OSA's Digital Library

Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 2, Iss. 5 — May. 1, 2011
  • pp: 1366–1376

Imaging skeletal muscle using second harmonic generation and coherent anti-Stokes Raman scattering microscopy

Christian P. Pfeffer, Bjorn R. Olsen, Feruz Ganikhanov, and François Légaré  »View Author Affiliations

Biomedical Optics Express, Vol. 2, Issue 5, pp. 1366-1376 (2011)

View Full Text Article

Enhanced HTML    Acrobat PDF (2132 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



We describe experimental results on label free imaging of striated skeletal muscle using second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy. The complementarity of the SHG and CARS data makes it possible to clearly identify the main sarcomere sub-structures such as actin, myosin, acto-myosin, and the intact T-tubular system as it emanates from the sarcolemma. Owing to sub-micron spatial resolution and the high sensitivity of the CARS microscopy technique we were able to resolve individual myofibrils. In addition, key organelles such as mitochondria, cell nuclei and their structural constituents were observed revealing the entire structure of the muscle functional units. There is a noticeable difference in the CARS response of the muscle structure within actin, myosin and t-tubule areas with respect to laser polarization. We attribute this to a preferential alignment of the probed molecular bonds along certain directions. The combined CARS and SHG microscopy approach yields more extensive and complementary information and has a potential to become an indispensable method for live skeletal muscle characterization.

© 2011 OSA

OCIS Codes
(190.4160) Nonlinear optics : Multiharmonic generation
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:

Original Manuscript: February 25, 2011
Revised Manuscript: April 11, 2011
Manuscript Accepted: April 18, 2011
Published: April 27, 2011

Christian P. Pfeffer, Bjorn R. Olsen, Feruz Ganikhanov, and François Légaré, "Imaging skeletal muscle using second harmonic generation and coherent anti-Stokes Raman scattering microscopy," Biomed. Opt. Express 2, 1366-1376 (2011)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. T. P. Burghardt, K. Ajtai, D. K. Chan, M. F. Halstead, J. Li, and Y. Zheng, “GFP-tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber,” Biophys. J. 93(6), 2226–2239 (2007). [CrossRef] [PubMed]
  2. M. E. Quinlan, J. N. Forkey, and Y. E. Goldman, “Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy,” Biophys. J. 89(2), 1132–1142 (2005). [CrossRef] [PubMed]
  3. E. Savio-Galimberti, J. Frank, M. Inoue, J. I. Goldhaber, M. B. Cannell, J. H. Bridge, and F. B. Sachse, “Novel features of the rabbit transverse tubular system revealed by quantitative analysis of three-dimensional reconstructions from confocal images,” Biophys. J. 95(4), 2053–2062 (2008). [CrossRef] [PubMed]
  4. R. M. Murphy, J. P. Mollica, and G. D. Lamb, “Plasma membrane removal in rat skeletal muscle fibers reveals caveolin-3 hot-spots at the necks of transverse tubules,” Exp. Cell Res. 315(6), 1015–1028 (2009). [CrossRef] [PubMed]
  5. F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005). [CrossRef] [PubMed]
  6. C. B. Schaffer, B. Friedman, N. Nishimura, L. F. Schroeder, P. S. Tsai, F. F. Ebner, P. D. Lyden, and D. Kleinfeld, “Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion,” PLoS Biol. 4(2), e22 (2006). [CrossRef] [PubMed]
  7. P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21(11), 1356–1360 (2003). [CrossRef] [PubMed]
  8. R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88(2), 1377–1386 (2005). [CrossRef] [PubMed]
  9. D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, “Depth-resolved structural imaging by third-harmonic generation microscopy,” J. Struct. Biol. 147(1), 3–11 (2004). [CrossRef] [PubMed]
  10. D. Débarre, W. Supatto, A. M. Pena, A. M. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3(1), 47–53 (2006). [CrossRef] [PubMed]
  11. C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A. 102(46), 16807–16812 (2005). [CrossRef] [PubMed]
  12. H. Wang, Y. Fu, P. Zickmund, R. Shi, and J. X. Cheng, “Coherent anti-stokes Raman scattering imaging of axonal myelin in live spinal tissues,” Biophys. J. 89(1), 581–591 (2005). [CrossRef] [PubMed]
  13. C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol. 164(1), 140–145 (2008). [CrossRef] [PubMed]
  14. U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, and T. Nann, “Quantum dots versus organic dyes as fluorescent labels,” Nat. Methods 5(9), 763–775 (2008). [CrossRef] [PubMed]
  15. A. Shiohara, A. Hoshino, K. Hanaki, K. Suzuki, and K. Yamamoto, “On the cyto-toxicity caused by quantum dots,” Microbiol. Immunol. 48(9), 669–675 (2004). [PubMed]
  16. S. M. Burrows, R. D. Reif, and D. Pappas, “Investigation of photobleaching and saturation of single molecules by fluorophore recrossing events,” Anal. Chim. Acta 598(1), 135–142 (2007). [CrossRef] [PubMed]
  17. J.H. Kim, W.H. Kong, H.J. Kim, S.W. Sco, “Limitation of Q Dot as an In Vivo Cell Tracer,” Tissue Engineering and regenerative medicine 6, 307–312 (2009).
  18. A. C. Kwan, D. A. Dombeck, and W. W. Webb, “Polarized microtubule arrays in apical dendrites and axons,” Proc. Natl. Acad. Sci. U.S.A. 105(32), 11370–11375 (2008). [CrossRef] [PubMed]
  19. S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90(2), 693–703 (2006). [CrossRef] [PubMed]
  20. M. D. Duncan, J. Reintjes, and T. J. Manuccia, “Scanning coherent anti-Stokes Raman microscope,” Opt. Lett. 7(8), 350–352 (1982). [CrossRef] [PubMed]
  21. A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett. 82(20), 4142–4145 (1999). [CrossRef]
  22. T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, “Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy,” Proc. Natl. Acad. Sci. U.S.A. 104(37), 14658–14663 (2007). [CrossRef] [PubMed]
  23. E. Ralston, B. Swaim, M. Czapiga, W.-L. Hwu, Y.-H. Chien, M. G. Pittis, B. Bembi, O. Schwartz, P. Plotz, and N. Raben, “Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence,” J. Struct. Biol. 162(3), 500–508 (2008). [CrossRef] [PubMed]
  24. S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, “Studies of χ(2)/χ(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy,” Biophys. J. 86(6), 3914–3922 (2004). [CrossRef] [PubMed]
  25. F. Ganikhanov, S. Carrasco, X. Sunney Xie, M. Katz, W. Seitz, and D. Kopf, “Broadly tunable dual-wavelength light source for coherent anti-Stokes Raman scattering microscopy,” Opt. Lett. 31(9), 1292–1294 (2006). [CrossRef] [PubMed]
  26. M. D. Abramoff, P. J. Magelhaes, and S. J. Ram, “Image Processing with ImageJ,” Biophotonics Int. 11(7), 36–42 (2004).
  27. G. Okano, H. Matsuzaka, and T. Shimojo, “A comparative study of the lipid composition of white, intermediate, red and heart muscle in rats,” Biochim. Biophys. Acta 619(1), 167–175 (1980). [PubMed]
  28. C. Hidalgo, “Lipid phase of transverse tubule membranes from skeletal muscle. An electron paramagnetic resonance study,” Biophys. J. 47(6), 757–764 (1985). [CrossRef] [PubMed]
  29. K. I. Popov, A. F. Pegoraro, A. Stolow, and L. Ramunno, “Image formation in CARS microscopy: effect of the Gouy phase shift,” Opt. Express 19(7), 5902–5911 (2011). [CrossRef] [PubMed]
  30. M. D. Levenson, C. Flytzanis, and N. Bloembergen, “Interference of resonant and nonresonant three-wave mixing in diamond,” Phys. Rev. B 6(10), 3962–3965 (1972). [CrossRef]
  31. H. M. Warrick and J. A. Spudich, “Myosin structure and function in cell motility,” Annu. Rev. Cell Biol. 3(1), 379–421 (1987). [CrossRef] [PubMed]
  32. R. Domunguez and K. C. Holmes, “Actin structure and function,” Ann. Rev. Biophys. 40, 169–186 (2011).

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.


Fig. 1 Fig. 2 Fig. 3
Fig. 4 Fig. 5

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited