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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 4, Iss. 5 — May. 1, 2013
  • pp: 652–658
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Three-photon excited fluorescence imaging of unstained tissue using a GRIN lens endoscope

David M. Huland, Kriti Charan, Dimitre G. Ouzounov, Jason S. Jones, Nozomi Nishimura, and Chris Xu  »View Author Affiliations


Biomedical Optics Express, Vol. 4, Issue 5, pp. 652-658 (2013)
http://dx.doi.org/10.1364/BOE.4.000652


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Abstract

We present a compact and portable three-photon gradient index (GRIN) lens endoscope system suitable for imaging of unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The lateral and axial resolution in water is 1.0 μm and 9.5 μm, respectively. The ~200 μm diameter field of view is imaged at 2 frames/s using a fiber-based excitation source at 1040 nm. Ex vivo imaging is demonstrated with unstained mouse lung at 5.9 mW average power. These results demonstrate the feasibility of three-photon GRIN lens endoscopy for optical biopsy.

© 2013 OSA

1. Introduction

In vivo two-photon (2P) microscopy has become a valuable tool for the study of subsurface features in intact tissues and organs [1

1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]

]. To be clinically useful, endoscopic 2P approaches are required. A number of different endoscopes and techniques have been demonstrated in the past [2

2. L. Fu, A. Jain, C. Cranfield, H. Xie, and M. Gu, “Three-dimensional nonlinear optical endoscopy,” J. Biomed. Opt. 12(4), 040501 (2007). [CrossRef] [PubMed]

9

9. D. R. Rivera, C. M. Brown, D. G. Ouzounov, W. W. Webb, and C. Xu, “Multifocal multiphoton endoscope,” Opt. Lett. 37(8), 1349–1351 (2012). [CrossRef] [PubMed]

], including in vivo imaging of unstained tissues [10

10. C. M. Brown, D. R. Rivera, I. Pavlova, D. G. Ouzounov, W. O. Williams, S. Mohanan, W. W. Webb, and C. Xu, “In vivo imaging of unstained tissues using a compact and flexible multiphoton microendoscope,” J. Biomed. Opt. 17(4), 040505 (2012). [CrossRef] [PubMed]

,11

11. D. M. Huland, C. M. Brown, S. S. Howard, D. G. Ouzounov, I. Pavlova, K. Wang, D. R. Rivera, W. W. Webb, and C. Xu, “In vivo imaging of unstained tissues using long gradient index lens multiphoton endoscopic systems,” Biomed. Opt. Express 3(5), 1077–1085 (2012). [CrossRef] [PubMed]

].

Three-photon (3P) microscopy was first demonstrated in the 1990s [17

17. C. Xu, W. R. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996). [CrossRef] [PubMed]

19

19. D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, “Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser,” Bioimaging 4(3), 208–214 (1996). [CrossRef]

]. 3P excitation is an effective approach to extend the spectral range of the excitation source. For example, 3P intrinsic fluorescence microscopy has been performed with deep UV-excitable intrinsic fluorophores such as serotonin and melatonin [20

20. 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(12), 7075–7080 (2003). [CrossRef] [PubMed]

,21

21. S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275(5299), 530–532 (1997). [CrossRef] [PubMed]

]. Here we demonstrate a GRIN lens endoscope that is capable of imaging unstained mouse lung tissues using 3P excitation by a fiber laser at 1040 nm. To the best of our knowledge, this is the first demonstration of 3P imaging of unstained tissues through a compact and portable system with potential for endoscopic tissue diagnosis.

2. Endoscope design and characterization

To compensate for the fiber’s anomalous dispersion, the pulses were pre-chirped by using a long piece of SF11 glass (Schott). Second order interferometric autocorrelations were performed to optimize the dispersion compensation by measuring the pulse-width at the sample for different lengths of SF11 glass before the fiber. We found that 65 cm of SF11 glass produced the shortest pulse with an intensity autocorrelation full-width at half-maximum (FWHM) of 509 fs. The resulting autocorrelation traces are shown in Fig. 2
Fig. 2 Second order interferometric autocorrelation traces of the pulse. (a) Directly from the source, inset: the corresponding intensity autocorrelation with a pulse width of 524 fs, (b) at the sample (i.e., after dispersion compensation using 65 cm of SF11 glass, the hollow core fiber, the optical components, and the GRIN lens), inset: the corresponding intensity autocorrelation with a pulse width of 509 fs.
.

