Analytical and Bioanalytical Chemistry

, Volume 387, Issue 5, pp 1691–1703 | Cite as

Resonance Raman spectroscopy of red blood cells using near-infrared laser excitation

  • Bayden R. Wood
  • Peter Caspers
  • Gerwin J. Puppels
  • Shveta Pandiancherri
  • Don McNaughton
Original Paper

Abstract

Resonance Raman spectra of oxygenated and deoxygenated functional erythrocytes recorded using 785 nm laser excitation are presented. The high-quality spectra show a mixture of enhanced A1g, A2g, B1g, B2g, Eu and vinyl modes. The high sensitivity of the Raman system enabled spectra from four oxygenation and deoxygenation cycles to be recorded with only 18 mW of power at the sample over a 60-minute period. This low power prevented photo-/thermal degradation and negated protein denaturation leading to heme aggregation. The large database consisting of 210 spectra from the four cycles was analyzed with principal components analysis (PCA). The PC1 loadings plot provided exquisite detail on bands associated with the oxygenated and deoxygenated states. The enhancement of a band at 567 cm−1, observed in the spectra of oxygenated cells and the corresponding PC1 loadings plot, was assigned to the Fe–O2 stretching mode, while a band appearing at 419 cm−1 was assigned to the Fe–O–O bending mode based on previous studies. For deoxygenated cells, the enhancement of B1g modes at 785 nm excitation is consistent with vibronic coupling between band III and the Soret transition. In the case of oxygenated cells, the enhancement of iron-axial out-of-plane modes and non-totally symmetric modes is consistent with enhancement into the y,z-polarized transition \({\text{a}}_{{{\text{iu}}}} {\left( {\text{ $ \pi $ }} \right)} \to {\text{d}}_{{{\text{xz}}}} + {\text{O}}_{{\text{2}}} {\left( {{\text{ $ \pi $ }}_{{\text{g}}} } \right)}\) centered at 785 nm. The enhancement of non-totally symmetric B1g modes in oxygenated cells suggests vibronic coupling between band IV and the Soret band. This study provides new insights into the vibrational dynamics, electronic structure and resonant enhancement of heme moieties within functional erythrocytes at near-IR excitation wavelengths.

Keywords

Raman spectroscopy Red blood cells Near-infrared excitation Ligand modes Vibronic coupling 

Abbreviations

PCA

principal components analysis

PC

principal component

RERS

resonance-enhanced Raman scattering

Hb

hemoglobin

RBCs

red blood cells

r.p.m

revolutions per minute

CCD

charged coupled device

NIR

near-infrared

HPRM

high-performance Raman module

Notes

Acknowledgment

This work is funded by an Australian Research Council Discovery Grant. Dr. Wood′s work is supported by an Australian Synchrotron Program Fellowship Grant and a Monash University Synchrotron Fellowship Grant.

