Abstract

With a micro-electro-mechanical system (MEMS) mirror, we successfully developed a miniaturized epi-third-harmonic-generation (epi-THG) fiber-microscope with a video frame rate (31Hz), which was designed for in vivo optical biopsy of human skin. With a large-mode-area (LMA) photonic crystal fiber (PCF) and a regular microscopic objective, the nonlinear distortion of the ultrafast pulses delivery could be much reduced while still achieving a 0.4μm lateral resolution for epi-THG signals. In vivo real time virtual biopsy of the Asian skin with a video rate (31Hz) and a sub-micron resolution was obtained. The result indicates that this miniaturized system was compact enough for the least invasive hand-held clinical use.

© 2010 OSA

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2010 (4)

S.-Y. Chen, S.-U. Chen, H.-Y. Wu, W.-J. Lee, Y.-H. Liao, and C.-K. Sun, “In vivo virtual biopsy of human skin by using noninvasive higher harmonic generation microscopy,” IEEE J. Sel. Top. Quantum Electron. 16, 478–492 (2010).
[CrossRef]

C.-F. Chang, C.-H. Yu, and C.-K. Sun, “Multi-photon resonance enhancement of third harmonic generation in human oxyhemoglobin and deoxyhemoglobin,” to be published in J. Biophoton. (2010).
[CrossRef]

C.-F. Chang, H.-C. Chen, M.-J. Chen, W.-R. Liu, W.-F. Hsieh, C.-H. Hsu, C.-Y. Chen, F.-H. Chang, C.-H. Yu, and C.-K. Sun, “Direct backward third-harmonic generation in nanostructures,” Opt. Express 18(7), 7397–7406 (2010).
[CrossRef] [PubMed]

K. G. Jespersen, T. Le, L. Grüner-Nielsen, D. Jakobsen, M. E. V. Pederesen, M. B. Smedemand, S. R. Keiding, and B. Palsdottir, “A higher-order-mode fiber delivery for Ti:Sapphire femtosecond lasers,” Opt. Express 18(8), 7798–7806 (2010).
[CrossRef] [PubMed]

2009 (8)

S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, “In vivo harmonic generation biopsy of human skin,” J. Biomed. Opt. 14(6), 060505 (2009).
[CrossRef]

J.-H. Lee, S.-Y. Chen, C.-H. Yu, S.-W. Chu, L.-F. Wang, C. K. Sun, and B. L. Chiang, “Noninvasive in vitro and in vivo assessment of epidermal hyperkeratosis and dermal fibrosis in atopic dermatitis,” J. Biomed. Opt. 14(1), 014008 (2009).
[CrossRef] [PubMed]

T. Yasui, Y. Takahashi, M. Ito, S. Fukushima, and T. Araki, “Ex vivo and in vivo second-harmonic-generation imaging of dermal collagen fiber in skin: comparison of imaging characteristics between mode-locked Cr:forsterite and Ti:sapphire lasers,” Appl. Opt. 48(10), D88–D95 (2009).
[CrossRef] [PubMed]

Y. Wu, Y. Leng, J. Xi, and X. Li, “Scanning all-fiber-optic endomicroscopy system for 3D nonlinear optical imaging of biological tissues,” Opt. Express 17, 7901–7915 (2009).
[CrossRef]

H. Bao and M. Gu, “A 0.4-mm-diameter probe for nonlinear optical imaging,” Opt. Express 17(12), 10098–10104 (2009).
[CrossRef] [PubMed]

R. Le Harzic, I. Riemann, M. Weinigel, K. König, and B. Messerschmidt, “Rigid and high-numerical-aperture two-photon fluorescence endoscope,” Appl. Opt. 48(18), 3396–3400 (2009).
[CrossRef] [PubMed]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009).
[CrossRef] [PubMed]

W. Piyawattanametha, E. D. Cocker, L. D. Burns, R. P. J. Barretto, J. C. Jung, H. Ra, O. Solgaard, and M. J. Schnitzer, “In vivo brain imaging using a portable 2.9 g two-photon microscope based on a microelectromechanical systems scanning mirror,” Opt. Lett. 34(15), 2309–2311 (2009).
[CrossRef] [PubMed]

2008 (7)

C.-H. Yu, S.-P. Tai, C.-T. Kung, W.-J. Lee, Y.-F. Chan, H.-L. Liu, J.-Y. Lyu, and C.-K. Sun, “Molecular third-harmonic-generation microscopy through resonance enhancement with absorbing dye,” Opt. Lett. 33(4), 387–389 (2008).
[CrossRef] [PubMed]

C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F. Helmchen, “Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo,” Opt. Express 16(8), 5556–5564 (2008).
[CrossRef] [PubMed]

C.-F. Chang, C.-Y. Chen, F.-H. Chang, S.-P. Tai, C.-Y. Chen, C.-H. Yu, Y.-B. Tseng, T.-H. Tsai, I.-S. Liu, W.-F. Su, and C.-K. Sun, “Cell tracking and detection of molecular expression in live cells using lipid-enclosed CdSe quantum dots as contrast agents for epi-third harmonic generation microscopy,” Opt. Express 16(13), 9534–9548 (2008).
[CrossRef] [PubMed]

