Abstract

An extremely compact all-fiber-optic scanning endomicroscopy system was developed for two-photon fluorescence (TPF) and second harmonic generation (SHG) imaging of biological samples. A conventional double-clad fiber (DCF) was employed in the endomicroscope for single-mode femtosecond pulse delivery, multimode nonlinear optical signals collection and fast two-dimensional scanning. A single photonic bandgap fiber (PBF) with negative group velocity dispersion at two-photon excitation wavelength (i.e. ~810 nm) was used for pulse prechirping in replacement of a bulky grating/lens -based pulse stretcher. The combined use of DCF and PBF in the endomicroscopy system made the endomicroscope basically a plug-and-play unit. The excellent imaging ability of the extremely compact all-fiber-optic nonlinear optical endomicroscopy system was demonstrated by SHG imaging of rat tail tendon and depth-resolved TPF imaging of epithelial tissues stained with acridine orange. The preliminary results suggested the promising potential of this extremely compact all-fiber-optic endomicroscopy system for realtime assessment of both epithelial and stromal structures in luminal organs.

© 2009 Optical Society of America

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2007 (1)

L. Fu, A. Jain, C. Cranfield, H. K. Xie, and M. Gu, "Three-dimensional nonlinear optical endoscopy," J. Biomed. Opt. 12, 040501 (2007).
[CrossRef] [PubMed]

2006 (1)

2005 (3)

2004 (1)

2003 (6)

M. T. Myaing, J. Y. Ye, T. B. Norris, T. Thomas, J. R. Baker, W. J. Wadsworth, G. Bouwmans, J. C. Knight, and P. S. J. Russell, "Enhanced two-photon biosensing with double-clad photonic crystal fibers," Opt. Lett. 28, 1224-1226 (2003).
[CrossRef] [PubMed]

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. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotechnol. 21, 1356-1360 (2003).
[CrossRef] [PubMed]

P. Russell, "Photonic crystal fibers," Science 299, 358-362 (2003).
[CrossRef] [PubMed]

J. C. Jung and M. J. Schnitzer, "Multiphoton endoscopy," Opt. Lett. 28, 902-904 (2003).
[CrossRef] [PubMed]

A. L. Clark, A. M. Gillenwater, T. G. Collier, R. Alizadeh-Naderi, A. K. El-Naggar, and R. R. Richards-Kortum, "Confocal microscopy for real-time detection of oral cavity neoplasia," Clin. Cancer Res. 9, 4714-4721 (2003).
[PubMed]

2002 (3)

T. R. Neu, U. Kuhlicke, and J. R. Lawrence, "Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms," Appl. Environ. Microbiol. 68, 901-909 (2002).
[CrossRef] [PubMed]

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. U.S.A. 99, 11014-11019 (2002).
[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, 493-508 (2002).
[CrossRef]

2001 (1)

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, "A miniature head-mounted two-photon microscope: High-resolution brain imaging in freely moving animals," Neuron 31, 903-912 (2001).
[CrossRef] [PubMed]

2000 (1)

B. M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, and L. B. Da Silva, "Collagen structure and nonlinear susceptibility: Effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity," Laser Surg. Med. 27, 329-335 (2000).
[CrossRef]

1993 (1)

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

1986 (1)

D. E. Birk and R. L. Trelstad, "Extracellular compartments in tendon morphogenesis - collagen fibril, bundle, and macroaggregate formation," J. Cell Biol. 103, 231-240 (1986).
[CrossRef] [PubMed]

1974 (1)

L. R. Adams, "Acridine-orange staining of epithelial-cells in strong salt solution," J. Histochem. Cytochem 22, 492-494 (1974).
[CrossRef] [PubMed]

1969 (1)

E. B. Treacy, "Optical pulse compression with diffraction gratings," IEEE J. Quantum Electron. 5, 454-458 (1969).
[CrossRef]

1965 (1)

D. H. Elliott, "Structure and function of mammalian tendon," Biol. Rev. Camb. Philos. Soc. 40, 392-421 (1965).
[CrossRef] [PubMed]

Adams, L. R.

