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Raman excitation wavelength investigation of single red blood cellsin vivo

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Raman spectra are reported for oxygenated and deoxygenated haemoglobin contained within a single red blood cell in vivo using excitation wavelengths of 488, 514, 568 and 632.8 nm. The peak assigned in previous work to ν4 is observed at 1376 cm−1 in
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   JOURNAL OF RAMAN SPECTROSCOPY  J. Raman Spectrosc.  2002;  33 : 517–523Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jrs.870 Raman excitation wavelength investigation of singlered blood cells  in vivo Bayden R. Wood and Don McNaughton ∗ Centre for Biospectroscopy and School of Chemistry, P.O. Box 23, Monash University, Victoria 3800, Australia Received 30 September 2001; Accepted 10 December 2001 Raman spectra are reported for oxygenated and deoxygenated haemoglobin contained within a single redbloodcell invivo usingexcitationwavelengthsof488,514,568and632.8 nm.Thepeakassignedinpreviouswork to  n 4  is observed at 1376 cm − 1 in oxygenated cells and 1356 cm − 1 in deoxygenated cells with theresults from 488 nm excitation consistent with earlier Raman studies on isolated haem proteins. Excitingthe cells with 514 nm radiation revealed two bands appearing in this region at 1372 and 1356 cm − 1 in theoxygenated state, whereas in the deoxygenated state only one band at 1356 cm − 1 is observed. At 632.8 nmexcitation bands in the  n 4  region appeared at 1367 and 1365 cm − 1 in the oxygenated and deoxygenatedstates, respectively. Our results clearly show that the enhancement of bands in the vicinity of  n 4  withinsingleerythrocytesisinfluencedbytheexcitationwavelength.Furthermore,manyotherbandsobservedinoxygenated erythrocytes using 632.8 nm excitation were dramatically enhanced compared with the bandsobserved with other excitation wavelengths. Ruling out other explanations, it is hypothesized that theenhancement observed at 632.8 nm results from excitonic coupling between aligned porphyrins. The highconcentration of haemoglobin in a single cell enables the porphyrins to be in close proximity to permitcharge transfer between the haem moieties. The high signal-to-noise ratio and excellent reproducibilityobtained using Raman water immersion microspectroscopy on single erythrocytes  in vivo  shows potentialas a diagnostic tool for a variety of haemopathies. However, judicious choice of the excitation wavelengthis a prerequisite especially if the technique is applied to diagnose oxidation status within erythrocytes.Copyright © 2002 John Wiley & Sons, Ltd. INTRODUCTION Resonance Raman (RR) spectroscopy is a particularly sensi-tive probe for studying the electronic and structural proper-ties of metalloporphyrin complexes including haemoglobin(Hb)andmyoglobin.Theinterpretationoftheintensespectraobtained from metalloporphyrin complexes has been basedon vibronically induced scattering from the B (Soret) orQ states from the porphyrin macrocycle. 1 In this paper,we report the application of the Raman technique tostudy single erythrocytes  in vivo  and outline the poten-tial of the method as an analytical and diagnostic tool inmedicine.EarlyresonantRamanstudiesonhaemproteinsreporteda bandthatappearedsensitive tooxidationstateofthehaemiron (Fe). 2–4 The local coordinate of this band (pyrrole half ring stretching vibration) involves principally C–N stretch-ing and was assigned by Abe  et al . 5 to   4 . Yamamoto andPalmer, 3 using 441.6 nm excitation (near the Soret band), Ł Correspondence to: Don McNaughton, Centre for Biospectroscopyand School of Chemistry, P.O. Box 23, Monash University, Victoria3800, Australia. E-mail: d.mcnaughton@sci.monash.edu.auContract/grant sponsor: Australian Research Council. notedthatthestrongestbandinthespectraofhaemproteinsoccurred between 1361 and 1356 cm  1 for reduced (ferrous)haem proteins, and between 1378 and 1370 cm  1 for oxi-dized (ferric) proteins. Moreover, based on these results,they concluded that the Fe atom is in the formal low-spin ferric state in oxygenated haemoglobin (oxyHb). TheRaman shift of    4  is thought to reflect the electron popu-lation in the porphyrin   Ł orbitals. Increasing the electronpopulation weakens the bonds resulting in a decrease invibrational wavenumber. The electron population in the   Ł orbitals is thought to be increased by back-donation of theelectrons from the Fe atom’s d   orbitals. Because back-donation is greater for Fe(II) than Fe(III), the oxidation statemarker bands are lower in wavenumber compared withthose of Fe(II). The reliability of the correlation of    4  as theferric/ferrous marker band has been questioned. 