C. L. Hoy, N. J. Durr, P. Chen, W. Piyawattanametha, H. Ra, O. Solgaard, and A. Ben-Yakar, “Miniaturized probe for femtosecond laser microsurgery and two-photon imaging,” Opt. Express 16(13), 9996–10005 (2008).
[CrossRef] [PubMed]

T.-M. Liu, M.-C. Chan, I.-H. Chen, S.-H. Chia, and C.-K. Sun, “Miniaturized multiphoton microscope with a 24Hz frame-rate,” Opt. Express 16(14), 10501–10506 (2008).
[CrossRef] [PubMed]

C.-S. Hsieh, S.-U. Chen, Y.-W. Lee, Y.-S. Yang, and C.-K. Sun, “Higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos,” Opt. Express 16(15), 11574–11588 (2008).
[PubMed]

R. Le Harzic, M. Weinigel, I. Riemann, K. König, and B. Messerschmidt, “Nonlinear optical endoscope based on a compact two axes piezo scanner and a miniature objective lens,” Opt. Express 16(25), 20588–20596 (2008).
[CrossRef] [PubMed]

2007 (3)

2006 (4)

2005 (4)

2004 (2)

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, “In vivo multiphoton microscopy of deep brain tissue,” J. Neurophysiol. 91(4), 1908–1912 (2004).
[CrossRef]

W. Göbel, J. N. D. Kerr, A. Nimmerjahn, and F. Helmchen, “Miniaturized two-photon microscope based on a flexible coherent fiber bundle and a gradient-index lens objective,” Opt. Lett. 29(21), 2521–2523 (2004).
[CrossRef] [PubMed]

2003 (4)

2002 (1)

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent cell damages in multi-photon confocal microscopy,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[CrossRef]

2001 (1)

P.-C. Cheng, B.-L. Lin, F.-J. Kao, M. Gu, M.-G. Xu, X. Gan, M.-K. Huang, and Y.-S. Wang, “Multi-photon fluorescence microscopy--the response of plant cells to high intensity illumination,” Micron 32(7), 661–669 (2001).
[CrossRef] [PubMed]

1999 (1)

1997 (1)

1994 (1)

1993 (1)

J. Schutz, W. Hodel, and H. Weber, “Nonlinear pulse distortion at the zero dispersion wavelength of an optical fibre,” Opt. Commun. 95(4-6), 357–365 (1993).
[CrossRef]

Anderson, E. P.

Araki, T.

Bao, H.

Barretto, R. P. J.

Ben-Yakar, A.

Bird, D.

Burns, L. D.

Campagnola, P. J.

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]

Chan, M.-C.

T.-M. Liu, M.-C. Chan, I.-H. Chen, S.-H. Chia, and C.-K. Sun, “Miniaturized multiphoton microscope with a 24Hz frame-rate,” Opt. Express 16(14), 10501–10506 (2008).
[CrossRef] [PubMed]

M.-C. Chan, T.-M. Liu, S.-P. Tai, and C.-K. Sun, “Compact fiber-delivered Cr:forsterite laser for nonlinear light microscopy,” J. Biomed. Opt. 10(5), 054006 (2005).
[CrossRef] [PubMed]

Chan, Y. F.

Chan, Y.-F.

Chang, C.-F.

Chang, F.-H.

Chen, C.-C.

Chen, C.-Y.

Chen, H.-C.

Chen, I.-H.

T.-M. Liu, M.-C. Chan, I.-H. Chen, S.-H. Chia, and C.-K. Sun, “Miniaturized multiphoton microscope with a 24Hz frame-rate,” Opt. Express 16(14), 10501–10506 (2008).
[CrossRef] [PubMed]

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent cell damages in multi-photon confocal microscopy,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[CrossRef]

Chen, M.-J.

Chen, P.

Chen, S.-U.

S.-Y. Chen, S.-U. Chen, H.-Y. Wu, W.-J. Lee, Y.-H. Liao, and C.-K. Sun, “In vivo virtual biopsy of human skin by using noninvasive higher harmonic generation microscopy,” IEEE J. Sel. Top. Quantum Electron. 16, 478–492 (2010).
[CrossRef]

C.-S. Hsieh, S.-U. Chen, Y.-W. Lee, Y.-S. Yang, and C.-K. Sun, “Higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos,” Opt. Express 16(15), 11574–11588 (2008).
[PubMed]

Chen, S.-Y.

S.-Y. Chen, S.-U. Chen, H.-Y. Wu, W.-J. Lee, Y.-H. Liao, and C.-K. Sun, “In vivo virtual biopsy of human skin by using noninvasive higher harmonic generation microscopy,” IEEE J. Sel. Top. Quantum Electron. 16, 478–492 (2010).
[CrossRef]

J.-H. Lee, S.-Y. Chen, C.-H. Yu, S.-W. Chu, L.-F. Wang, C. K. Sun, and B. L. Chiang, “Noninvasive in vitro and in vivo assessment of epidermal hyperkeratosis and dermal fibrosis in atopic dermatitis,” J. Biomed. Opt. 14(1), 014008 (2009).
[CrossRef] [PubMed]

S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, “In vivo harmonic generation biopsy of human skin,” J. Biomed. Opt. 14(6), 060505 (2009).
[CrossRef]

M.-R. Tsai, S.-Y. Chen, D.-B. Shieh, P.-J. Lou, and C.-K. Sun, “In vivo optical virtual biopsy of human oral cavity with harmonic generation microscopy,” Proc. SPIE (to be published).

