Histochemistry and Cell Biology

, Volume 139, Issue 1, pp 181–193 | Cite as

Label-free detection of peripheral nerve tissues against adjacent tissues by spontaneous Raman microspectroscopy

  • Takeo Minamikawa
  • Yoshinori Harada
  • Noriaki Koizumi
  • Koji Okihara
  • Kazumi Kamoi
  • Akio Yanagisawa
  • Tetsuro TakamatsuEmail author
Original Paper


Detection of peripheral nerve tissues during surgery is required to avoid neural disturbance following surgery as an aspect of realizing better functional outcome. We provide a proof-of-principle demonstration of a label-free detection technique of peripheral nerve tissues, including myelinated and unmyelinated nerves, against adjacent tissues that employ spontaneous Raman microspectroscopy. To investigate the Raman spectral features of peripheral nerves in detail, we used unfixed sectioned samples. Raman spectra of myelinated nerve, unmyelinated nerve, fibrous connective tissue, skeletal muscle, tunica media of blood vessel, and adipose tissue of Wistar rats were analyzed, and Raman images of the tissue distribution were constructed using the map of the ordinary least squares regression (OLSR) estimates. We found that nerve tissues exhibited a specific Raman spectrum arising from axon or myelin sheath, and that the nerve tissues can be selectively detected against the other tissues. Moreover, myelinated and unmyelinated nerves can be distinguished by the intensity differences of 2,855 cm−1, and 2,945 cm−1, which are mainly derived from lipid and protein contents of nerve fibers. We applied this method to unfixed section samples of human periprostatic tissues excised from prostatic cancer patients. Myelinated nerves, unmyelinated nerves, fibrous connective tissues, and adipose tissues of the periprostatic tissues were separately detected by OLSR analysis. These results suggest the potential of the Raman spectroscopic observation for noninvasive and label-free nerve detection, and we expect this method could be a key technique for nerve-sparing surgery.


Raman microspectroscopy Unmyelinated and myelinated nerves Tissue imaging Nerve detection Nerve-sparing surgery 



We thank T. Okuda and T. Kawamura of the Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, for histological staining. A portion of this work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and Research for Promoting Technological Seeds from Japan Science and Technology Agency (JST). One of the authors (T.M.) acknowledges support by a Grant-in-Aid for JSPS Fellows from the Japan Society for the Promotion of Science (JSPS).

Supplementary material

418_2012_1015_MOESM1_ESM.pdf (70 kb)
Supplementary material 1 (PDF 69 kb)