L. R. Adams, "Acridine-orange staining of epithelial-cells in strong salt solution," J. Histochem. Cytochem 22, 492-494 (1974).
[CrossRef] [PubMed]

Alizadeh-Naderi, R.

A. L. Clark, A. M. Gillenwater, T. G. Collier, R. Alizadeh-Naderi, A. K. El-Naggar, and R. R. Richards-Kortum, "Confocal microscopy for real-time detection of oral cavity neoplasia," Clin. Cancer Res. 9, 4714-4721 (2003).
[PubMed]

Anderson, E. P.

Baker, J. R.

Birk, D. E.

D. E. Birk and R. L. Trelstad, "Extracellular compartments in tendon morphogenesis - collagen fibril, bundle, and macroaggregate formation," J. Cell Biol. 103, 231-240 (1986).
[CrossRef] [PubMed]

Bouwmans, G.

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, 1356-1360 (2003).
[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, 493-508 (2002).
[CrossRef]

Chang, R. L.

Chen, Y. C.

Christie, R.

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. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Clark, A. L.

A. L. Clark, A. M. Gillenwater, T. G. Collier, R. Alizadeh-Naderi, A. K. El-Naggar, and R. R. Richards-Kortum, "Confocal microscopy for real-time detection of oral cavity neoplasia," Clin. Cancer Res. 9, 4714-4721 (2003).
[PubMed]

Cobb, M. J.

Cocker, E. D.

Collier, T. G.

A. L. Clark, A. M. Gillenwater, T. G. Collier, R. Alizadeh-Naderi, A. K. El-Naggar, and R. R. Richards-Kortum, "Confocal microscopy for real-time detection of oral cavity neoplasia," Clin. Cancer Res. 9, 4714-4721 (2003).
[PubMed]

Cranfield, C.

L. Fu, A. Jain, C. Cranfield, H. K. Xie, and M. Gu, "Three-dimensional nonlinear optical endoscopy," J. Biomed. Opt. 12, 040501 (2007).
[CrossRef] [PubMed]

Da Silva, L. B.

B. M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, and L. B. Da Silva, "Collagen structure and nonlinear susceptibility: Effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity," Laser Surg. Med. 27, 329-335 (2000).
[CrossRef]

Denk, W.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, "A miniature head-mounted two-photon microscope: High-resolution brain imaging in freely moving animals," Neuron 31, 903-912 (2001).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Eichler, J.

B. M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, and L. B. Da Silva, "Collagen structure and nonlinear susceptibility: Effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity," Laser Surg. Med. 27, 329-335 (2000).
[CrossRef]

Elliott, D. H.

D. H. Elliott, "Structure and function of mammalian tendon," Biol. Rev. Camb. Philos. Soc. 40, 392-421 (1965).
[CrossRef] [PubMed]

El-Naggar, A. K.

A. L. Clark, A. M. Gillenwater, T. G. Collier, R. Alizadeh-Naderi, A. K. El-Naggar, and R. R. Richards-Kortum, "Confocal microscopy for real-time detection of oral cavity neoplasia," Clin. Cancer Res. 9, 4714-4721 (2003).
[PubMed]

Fee, M. S.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, "A miniature head-mounted two-photon microscope: High-resolution brain imaging in freely moving animals," Neuron 31, 903-912 (2001).
[CrossRef] [PubMed]

Flusberg, B. A.

Fu, L.

L. Fu, A. Jain, C. Cranfield, H. K. Xie, and M. Gu, "Three-dimensional nonlinear optical endoscopy," J. Biomed. Opt. 12, 040501 (2007).
[CrossRef] [PubMed]

L. Fu, X. S. Gan, and M. Gu, "Nonlinear optical microscopy based on double-clad photonic crystal fibers," Opt. Express 13, 5528-5534 (2005).
[CrossRef] [PubMed]

Gan, X. S.

Gillenwater, A. M.

A. L. Clark, A. M. Gillenwater, T. G. Collier, R. Alizadeh-Naderi, A. K. El-Naggar, and R. R. Richards-Kortum, "Confocal microscopy for real-time detection of oral cavity neoplasia," Clin. Cancer Res. 9, 4714-4721 (2003).
[PubMed]

Gu, M.