Chen, Y.-C.

Cheng, P.-C.

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent cell damages in multi-photon confocal microscopy,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[CrossRef]

P.-C. Cheng, B.-L. Lin, F.-J. Kao, M. Gu, M.-G. Xu, X. Gan, M.-K. Huang, and Y.-S. Wang, “Multi-photon fluorescence microscopy--the response of plant cells to high intensity illumination,” Micron 32(7), 661–669 (2001).
[CrossRef] [PubMed]

Chia, S.-H.

Chiang, B. L.

J.-H. Lee, S.-Y. Chen, C.-H. Yu, S.-W. Chu, L.-F. Wang, C. K. Sun, and B. L. Chiang, “Noninvasive in vitro and in vivo assessment of epidermal hyperkeratosis and dermal fibrosis in atopic dermatitis,” J. Biomed. Opt. 14(1), 014008 (2009).
[CrossRef] [PubMed]

Chu, S.-W.

J.-H. Lee, S.-Y. Chen, C.-H. Yu, S.-W. Chu, L.-F. Wang, C. K. Sun, and B. L. Chiang, “Noninvasive in vitro and in vivo assessment of epidermal hyperkeratosis and dermal fibrosis in atopic dermatitis,” J. Biomed. Opt. 14(1), 014008 (2009).
[CrossRef] [PubMed]

C.-K. Sun, C.-C. Chen, S.-W. Chu, T.-H. Tsai, Y.-C. Chen, and B.-L. Lin, “Multiharmonic-generation biopsy of skin,” Opt. Lett. 28(24), 2488–2490 (2003).
[CrossRef] [PubMed]

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent cell damages in multi-photon confocal microscopy,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[CrossRef]

Cocker, E. D.

Cranfield, C.

Denk, W.

J. Sawinski and W. Denk, “Miniature random-access fiber scanner for in vivo multiphoton imaging,” J. Appl. Phys. 102(3), 034701 (2007).
[CrossRef]

Dombeck, D. A.

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, “In vivo multiphoton microscopy of deep brain tissue,” J. Neurophysiol. 91(4), 1908–1912 (2004).
[CrossRef]

Durr, N. J.

Durst, M. E.

Engelbrecht, C. J.

Flusberg, B. A.

Fu, L.

Fukushima, S.

Gan, X.

P.-C. Cheng, B.-L. Lin, F.-J. Kao, M. Gu, M.-G. Xu, X. Gan, M.-K. Huang, and Y.-S. Wang, “Multi-photon fluorescence microscopy--the response of plant cells to high intensity illumination,” Micron 32(7), 661–669 (2001).
[CrossRef] [PubMed]

Göbel, W.

Gratton, E.

Grüner-Nielsen, L.

Gu, M.

Helmchen, F.

Hodel, W.

J. Schutz, W. Hodel, and H. Weber, “Nonlinear pulse distortion at the zero dispersion wavelength of an optical fibre,” Opt. Commun. 95(4-6), 357–365 (1993).
[CrossRef]

Hoy, C. L.

Hsieh, C.-S.

Hsieh, W.-F.

Hsu, C.-H.

Huang, H. J.

Huang, H.-Y.

Huang, M.-K.

P.-C. Cheng, B.-L. Lin, F.-J. Kao, M. Gu, M.-G. Xu, X. Gan, M.-K. Huang, and Y.-S. Wang, “Multi-photon fluorescence microscopy--the response of plant cells to high intensity illumination,” Micron 32(7), 661–669 (2001).
[CrossRef] [PubMed]

Ito, M.

Jain, A.

Jakobsen, D.

Jespersen, K. G.

Johnston, R. S.

Jung, J. C.

Kao, F.-J.

P.-C. Cheng, B.-L. Lin, F.-J. Kao, M. Gu, M.-G. Xu, X. Gan, M.-K. Huang, and Y.-S. Wang, “Multi-photon fluorescence microscopy--the response of plant cells to high intensity illumination,” Micron 32(7), 661–669 (2001).
[CrossRef] [PubMed]

Kasischke, K. A.

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, “In vivo multiphoton microscopy of deep brain tissue,” J. Neurophysiol. 91(4), 1908–1912 (2004).
[CrossRef]

Keiding, S. R.

Kerr, J. N. D.

Ko, T. H.

Kobat, D.

König, K.

Kung, C. T.

Kung, C.-T.

Le, T.

Le Harzic, R.

Lee, D.

Lee, J.-H.

J.-H. Lee, S.-Y. Chen, C.-H. Yu, S.-W. Chu, L.-F. Wang, C. K. Sun, and B. L. Chiang, “Noninvasive in vitro and in vivo assessment of epidermal hyperkeratosis and dermal fibrosis in atopic dermatitis,” J. Biomed. Opt. 14(1), 014008 (2009).
[CrossRef] [PubMed]

Lee, W. J.

Lee, W.-J.

Lee, Y.-W.

Leng, Y.

Levene, M. J.

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, “In vivo multiphoton microscopy of deep brain tissue,” J. Neurophysiol. 91(4), 1908–1912 (2004).
[CrossRef]

Li, X.

Liao, Y.-H.

Lin, B.-L.