We imaged fluorescent beads (0.1 μm diameter, absorption peak 350 nm, emission peak 440 nm, Invitrogen) embedded in agarose gel and with water immersion to characterize the lateral and axial 3P resolution. The FWHM for the lateral and axial resolution is 1.0 μm and 9.5 μm, respectively (Fig. 3
Fig. 3 Three-photon lateral and axial resolution of the GRIN lens endoscope system. (a) Lateral and (b) axial intensity line profile across a subresolution fluorescent bead (blue diamonds). The Gaussian fits are indicated by the red lines. (c) Log-log plot of fluorescence signal as a function of excitation power at the sample. The slope is 2.9, indicating that the signal is generated by 3P excitation. Data in (c) was acquired using an ultrafast fiber laser at 1030 nm (Satsuma, Amplitude Systems, 5.7 MHz repetition rate).
). To confirm 3P excitation, fluorescence signal was measured at 5 different excitation powers while the laser beam was fixed on a bead. Figure 3(c) shows that the fluorescence signal generated closely follows a cubic dependence on the excitation power. To demonstrate the capability of our device for imaging intrinsic fluorescence, we imaged unstained mouse lung tissue ex vivo. A 3 month old female, wild type mouse (Jackson Labs) was euthanized and a lung lobe was removed, embedded in agarose gel and plated on a standard glass microscope slide. The tissue was imaged within 1 hour of euthanasia using 5.9 mW at the sample and at a frame rate of 2 frames/s (512 by 512 pixels). Representative images are shown in Figs. 4(a)
Fig. 4 Unaveraged image of ex vivo unstained mouse lung acquired at 2 frames/s. Green: 3P autofluorescence. Red: SHG. Scale bar is 20 μm. Images taken at (a) 20 μm, (b) 30 μm, and (c) 40 μm below the tissue surface. (d) Log-log plot of autofluorescence signal as a function of excitation power at the sample. The slope is 2.9, indicating that the signal is generated by 3P excitation.
-4(c). We can identify the surface of the lung with strong SHG signal coming presumably from the pleura (Fig. 4(a)). Below that, we can identify individual circular alveoli Figs. 4(b)-4(c), showing that the images could potentially provide diagnostic information. To confirm 3P excitation, fluorescence photons of the autofluorescence channel were measured at 5 different excitation powers at the sample by photon counting while scanning the laser beam at a fixed area in the tissue. Figure 4(d) shows that the fluorescence signal generated from the unstained tissue closely follows a cubic dependence on the excitation power, confirming that the image contrast is indeed generated by 3P excitation of intrinsic fluorescence.

3. Discussion

The main disadvantage of 3P endoscopy would be an increase in chromatic aberrations in the endoscope optics due to the larger difference between the excitation and signal wavelengths. The impact of this, however, can be reduced by carefully designing the optics for specific applications. Fiber delivery of the energetic femtosecond pulses for 3P excitation is another concern. The use of hollow core fibers, as shown in this paper, overcomes this difficulty. While hollow core fibers cannot effectively collect the fluorescence signal back through the excitation path, efficient signal collection through non-reciprocal optical path (e.g., using large core multimode fibers) has been demonstrated in the past [25

25. D. G. Ouzounov, D. R. Rivera, C. M. Brown, W. W. Webb, and C. Xu, “Dual modality microendoscope with optical zoom capability,” in CLEO 2012, San Jose, CA (Optical Society of America, 2012), postdeadline paper ATh5A.2.

,26

26. 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]

]. Thus, 3P excitation can be implemented in a flexible endoscope with a small rigid tip.