6 , 7 Spauld-ing  et al . 6 investigated a wide range of metalloporphyrinsand found   4  to be relatively invariant despite the largeanticipated differences in charge density migration fromthe various metals to the conjugated porphyrin ring. Theresultimpliedthatthechargedistributionaroundthecentraliron atom is not a fundamental factor in the positioning of this band. Copyright © 2002 John Wiley & Sons, Ltd.  518 B. R. Wood and D. McNaughton Raman techniques to probe single living cells werepioneered in the late 1990s by Greve and co-workers. 8–10 Their studies primarily focused on lymphocytes 9 , 11 andgranulocytes. 10 , 12 , 13 The group identified haem moietiesincluding eosinophil peroxidase (EPO) and myeloperox-idase in eosinophils and neutrophils, respectively, 10 andconfirmed the high-spin six-coordinated structure of thesehaems. Recently, Schuster and co-workers 14 , 15 analysed sin-gle  Clostridium  cells at 632.8 nm excitation and observed bands associated with the major macromolecules includinglipids, proteins and carbohydrates.In a recent study, 16 we reported the micro-Raman char-acterizationofHb in boththeoxygenatedand deoxygenatedstates using 632.8 nm excitation. The major differences inthe spectra were attributed to changes in the spin state between the oxygenated haem in the ferric high-spin ( S D 2)state and the deoxygenated haem in the ferrous low-spin( S  D  0 . 5) state. We also reported that a number of bandsappearedenhancedat632.8 nmindependentofresonanceorpre-resonance Raman scattering.Excitons in molecular crystals and aggregates have beenextensively studied over the last decade and have beenimportantinelucidatingthefunctionalityofphoto-pigmentsin photosynthesis. 17 In biological systems such as bacteriaand higher plants, light energy is absorbed by specialchlorophyll (or bacteriochlorophyll) pigments assembledin protein matrixes in so-called antenna complexes andis transferred via an exciton mechanism to the reactioncentre. 18 Recently,considerableattentionhasbeenfocusedoncreating artificial mimics of the light-harvesting complexesfor nanoarchitectures in molecular-scale electronics, sensorsand other optical electronic devices. 19 Most studies havefocused on covalently linked porphyrin arrays in whichthe extent of electronic interaction is dependent on both thelengthandtypeoflinker. 20 Thelargestelectronicinteractionsare observed in such systems that contain direct  meso,meso -linked porphyrins resulting in excitonic interactions that areseveral tenths of an electronvolt. 21 Although most excitonic coupling usually involves adirect covalent linkage from one porphyrin to the next, it isconceivablethatsuchcouplingcanalsooccurinsystemssuchas haems contained within the erythrocyte protein matrix,given the extremely high concentration of the Hb moiety.Goldbeck et al ., 22 usingSoretcirculardichroism(SCD),notedthat at high concentration Hb formed tetramers that had adifferentSCDprofilethanatlowconcentrationsofHb,whichapparently formed dimers. Goldbeck  et al . 22 attributed thesechanges to excitonic coupling between adjacent porphyrins based on theoretical calculations by R. W. Woody.In this paper, we report the excitation wavelengthdependence of bands in the   4  region in Hb encapsulatedwithinasingleredbloodcellusing488,514,568and632.8 nmexcitation. We also report the unusual enhancement of anumber of vibrational modes at 632.8 nm compared withthe other wavelengths investigated. A tentative hypothesis based on excitonic coupling is put forward to explain theunusual enhancement observed at this wavelength. EXPERIMENTALPreparation of blood Blood (1 ml) was obtained by venipuncture from healthyvolunteers and placed in glass tubes containing acid citratedextrose as an anticoagulant. A 20  µ l aliquot of blood wasdiluted to 10 ml with RPMI 1640 medium (pH 7.4) at300 K. The cells were transferred to an 80 mm diameterglass Petri dish sputter coated with aluminium. The Petridish was further coated with poly- L -lysine, dried with ahair dryer prior to the addition of the suspension. Thecells were allowed to settle ( ¾ 10 min) before spectra