  1. Beljebbar A, Dukic S, Amharref N, Manfait M (2010) Ex vivo and in vivo diagnosis of C6 glioblastoma development by Raman spectroscopy coupled to a microprobe. Anal Bioanal Chem 398(1):477–487. doi: 10.1007/s00216-010-3910-6 PubMedCrossRefGoogle Scholar
  2. Brennan JF 3rd, Romer TJ, Lees RS, Tercyak AM, Kramer JR Jr, Feld MS (1997) Determination of human coronary artery composition by Raman spectroscopy. Circulation 96(1):99–105PubMedCrossRefGoogle Scholar
  3. Buschman HP, Marple ET, Wach ML, Bennett B, Schut TC, Bruining HA, Bruschke AV, van der Laarse A, Puppels GJ (2000) In vivo determination of the molecular composition of artery wall by intravascular Raman spectroscopy. Anal Chem 72(16):3771–3775PubMedCrossRefGoogle Scholar
  4. Dumont P, Denoyer A, Robin P (2004) Long-term results of thoracoscopic sympathectomy for hyperhidrosis. Ann Thorac Surg 78(5):1801–1807. doi: 10.1016/j.athoracsur.2004.03.012 Google Scholar
  5. Fu Y, Wang H, Huff TB, Shi R, Cheng JX (2007) Coherent anti-Stokes Raman scattering imaging of myelin degradation reveals a calcium-dependent pathway in lyso-PtdCho-induced demyelination. J Neurosci Res 85(13):2870–2881. doi: 10.1002/jnr.21403 PubMedCrossRefGoogle Scholar
  6. Gao L, Zhou H, Thrall MJ, Li F, Yang Y, Wang Z, Luo P, Wong KK, Palapattu GS, Wong ST (2011) Label-free high-resolution imaging of prostate glands and cavernous nerves using coherent anti-Stokes Raman scattering microscopy. Biomedical Opt Express 2(4):915–926. doi: 10.1364/BOE.2.000915 CrossRefGoogle Scholar
  7. Georgakoudi I, Jacobson BC, Müller MG, Sheets EE, Badizadegan K, Carr-Locke DL, Crum CP, Boone CW, Dasari RR, Van Dam J (2002) NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes. Cancer Res 62(3):682PubMedGoogle Scholar
  8. Giarola M, Rossi B, Mosconi E, Fontanella M, Marzola P, Scambi I, Sbarbati A, Mariotto G (2011) Fast and minimally invasive determination of the unsaturation index of white fat depots by micro-Raman spectroscopy. Lipids 46(7):659–667. doi: 10.1007/s11745-011-3567-8 PubMedCrossRefGoogle Scholar
  9. Haka AS, Shafer-Peltier KE, Fitzmaurice M, Crowe J, Dasari RR, Feld MS (2005) Diagnosing breast cancer by using Raman spectroscopy. Proc Natl Acad Sci USA 102(35):12371–12376. doi: 10.1073/pnas.0501390102 PubMedCrossRefGoogle Scholar
  10. Hamada K, Fujita K, Smith NI, Kobayashi M, Inouye Y, Kawata S (2008) Raman microscopy for dynamic molecular imaging of living cells. J Biomed Opt 13(4):044027. doi: 10.1117/1.2952192 PubMedCrossRefGoogle Scholar
  11. Han M, Kim C, Mozer P, Schafer F, Badaan S, Vigaru B, Tseng K, Petrisor D, Trock B, Stoianovici D (2011) Tandem-robot assisted laparoscopic radical prostatectomy to improve the neurovascular bundle visualization: a feasibility study. Urology 77(2):502–506. doi: 10.1016/j.urology.2010.06.064 PubMedCrossRefGoogle Scholar
  12. Harada Y, Takamatsu T (2012) Raman molecular imaging of cells and tissues: towards functional diagnostic imaging without labeling. Curr Pharm Biotechnol (E-pub ahead of print)Google Scholar
  13. Harada Y, Dai P, Yamaoka Y, Ogawa M, Tanaka H, Nosaka K, Akaji K, Takamatsu T (2009) Intracellular dynamics of topoisomerase I inhibitor, CPT-11, by slit-scanning confocal Raman microscopy. Histochem Cell Biol 132(1):39–46. doi: 10.1007/s00418-009-0594-0 PubMedCrossRefGoogle Scholar
  14. Hashimoto K, Hisasue S, Masumori N, Kobayashi K, Kato R, Fukuta F, Takahashi A, Hasegawa T, Tsukamoto T (2010) Clinical safety and feasibility of a newly developed, simple algorithm for decision-making on neurovascular bundle preservation in radical prostatectomy. Jpn J Clin Oncol 40(4):343–348. doi: 10.1093/jjco/hyp157 PubMedCrossRefGoogle Scholar
  15. Hattori Y, Komachi Y, Asakura T, Shimosegawa T, Kanai G, Tashiro H, Sato H (2007) In vivo Raman study of the living rat esophagus and stomach using a micro-Raman probe under an endoscope. Appl Spectrosc 61(6):579–584. doi: 10.1366/000370207781269747 PubMedCrossRefGoogle Scholar
  16. Huff TB, Cheng JX (2007) In vivo coherent anti-Stokes Raman scattering imaging of sciatic nerve tissue. J Microsc 225(Pt 2):175–182. doi: 10.1111/j.1365-2818.2007.01729.x PubMedCrossRefGoogle Scholar
  17. Imaizumi K, Harada Y, Wakabayashi N, Yamaoka Y, Konishi H, Dai P, Tanaka H, Takamatsu T (2012) Dual-wavelength excitation of mucosal autofluorescence for precise detection of diminutive colonic adenomas. Gastrointest Endosc 75(1):110–117. doi: 10.1016/j.gie.2011.08.012 PubMedCrossRefGoogle Scholar
  18. Katagiri T, Yamamoto YS, Ozaki Y, Matsuura Y, Sato H (2009) High axial resolution Raman probe made of a single hollow optical fiber. Appl Spectrosc 63(1):103–107. doi: 10.1366/000370209787169650 PubMedCrossRefGoogle Scholar