It should be noted that although we used a fiber laser that is capable of producing average power up to 1 W (i.e., 1 μJ pulses), less than 6 mW (i.e., 6 nJ pulses) was used at the sample in our experiments. Compact, fiber based femtosecond oscillators producing >40 nJ pulse energy are commercially available. These sources are adequate for 3P excitation of intrinsic fluorophores assuming a reasonable system throughput of ~25%. Furthermore, our frame rate (2 frames per second) was limited by the low repetition rate of the laser (1MHz). Oscillators providing higher repetition rates (e.g., 3 MHz) and shorter pulses (e.g., 150 fs) can significantly increase the imaging acquisition rate without increasing the average excitation power, which will be valuable for overcoming motion artifacts for in vivo applications. Alternatively, the pixel clock of the image acquisition system could be synchronized to the laser pulses to maximize the frame rate [27

27. P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 µm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett. 28(12), 1022–1024 (2003). [CrossRef] [PubMed]

], For example, 3 MHz repetition rate can provide a maximum frame rate of ~12 frames/s at 512 pixels by 512 pixels per frame, which is adequate to overcome motion artifacts in in vivo imaging. Such a frame rate was shown to be adequate for in vivo imaging of Fluorescein stained human bladders using a confocal laser endoscope [28

28. G. A. Sonn, S. N. Jones, T. V. Tarin, C. B. Du, K. E. Mach, K. C. Jensen, and J. C. Liao, “Optical biopsy of human bladder neoplasia with in vivo confocal laser endomicroscopy,” J. Urol. 182(4), 1299–1305 (2009). [CrossRef] [PubMed]

].

4. Conclusion

We have demonstrated the feasibility of 3P intrinsic fluorescence endoscopy using a GRIN lens endoscope and a fiber-based excitation source at 1040 nm. The compact and portable device can acquire 3P intrinsic fluorescence and SHG images at a rate of 2 frames/s with a field-of-view of ~200 μm diameter with subcellular resolution. The presented ex vivo results of unstained mouse lung tissue show great promise for using 3P GRIN lens endoscopy for optical biopsy. The combination of longer wavelength and 3P excitation, together with the convenient fiber-based excitation source, may make 3P endoscopy a valuable alternative to the conventional 2P approach.

Acknowledgments

This research was made possible by National Institutes of Health/National Cancer Institute Grant Number R01-CA133148 and National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering Grant Numbers R01EB014873 and R01-EB006736. We thank members of the Xu and Schaffer-Nishimura research groups for discussions and technical suggestions.

References and links

1.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]

2.

L. Fu, A. Jain, C. Cranfield, H. Xie, and M. Gu, “Three-dimensional nonlinear optical endoscopy,” J. Biomed. Opt. 12(4), 040501 (2007). [CrossRef] [PubMed]

3.

M. T. Myaing, D. J. MacDonald, and X. Li, “Fiber-optic scanning two-photon fluorescence endoscope,” Opt. Lett. 31(8), 1076–1078 (2006). [CrossRef] [PubMed]

4.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, “Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(43), 17598–17603 (2011). [CrossRef] [PubMed]

5.

S. Tang, W. Jung, D. McCormick, T. Xie, J. Su, Y.-C. Ahn, B. J. Tromberg, and Z. Chen, “Design and implementation of fiber-based multiphoton endoscopy with microelectromechanical systems scanning,” J. Biomed. Opt. 14(3), 034005 (2009). [CrossRef] [PubMed]

6.

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(10), 7907–7915 (2009). [CrossRef] [PubMed]

7.

E. J. Seibel and Q. Y. J. Smithwick, “Unique features of optical scanning, single fiber endoscopy,” Lasers Surg. Med. 30(3), 177–183 (2002). [CrossRef] [PubMed]

8.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, W. W. Webb, and C. Xu, “Use of a lensed fiber for a large-field-of-view, high-resolution, fiber-scanning microendoscope,” Opt. Lett. 37(5), 881–883 (2012). [CrossRef] [PubMed]

9.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, W. W. Webb, and C. Xu, “Multifocal multiphoton endoscope,” Opt. Lett. 37(8), 1349–1351 (2012). [CrossRef] [PubMed]

10.

C. M. Brown, D. R. Rivera, I. Pavlova, D. G. Ouzounov, W. O. Williams, S. Mohanan, W. W. Webb, and C. Xu, “In vivo imaging of unstained tissues using a compact and flexible multiphoton microendoscope,” J. Biomed. Opt. 17(4), 040505 (2012). [CrossRef] [PubMed]

11.

D. M. Huland, C. M. Brown, S. S. Howard, D. G. Ouzounov, I. Pavlova, K. Wang, D. R. Rivera, W. W. Webb, and C. Xu, “In vivo imaging of unstained tissues using long gradient index lens multiphoton endoscopic systems,” Biomed. Opt. Express 3(5), 1077–1085 (2012). [CrossRef] [PubMed]

12.

