonic tissues the observed differences between spectra of homogenate and sections mainly regard intensity of bands assigned to aromatic amino acid residues in proteins of connective tissue [4,23]. For other tissues prepared without fixation like kidney [11,20], liver [7], lung [3,5,12], the main differences between Raman spectra of fresh and fixed tissues and our spectra of homogenates are associated with much more lower intensity of bands in the region of 1600–1750 cm 1 in spectral profile of homogenates. This observation likely results from the preparation method of tissue sections and homogenates. In summary, the spectral changes observed in Raman spectra of homogenized tissue are specific enough for future investigations toward recognition of a disease state, in particular in conjunction with the complementary FTIR spectroscopy. Acknowledgements This work was supported by National Center of Science (DEC2013/08/A/ST4/00308) and by the European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (Grant coordinated by JCET-UJ, No POIG.01.01.02-00-069/09). Authors would like also thank Analytical and Pharmacokinetic Laboratory of JCET for supplying samples of tissue homogenates. References [1] M. Diem, P.R. Griffiths, J.M. Chalmers, Vibrational Spectroscopy for Medical Diagnosis, Wiley, Chichester, 2008. [2] M. Diem, A. Mazur, K. Lenau, J. Schubert, B. Bird, M. Milijkovic, C. Krafft, J. Popp, J. Biophotonics 6 (2013) 855. [3] C. Krafft, D. Codrich, G. Pelizzo, V. Sergo, Vib. Spectrosc. 133 (2008) 361. [4] C. Krafft, D. Codrich, G. Pelizzo, V. Sergo, J. Biophotonics 1 (2008) 154. [5] C. Krafft, D. Codrich, G. Pelizzo, V. Sergo, Analyst 133 (2008) 361. [6] C. Krafft, S.B. Sobottka, G. Schackert, R. Salzer, Analyst 130 (2005) 1070. [7] C. Krafft, M.A. Diderhoshan, P. Recknagel, M. Miljkovic, M. Bauer, J. Popp, Vib. Spectrosc. 55 (2011) 90. [8] M. Ogawa, Y. Harada, Y. Yamaoka, K. Fujita, H. Yaku, T. Takamatsu, Biochem. Biophys. Res. Commun. 382 (2009) 370. [9] P.O. Andrade, R.A. Bitar, K. Yassoyama, H. Martinho, A.M. Santo, P.M. Bruno, A.A. Martin, Anal. Bioanal. Chem. 387 (2007) 1643. [10] R.M. Palaniappan, K.S. Pramod, Vib. Spectrosc. 56 (2011) 146. [11] A.W. Auner, R.E. Kast, R. Rabah, J.M. Poulik, M.D. Klein, Pediatr. Surg. Int. 29 (2013) 129. [12] Z. Huang, A. McWilliams, H. Lui, D.I. McLean, S. Lam, H. Zeng, Int. J. Cancer 107 (2003) 1047. [13] A. Lorincz, D. Haddad, R. Naik, V. Naik, A. Fung, A. Cao, P. Manda, A. Pandya, G. Auner, R. Rabah, S.E. Langenburg, M.D. Klein, J. Pediatr. Surg. 39 (2004) 953.

