INSTITUTO SUPERIOR TECNICO

Raman Spectroscopy
Raman spectroscopy in stomach cancer diagnose
Ana Filipa Santos ( n 54848)
15-07-2010

Structural Biology

Contents
Abstract ........................................................................................................................ 3 Introduction to Raman spectroscopy ........................................................................... 3 Principals of Raman Spectroscopy........................................................................... 3 Instrumentation........................................................................................................ 4 Raman spectra .............................................................................................................. 6 Interpretation of Raman spectra .............................................................................. 6 Cancer Biology .............................................................................................................. 7 Raman spectroscopy in stomach cancer diagnose ...................................................... 7 Proteins secundary structure ................................................................................... 9 Amide vibrations....................................................................................................... 9 Protein secondary structure in cancer tissues ....................................................... 10 Conclusion .................................................................................................................. 12 Perspectives on the Future ......................................................................................... 12

Abstract
Despite the success of fluorescence spectroscopy in cancer diagnose, the results indicate that fluorescence spectra of precancerous tissues and benign abnormalities such as inflamation and metaplasia are similar in many patients . This suggests that the use of fluorescence diagnosis in a screening setting, where the incidence of precancer is expected to be low, may result in an unacceptably high false positive rate. To enhance the specificity of spectroscopic diagnosis, vibrational spectroscopy has been considered in the last years

Introduction to Raman spectroscopy

Raman spectroscopy has been used for many years to probe into the biochemistry of various biological molecules. In recent years there has been interest in using this technique in diagnostics. Raman spectroscopy probes different characteristics of materials than fluorescence. Only a limited number of biological molecules such as flavins, and structural proteins (collagen and elastic) contribute to tissue fluorescence, most with overlapping, broadband emission. In contrast, most biological molecules are Raman active with fingerprint spectral characteristics, because of this, vibrational spectroscopy may overcome some of the limitations of fluorescence diagnosis of preancers and cancers When a photon is incident on a molecule, it may be transmitted, absorbed or scattered.

Figure 1 : Raman Scattering

Raman scattering arises from perturbations of the molecule that induces vibrational or rotational transitions.

Principals of Raman Spectroscopy Classically speaking, when the energy of the incident photon is unaltered after collision with a molecule, the scattered photon has the same frequency as the incident photon. This is Rayleigh or elastic scattering When energy is transferred either from the molecule to the photon or vice versa, the scattered photon has less or more than the energy of the incident photon. This is inelastic or Raman scattering.

Figure 2 : Raman energy levels

If the scattered photon has less energy then the incident photon we call stokes Raman Scatering. If the scattered photon has more energy then the incident photon we call stokes AntiRaman Scatering. About 99.999% of all incident photons in spontaneous Raman undergo elastic Rayleigh scattering. This type of signal is useless for practical purposes of molecular characterization. Only about 0.001% of the incident light produces inelastic Raman signal with frequencies 0 m. Spontaneous Raman scattering is very weak and special measures should be taken to distinguish it from the predominant Rayleigh scattering. Instruments such as notch filters, tunable filters, laser stop apertures, double and triple spectrometric systems are used to reduce Rayleigh scattering and obtain high-quality Raman spectra. Instrumentation A Raman system typically consists of four major components: 1. Excitation source (Laser). 2. Sample illumination system and light collection optics. 3. Wavelength selector (Filter or Spectrophotometer). 4. Detector (Photodiode array, CCD or PMT).

Figure 3 : Schematic drawing of an Raman microscope (not scale)

A large variety of laser beams are in use, UV (250 nm), visible (green, 514 nm, or red, 633 nm), near-IR (780 nm or 1064 nm). The intensity of the Raman signal is proportional to 1/4, where is the laser wavelength. As a consequence, UV lasers produce Raman bands of a higher intensity and IR lasers produce the least intense Raman spectra. Unfortunately, selection of the ideal laser is not easy because UV lasers are expensive and spectral resolution is not optimal. On the other hand, IR lasers produce a weak Raman signal. In the middle, visible lasers give a good signal/background ratio but in some cases fluorescence spectra of a sample overlap with the Raman spectrum which prevents assignation of Raman bands. In general, green laser (514 nm) leads to more intensive fluorescence than red laser (780 nm.

Figure 4: Electromagnetic spectrum

A sample is normally illuminated with a laser beam in the ultraviolet (UV), visible (Vis) or near infrared (NIR) range. Scattered light is collected with a lens and is sent through interference filter or spectrophotometer to obtain Raman spectrum of a sample. Since spontaneous Raman scattering is very weak the main difficulty of Raman spectroscopy is separating it from the intense Rayleigh scattering. More precisely, the major problem here is not the Rayleigh scattering itself, but the fact that the intensity of stray light from the Rayleigh scattering may greatly exceed the intensity of the useful Raman signal in the close proximity to the laser wavelength. People use commercially available interference (notch) filters which cut-off spectral range of 80-120 cm-1 from the laser line. This method is efficient in stray light elimination but it does not allow detection of low-frequency Raman modes in the range below 100 cm-1. Stray light is generated in the spectrometer mainly upon light dispersion on gratings and strongly depends on grating quality. Raman spectrometers typically use holographic gratings which normally have much less manufacturing defects in their structure then the ruled once. Stray light produced by holographic gratings is about an order of magnitude less intense then from ruled gratings of the same groove density. In earlier times people primarily used single-point detectors such as photon-counting Photomultiplier Tubes (PMT). However, a single Raman spectrum obtained with a PMT detector in wavenumber scanning mode was taking substantial period of time, slowing down any research or industrial activity based on Raman analytical technique. Nowadays, more and more often researchers use multi-channel detectors like Photodiode Arrays (PDA) or, more commonly, a Charge-Coupled Devices (CCD) to detect the Raman scattered light. Sensitivity and performance of modern CCD detectors are rapidly improving. In many cases CCD is becoming the detector of choice for Raman spectroscopy.

Raman spectra
What kind of information can we get from Raman Spectroscopy? 1. Characteristic Raman Frequencies Composition of material 2. Changes in frequency of Raman peak Stress/strain n state

3. Polarisation of Raman peak crystal symmetry and orientation

4. Width of Raman peak Quality of crystal

5. Intensity of Raman peak Amout of material

Interpretation of Raman spectra A Raman spectrum is a plot of scattered intensity as a function of the energy difference between the incident and scattered photons. The loss (or gain) in photon energies corresponds to the difference in the final and initial vibrational energy levels of molecules participating in the interaction. The resultant spectra are characterized by shifts in wavenumbers (inverse of wavelength in cm-1) from the incident frequency.

Figure 5: Characteristic group frequencies

Typically Raman peaks are spectrally narrow (a flew wavenumbers), and in many cases can be associated with the vibration of a particular chemical bond (or normal mode dominated by the vibration of single functional group) in a molecule. Raman spectra provide "fingerprints" of the molecular structure and, as such, permit qualitative analysis of individual compounds, either by direct comparison of the spectra of the known and unknown materials run consecutively, or by comparison of the spectrum of the unknown compound with catalogues of reference spectra. By comparisons with the spectra of a considerable number of compounds of known structure, it may be possible to recognize bands at specific positions in the spectrum, which can be identified as "characteristic group frequencies" associated with the presence of a particular molecular structure, such as methyl, carbonyl, or hydroxyl groups.

Cancer Biology
Literaly, the word neoplasia (or cancer) means new growth, and its clinically used to describe pathologic tissue masses which grow independent of and faster than normal tissues. Neoplastic cells are characterized by increased nuclear material, an increased nuclear to cytoplasmic ratio, increase mitotic activity, abnormal chromatin distribution, and an decreased diferentiation. There is a progressive loss of cell maturation, and proliferation of these undifferentiated cells results in increased metabolic activity. These general features of neoplastic cells result in specific changes in nucleic acid, protein, lipid and carbohydrate quantizes and/or conformation. The morphologic and biochemical changes that occur with neoplasia are numerous and in many cases, they depend on the specific type and localization of the cancer. Several biological molecules such as nucleic acids, proteins and lipids have distinctive Raman features that yield structural and environmental information. Hence, the molecular and cellular changes that occur with cancer may result in distinct Raman spectra from normal and cancerous tissues.

Raman spectroscopy in stomach cancer diagnose

There are specific spectral differences in Raman spectra between dysplasia and normal tissue, demonstrating the utility of NIR Raman spectroscopy in gastric precancer detection. Figure 6A shows the comparison of mean normalised Raman spectra between normal and cancer tissue and figure 6B represent the spectral differences between normal and dysplasia tissues. The difference spectrum reveals the changes of prominent Raman peaks occurring in dysplasia gastric tissue, confirming a potential role of Raman spectroscopy for precancer diagnosis in the stomach.

Figure 6: (A) Comparison of the mean normalised Raman spectra of normal (n=55) and dysplasia (n=21) tissues. (B) Difference spectrum calculated from the mean Raman spectra of normal and dysplasia tissue (i.e., the mean normalised Raman spectrum of dysplasia tissue minus the mean normalised Raman spectrum of normal tissue).

875 cm 1004 cm1 1100 cm1 1210 cm1 1335 cm1 1450 cm1 1745 cm1 1265 cm1 1660 cm1 1655 cm1

Biomolecule Hydroxyproline of collagen Ring breathing mode Phenylalanine Stretching C-C Phospholipids Stretching C-C6H5 Phenylalanine , tryptophan Bending CH3CH2 Proteins; nucleic acid Bending CH2 Proteins and lipids Stretching C=O Phospholipids Stretching C-N Amide III, -helix Amide I , -pleated Amide I , -helix

Vibrational modes

Raman peak intensity at 875 cm1 (hydroxyproline of collagen) was found to be much reduced in dysplastic tissue, and this was probably due to the elevated concentration of metalloproteinase, which cleaved collagen in the stroma layer in gastric dysplasia.

Figure 7: Hydroxyproline

The Raman peaks at 1004 cm1 ( phenylalanine, ring breathing mode) and 1210 cm1 (stretching C-C6H5 mode) for phenylalanine and tryptophan, showed lower percentage signals for dysplasia tissue compared with the normal tissue, suggesting a decrease in the

percentage of phenylalanine relative to the total Raman-active constituents in the dysplasia .

Figure 8 :Phenylalanine and tryptophan

We can also conclude that there is a decrease in the percentage of phospholipids due to the low percentage signals for the Raman peaks at 1100 cm1 (stretching C-C skeletal vibrations in the gauche conformation) and 1745 cm1 (C=O stretching mode of phospholipids). The relative peak intensities at 1450 cm1 (CH2 bending mode of proteins and lipids) and 1335 cm1 (bending mode of CH3CH2 twisting of protein) were found to be higher for dysplasia tissues, indicating the elevated concentration of biomolecules (nucleic acid , protein and, histones) due to hyperchromatism in tissue with dysplastic transformation. Proteins secundary structure The Raman spectra represented in figure 6 can also give us some information about the secondary structure of proteins in the tissue. Characteristic groups of atoms give rise to vibrational bands near the same frequency regardless of the molecule in which they are found. The precise wave numbers of bands within this range depend on inter and intra-molecular effects, including peptide-bond angles and hydrogen-bonding patterns. Thus, vibrational spectra can be used to estimate the secondary structure of proteins by inspection of the frequencies at which the amide bonds absorb radiation. Amide vibrations Nine normal modes are allowed for the amide band of proteins. These are called A, B, and I-VII in order of decreasing frequency. The amide A band (about 3500 cm-1) and amide B (about 3100 cm-1) originate from a Fermi resonance between the first overtone of amide II and and the N-H stretching vibration. Amide I and amide II bands are two major bands of the protein infrared spectrum. The amide I band (between 1600 and 1700 cm-1) is mainly associated with the C=O stretching vibration (70-85%) and is directly related to the backbone conformation. Amide II results from the N-H bending vibration (40-60%) and from the C-N stretching vibration (1840%). This band is conformationally sensitive. Amide III and IV are very complex bands resulting from a mixture of several coordinate displacements. The out-of-plane motions are found in amide V, VI andVIII.