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]

13.

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011). [CrossRef] [PubMed]

14.

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent damage in biological multi-photon confocal microscopy: A micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser,” Opt. Quantum Electron. 34(12), 1251–1266 (2002). [CrossRef]

15.

Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, “Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy,” Opt. Express 14(9), 3942–3951 (2006). [CrossRef] [PubMed]

16.

G. Liu, K. Kieu, F. W. Wise, and Z. Chen, “Multiphoton microscopy system with a compact fiber-based femtosecond-pulse laser and handheld probe,” J. Biophotonics 4(1-2), 34–39 (2011). [CrossRef] [PubMed]

17.

C. Xu, W. R. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996). [CrossRef] [PubMed]

18.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. M. Malak, I. Gryczynski, and J. R. Lakowicz, “Three-photon excitation in fluorescence microscopy,” J. Biomed. Opt. 1(1), 71–74 (1996). [CrossRef] [PubMed]

19.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, “Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser,” Bioimaging 4(3), 208–214 (1996). [CrossRef]

20.

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(12), 7075–7080 (2003). [CrossRef] [PubMed]

21.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275(5299), 530–532 (1997). [CrossRef] [PubMed]

22.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013). [CrossRef]

23.

J. G. Fujimoto, C. Pitris, S. A. Boppart, and M. E. Brezinski, “Optical coherence tomography: an emerging technology for biomedical imaging and optical biopsy,” Neoplasia 2(1-2), 9–25 (2000). [CrossRef] [PubMed]

24.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17(8), 763–767 (1999). [CrossRef] [PubMed]

25.

D. G. Ouzounov, D. R. Rivera, C. M. Brown, W. W. Webb, and C. Xu, “Dual modality microendoscope with optical zoom capability,” in CLEO 2012, San Jose, CA (Optical Society of America, 2012), postdeadline paper ATh5A.2.

26.

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]

27.

P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 µm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett. 28(12), 1022–1024 (2003). [CrossRef] [PubMed]

28.

G. A. Sonn, S. N. Jones, T. V. Tarin, C. B. Du, K. E. Mach, K. C. Jensen, and J. C. Liao, “Optical biopsy of human bladder neoplasia with in vivo confocal laser endomicroscopy,” J. Urol. 182(4), 1299–1305 (2009). [CrossRef] [PubMed]

OCIS Codes
(110.2760) Imaging systems : Gradient-index lenses
(170.2150) Medical optics and biotechnology : Endoscopic imaging
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Endoscopes, Catheters and Micro-Optics

History
Original Manuscript: January 15, 2013
Revised Manuscript: March 7, 2013
Manuscript Accepted: March 15, 2013
Published: April 1, 2013

Citation
David M. Huland, Kriti Charan, Dimitre G. Ouzounov, Jason S. Jones, Nozomi Nishimura, and Chris Xu, "Three-photon excited fluorescence imaging of unstained tissue using a GRIN lens endoscope," Biomed. Opt. Express 4, 652-658 (2013)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-4-5-652