C.-K. Sun, C.-C. Chen, S.-W. Chu, T.-H. Tsai, Y.-C. Chen, and B.-L. Lin, “Multiharmonic-generation biopsy of skin,” Opt. Lett. 28(24), 2488–2490 (2003).
[CrossRef] [PubMed]

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent cell damages in multi-photon confocal microscopy,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[CrossRef]

P.-C. Cheng, B.-L. Lin, F.-J. Kao, M. Gu, M.-G. Xu, X. Gan, M.-K. Huang, and Y.-S. Wang, “Multi-photon fluorescence microscopy--the response of plant cells to high intensity illumination,” Micron 32(7), 661–669 (2001).
[CrossRef] [PubMed]

Liu, H.-L.

Liu, I.-S.

Liu, T.-M.

T.-M. Liu, M.-C. Chan, I.-H. Chen, S.-H. Chia, and C.-K. Sun, “Miniaturized multiphoton microscope with a 24Hz frame-rate,” Opt. Express 16(14), 10501–10506 (2008).
[CrossRef] [PubMed]

M.-C. Chan, T.-M. Liu, S.-P. Tai, and C.-K. Sun, “Compact fiber-delivered Cr:forsterite laser for nonlinear light microscopy,” J. Biomed. Opt. 10(5), 054006 (2005).
[CrossRef] [PubMed]

Liu, W.-R.

Loew, L. M.

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]

Lou, P.-J.

M.-R. Tsai, S.-Y. Chen, D.-B. Shieh, P.-J. Lou, and C.-K. Sun, “In vivo optical virtual biopsy of human oral cavity with harmonic generation microscopy,” Proc. SPIE (to be published).

Lyu, J.-Y.

Mantulin, W. W.

Messerschmidt, B.

Molloy, R. P.

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, “In vivo multiphoton microscopy of deep brain tissue,” J. Neurophysiol. 91(4), 1908–1912 (2004).
[CrossRef]

Nimmerjahn, A.

Nishimura, N.

Palsdottir, B.

Pederesen, M. E. V.

Pierce, M. C.

Piyawattanametha, W.

Ra, H.

Richards-Kortum, R.

Riemann, I.

Sawinski, J.

J. Sawinski and W. Denk, “Miniature random-access fiber scanner for in vivo multiphoton imaging,” J. Appl. Phys. 102(3), 034701 (2007).
[CrossRef]

Schaffer, C. B.

Schnitzer, M. J.

Schutz, J.

J. Schutz, W. Hodel, and H. Weber, “Nonlinear pulse distortion at the zero dispersion wavelength of an optical fibre,” Opt. Commun. 95(4-6), 357–365 (1993).
[CrossRef]

Seibel, E. J.

Shieh, D.-B.

Shin, H. J.

Silberberg, Y.

Smedemand, M. B.

So, P. T. C.

Solgaard, O.

Su, W.-F.

Sun, C. K.

J.-H. Lee, S.-Y. Chen, C.-H. Yu, S.-W. Chu, L.-F. Wang, C. K. Sun, and B. L. Chiang, “Noninvasive in vitro and in vivo assessment of epidermal hyperkeratosis and dermal fibrosis in atopic dermatitis,” J. Biomed. Opt. 14(1), 014008 (2009).
[CrossRef] [PubMed]

C. K. Sun, C. H. Yu, S. P. Tai, C. T. Kung, I. J. Wang, H. C. Yu, H. J. Huang, W. J. Lee, and Y. F. Chan, “In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy,” Opt. Express 15(18), 11167–11177 (2007).
[CrossRef] [PubMed]

Sun, C.-K.

S.-Y. Chen, S.-U. Chen, H.-Y. Wu, W.-J. Lee, Y.-H. Liao, and C.-K. Sun, “In vivo virtual biopsy of human skin by using noninvasive higher harmonic generation microscopy,” IEEE J. Sel. Top. Quantum Electron. 16, 478–492 (2010).
[CrossRef]

C.-F. Chang, C.-H. Yu, and C.-K. Sun, “Multi-photon resonance enhancement of third harmonic generation in human oxyhemoglobin and deoxyhemoglobin,” to be published in J. Biophoton. (2010).
[CrossRef]

C.-F. Chang, H.-C. Chen, M.-J. Chen, W.-R. Liu, W.-F. Hsieh, C.-H. Hsu, C.-Y. Chen, F.-H. Chang, C.-H. Yu, and C.-K. Sun, “Direct backward third-harmonic generation in nanostructures,” Opt. Express 18(7), 7397–7406 (2010).
[CrossRef] [PubMed]

S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, “In vivo harmonic generation biopsy of human skin,” J. Biomed. Opt. 14(6), 060505 (2009).
[CrossRef]

C.-H. Yu, S.-P. Tai, C.-T. Kung, W.-J. Lee, Y.-F. Chan, H.-L. Liu, J.-Y. Lyu, and C.-K. Sun, “Molecular third-harmonic-generation microscopy through resonance enhancement with absorbing dye,” Opt. Lett. 33(4), 387–389 (2008).
[CrossRef] [PubMed]

C.-F. Chang, C.-Y. Chen, F.-H. Chang, S.-P. Tai, C.-Y. Chen, C.-H. Yu, Y.-B. Tseng, T.-H. Tsai, I.-S. Liu, W.-F. Su, and C.-K. Sun, “Cell tracking and detection of molecular expression in live cells using lipid-enclosed CdSe quantum dots as contrast agents for epi-third harmonic generation microscopy,” Opt. Express 16(13), 9534–9548 (2008).
[CrossRef] [PubMed]

C.-S. Hsieh, S.-U. Chen, Y.-W. Lee, Y.-S. Yang, and C.-K. Sun, “Higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos,” Opt. Express 16(15), 11574–11588 (2008).
[PubMed]

T.-M. Liu, M.-C. Chan, I.-H. Chen, S.-H. Chia, and C.-K. Sun, “Miniaturized multiphoton microscope with a 24Hz frame-rate,” Opt. Express 16(14), 10501–10506 (2008).
[CrossRef] [PubMed]

S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, “In vivo optical biopsy of hamster oral cavity with epi-third-harmonic-generation microscopy,” Opt. Express 14(13), 6178–6187 (2006).
[CrossRef] [PubMed]

T.-H. Tsai, S.-P. Tai, W.-J. Lee, H.-Y. Huang, Y.-H. Liao, and C.-K. Sun, “Optical signal degradation study in fixed human skin using confocal microscopy and higher-harmonic optical microscopy,” Opt. Express 14(2), 749–758 (2006).
[CrossRef] [PubMed]

S.-P. Tai, T.-H. Tsai, W.-J. Lee, D.-B. Shieh, Y.-H. Liao, H.-Y. Huang, K. Zhang, H.-L. Liu, and C.-K. Sun, “Optical biopsy of fixed human skin with backward-collected optical harmonics signals,” Opt. Express 13(20), 8231–8242 (2005).
[CrossRef] [PubMed]

C.-K. Sun, “Higher harmonic generation microscopy,” Adv. Biochem. Engin, Biotechnol. 95, 17–56 (2005).

M.-C. Chan, T.-M. Liu, S.-P. Tai, and C.-K. Sun, “Compact fiber-delivered Cr:forsterite laser for nonlinear light microscopy,” J. Biomed. Opt. 10(5), 054006 (2005).
[CrossRef] [PubMed]

C.-K. Sun, C.-C. Chen, S.-W. Chu, T.-H. Tsai, Y.-C. Chen, and B.-L. Lin, “Multiharmonic-generation biopsy of skin,” Opt. Lett. 28(24), 2488–2490 (2003).
[CrossRef] [PubMed]

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent cell damages in multi-photon confocal microscopy,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[CrossRef]

M.-R. Tsai, S.-Y. Chen, D.-B. Shieh, P.-J. Lou, and C.-K. Sun, “In vivo optical virtual biopsy of human oral cavity with harmonic generation microscopy,” Proc. SPIE (to be published).

Tai, S. P.

Tai, S.-P.

C.-H. Yu, S.-P. Tai, C.-T. Kung, W.-J. Lee, Y.-F. Chan, H.-L. Liu, J.-Y. Lyu, and C.-K. Sun, “Molecular third-harmonic-generation microscopy through resonance enhancement with absorbing dye,” Opt. Lett. 33(4), 387–389 (2008).
[CrossRef] [PubMed]

C.-F. Chang, C.-Y. Chen, F.-H. Chang, S.-P. Tai, C.-Y. Chen, C.-H. Yu, Y.-B. Tseng, T.-H. Tsai, I.-S. Liu, W.-F. Su, and C.-K. Sun, “Cell tracking and detection of molecular expression in live cells using lipid-enclosed CdSe quantum dots as contrast agents for epi-third harmonic generation microscopy,” Opt. Express 16(13), 9534–9548 (2008).
[CrossRef] [PubMed]

S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, “In vivo optical biopsy of hamster oral cavity with epi-third-harmonic-generation microscopy,” Opt. Express 14(13), 6178–6187 (2006).
[CrossRef] [PubMed]

T.-H. Tsai, S.-P. Tai, W.-J. Lee, H.-Y. Huang, Y.-H. Liao, and C.-K. Sun, “Optical signal degradation study in fixed human skin using confocal microscopy and higher-harmonic optical microscopy,” Opt. Express 14(2), 749–758 (2006).
[CrossRef] [PubMed]

S.-P. Tai, T.-H. Tsai, W.-J. Lee, D.-B. Shieh, Y.-H. Liao, H.-Y. Huang, K. Zhang, H.-L. Liu, and C.-K. Sun, “Optical biopsy of fixed human skin with backward-collected optical harmonics signals,” Opt. Express 13(20), 8231–8242 (2005).
[CrossRef] [PubMed]

M.-C. Chan, T.-M. Liu, S.-P. Tai, and C.-K. Sun, “Compact fiber-delivered Cr:forsterite laser for nonlinear light microscopy,” J. Biomed. Opt. 10(5), 054006 (2005).
[CrossRef] [PubMed]

Takahashi, Y.

Tsai, M.-R.

M.-R. Tsai, S.-Y. Chen, D.-B. Shieh, P.-J. Lou, and C.-K. Sun, “In vivo optical virtual biopsy of human oral cavity with harmonic generation microscopy,” Proc. SPIE (to be published).

Tsai, T.-H.

Tseng, Y.-B.

Wang, I. J.

Wang, L.-F.

J.-H. Lee, S.-Y. Chen, C.-H. Yu, S.-W. Chu, L.-F. Wang, C. K. Sun, and B. L. Chiang, “Noninvasive in vitro and in vivo assessment of epidermal hyperkeratosis and dermal fibrosis in atopic dermatitis,” J. Biomed. Opt. 14(1), 014008 (2009).
[CrossRef] [PubMed]

Wang, Y.-S.

P.-C. Cheng, B.-L. Lin, F.-J. Kao, M. Gu, M.-G. Xu, X. Gan, M.-K. Huang, and Y.-S. Wang, “Multi-photon fluorescence microscopy--the response of plant cells to high intensity illumination,” Micron 32(7), 661–669 (2001).
[CrossRef] [PubMed]

Webb, W. W.

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, “In vivo multiphoton microscopy of deep brain tissue,” J. Neurophysiol. 91(4), 1908–1912 (2004).
[CrossRef]

Weber, H.

J. Schutz, W. Hodel, and H. Weber, “Nonlinear pulse distortion at the zero dispersion wavelength of an optical fibre,” Opt. Commun. 95(4-6), 357–365 (1993).
[CrossRef]

Weinigel, M.

Wise, F. W.

Wong, A. W.

Wu, H.-Y.

S.-Y. Chen, S.-U. Chen, H.-Y. Wu, W.-J. Lee, Y.-H. Liao, and C.-K. Sun, “In vivo virtual biopsy of human skin by using noninvasive higher harmonic generation microscopy,” IEEE J. Sel. Top. Quantum Electron. 16, 478–492 (2010).
[CrossRef]

S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, “In vivo harmonic generation biopsy of human skin,” J. Biomed. Opt. 14(6), 060505 (2009).
[CrossRef]

Wu, P.-C.

Wu, Y.

Xi, J.

Xie, H.

Xu, C.

Xu, M.-G.

P.-C. Cheng, B.-L. Lin, F.-J. Kao, M. Gu, M.-G. Xu, X. Gan, M.-K. Huang, and Y.-S. Wang, “Multi-photon fluorescence microscopy--the response of plant cells to high intensity illumination,” Micron 32(7), 661–669 (2001).
[CrossRef] [PubMed]

Yang, Y.-S.

Yanovsky, V. P.

Yasui, T.

Yelin, D.

Yu, C. H.

Yu, C.-H.

C.-F. Chang, C.-H. Yu, and C.-K. Sun, “Multi-photon resonance enhancement of third harmonic generation in human oxyhemoglobin and deoxyhemoglobin,” to be published in J. Biophoton. (2010).
[CrossRef]

C.-F. Chang, H.-C. Chen, M.-J. Chen, W.-R. Liu, W.-F. Hsieh, C.-H. Hsu, C.-Y. Chen, F.-H. Chang, C.-H. Yu, and C.-K. Sun, “Direct backward third-harmonic generation in nanostructures,” Opt. Express 18(7), 7397–7406 (2010).
[CrossRef] [PubMed]

J.-H. Lee, S.-Y. Chen, C.-H. Yu, S.-W. Chu, L.-F. Wang, C. K. Sun, and B. L. Chiang, “Noninvasive in vitro and in vivo assessment of epidermal hyperkeratosis and dermal fibrosis in atopic dermatitis,” J. Biomed. Opt. 14(1), 014008 (2009).
[CrossRef] [PubMed]

C.-H. Yu, S.-P. Tai, C.-T. Kung, W.-J. Lee, Y.-F. Chan, H.-L. Liu, J.-Y. Lyu, and C.-K. Sun, “Molecular third-harmonic-generation microscopy through resonance enhancement with absorbing dye,” Opt. Lett. 33(4), 387–389 (2008).
[CrossRef] [PubMed]

C.-F. Chang, C.-Y. Chen, F.-H. Chang, S.-P. Tai, C.-Y. Chen, C.-H. Yu, Y.-B. Tseng, T.-H. Tsai, I.-S. Liu, W.-F. Su, and C.-K. Sun, “Cell tracking and detection of molecular expression in live cells using lipid-enclosed CdSe quantum dots as contrast agents for epi-third harmonic generation microscopy,” Opt. Express 16(13), 9534–9548 (2008).
[CrossRef] [PubMed]

S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, “In vivo optical biopsy of hamster oral cavity with epi-third-harmonic-generation microscopy,” Opt. Express 14(13), 6178–6187 (2006).
[CrossRef] [PubMed]

Yu, H. C.

Zhang, K.

Adv. Biochem. Engin, Biotechnol. (1)

C.-K. Sun, “Higher harmonic generation microscopy,” Adv. Biochem. Engin, Biotechnol. 95, 17–56 (2005).

Appl. Opt. (2)

IEEE J. Sel. Top. Quantum Electron. (1)

S.-Y. Chen, S.-U. Chen, H.-Y. Wu, W.-J. Lee, Y.-H. Liao, and C.-K. Sun, “In vivo virtual biopsy of human skin by using noninvasive higher harmonic generation microscopy,” IEEE J. Sel. Top. Quantum Electron. 16, 478–492 (2010).
[CrossRef]

J. Appl. Phys. (1)

J. Sawinski and W. Denk, “Miniature random-access fiber scanner for in vivo multiphoton imaging,” J. Appl. Phys. 102(3), 034701 (2007).
[CrossRef]

J. Biomed. Opt. (3)

M.-C. Chan, T.-M. Liu, S.-P. Tai, and C.-K. Sun, “Compact fiber-delivered Cr:forsterite laser for nonlinear light microscopy,” J. Biomed. Opt. 10(5), 054006 (2005).
[CrossRef] [PubMed]

J.-H. Lee, S.-Y. Chen, C.-H. Yu, S.-W. Chu, L.-F. Wang, C. K. Sun, and B. L. Chiang, “Noninvasive in vitro and in vivo assessment of epidermal hyperkeratosis and dermal fibrosis in atopic dermatitis,” J. Biomed. Opt. 14(1), 014008 (2009).
[CrossRef] [PubMed]

S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, “In vivo harmonic generation biopsy of human skin,” J. Biomed. Opt. 14(6), 060505 (2009).
[CrossRef]

J. Biophoton. (1)

C.-F. Chang, C.-H. Yu, and C.-K. Sun, “Multi-photon resonance enhancement of third harmonic generation in human oxyhemoglobin and deoxyhemoglobin,” to be published in J. Biophoton. (2010).
[CrossRef]

J. Neurophysiol. (1)

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, “In vivo multiphoton microscopy of deep brain tissue,” J. Neurophysiol. 91(4), 1908–1912 (2004).
[CrossRef]

Micron (1)

P.-C. Cheng, B.-L. Lin, F.-J. Kao, M. Gu, M.-G. Xu, X. Gan, M.-K. Huang, and Y.-S. Wang, “Multi-photon fluorescence microscopy--the response of plant cells to high intensity illumination,” Micron 32(7), 661–669 (2001).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

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]

Opt. Commun. (1)

J. Schutz, W. Hodel, and H. Weber, “Nonlinear pulse distortion at the zero dispersion wavelength of an optical fibre,” Opt. Commun. 95(4-6), 357–365 (1993).
[CrossRef]

Opt. Express (18)

D. Yelin and Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Express 5(8), 169–175 (1999).
[CrossRef] [PubMed]

Y. Wu, Y. Leng, J. Xi, and X. Li, “Scanning all-fiber-optic endomicroscopy system for 3D nonlinear optical imaging of biological tissues,” Opt. Express 17, 7901–7915 (2009).
[CrossRef]

H. Bao and M. Gu, “A 0.4-mm-diameter probe for nonlinear optical imaging,” Opt. Express 17(12), 10098–10104 (2009).
[CrossRef] [PubMed]

C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F. Helmchen, “Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo,” Opt. Express 16(8), 5556–5564 (2008).
[CrossRef] [PubMed]

C.-F. Chang, C.-Y. Chen, F.-H. Chang, S.-P. Tai, C.-Y. Chen, C.-H. Yu, Y.-B. Tseng, T.-H. Tsai, I.-S. Liu, W.-F. Su, and C.-K. Sun, “Cell tracking and detection of molecular expression in live cells using lipid-enclosed CdSe quantum dots as contrast agents for epi-third harmonic generation microscopy,” Opt. Express 16(13), 9534–9548 (2008).
[CrossRef] [PubMed]

C. L. Hoy, N. J. Durr, P. Chen, W. Piyawattanametha, H. Ra, O. Solgaard, and A. Ben-Yakar, “Miniaturized probe for femtosecond laser microsurgery and two-photon imaging,” Opt. Express 16(13), 9996–10005 (2008).
[CrossRef] [PubMed]

T.-M. Liu, M.-C. Chan, I.-H. Chen, S.-H. Chia, and C.-K. Sun, “Miniaturized multiphoton microscope with a 24Hz frame-rate,” Opt. Express 16(14), 10501–10506 (2008).
[CrossRef] [PubMed]

C.-S. Hsieh, S.-U. Chen, Y.-W. Lee, Y.-S. Yang, and C.-K. Sun, “Higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos,” Opt. Express 16(15), 11574–11588 (2008).
[PubMed]

R. Le Harzic, M. Weinigel, I. Riemann, K. König, and B. Messerschmidt, “Nonlinear optical endoscope based on a compact two axes piezo scanner and a miniature objective lens,” Opt. Express 16(25), 20588–20596 (2008).
[CrossRef] [PubMed]

S.-P. Tai, T.-H. Tsai, W.-J. Lee, D.-B. Shieh, Y.-H. Liao, H.-Y. Huang, K. Zhang, H.-L. Liu, and C.-K. Sun, “Optical biopsy of fixed human skin with backward-collected optical harmonics signals,” Opt. Express 13(20), 8231–8242 (2005).
[CrossRef] [PubMed]

T.-H. Tsai, S.-P. Tai, W.-J. Lee, H.-Y. Huang, Y.-H. Liao, and C.-K. Sun, “Optical signal degradation study in fixed human skin using confocal microscopy and higher-harmonic optical microscopy,” Opt. Express 14(2), 749–758 (2006).
[CrossRef] [PubMed]

L. Fu, A. Jain, H. Xie, C. Cranfield, and M. Gu, “Nonlinear optical endoscopy based on a double-clad photonic crystal fiber and a MEMS mirror,” Opt. Express 14(3), 1027–1032 (2006).
[CrossRef] [PubMed]

S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, “In vivo optical biopsy of hamster oral cavity with epi-third-harmonic-generation microscopy,” Opt. Express 14(13), 6178–6187 (2006).
[CrossRef] [PubMed]

H. J. Shin, M. C. Pierce, D. Lee, H. Ra, O. Solgaard, and R. Richards-Kortum, “Fiber-optic confocal microscope using a MEMS scanner and miniature objective lens,” Opt. Express 15(15), 9113–9122 (2007).
[CrossRef] [PubMed]

C. K. Sun, C. H. Yu, S. P. Tai, C. T. Kung, I. J. Wang, H. C. Yu, H. J. Huang, W. J. Lee, and Y. F. Chan, “In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy,” Opt. Express 15(18), 11167–11177 (2007).
[CrossRef] [PubMed]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009).
[CrossRef] [PubMed]

C.-F. Chang, H.-C. Chen, M.-J. Chen, W.-R. Liu, W.-F. Hsieh, C.-H. Hsu, C.-Y. Chen, F.-H. Chang, C.-H. Yu, and C.-K. Sun, “Direct backward third-harmonic generation in nanostructures,” Opt. Express 18(7), 7397–7406 (2010).
[CrossRef] [PubMed]

K. G. Jespersen, T. Le, L. Grüner-Nielsen, D. Jakobsen, M. E. V. Pederesen, M. B. Smedemand, S. R. Keiding, and B. Palsdottir, “A higher-order-mode fiber delivery for Ti:Sapphire femtosecond lasers,” Opt. Express 18(8), 7798–7806 (2010).
[CrossRef] [PubMed]

Opt. Lett. (10)

W. Piyawattanametha, E. D. Cocker, L. D. Burns, R. P. J. Barretto, J. C. Jung, H. Ra, O. Solgaard, and M. J. Schnitzer, “In vivo brain imaging using a portable 2.9 g two-photon microscope based on a microelectromechanical systems scanning mirror,” Opt. Lett. 34(15), 2309–2311 (2009).
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C.-H. Yu, S.-P. Tai, C.-T. Kung, W.-J. Lee, Y.-F. Chan, H.-L. Liu, J.-Y. Lyu, and C.-K. Sun, “Molecular third-harmonic-generation microscopy through resonance enhancement with absorbing dye,” Opt. Lett. 33(4), 387–389 (2008).
[CrossRef] [PubMed]

W. Piyawattanametha, R. P. J. Barretto, T. H. Ko, B. A. Flusberg, E. D. Cocker, H. Ra, D. Lee, O. Solgaard, and M. J. Schnitzer, “Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two- dimensional scanning mirror,” Opt. Lett. 31(13), 2018–2020 (2006).
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C.-K. Sun, C.-C. Chen, S.-W. Chu, T.-H. Tsai, Y.-C. Chen, and B.-L. Lin, “Multiharmonic-generation biopsy of skin,” Opt. Lett. 28(24), 2488–2490 (2003).
[CrossRef] [PubMed]

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Opt. Quantum Electron. (1)

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent cell damages in multi-photon confocal microscopy,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[CrossRef]

Proc. SPIE (1)

M.-R. Tsai, S.-Y. Chen, D.-B. Shieh, P.-J. Lou, and C.-K. Sun, “In vivo optical virtual biopsy of human oral cavity with harmonic generation microscopy,” Proc. SPIE (to be published).

Other (1)

http://www.imaginis.com/biopsy/biopsy_risks.asp

Supplementary Material (2)

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Figures (7)

Fig. 1
Fig. 1

The counts of sampling points per pixel in a frame trigger period.

Fig. 2
Fig. 2

The optical path differences of the designed result.

Fig. 3
Fig. 3

(a) The spectra and (b) the autocorrelation traces of the Cr:forsterite laser (the dotted lines) and the fiber-based femtosecond source (the solid lines).

Fig. 4
Fig. 4

The schematic diagram of the miniaturized THG fiber-microscope. DM: dichroic mirror; MMF: multi-mode fiber; Dot line: electronic line. The inset shows the detailed schematic layout of the designed microscope. A: LMA PCF collimator; B: MEMS mirror; C: relay lenses; D: dichroic mirror; E: broadband mirror; F: objective; G: MMF coupler.

Fig. 5
Fig. 5

(a) The averaged image of the 1μm fluorescent beads. Red: 2PF; White: THG. (b) Intensity distributive THG profiles of (a) along the center of the bead (blue), along the red line, where the THG pattern of the bead was just resolved (red), and along the green line, where the THG pattern could not be resolved (green).

Fig. 6
Fig. 6

In vivo horizontal-sectioned epi-THG and epi-SHG images of Asian forearn skin in different layers. In the epidermis, the morphology of (a) the stratum corneum, (b) the stratum spinosum, (c) the upper section and (d) the deeper section of the stratum basale (Media 1) could all be clearly distinguished using this epi-THG fiber-microscopy. Combined with the epi-SHG modality, the collagen fibers in the dermal papilla [arrowhead in (d)] were revealed; (e) and (f) the collagenous distribution in the dermis was also observed through epi-SHG. Yellow: THG; Green: SHG. Image size: 100μm×70μm. The integration time of each image except (b) is 0.33 second and 2 second in (b).

Fig. 7
Fig. 7

The five times slower movie showing the in vivo blood flow with a speed of ~300 µm/s (Media 2). Yellow: THG; Green: SHG. Image size: 100μm×70μm. Scale bar: 20 μm. Actual size of the recorded movie: 512×512 pixels

Equations (1)

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d m a x ~ π [ ( f x l x ) 2 + ( f y l y ) 2 ] 1 / 2 f s a m p l i n g ,

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