Figure 9: Amida vibration modes

Between the four normal connection modes between the residual amino acids, there are two Raman active: The Amide I mode, whose structure is responsible for the stretching vibration between the atoms of carbon and oxygen (C = O), and the Amide III mode involving two types of vibration: (i) the bending vibration of nitrogen and hydrogen (NH) (ii) stretching vibration corresponding to the binding of carbon and nitrogen (CN). Generally, proteins with high -helical content show an amide I band centred around 16501655 cm1 while those with predominantly -sheets structures show the band at 1660 cm1 The intensity of the amide III band for -helix structure appears around 13001260 cm1 which overlaps with the region assigned for -turns. The -structure and random coil bands are overlapped in the Raman spectra so that the intensity in the amide III region between 1250 and 1240 cm1 due to contributions from both the random coil and the -structure. Protein secondary structure in cancer tissues In Figure 7 there is a relative increase of amide III band 1265 cm1(C-N stretching mode of proteins) and amide I band (1655 cm1) in intensity, suggest that dysplasia tissue may be associated with an increase in the relative amount of proteins in the -helix conformation. A shoulder band at 1660 cm1 (amide I, -pleated sheet conformation) was also revealed in the difference spectra (B), suggesting that dysplastic transformation may also be associated with an increase in the relative amount of protein in the -pleated sheet .

Figure 5: Amida I mode

The appearance of these proteins in the -pleated sheet conformation may signify more chemical interaction between the proteins and the microenvironment occurring in the cells, which could be related to increase of mitotic activity, one of the cellular alteration characteristics of gastric dysplasia .

Conclusion
Raman spectroscopy is a promising new tool for noninvasive, real-time diagnosis of tissue abnormalities. Several other cancers also have been studied with Raman spectroscopy, such as those in the ovary, brain, and lung, with similar results. Thus, many researchers have applied NIR Raman spectroscopy in vitro, ex vivo as well as in vivo for the diagnosis of cancer with varying degrees of success. Despite this research for more than 20 years, the technique has not been incorporated into routine clinical care for several reasons. There is a continued tendency by researchers to use empirical or several different classification techniques after many spectra are recorded demonstrating varying degrees of success. Although limitations exist, the technique shows every indication of being an exciting prospect in the management of cancer in a clinical setting.

Perspectives on the Future

The success of Raman spectroscopy for precancer and cancer detection has led to the development of feasible clinical systems that can measure Raman signals from tissue with short collection times . The complexity of tissue structure and environment make the interpretation of tissue Raman spectra difficult to achieve the maximum benefit from Raman based diagnostic systems, an understanding of the molecular, microscopic and macroscopic origin of observed tissue Raman signals is required. In vitro results have demonstrated contributions from proteins, lipids and nucleic acids which are altered under neoplastic transformations. However, extracting more detailed information will require more detailed chemical and microscopic studies to confirm the molecular basis of tissue signal and the development of models to relate the microscopic signal to its microscopic origins.

References
Farinha, J. P., Espectroscopia de Raman, in notes of Qumica Fsica, MIEB 2009-2010, available at http://web.ist.utl.pt/farinha/QF/pdf_files/QF_MIEB%200910_Espectr_Raman.pdf. Lyon, L. A., Keating, C. D., Fox, A. P., Baker, B. E., He, L., Nicewarner, S. R., Mulvaney, S. P., and Natan, M. J. (1998), Raman Spectroscopy, in Analytical Chemistry, Vol. 70, No. 12, pp. 341R-361R. Mahadevan-Jansen, A., Richards-Kortum, R. (1997), Raman Spectroscopy for cancer detection: A review, in Proceedings - 19 International Conference, p.2722-2828. Teh, SK, Zheng, W, Ho, KY, Teh, M, Yeoh, KG, e Huang, Z (2008), Diagnostic potential of near-infrared Raman spectroscopy in the stomach: differentiating dysplasia from normal tissue, in British Journal of Cancer, 98, pp. 457-465. Other type of references Princeton Instruments, Raman Spectroscopy Basics, available at http://content.piacton.com/Uploads/Princeton/Documents/Library/UpdatedLibrary/Ram an_Spectroscopy_Basics.pdf
th