OSA's Digital Library

Optics Express

Optics Express

  • Editor: Michael Duncan
  • Vol. 13, Iss. 20 — Oct. 3, 2005
  • pp: 8263–8276

Visualization of mitochondria in cardiomyocytes

Virginijus Barzda, Catherine Greenhalgh, Jürg Aus der Au, Steven Elmore, Johannes HGM van Beek, and Jeff Squier  »View Author Affiliations

Optics Express, Vol. 13, Issue 20, pp. 8263-8276 (2005)

View Full Text Article

Enhanced HTML    Acrobat PDF (401 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



The simultaneous detection of third harmonic (THG), and multiphoton excitation fluorescence (MPF) or second harmonic (SHG) from the same focal volume has led us to the development of a nonlinear multimodal microscopic biological imaging tool. The multimodal microscope has been applied for imaging of isolated live cardiomyocytes, and investigation of structural origin of the THG and SHG signals has been performed. By employing the different image contrast mechanisms, differentiation of structures inside a single live adult rat cardiomyocyte has been achieved. Based on structural crosscorrelation image analysis between NAD(P)H fluorescence and THG, and morphology of cardiomyocytes we were able to assign large part of the structure revealed by THG to the mitochondria. The crosscorrelation of THG with fluorescence of tetramethylrhodamine methyl ester (TMRM) labeled cardiomyocytes confirmed the mitochondrial origin of THG. The SHG generated structures were anticorrelated with THG and possessed the characteristic pattern of the myofibrils in the myocyte in accordance with the literature. Possible visualization of mitochondria with THG microscopy appeared due to enhancement of the third harmonic by multilayer arrangement of cristae.

© 2005 Optical Society of America

OCIS Codes
(110.2960) Imaging systems : Image analysis
(110.6880) Imaging systems : Three-dimensional image acquisition
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(180.5810) Microscopy : Scanning microscopy
(190.4160) Nonlinear optics : Multiharmonic generation

ToC Category:
Research Papers

Original Manuscript: July 18, 2005
Revised Manuscript: September 21, 2005
Published: October 3, 2005

Virginijus Barzda, Catherine Greenhalgh, Jürg Aus der Au, Steven Elmore, Johannes van Beek, and Jeff Squier, "Visualization of mitochondria in cardiomyocytes by simultaneous harmonic generation and fluorescence microscopy," Opt. Express 13, 8263-8276 (2005)

