Abstract

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

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References

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Am J Physiol-Heart C

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).

Appl Phys. B-Lasers O

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]

Appl. Optics

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]

Appl. Phys. Lett.

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]

Biophys J

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]

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]

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]

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]

Biophys. J

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]

Biophys. J.

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]

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]

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]

IEE J. Sel. Top. Quantum Electron.

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).

J Microsc-Oxford

K. Konig, "Multiphoton microscopy in life sciences," J Microsc-Oxford 200, 83-104 (2000).
[CrossRef]

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]

J Opt. Soc. Am B

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]

J. Biological Chem.

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).

J. Microsc-Oxford

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]

J. Microsc.-Oxford

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]

Opt. Express

Opt. Lett.

P Natl Acad Sci

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]

P Natl Acad Sci USA

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]

Phys. Rev. A

T. Y. F. Tsang, "Optical 3rd-Harmonic Generation at Interfaces," Phys Rev A 52(5), 4116-4125 (1995).
[CrossRef] [PubMed]

Physiol Res

L. Skarka and B. Ostadal, "Mitochondrial membrane potential in cardiac myocytes," Physiol Res 51(5), 425-434 (2002).
[PubMed]

Proc. Natl. Acad. Sci.

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]

Proc. of SPIE

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).

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]

Other

R. W. Boyd, Nonlinear optics, 2nd ed. (Academic Press, San Diego, CA, 2003).

S. Inouâe and R. J. Walter, Video microscopy (Plenum Press, New York, 1986).

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).

Supplementary Material (4)

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Figures (7)

Fig. 1.
Fig. 1.

Schematic diagram of the multimodal microscope. F-denotes filters, L-lenses, M-mirrors, DM-dichroic mirrors, and PMT stands for photomultipliers.

Fig. 2.
Fig. 2.

A sample image of an isolated cardiomyocyte in white light. The rectangular region represents a typical scanning area for the nonlinear multimodal microscope. The length of the entire cardiomyocye shown here is approximately 100 μm.

Fig. 3.
Fig. 3.

(637KB) Comparison of NAD(P)H MPF and THG images in a typical cardiomyocyte excited with 837nm laser pulses. (a) A 2D optical section of NAD(P)H MPF of a cardiomyocyte; (b) The same 2D optical section simultaneously imaged with THG. The intensity profiles along the same line shown in (a and b) are plotted in (c), where the green diamonds indicate MPF and blue triangles THG intensities. (d) The 2D SCIA output for the slice shown in (a) and (b), red color indicates the correlated region while green and blue are the uncorrelated MPF and THG, respectively. (e) The rendered NAD(P)H MPF. (f) The rendered THG image obtained simultaneously with the MPF. (g) Results of the SCIA where red represents the correlated signal, while green is the uncorrelated NAD(P)H MPF and blue is the uncorrelated THG signal. (h) The same SCIA as (g) but showing only the correlated volume in red. The laser beam propagation is parallel to the z direction while the origin of the axis is placed at the perimeter of the cardiomyocyte. The size of a pixel is 0.24μm

Fig. 4.
Fig. 4.

(565KB) The comparison of autofluorescence and THG of a typical cardiomyocyte with 1064nm excitation. (a) A 2D slice of the autofluorescence emitted at 630-700nm; (b) The THG generated simultaneously with the MPF. (c) The 3D rendered image of autofluorescence; (d) the rendered image of simultaneously generated THG. (e) The SCIA where red is the correlated signal, while green, which is barely noticeable, is the uncorrelated autofluorescence and blue is the uncorrelated THG signal. (f) The same SCIA as (e) but showing only the correlated volume in red. The laser beam propagation is parallel to the z direction while the origin of the axis is placed at the perimeter of the cardiomyocyte. The size of a pixel is 0.24μm

Fig. 5.
Fig. 5.

(775KB) Comparison of MPF and THG images of TMRM labeled cardiomyocyte excited with 1064nm laser pulses. (a) A 2D optical section of TMRM MPF of a cardiomyocyte collected at 630-700 nm; (b) The same 2D optical section simultaneously imaged with THG. The intensity profiles along the same line shown in (a and b) are plotted in (c), where the green diamonds indicate MPF and blue triangles show THG intensities. (d) The 2D SCIA output for the slice shown in (a) and (b), red color indicates the correlated region while green and blue are the uncorrelated MPF and THG, respecitively. (e) The rendered 3D image of TMRM MPF. (f) The rendered THG image obtained simultaneously with the MPF. (g) Results of the SCIA where red represents the correlated signal, while green is represents uncorrelated NAD(P)H MPF, and blue is the uncorrelated THG signal. (h) The same SCIA as (g) but showing only the correlated volume in red. The laser beam propagation is parallel to the z direction while the origin of the axis is placed at the perimeter of the cardiomyocyte. The size of a pixel is 0.12μm

Fig. 6.
Fig. 6.

(600KB) Comparison of SHG and THG images of cardiomyocyte excited with 1064nm laser pulses. (a) A 2D optical section of SHG of a cardiomyocyte; (b) The same 2D optical section simultaneously imaged with THG. (c) The rendered 3D image of SHG. (d) The rendered THG image obtained simultaneously with the SHG. (e) Results of the SCIA where red, which is barely visible, represents the correlated signal, while green represents uncorrelated SHG, and blue is the uncorrelated THG signal. (f) The same SCIA as (e) but showing only the only the minute correlated volume in red. The laser beam propagation is parallel to the z direction while the origin of the axis is placed at the perimeter of the cardiomyocyte. The size of a pixel is 0.24μm

Fig. 7.
Fig. 7.

Pearson’s coefficient ( r ) analysis of optical slices at different depths of typical cardiomyocytes. Graph shows how THG and MPF are correlated (r >0) through most depths of the sample, while THG and SHG are anticorrelated (r < 0) for much of the sample. In the middle of cardiomyocyte, the coefficient shifts closer to 0, where correlation is difficult to determine.

Equations (2)

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r = A i B i A i B i N ( A i 2 ( A i ) 2 N ) ( B i 2 ( B i ) 2 N )
For a i > a min b i > b min : C i = a i a max b i b max ( a i a max b i b max ) max , otherwise C i = 0

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