L. Fu, A. Jain, C. Cranfield, H. K. Xie, and M. Gu, "Three-dimensional nonlinear optical endoscopy," J. Biomed. Opt. 12, 040501 (2007).
[CrossRef] [PubMed]

L. Fu, X. S. Gan, and M. Gu, "Nonlinear optical microscopy based on double-clad photonic crystal fibers," Opt. Express 13, 5528-5534 (2005).
[CrossRef] [PubMed]

Helmchen, F.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, "A miniature head-mounted two-photon microscope: High-resolution brain imaging in freely moving animals," Neuron 31, 903-912 (2001).
[CrossRef] [PubMed]

Hoppe, P. E.

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, 493-508 (2002).
[CrossRef]

Hyman, B. T.

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. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Jain, A.

L. Fu, A. Jain, C. Cranfield, H. K. Xie, and M. Gu, "Three-dimensional nonlinear optical endoscopy," J. Biomed. Opt. 12, 040501 (2007).
[CrossRef] [PubMed]

Jung, J. C.

Kim, B. M.

B. M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, and L. B. Da Silva, "Collagen structure and nonlinear susceptibility: Effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity," Laser Surg. Med. 27, 329-335 (2000).
[CrossRef]

Kimmey, M. B.

Knight, J. C.

Kuhlicke, U.

T. R. Neu, U. Kuhlicke, and J. R. Lawrence, "Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms," Appl. Environ. Microbiol. 68, 901-909 (2002).
[CrossRef] [PubMed]

Lawrence, J. R.

T. R. Neu, U. Kuhlicke, and J. R. Lawrence, "Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms," Appl. Environ. Microbiol. 68, 901-909 (2002).
[CrossRef] [PubMed]

Li, X. D.

Liu, X. M.

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, 1356-1360 (2003).
[CrossRef] [PubMed]

Lung, J. C.

MacDonald, D. J.

Malone, C. J.

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, 493-508 (2002).
[CrossRef]

Millard, A. C.

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, 493-508 (2002).
[CrossRef]

Mohler, W. A.

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, 493-508 (2002).
[CrossRef]

Myaing, M. T.

Neu, T. R.

T. R. Neu, U. Kuhlicke, and J. R. Lawrence, "Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms," Appl. Environ. Microbiol. 68, 901-909 (2002).
[CrossRef] [PubMed]

Nikitin, A. Y.

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. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Norris, T. B.

Reiser, K. M.

B. M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, and L. B. Da Silva, "Collagen structure and nonlinear susceptibility: Effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity," Laser Surg. Med. 27, 329-335 (2000).
[CrossRef]

Richards-Kortum, R. R.

A. L. Clark, A. M. Gillenwater, T. G. Collier, R. Alizadeh-Naderi, A. K. El-Naggar, and R. R. Richards-Kortum, "Confocal microscopy for real-time detection of oral cavity neoplasia," Clin. Cancer Res. 9, 4714-4721 (2003).
[PubMed]

Rubenchik, A. M.

B. M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, and L. B. Da Silva, "Collagen structure and nonlinear susceptibility: Effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity," Laser Surg. Med. 27, 329-335 (2000).
[CrossRef]

Russell, P.

P. Russell, "Photonic crystal fibers," Science 299, 358-362 (2003).
[CrossRef] [PubMed]

Russell, P. S. J.

Schnitzer, M. J.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Tank, D. W.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, "A miniature head-mounted two-photon microscope: High-resolution brain imaging in freely moving animals," Neuron 31, 903-912 (2001).
[CrossRef] [PubMed]

Terasaki, M.

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, 493-508 (2002).
[CrossRef]

Thomas, T.

Treacy, E. B.

E. B. Treacy, "Optical pulse compression with diffraction gratings," IEEE J. Quantum Electron. 5, 454-458 (1969).
[CrossRef]

Trelstad, R. L.

D. E. Birk and R. L. Trelstad, "Extracellular compartments in tendon morphogenesis - collagen fibril, bundle, and macroaggregate formation," J. Cell Biol. 103, 231-240 (1986).
[CrossRef] [PubMed]

Tromberg, B. J.

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. U.S.A. 99, 11014-11019 (2002).
[CrossRef] [PubMed]

Wadsworth, W. J.

Wang, J. P.

Webb, W. W.

R. M. Williams, W. R. Zipfel, and W. W. Webb, "Interpreting second-harmonic generation images of collagen I fibrils," Biophys. J. 88, 1377-1386 (2005).
[CrossRef]

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. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Williams, R. M.

R. M. Williams, W. R. Zipfel, and W. W. Webb, "Interpreting second-harmonic generation images of collagen I fibrils," Biophys. J. 88, 1377-1386 (2005).
[CrossRef]

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. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Xie, H. K.

L. Fu, A. Jain, C. Cranfield, H. K. Xie, and M. Gu, "Three-dimensional nonlinear optical endoscopy," J. Biomed. Opt. 12, 040501 (2007).
[CrossRef] [PubMed]

Ye, J. Y.

Yeh, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. U.S.A. 99, 11014-11019 (2002).
[CrossRef] [PubMed]

Zipfel, W. R.

R. M. Williams, W. R. Zipfel, and W. W. Webb, "Interpreting second-harmonic generation images of collagen I fibrils," Biophys. J. 88, 1377-1386 (2005).
[CrossRef]

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. U.S.A. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Zoumi, A.

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Supplementary Material (2)

» Media 1: MOV (3183 KB)     
» Media 2: MOV (2712 KB)     

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Figures (7)

Fig. 1.
Fig. 1.

Schematic (a) and photo (b) of the distal end of the fiber-optic scanning nonlinear optical endomicroscope probe. The piezoelectric transducer tube (PZT), fiber-optic scanner, and GRIN lens were encased in a hypodermic tube with an overall outer diameter of 2.4 mm.

Fig. 2.
Fig. 2.

(a). Schematic of the all-fiber-optic scanning nonlinear optical endomicroscope imaging system. The combination of a DCF and PBF made the system all-fiber-optic, in which the DCF was used for both nonlinear optical excitation and TPF/SHG collection, whereas the PBF was employed for dispersion management. FL: Fiber launcher; PBF: Photonic bandgap fiber; M: Mirror; DM: Dichroic mirror; DCF: Double-clad fiber; L: Focusing lens; F: Short-pass filter and/or band-pass filter; PMT: Photomultiplier tube; DAQ: Data acquisition. (b) Photo of the all-fiber-optic scanning nonlinear optical endomicroscope imaging system inside a box with the flexible endomicroscope probe extended outside the box and placed on the cover. The endomicroscope probe can be easily plugged into the system and becomes freely accessible.

Fig. 3.
Fig. 3.

Second-order intensity autocorrelation curves of laser pulses existing from the endomicroscope with different powers delivered through the core of the DCF. A pulse width of less than 200 fs could be achieved even with a power of 70 mW in the DCF core.

Fig. 4.
Fig. 4.

Fluorescence intensity profiles (dots) across the center of a 0.1-μm fluorescent bead along (a) the lateral and (b) axial dimension. Black traces are Gaussian-fitted curves. The measured lateral and axial resolution given by the FWHM in (a) and (b) are ~1.6 μm and 11.4 μm, respectively.

Fig. 5.
Fig. 5.

Representative intrinsic SHG images of rat tail tendon: (a) real-time image (3.3 frames/second) and (b) 10 frames averaged image. Media 1. (c) animation of layered intrinsic SHG images as a function of depth. The SHG signals are solely attributed to type I collagen.

Fig. 6.
Fig. 6.

Exogenous TPF images of pig cornea tissue stained with acridine orange. (a) corneal limbus and (b) corneal stroma. The densely packed epithelial cells in the limbus and sparsely distributed keratocytes in the stroma can be clearly identified from the images.

Fig. 7.
Fig. 7.

Typical depth-resolved TPF images of rat oral tissue stained with acridine orange: (a) at the depth of 10 μm; (b) at the depth of 50 μm; and (c) movie of stacked 3D images. Media 2. The depth-resolved TPF images reveal that the nucleus density increases from the superficial layer to the basal layer.

Tables (1)

Tables Icon

Table 1. Measured GVD parameter (β2) and dispersion parameter (D) of DCF and PBF.

Metrics