Sort:  Journal  |  Reset  


  1. K. Konig, "Multiphoton microscopy in life sciences," J Microsc-Oxford 200, 83-104 (2000). [CrossRef]
  2. L. Moreaux, O. Sandre, and J. Mertz, "Membrane imaging by second-harmonic generation microscopy," J Opt Soc Am B 17(10), 1685-1694 (2000). [CrossRef]
  3. L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, and J. Mertz, "Coherent scattering in multi-harmonic light microscopy," Biophys J 80(3), 1568-1574 (2001). [CrossRef] [PubMed]
  4. P. J. Campagnola, M. D. Wei, A. Lewis, and L. M. Loew, "High-resolution nonlinear optical imaging of live cells by second harmonic generation," Biophys J 77(6), 3341-3349 (1999). [CrossRef] [PubMed]
  5. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, "Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues," Biophys. J. 82(1), 493-508 (2002). [CrossRef]
  6. R. Barille, L. Canioni, S. Rivet, L. Sarger, P. Vacher, and T. Ducret, "Visualization of intracellular Ca2+dynamics with simultaneous two-photon-excited fluorescence and third-harmonic generation microscopes," Appl Phys Lett 79(24), 4045-4047 (2001). [CrossRef]
  7. S. W. Chu, S. Y. Chen, T. H. Tsai, T. M. Liu, C. Y. Lin, H. J. Tsai, and C. K. Sun, "In vivo developmental biology study using noninvasive multi-harmonic generation microscopy," Opt Express 11(23), 3093-3099 (2003). <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-11-23-3093">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-11-23-3093</a> [CrossRef] [PubMed]
  8. S. W. Chu, S. Y. Chen, G. W. Chern, T. H. Tsai, Y. C. Chen, B. L. Lin, and C. K. Sun, "Studies of x((2))/x((3)) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86(6), 3914-3922 (2004). [CrossRef] [PubMed]
  9. S. W. Chu, I. H. Chen, T. M. Liu, P. C. Chen, C. K. Sun, and B. L. Lin, "Multimodal nonlinear spectral microscopy based on a femtosecond Cr : forsterite laser," Opt Lett 26(23), 1909-1911 (2001). [CrossRef]
  10. V. Barzda, C. Greenhalgh, J. Aus der Au, J. A. Squier, S. Elmore, and J. H. van Beek, "Second- and third-harmonic generation and multiphoton excitation fluorescence microscopy for simultaneous imaging of cardiomyocytes," in Commercial and Biomedical Applications of Ultrafast Lasers IV, J. Neev, C. B. Schaffer, and A. Ostendorf eds, Proc. of SPIE 5340, 96-103 (2004). [CrossRef]
  11. R. W. Boyd, Nonlinear optics, 2nd ed. (Academic Press, San Diego, CA, 2003).
  12. I. Freund and M. Deutsch, "2nd-Harmonic Microscopy of Biological Tissue," Opt. Lett. 11(2), 94-96 (1986). [CrossRef] [PubMed]
  13. L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, "Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy," Opt Lett 25(5), 320-322 (2000). [CrossRef]
  14. I. Freund, M. Deutsch, and A. Sprecher, "Connective-Tissue Polarity - Optical 2nd-Harmonic Microscopy, Crossed-Beam Summation, and Small-Angle Scattering in Rat-Tail Tendon," Biophys J 50(4), 693-712 (1986). [CrossRef] [PubMed]
  15. Y. C. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, "Second-harmonic tomography of tissues," Opt. Lett. 22 (17), 1323-1325 (1997). [CrossRef]
  16. J. A. Squier, M. Muller, G. J. Brakenhoff, and K. R. Wilson, "Third harmonic generation microscopy," Opt Express 3(9), 315-324 (1998). <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-3-9-315">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-3-9-315</a> [CrossRef] [PubMed]
  17. T. Y. F. Tsang, "Optical 3rd-Harmonic Generation at Interfaces," Phys Rev A 52(5), 4116-4125 (1995). [CrossRef] [PubMed]
  18. M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, "3D microscopy of transparent objects using third-harmonic generation," J Microsc-Oxford 191, 266-274 (1998). [CrossRef]
  19. A. C. Millard, P. W. Wiseman, D. N. Fittinghoff, K. R. Wilson, J. A. Squier, and M. Muller, "Third-harmonic generation microscopy by use of a compact, femtosecond fiber laser source," Appl Optics 38(36), 7393-7397 (1999). [CrossRef]