  19. Koljenovic S, Bakker Schut TC, Wolthuis R, de Jong B, Santos L, Caspers PJ, Kros JM, Puppels GJ (2005) Tissue characterization using high wave number Raman spectroscopy. J Biomed Opt 10(3):031116. doi: 10.1117/1.1922307 PubMedCrossRefGoogle Scholar
  20. Li QB, Xu Z, Zhang NW, Zhang L, Wang F, Yang LM, Wang JS, Zhou S, Zhang YF, Zhou XS, Shi JS, Wu JG (2005) In vivo and in situ detection of colorectal cancer using Fourier transform infrared spectroscopy. World J Gastroenterol 11(3):327–330PubMedGoogle Scholar
  21. Lieber CA, Mahadevan-Jansen A (2003) Automated method for subtraction of fluorescence from biological Raman spectra. Appl Spectrosc 57(11):1363–1367PubMedCrossRefGoogle Scholar
  22. Matousek P (2007) Deep non-invasive Raman spectroscopy of living tissue and powders. Chem Soc Rev 36(8):1292–1304. doi: 10.1039/b614777c PubMedCrossRefGoogle Scholar
  23. Montorsi F, Salonia A, Suardi N, Gallina A, Zanni G, Briganti A, Deho F, Naspro R, Farina E, Rigatti P (2005) Improving the preservation of the urethral sphincter and neurovascular bundles during open radical retropubic prostatectomy. Eur Urol 48(6):938–945. doi: 10.1016/j.eururo.2005.09.004 PubMedCrossRefGoogle Scholar
  24. Nakano K, Harada Y, Yamaoka Y, Miyawaki K, Imaizumi K, Takaoka H, Nakaoka M, Wakabayashi N, Yoshikawa T, Takamatsu T (2012) Precise analysis of the autofluorescence characteristics of rat colon under UVA and violet light excitation. Curr Pharm Biotechnol (E-pub ahead of print)Google Scholar
  25. Ogawa M, Harada Y, Yamaoka Y, Fujita K, Yaku H, Takamatsu T (2009) Label-free biochemical imaging of heart tissue with high-speed spontaneous Raman microscopy. Biochem Biophys Res Commun 382(2):370–374. doi: 10.1016/j.bbrc.2009.03.028 PubMedCrossRefGoogle Scholar
  26. Okada M, Smith NI, Palonpon AF, Endo H, Kawata S, Sodeoka M, Fujita K (2011) Label-free Raman observation of cytochrome c dynamics during apoptosis. Proc Natl Acad Sci USA. doi: 10.1073/pnas.1107524108
  27. Peres MB, Silveira L Jr, Zangaro RA, Pacheco MT, Pasqualucci CA (2011) Classification model based on Raman spectra of selected morphological and biochemical tissue constituents for identification of atherosclerosis in human coronary arteries. Lasers Med Sci 26(5):645–655. doi: 10.1007/s10103-011-0908-z PubMedCrossRefGoogle Scholar
  28. Pezolet M, Georgescauld D (1985) Raman spectroscopy of nerve fibers. A study of membrane lipids under steady state conditions. Biophys J 47(3):367–372. doi: 10.1016/S0006-3495(85)83927-8 PubMedCrossRefGoogle Scholar
  29. Pietrangeli A, Pugliese P, Perrone M, Sperduti I, Cosimelli M, Jandolo B (2009) Sexual dysfunction following surgery for rectal cancer—a clinical and neurophysiological study. J Exp Clin Cancer Res 28:128. doi: 10.1186/1756-9966-28-128 CrossRefGoogle Scholar
  30. Schafer KC, Denes J, Albrecht K, Szaniszlo T, Balog J, Skoumal R, Katona M, Toth M, Balogh L, Takats Z (2009) In vivo, in situ tissue analysis using rapid evaporative ionization mass spectrometry. Angew Chem Int Ed Engl 48(44):8240–8242. doi: 10.1002/anie.200902546 PubMedCrossRefGoogle Scholar
  31. Schiefke F, Akdemir M, Weber A, Akdemir D, Singer S, Frerich B (2009) Function, postoperative morbidity, and quality of life after cervical sentinel node biopsy and after selective neck dissection. Head & neck 31(4):503–512. doi: 10.1002/hed.21001 Google Scholar
  32. Shabsigh R (2001) Prevalence of and recent developments in female sexual dysfunction. Curr Psychiatry Rep 3(3):188–194PubMedCrossRefGoogle Scholar
  33. Stoffel W, Bosio A (1997) Myelin glycolipids and their functions. Curr Opin Neurobiol 7(5):654–661PubMedCrossRefGoogle Scholar
  34. Weiss DG (1982) Axoplasmic transport. Springer, New YorkCrossRefGoogle Scholar
  35. Williams PL, Warwick R, Dyson M, Bannister LH (1989) Gray’s anatomy. Churchill Livingstone, LondonGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Takeo Minamikawa
    • 1
  • Yoshinori Harada
    • 1
  • Noriaki Koizumi
    • 1
    • 2
  • Koji Okihara
    • 3
  • Kazumi Kamoi
    • 3
  • Akio Yanagisawa
    • 4
  • Tetsuro Takamatsu
    • 1
    Email author
  1. 1.Department of Pathology and Cell RegulationGraduate School of Medical Science, Kyoto Prefectural University of MedicineKyotoJapan
  2. 2.Division of Digestive Surgery, Department of SurgeryGraduate School of Medical Science, Kyoto Prefectural University of MedicineKyotoJapan
  3. 3.Department of UrologyGraduate School of Medical Science, Kyoto Prefectural University of MedicineKyotoJapan
  4. 4.Department of Surgical PathologyGraduate School of Medical Science, Kyoto Prefectural University of MedicineKyotoJapan

Personalised recommendations