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References

  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990). [CrossRef] [PubMed]
  2. L. Fu, A. Jain, C. Cranfield, H. Xie, and M. Gu, “Three-dimensional nonlinear optical endoscopy,” J. Biomed. Opt.12(4), 040501 (2007). [CrossRef] [PubMed]
  3. M. T. Myaing, D. J. MacDonald, and X. Li, “Fiber-optic scanning two-photon fluorescence endoscope,” Opt. Lett.31(8), 1076–1078 (2006). [CrossRef] [PubMed]
  4. D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, “Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue,” Proc. Natl. Acad. Sci. U.S.A.108(43), 17598–17603 (2011). [CrossRef] [PubMed]
  5. S. Tang, W. Jung, D. McCormick, T. Xie, J. Su, Y.-C. Ahn, B. J. Tromberg, and Z. Chen, “Design and implementation of fiber-based multiphoton endoscopy with microelectromechanical systems scanning,” J. Biomed. Opt.14(3), 034005 (2009). [CrossRef] [PubMed]
  6. Y. Wu, Y. Leng, J. Xi, and X. Li, “Scanning all-fiber-optic endomicroscopy system for 3D nonlinear optical imaging of biological tissues,” Opt. Express17(10), 7907–7915 (2009). [CrossRef] [PubMed]
  7. E. J. Seibel and Q. Y. J. Smithwick, “Unique features of optical scanning, single fiber endoscopy,” Lasers Surg. Med.30(3), 177–183 (2002). [CrossRef] [PubMed]
  8. D. R. Rivera, C. M. Brown, D. G. Ouzounov, W. W. Webb, and C. Xu, “Use of a lensed fiber for a large-field-of-view, high-resolution, fiber-scanning microendoscope,” Opt. Lett.37(5), 881–883 (2012). [CrossRef] [PubMed]
  9. D. R. Rivera, C. M. Brown, D. G. Ouzounov, W. W. Webb, and C. Xu, “Multifocal multiphoton endoscope,” Opt. Lett.37(8), 1349–1351 (2012). [CrossRef] [PubMed]
  10. C. M. Brown, D. R. Rivera, I. Pavlova, D. G. Ouzounov, W. O. Williams, S. Mohanan, W. W. Webb, and C. Xu, “In vivo imaging of unstained tissues using a compact and flexible multiphoton microendoscope,” J. Biomed. Opt.17(4), 040505 (2012). [CrossRef] [PubMed]
  11. D. M. Huland, C. M. Brown, S. S. Howard, D. G. Ouzounov, I. Pavlova, K. Wang, D. R. Rivera, W. W. Webb, and C. Xu, “In vivo imaging of unstained tissues using long gradient index lens multiphoton endoscopic systems,” Biomed. Opt. Express3(5), 1077–1085 (2012). [CrossRef] [PubMed]
  12. 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. Express17(16), 13354–13364 (2009). [CrossRef] [PubMed]
  13. D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt.16(10), 106014 (2011). [CrossRef] [PubMed]
  14. I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent damage in biological multi-photon confocal microscopy: A micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser,” Opt. Quantum Electron.34(12), 1251–1266 (2002). [CrossRef]
  15. Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, “Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy,” Opt. Express14(9), 3942–3951 (2006). [CrossRef] [PubMed]
  16. G. Liu, K. Kieu, F. W. Wise, and Z. Chen, “Multiphoton microscopy system with a compact fiber-based femtosecond-pulse laser and handheld probe,” J. Biophotonics4(1-2), 34–39 (2011). [CrossRef] [PubMed]
  17. C. Xu, W. R. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A.93(20), 10763–10768 (1996). [CrossRef] [PubMed]
  18. S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. M. Malak, I. Gryczynski, and J. R. Lakowicz, “Three-photon excitation in fluorescence microscopy,” J. Biomed. Opt.1(1), 71–74 (1996). [CrossRef] [PubMed]
  19. D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, “Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser,” Bioimaging4(3), 208–214 (1996). [CrossRef]
  20. 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(12), 7075–7080 (2003). [CrossRef] [PubMed]
  21. S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science275(5299), 530–532 (1997). [CrossRef] [PubMed]
  22. N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics7(3), 205–209 (2013). [CrossRef]
  23. J. G. Fujimoto, C. Pitris, S. A. Boppart, and M. E. Brezinski, “Optical coherence tomography: an emerging technology for biomedical imaging and optical biopsy,” Neoplasia2(1-2), 9–25 (2000). [CrossRef] [PubMed]
  24. J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol.17(8), 763–767 (1999). [CrossRef] [PubMed]
  25. D. G. Ouzounov, D. R. Rivera, C. M. Brown, W. W. Webb, and C. Xu, “Dual modality microendoscope with optical zoom capability,” in CLEO 2012, San Jose, CA (Optical Society of America, 2012), postdeadline paper ATh5A.2.
  26. 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. Express16(8), 5556–5564 (2008). [CrossRef] [PubMed]
  27. P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 µm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett.28(12), 1022–1024 (2003). [CrossRef] [PubMed]
  28. G. A. Sonn, S. N. Jones, T. V. Tarin, C. B. Du, K. E. Mach, K. C. Jensen, and J. C. Liao, “Optical biopsy of human bladder neoplasia with in vivo confocal laser endomicroscopy,” J. Urol.182(4), 1299–1305 (2009). [CrossRef] [PubMed]

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