References

  1. 1.
    Spiro TG (ed) (1988) Biological applications of Raman spectroscopy. Wiley, New YorkGoogle Scholar
  2. 2.
    Puppels GJ, Olminkhof JHF, Segers-Nolten GMJ, Otto C, Mul de FFM, Greve J (1991) Exp Cell Res 195:361–367CrossRefGoogle Scholar
  3. 3.
    Puppels GJ, Garritsen GMJ, Kummer JA, Greve J (1993) Cytometry 14:251–256CrossRefGoogle Scholar
  4. 4.
    Van Manen H-J, Kraan YM, Roos D, Otto C (2004) J Phys Chem B 108:18762–18771CrossRefGoogle Scholar
  5. 5.
    Puppels GJ, Garritsen GMJ, Segers-Nolten GMJ, De Mul FFM, Greve J (1991) Biophys J 60:436–446CrossRefGoogle Scholar
  6. 6.
    Salmaso BLN, Puppels GJ, Caspers PJ, Floris R, Greve J (1994) Biophys J 67:36–446Google Scholar
  7. 7.
    Otto C, Sijisema NM, Greve J (1998) Eur Biophys J 271:582–589CrossRefGoogle Scholar
  8. 8.
    Wood BR, Hammer L, Davis L, McNaughton D (2004) J Biomed Opt 10:14005CrossRefGoogle Scholar
  9. 9.
    Wood BR, Hammer L, McNaughton D (2005) Vib Spectrosc 78:71–78Google Scholar
  10. 10.
    Wood BR, Langford S, Cooke BM, Glenister FK, Lim J, Duriska M, McNaughton D (2003) FEBS Lett 554:247–252CrossRefGoogle Scholar
  11. 11.
    Wood BR, McNaughton D (2002) Biopolymers (Biospectroscopy) 67:259–262CrossRefGoogle Scholar
  12. 12.
    Wood BR, McNaughton D (2002) J Raman Spectrosc 33:517–523CrossRefGoogle Scholar
  13. 13.
    Wood BR, Tait B, McNaughton D (2001) Biochim Biophys Acta 1539:58–70CrossRefGoogle Scholar
  14. 14.
    McNaughton D, Lim J, Langford S, Collie J, Wood BR (2005) Proc SPIE 5651:52–60CrossRefGoogle Scholar
  15. 15.
    Wood BR, McNaughton D (2006) In: O’Malley PD (ed) New developments in sickle cell disease. Nova, New York, pp 63–119Google Scholar
  16. 16.
    Yan X-l, Dong R-X, Wang Q-G (2004) Spectrosc Spect Anal 24:576–578Google Scholar
  17. 17.
    Hoey S, Brown DH, McConnell AA, Smith WE, Marabani M, Sturrock RD (1988) J Inorg Biochem 34:189–199CrossRefGoogle Scholar
  18. 18.
    Brunner H, Mayer A, Sussner H (1972) J Mol Biol 70:153–156CrossRefGoogle Scholar
  19. 19.
    Spiro TG, Streakas TC (1973) J Am Chem Soc 96:338–345CrossRefGoogle Scholar
  20. 20.
    Spiro TG (1975) Biochim Biophys Acta 416:169–189Google Scholar
  21. 21.
    Yamamoto T, Palmer G (1973) J Biol Chem 248:5211–5213Google Scholar
  22. 22.
    Brunner H (1974) Naturwissenschaften 61:129–130CrossRefGoogle Scholar
  23. 23.
    Jeyarajah S, Proniewicz LM, Bronder H, Kincaid JR (1994) J Biol Chem 269:31047–31050Google Scholar
  24. 24.
    Hu S, Kincaid JR (1991) J Am Chem Soc 113:7189–7194CrossRefGoogle Scholar
  25. 25.
    Abe M, KitagawaT, Kyogoku K (1978) J Chem Phys 69:4526–4534CrossRefGoogle Scholar
  26. 26.
    Hu S, Smith KM, Spiro TG (1996) J Am Chem Soc 118:12638–12646CrossRefGoogle Scholar
  27. 27.
    Choi S, Spiro TG, Langry KC, Smith KM (1982) J Am Chem Soc 104:4337Google Scholar
  28. 28.
    Franzen S, Wallace-Williams SE, Shreve AP (2001) J Am Chem Soc 124:7146–7155CrossRefGoogle Scholar
  29. 29.
    Eaton WA, Hanson LK, Stephens PJ, Sutherland JC, Dunn JBR (1978) J Am Chem Soc 100:4991–5003CrossRefGoogle Scholar
  30. 30.
    Srajer V, Champion PM (1991) Biochemistry 30:7390–7402CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Bayden R. Wood
    • 1
  • Peter Caspers
    • 2
  • Gerwin J. Puppels
    • 2
  • Shveta Pandiancherri
    • 1
  • Don McNaughton
    • 1
  1. 1.Centre for Biospectroscopy and School of ChemistryMonash UniversityClaytonAustralia
  2. 2.Center for Optical Diagnostics and Therapy, Erasmus MCUniversity Medical CenterRotterdamThe Netherlands

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