wererecorded. To prepare deoxygenated erythrocytes, 50 mg of sodium dithionite (Na 2 S 2 O 4 , Sigma-Aldrich) was sprinkledinto the supernatant and measurements were recorded after5 min. Na 2 S 2 O 4  rapidly deoxygenatesthe cells and producesidenticalspectratocellsdeoxygenatedwithN 2 .Ourpreviousstudy 16 demonstrated this and hence we used this simplerprocedure in this study. Raman microscopy Raman spectra of viable erythrocytes were recorded ona Renishaw System 2000 instrument (equipped with aPeltier-cooled CCD detector) using 632.8 nm excitationradiation from a helium–neon laser and a modified BH2-UMA Olympus optical microscope with a Zeiss ð 60 waterimmersion objective.Spectra were also recorded using 488 and 514 nmexcitation radiation generated by a Spectra Physics Stabilite2017 argon ion laser system and also 568 nm excitationradiation generated by a Spectra-Physics Beamlock 2060krypton ion laser. In this configuration both lasers werecoupled to a Renishaw Raman 2000 spectrometer andinterfacedtoaLeicaRamanmicroscopeusingthesamewaterimmersion objective as mentioned above. The power at thesamplewas ¾ 2 mWfora1–2  µ mlaserspotsize.Spectrawererecorded between 1800 and 200 cm  1 with a resolution of  ¾ 1–2 cm  1 . For each spectrum, 10 scans were accumulatedand the laser exposure for each scan was 10 s. Constantexposure resulted in haemolysis and photodissociation, asdescribed in our previous study, 16 so the laser exposurewas stopped while the grating re-positioned to its startingposition between scans. In this way the cells remained fullyviable and alive.Unlessstatedotherwise,spectrawereaveragedfromfourrecorded spectra using OPUS spectroscopic software. RESULTS Figure 1 depicts spectra recorded of oxygenated single red blood cells at 488,514,568 and 632.8 nm and Fig. 2 comparesthe corresponding deoxygenated cells at the same excitation Copyright  ©  2002 John Wiley & Sons, Ltd.  J. Raman Spectrosc.  2002;  33 : 517–523  In vivo  investigation of single red blood cells 519 6008001000120014001600         1        6        3        8        1        6        0        4        1       5        8        6        1       5        0       7        1        4        3        2        1        3       7        6        1        3        0        8        1        1       7        3        1        2        2       7        1        0        0        3       7       5       5        1        2        4        8 488 nm         1        1        2       7        1        3        4        4        9       7       7 1800 Wavenumber/cm -1         1        6        3        8 1        6        0        4        1       5        8       5        1       5        4       7        1        4       7        1        1        4        3        0        1        3       5        6        1        3        3        6        1        3        9       7        1        2        4       5 1        1       7        1        1        0        9        0        1        0        0        1       7       5       5        6       7        6        1        2        2        8        1        3        0        1        1        1        3        4        9       7       7 514 nm         1        6        4        0        1       5        8        8        1       5        6       7        1        4        6        9        1        4        3        1        1        3        9        8        1        3        6        6        1        3        4        1        1        3        0       5        1        2        2        3        1        1       7        1        1        1        3        1        1        0        9        1        9        9       5        9       7       7        9        2        8       7       5        3        6       7       5       7        9        2        1        2        4        8 568 nm         1        6        3        8        1        6        1        8        1       5        4        6        1        4        2        8        1        3        9        8        1        3        6       7        1        3        4        2        1        2        4        9         ∗         1        2        2        6         ∗         1        1       7        2         ∗         1        1        2        9        9        9        6         ∗         9       7        8         ∗        7        8       7         ∗        7       5        3         ∗         6        6        8         ∗         1       5        6       5         ∗ 633 nm         1        6        0        4        1       5        6        8        8        2       7        8        2       7 Figure 1.  