  20. D. Yelin, D. Oron, E. Korkotian, M. Segal, and Y. Silbergerg, "Third-harmonic microscopy with a titanium-sapphire laser," Appl Phys B-Lasers O 74, S97-S101 (2002). [CrossRef]
  21. D. Oron, E. Tal, and Y. Silberberg, "Depth-resolved multiphoton polarization microscopy by third-harmonic generation," Opt Lett 28(23), 2315-2317 (2003). [CrossRef] [PubMed]
  22. S. H. Huang, A. A. Heikal, and W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys J 82(5), 2811-2825 (2002). [CrossRef] [PubMed]
  23. C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, "Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy," P Natl Acad Sci USA 93(20), 10763-10768 (1996). [CrossRef]
  24. S. W. Chu, I. H. Chen, T. M. Liu, C. K. Sun, S. P. Lee, B. L. Lin, P. C. Cheng, M. X. Kuo, D. J. Lin, and H. L. Liu, "Nonlinear bio-photonic crystal effects revealed with multimodal nonlinear microscopy," J Microsc-Oxford 208, 190-200 (2002). [CrossRef]
  25. D. Kopf, F. X. Kartner, U. Keller, and K. J. Weingarten, "Diode-Pumped Mode-Locked Nd-Glass Lasers with an Antiresonant Fabry-Perot Saturable Absorber," Opt Lett 20(10), 1169-1171 (1995). [CrossRef] [PubMed]
  26. C. Greenhalgh, R. Cisek, N. Prent, A. Major, J. Aus der Au, J. Squier, and V. Barzda, "Time and structural crosscorrelation image analysis of microscopic volumes, simultaneously recorded with second harmonic generation, third harmonic generation, and multiphoton excitation fluorescence microscopy," in Photonic Applications in Biosensing and Imaging, W.C.W. Chan, K. Yu, U.J. Krull, R.I. Hornsey, B.C. Wilson, and R.A. Weersink. eds., Proc. of SPIE 5969, 557-564 (2005).
  27. H. L. Li, J. Suzuki, E. Bayna, F. M. Zhang, E. D. Molle, A. Clark, R. L. Engler, and W. Y. W. Lew, "Lipopolysaccharide induces apoptosis in adult rat ventricular myocytes via cardiac AT(1) receptors," Am J Physiol-Heart C 283(2), H461-H467 (2002).
  28. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci. USA 100(12), 7075-7080 (2003). [CrossRef] [PubMed]
  29. K. Blinova, C. Combs, P. Kellman, and R. S. Balaban, "Fluctuation analysis of mitochondrial NADH fluorescence signals in confocal and two-photon microscopy images of living cardiac myocytes," J. Microsc.-Oxford 213, 70-75 (2004). [CrossRef]
  30. J. Eng, R. M. Lynch, and R. S. Balaban, "Nicotinamide Adenine-Dinucleotide Fluorescence Spectroscopy and Imaging of Isolated Cardiac Myocytes," Biophys. J. 55(4), 621-630 (1989). [CrossRef] [PubMed]
  31. D. L. Farkas, M. D. Wei, P. Febbroriello, J. H. Carson, and L. M. Loew, "Simultaneous Imaging of Cell and Mitochondrial-Membrane Potentials," Biophys J 56(6), 1053-1069 (1989). [CrossRef] [PubMed]
  32. D. N. Romashko, E. Marban, and B. O'Rourke, "Subcellular metabolic transients and mitochondrial redox waves in heart cells," P Natl Acad Sci USA 95(4), 1618-1623 (1998). [CrossRef]
  33. S. Inouâe and R. J. Walter, Video microscopy (Plenum Press, New York, 1986).
  34. F. Martini and W. C. Ober, Fundamentals of anatomy & physiology, 5th ed. (Prentice Hall, Upper Saddle River, N.J. ; Toronto, 2001), p. 1 v. (various pagings).
  35. L. Skarka and B. Ostadal, "Mitochondrial membrane potential in cardiac myocytes," Physiol Res 51(5), 425-434 (2002). [PubMed]
  36. J. W. Palmer, B. Tandler, and C. L. Hoppel, "Biochemical Properties of Subsarcolemmal and Inter-Fibrillar Mitochondria Isolated from Rat Cardiac-Muscle," J. Biological Chem. 252(23), 8731-8739 (1977).
  37. U. Keller, K. J. Weingarten, F. X. Kartner, D. Kopf, B. Braun, I. D. Jung, R. Fluck, C. Honninger, N. Matuschek, and J. A. derAu, "Semiconductor saturable absorber mirrors (SESAM's) for femtosecond to nanosecond pulse generation in solid-state lasers," IEEE J. Sel. Top. Quantum Electron. 2(3), 435-453 (1996).

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.

Supplementary Material

» Media 1: AVI (637 KB)     
» Media 2: AVI (564 KB)     
» Media 3: AVI (775 KB)     
» Media 4: AVI (600 KB)     

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited