Abstract

Two-photon fluorescence and second harmonic generation microscopy have enabled functional and morphological in vivo imaging. However, in vivo applications of those techniques to living animals are limited by bulk optics on a bench top. Fortunately, growing functionality of fiber-optic devices and miniaturization of scanning mirrors stimulate the race to develop nonlinear optical endoscopy. In this paper, we report on a prototype of a nonlinear optical endoscope based on a double-clad photonic crystal fiber to improve the detection efficiency and a MEMS mirror to steer the light at the fiber tip. The miniaturized fiber-optic nonlinear microscope is characterized by rat esophagus imaging. Line profiles from the rat tail tendon and esophagus prove the potential of the technology in in vivo applications.

© 2006 Optical Society of America

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References

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Appl. Opt.

Conference on Lasers and Electro Optics

H. Xie, A. Jain, T. Xie, Y. Pan, and G. K. Fedder, “A single-crystal silicon-based micromirror with large scanning angle for biomedical applications,” Conference on Lasers and Electro Optics 2003, Baltimore, MD (2003).

IEEE journal of selected topics in Quantum electronics

A. Jain, A. Kopa, Y. Pan, G. K. Fedder and H. Xie, “A two-axis electrothermal micromirror for endoscopic optical coherence tomography,” IEEE journal of selected topics in Quantum electronics 10, 636-642 (2004).
[CrossRef]

J. Neurophysiology

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski and M. J. Schnitzer, “In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy,” J. Neurophysiology 92, 3121-3133 (2004).
[CrossRef]

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. Neurophysiology 91, 1908-1912 (2004).
[CrossRef]

Min. Invas. Ther. & Allied. Technol.

M. George, “Optical methods and sensors for in situ histology in surgery and endoscopy,” Min. Invas. Ther. & Allied. Technol. 13, 95-104 (2004).
[CrossRef]

Nat. Biotechnol.

W. R. Zipfel, R. M. Williams and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369-1377 (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]

Neuron

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]

Opt. Express

Opt. Lett.

Proc. SPIE

J. Y. Ye, M. T. Myaing , T. P. Thomas, I. Majoros, A. Koltyar, J. R. Baker, W. J. Wadsworth, G. Bouwmans, J. C. Knight, P. St. J. Russell, and T. B. Norris, “Development of a double-clad photonic crystal fiber based scanning microscope,” in Multiphoton Microscopy in the Biomedical Sciences V , A. Periasamy and P. T. C. So, eds., Proc. SPIE 5700, 23-27 (2005).
[CrossRef]

A. D. Aguirre, P. R. Herz, Y. Chen, J. G. Fujimoto, W. Piyawattanametha, L. Fan, S. Hsu, M. Fujino, M. C. Wu, and D. Kopf, “Ultrahigh resolution OCT imaging with a two-dimensional MEMS scanning endoscope,” in Advanced Biomedical and Clinical Diagnostic Systems III, T. Vo-Dinh, W. S. Grundfest , D. A. Benaron, and G. E. Cohn, eds., Proc. SPIE 5692, 277-282 (2005).
[CrossRef]

D. Kim, K. H. Kim, S. Yazdanfar and P. T. C. So, “Optical biopsy in high-speed handheld miniaturized multifocal multiphoton microscopy,” in Multiphoton Microscopy in the Biomedical Sciences V, A. Periasamy and P. T. C. So, eds., Proc. SPIE 5700, 14-22 (2005).
[CrossRef]

Sens. Actuators

H. Xie, Y. Pan, and G. K. Fedder, “Endoscopic optical coherence tomographic imaging with a CMOS-MEMS micromirror,” Sens. Actuators 103, 237-241 (2003).
[CrossRef]

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Figures (4)

Fig. 1.
Fig. 1.

(a) Schematic diagram of the nonlinear optical endoscope. The endoscope probe is based on a double-clad PCF, a MEMS mirror, and a GRIN lens. (b) A far-field output pattern from a double-clad PCF at wavelength 800 nm overlaid on a SEM image. (c) A SEM image of the MEMS mirror.

Fig. 2.
Fig. 2.

(a) Detected intensity and axial resolution of two-photon fluorescence as a function of gap length. (b). Quadratic dependence of TPEF and SHG intensity on the excitation power. The inset show the axial responses of TPEF and SHG at 800 nm. A GRIN lens used for imaging has a diameter of 0.5 mm and a NA of 0.5.

Fig. 3.
Fig. 3.

Z projection of 8 slices through the rat esophagus tissue stained with Acridine Orange. 3-D movie for rat eshophagus tissue imaging is shown as m1 in supporting online material. Two-photon fluorescence (red) and SHG (green) visualize cell nuclei and connective tissue, respectively. A GRIN lens used for imaging has a diameter of 0.5 mm and a NA of 0.5. The excitation power on the sample resulting in two-photon fluorescence and SHG signals is 10 mW and 25 mW, respectively. Slice spacing is 5 μm. Scale bar represents 20 μm. [Media 1]

Fig. 4.
Fig. 4.

(a) A series of SHG line profiles taken at a 10-μm step into rat tail tendon. (b) A SHG line profile from unstained rat esophagus tissue.

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