Raman spectra of oxygenated single erythrocytesrecorded using different excitation wavelengths. Bandsmarked with asterisks are those which appear relativelyenhanced at 632.8 nm compared with other excitationwavelengths. The spectra presented are averaged from fourspectra recorded of different erythrocytes under the sameconditions. For each spectrum 10 intermittent scans wereaccumulated with a 10 s exposure time for each accumulation. wavelengths. The following assignments, which are detailedin Table 1, are based on the notation system proposed byAbe  et al . 5 with some amendments by Hu  et al . 23 Core size or spin state marker band region(1650–1500 cm − 1 ) Bands in the core size (or spin state marker) band region(1650–1500 cm  1 ) for deoxygenated cells show a similar pat-tern both in terms of band position and relative intensity atthe different excitation wavelengths investigated. In deoxy-genatedcellsthisregioniscomprisedofthreeprincipalbandsappearing at 1608–1604, 1582–1580 and 1547–1544 cm  1 assigned to   19 ,   37  and   11 , respectively. In the oxygenatedcells this region shows more variation for the different exci-tation wavelengths especially in terms of the relative bandintensity. Both the 488 and 568 nm spectra show similar pro-files with the most intense bands appearing at 1640–1638,1588–1586 and 1568–1567 cm  1 assigned to   10 ,   37  and   2 , 6008001000120014001600 488 nm         1        6        0       5        1       5        8        2        1       5        4       7        1        4       7        3        1        4        2       5        1        3        9        3        1        3       5       7        1        3        3       7        1        3        0        2        1        2        1        3        1        1       7        2        1        1        2       5        9       7       5       7       5       5        6       7        3        9        9        4 1800 Wavenumber/cm -1         1        6        0        4           1       5        8        0        1       5        4        6        1        4       7        1        1        3        9        4        1        3       5        6        1        3        3        6        1        3        0        1        1        2        1        2        1        1       7        2        1        1        2        4        9        9        3       7       5        4        6       7        2        1        4        2       5 514 nm         1       5        4       5        1        6        0        3        1       5        8        1        1        4        2       5        1        3        9        1        1        3       5        9        1        3        3       5        1        3        0        1        1        2        1        1        1        1        6        9        1        1        2        2        1        0        8        4        9        9        3       7        8        9       7       5       5        6       7        2        9       7       5        1        2        2        3 568 nm         1       5        8        1        1        4        2        6        1        3        6       5        1        3        9        8        1        3        4        0        1        3        0        6        1        2        1        3        1        2        2       7        8        2       7 6       7        2       7        9        0       7       5        2        1        1       7        1        1        1        2        3        9        9        8        9       7       7        1        6        0        8        1       5        4        4 633 nm         9       7        3        8        2       7 Figure 2.  Same as Fig. 1 for cells deoxygenated with sodiumdithionite. respectively. The 488 nm spectrum differs from the 568 nmspectrum in that   19  appears as a distinct shoulder featurein the former spectrum at 1604 cm  1 . Both of these spectradiffer markedly from the 514 and 632.8 nm spectra, whichhave similar profiles in this region. The 514 nm spectrumhas four principal bands appearing at 1638, 1604, 1585 and1547 cm  1 , and two shoulder bands at 1621 and 1557 cm  1 that are clearly visible in the second-derivative spectrum(data not shown). The 632.8 nm spectrum has five principal bandsappearingat1638,1618,1604,1565and1546 cm  1 ,anda shoulder feature at 1581 cm  1 that is clearly observed inthe second-derivative spectrum (data not shown). The bandat 1621–1618 cm  1 that appearsin both the 514 and 632.8 nmspectra is assigned to the   (C C) mode of the porphyrinmacrocycle. Pyrrole ring stretching region (1400–1300 cm − 1 ) The region between 1400 and 1300 cm  1 contains three prin-cipal modes assigned to pyrrole ring stretching vibrationswithdifferentphasing. 23 Theseinclude  20  (1398–1391 cm  1 ),  4  (1367–1356 cm  1 ) and   41  (1342–1335 cm  1 ). The bandassigned to   4  is the most intense band observed in the 488 Copyright © 2002 John Wiley & Sons, Ltd.  J. Raman Spectrosc.  2002;  33 : 517–523  520 B. R. Wood and D. McNaughton Table 1.  Observed wavenumber (cm  1  ), assignments and local coordinates for haemoglobin at different excitation wavelengths Oxy488 nmDeoxy488 nmOxy514 nmDeoxy514 nmOxy568 nmDeoxy568 nmOxy632.8 nmDeoxy632.8 nm Assignment a Localcoordinate  b 1638 Absent 1638 Absent 1640 Absent 1638 Absent   10   C ˛ C m  asym Absent Absent 1627 c Absent Absent Absent 1618 Absent    C C    C a  C  b  1604 1605 1604 1604 Absent 1603 1604 1608   19   C ˛ C m  asym 1586 1582 1585 1580 1588 1581 1581 c 1585   37   C ˛ C m  asym 1568 Absent 1557 c Absent 1567 Absent 1565 Absent   2   C ˇ C ˇ  Absent 1547 1547 1546 1545 1546 1544   11   C ˇ C ˇ  1507 Absent Absent Absent 1505 Absent Absent Absent 2  15   (pyr breathing)1473 1471 1471 1469 Absent Absent Absent –CH 2  (scissor) –CH 2  (scissor)1432 1425 1430 1425 1431 1425 1428 1426   28   (C ˛ C m  sym 1397 1394 1397 1394 1398 1391 1398 1398   20   (pyr quarter-ring)1376 1357 1371 (m) 1356 1366 1359 1367 1365   4d  (pyr half-ring) sym 1356 (s)1344 1337 1336 1336 1341 1335 1342 1340   41   (pyr half-ring) sym 1308 1302 1301 1301 1305 1301 1306 1306   21  υ C m H  1248 Absent 1245 Absent 1248 Absent 1249 Absent   13 1227 1221 1228 1220 1223 1223 1226 1223   13  or   42  υ C m H  Absent 1213 Absent 1212 Absent 1211 Absent 1213   5 C  18  υ C m H  1173 1172 1171 1172 1171 1169 1172 1171   30   (pyr half-ring) asym 1127 1124 1134 1124 1131 1122 1129 1123   22   (pyr half-ring) asym Absent Absent 1090 1082 1091 1084 1090 1084   23   C ˇ C 1  asym 1003 994 1001 993 995 998 996 996   47   C ˇ C 1  asym 977 975 972 973 977 977 978 972   46  υ (pyr deform) asym and/or     ( C  b H 2 ) sym Absent Absent Absent Absent 827 827 827 827    10    (C m H)Absent Absent Absent Absent 792 790 787 790   6   (pyr breathing)755 755 755 754 753 752 753 752   15   (pyr breathing)675 673 676 672 675 672 668 672   7  υ (pyr deform) syma Assignments are based mainly on labeling scheme srcinally devised by Abe  et al . 5 for octaethylporphyrinato-Ni(II).  b Local coordinates based mainly on studies by Hu  et al . 23 for myoglobin. c Only observed after calculating second derivative. d Position influenced by excitonic enhancement. and 514 nm spectra for both the oxygenated and deoxy-genated states. However, at the longer wavelengths (568and 632.8 nm) the intensity of this band diminishes dra-matically. Figure 3 shows oxygenated and deoxygenatedspectra of multiple cells recorded at the different excita-tion wavelengths centred in the vicinity of the   4  mode(1450–1300 cm  1 ). At 488 nm   4  appears at 1376 cm  1 inthe oxygenated state and 1357 cm  1 in the deoxygenatedstate, consistent with previous studies on Hb solutions. 3 , 24 Spectra recorded at 514 nm show two bands appearing inthe oxygenated state at 1372 and 1357 cm  1 , whereas in thedeoxygenated state a single band is observed at 1356 cm  1 .At 568 nm bands appear at 1366 and 1359 cm  1 in theoxygenated and deoxygenated states, respectively. Spectrarecordedat632.8 nmshowbandsinthevicinityof   4  appear-ing at almost the same Raman wavenumber value for boththe oxygenated (1367 cm  1 ) and deoxygenated (1365 cm  1 )states. Figure 4 depicts a plot of the position of bands inthe vicinity of    4  versus excitation wavelength for both oxy-genated and deoxygenated cells. It is clear from the plotthat as the excitation wavelength increases the values of the bands in the vicinity of    4  for oxygenated and deoxygenatedcells approach one another. Methine C–H deformation region(1300–1200 cm − 1 ) The C–H methine deformation region consists of three bands tentatively assigned to   13  (1249–1248 cm  1 ),   13  or  42  (1228–1223 cm  1 ) and   5 C  18  (1213–1211 cm  1 ). Thelatter assignments are based on the work of Salmaso  et al . 12 for EPO. In the oxygenated cells a doublet dominates thisregion occurring at 1249–1248 and 1228–1223 cm  1 for allwavelengths investigated. In the deoxygenated state thisdoublet appears to shift to 1228–1223 and 1213–1211 cm  1 Copyright © 2002 John Wiley & Sons, Ltd.  J. Raman Spectrosc.  2002;  33 : 517–523  In vivo  investigation of single red blood cells 521 488 nm 13761357 514 nm 13561372 564 nm 13591366 633 nm 13671365 Figure 3.  Raman spectra showing the pyrrole breathing moderegion (1450–1300 cm  1  ) with bands in the vicinity of   4 highlighted for both the oxygenated and deoxygenated statesfor all excitation wavelengths investigated. For each spectrumpresented, 10 intermittent scans were accumulated with a 10 sexposure time for each accumulation. 1350135513601365137013751380480 520 560 600 640 Excitation wavelength/nm     W   a   v   e   n   u   m    b   e   r    /   c   m   -    1 OxyDeoxy Figure 4.  Plot depicting wavenumber versus excitationwavelength of bands in the vicinity of   4  that are highlighted inFig. 3. for all four excitation wavelengths. The proximity of themethine C-H vibrations to the protein subunits wouldinfluence the deformation angle and consequently theRaman shifts of these bands between the oxygenated anddeoxygenated states. 16 Low-wavenumber region (1200–600 cm − 1 ) The bands appearing at 1173–1171 and 1134–1127 cm  1 in the oxygenated cells for all wavelengths investigatedare assigned to asymmetric pyrrole half-ring stretchingvibrations,  30  and  22 ,respectively.Thecorrespondingbandsin the deoxygenated cells are marginally red shifted andappear at 1172–1169 and 1125–1121 cm  1 . The intensityratio of these bands varies between the various excitationwavelengths for both oxygenated and deoxygenated cells.The bands observed at 1003–995 cm  1 (  47 ) and 978–977cm  1 (  46 ) in oxygenated cells, and the bands appearing at998–993 cm  1 (  47 ) and 977–973 cm  1 (  46 ) in deoxygenatedcells are associated with C–C asymmetric stretching vibra-tions of the porphyrin macrocycle. At 632.8 nm the relativeintensity of these bands is greater than those of the corre-sponding bands at the shorter excitation wavelengths forthe oxygenated cells. In the deoxygenated cells these bandsappear enhanced with 632.8 and 568 nm excitation wave-lengths compared with the 514 and 488 nm wavelengths.Other bands that appear to be enhanced as the excita-tion wavelength becomes longer in both oxygenated anddeoxygenated cells include the bands appearing at 827and 792–787 cm  1 assigned to    10  (methine out-of-planedeformation mode) and   6  (pyrrole breathing mode), respec-tively. Bands observed at 755–753 and 678–668 cm  1 inoxygenated cells and assigned to   15  (a pyrrole breathingmode) and   7  (symmetric pyrrole deformation mode) also become enhanced as the excitation wavelength increases,although for the latter mode this enhancement is not as dra-matic. In deoxygenated erythrocytes   15  appears the mostenhanced at 568 nm followed by 632.8 nm and is much lessintense for the shorter wavelengths. DISCUSSION In this study we have probed single erythrocytes  in vivo with a variety of excitation wavelengths. A number of important points can be made concerning the relationshipof excitation wavelength with Raman wavenumbers and theobserved band intensity for oxygenated and deoxygenatedcells.(1) Thespinstatemarkerbandregion(1650–1500 cm  1 )alters in terms of relative intensity and the number of observed bands for oxygenated cells, whereas this region isgenerally consistent for deoxygenated cells for all excitationwavelengths investigated. (2) The band appearing between1375 and 1355 cm  1 tentatively assigned to   4  is dramaticallymodified depending on the excitation wavelength. (3) Thetwo bands that are associated with the methine deformationmodes between 1250 and 1200 cm  1 are red shifted on goingfrom the oxygenated to the deoxygenated state, whichis consistent for all excitation wavelengths investigated.(4) In the low-wavenumber region bands including   30 ,   22 ,   10 ,   15  and   7 , generally become enhanced at the longerwavelengths. Copyright © 2002 John Wiley & Sons, Ltd.  J. Raman Spectrosc.  2002;  33 : 517–523
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