256

E. Staniszewska-Slezak et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 147 (2015) 245–256

[14] C. Krafft, D. Codrich, G. Pelizzo, V. Sergo, J. Raman Spectrosc. 40 (2008) 595. [15] K. Kochan, K.M. Marzec, K. Chruszcz-Lipska, A. Jasztal, E. Maslak, H. Musiolik, S. Chlopicki, M. Baranska, Analyst 138 (2013) 3885. [16] L.L. Tay, R.G. Tremblay, J. Hulse, B. Zurakowski, M. Thompson, M. BaniYaghoub, Analyst 136 (2011) 1620. [17] M. Köhler, S. Machill, R. Salzer, C. Krafft, Anal. Bioanal. Chem. 393 (2009) 1513. [18] N. Amharref, A. Beljebbar, S. Dukic, L. Venteo, L. Schneider, M. Pluot, M. Manfait, Biochim. Biophys. Acta 1768 (2007) 2605. [19] K. Gajjar, L.D. Heppenstall, W. Pang, K.M. Ashton, J. Trevisan, I.I. Patel, V. Llabjani, H.F. Stringfellow, P.L. Martin-Hisch, T. Dawson, F.L. Martin, Anal. Methods 5 (2013) 89. [20] C.A. Lieber, M.H. Kaberr, J. Pediatr. Surg. 45 (2010) 549. [21] F.M. Lyng, E.Ó. Faolain, J. Conroy, A.D. Meade, P. Kneif, B. Duffy, M.B. Hunter, J.M. Byrne, P. Kelehan, H.J. Byrne, Exp. Mol. Pathol. 82 (2007) 121. [22] C. Krafft, L. Neudert, T. Simat, R. Salzer, Spectrochim. Acta A 61 (2005) 1529. [23] A. Beljebbar, O. Bouché, M.D. Diébold, P.J. Guillou, J.P. Palot, D. Eudes, M. Manfait, CRC Cr. Rev. Oncol-Hem. 72 (2009) 255. [24] C. Krafft, S.B. Sobottka, G. Schackert, R. Salzer, J. Raman Spectrosc. 36 (2006) 367. [25] J. Fleureau, K. Bensalah, D. Rolland, O. Lavastre, N. Rioux-Leclercq, F. Guillé, J.J. Patard, R. de Crevoisier, L. Senhadji, Expert Syst. Appl. 38 (2011) 14301. [26] A. Pavic´evic´, S. Glumac, J. Sopta, A. Mojovic´-Bijelic´, M. Mojovic´, G. Bacˇic´, Croat. Med. J. 53 (2012) 551. [27] T. Ingle, E. Dervishi, A.R. Biris, T. Mustafa, R.A. Buchanan, A.S. Biris, J. Appl. Toxicol. 33 (2013) 1044. [28] N.A. Brazhe, M. Treiman, B. Faricelli, J.H. Vestergaard, O. Sosnovtseva, PLoS One 8 (2013) e70488. [29] K. Majzner, K. Kochan, N. Kachamakova-Tojanowska, E. Maslak, S. Chlopicki, M. Baranska, Anal. Chem. 86 (2014) 6666.

[30] L.M. Fullwood, G. Clemens, D. Griffiths, K. Ashton, T.P. Dawson, R.W. Lea, C. Davis, F. Bonnier, H.J. Byrne, M.J. Baker, Anal. Methods 6 (2014) 3948. [31] S.N. Kalkanis, R.E. Kast, M.L. Rosenblum, T. Mikkelsen, S.M. Yurgelevic, K.M. Nelson, A. Raghunathan, L.M. Poisson, G.W. Auner, J. Neurooncol. 116 (2014) 477. [32] E. Staniszewska, K. Malek, M. Baranska, Spectrochim. Acta A 118 (2014) 981. [33] B.R. Wood, P.J. Caspers, J. Puppels, S. Pandiancherri, D. McNaghthon, Anal. Bioanal. Chem. 387 (2007) 1691. [34] B.R. Wood, A. Hermelink, P. Lasch, K.R. Bambery, G.T. Webster, M.A. Khiavi, B.M. Cooke, S. Deed, D. Naumann, D. McNaughton, Analyst 134 (2009) 1119. [35] T.G. Spiro, Adv. Protein Chem. 37 (1985) 111. [36] A. Jaworska, L.E. Jamieson, K. Malek, C.J. Campbell, J. Choo, S. Chlopicki, M. Baranska, Analyst (2015), http://dx.doi.org/10.1039/c4an01988a. [37] E.F. Olsen, E.O. Rukke, A. Flåtten, T. Isaksson, Meat Sci. 76 (2007) 628. [38] E.A. Carter, H.G.M. Edwards, Infrared and Raman Spectroscopy of Biological Materials, Marcel Dekker, New York, 2001. p. 421. [39] A. Synytsya, M. Judexová, T. Hruby´, M. Tatarkovicˇ, M. Miškovicˇová, L. Petruzˇelka, V. Setnicˇka, Anal. Bioanal. Chem. 405 (2013) 5441. [40] C. Yu, E. Gestl, K. Eckert, D. Allara, J. Irudayaraj, Cancer Detect. Prev. 30 (2006) 515. [41] M. Leroy, J.-F. Labbé, M. Ouellet, J. Jean, T. Lefèvre, G. Laroche, M. Auger, R. Pouliot, Acta Biomater. 10 (2014) 2703. [42] Z. Movasaghi, S. Rehman, I.U. Rehman, Appl. Spectrosc. Rev. 42 (2007) 493. [43] N.C. Maiti, M.M. Apetri, M.G. Zagorski, P.R. Carey, V.E. Anderson, J. Am. Chem. Soc. 126 (2004) 2399. [44] J.L. Pichardo-Molina, C. Frausto-Reyes, O. Barbosa-García, R. Huerta-Franco, J.L. González-Trujillo, C.A. Ramírez-Alvarado, G. Gutiérrez-Juárez, C. MedinaGutiérre, Lasers Med. Sci. 22 (2007) 229. [45] N. Huang, M. Short, J. Zhao, H. Wang, H. Lui, M. Korbelih, H. Zeng, Opt. Express 19 (2011) 22892.