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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 3, Iss. 5 — May. 1, 2012
  • pp: 1077–1085
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In vivo imaging of unstained tissues using long gradient index lens multiphoton endoscopic systems

David M. Huland, Christopher M. Brown, Scott S. Howard, Dimitre G. Ouzounov, Ina Pavlova, Ke Wang, David R. Rivera, Watt W. Webb, and Chris Xu  »View Author Affiliations


Biomedical Optics Express, Vol. 3, Issue 5, pp. 1077-1085 (2012)
http://dx.doi.org/10.1364/BOE.3.001077


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Abstract

We characterize long (up to 285 mm) gradient index (GRIN) lens endoscope systems for multiphoton imaging. We fabricate a portable, rigid endoscope system suitable for imaging unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The portable device is capable of imaging a ~200 µm diameter field of view at 4 frames/s. The lateral and axial resolution in water is 0.85 µm and 7.4 µm respectively. In vivo images of unstained tissues in live, anesthetized rats using the portable device are presented. These results show great promise for GRIN endoscopy to be used clinically.

© 2012 OSA

1. Introduction

Two-photon fluorescence (TPF) and second-harmonic generation (SHG) microscopy are powerful tools for imaging unstained biological tissues [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]

]. These label-free techniques are capable of producing high-resolution real-time in vivo images and have shown great promise for medical diagnostics of various diseases, potentially replacing surgical biopsies [2

2. S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, “Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging,” Opt. Lett. 31(18), 2756–2758 (2006). [CrossRef] [PubMed]

7

7. P. Wilder-Smith, K. Osann, N. Hanna, N. El Abbadi, M. Brenner, D. Messadi, and T. Krasieva, “In vivo multiphoton fluorescence imaging: a novel approach to oral malignancy,” Lasers Surg. Med. 35(2), 96–103 (2004). [CrossRef] [PubMed]

]. The maximum imaging depth, however, is limited in most tissues to ~1 mm [8

8. 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]

,9

9. 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]

].

One strategy to overcome the depth limitation is to develop miniaturized TPF microscopes that could be used as endoscopes in a clinical setting. A number of different endoscopes and techniques have been demonstrated [10

10. 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]

17

17. 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]

], including in vivo imaging of unstained tissues [18

18. 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]

]. These devices are typically composed of a miniaturized scanning mechanism and focusing optics in a protective housing. The scanners proposed generally use either a miniaturized fiber scanning mechanism or microelectromechanical systems scanning mirror. The need to encapsulate a scanning mechanism into a housing of suitable size for minimally invasive procedures poses several challenges including: (1) uniformity of scan, (2) sensitivity, durability and reliability of the scanner, and (3) miniaturization of the distal scan mechanism and optics. A different approach has been to use gradient index (GRIN) lenses to relay the excitation light and TPF/SHG emission to and from an external microscope deep into soft tissue [19

19. B. A. Flusberg, J. C. Jung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, “In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope,” Opt. Lett. 30(17), 2272–2274 (2005). [CrossRef] [PubMed]

22

22. J. C. Jung and M. J. Schnitzer, “Multiphoton endoscopy,” Opt. Lett. 28(11), 902–904 (2003). [CrossRef] [PubMed]

]. Since only the GRIN lens penetrates the tissue, the excitation, scanning, and collection optics need not be miniaturized for in vivo imaging. GRIN lenses have been shown to be biocompatible and previously used in a clinical setting for non-penetrative imaging of chronic leg ulcers with delayed wound healing [23

23. K. König, A. Ehlers, I. Riemann, S. Schenkl, R. Bückle, and M. Kaatz, “Clinical two-photon microendoscopy,” Microsc. Res. Tech. 70(5), 398–402 (2007). [CrossRef] [PubMed]

]. Use of a small diameter lens to penetrate deeply within tissue has been demonstrated with a hypodermic needle GRIN system [24

24. R. S. Pillai, D. Lorenser, and D. D. Sampson, “Deep-tissue access with confocal fluorescence microendoscopy through hypodermic needles,” Opt. Express 19(8), 7213–7221 (2011). [CrossRef] [PubMed]

]. These studies show great promise for GRIN endoscopy to be used as either a guide for or a replacement of traditional surgical biopsies.

2. GRIN endoscopes

3. Experimental setup

Figure 1
Fig. 1 Experimental setup used for the optical characterization of the long gradient index endoscope systems and close-up of the doublet GRIN system design (shown here using a 0.75 relay lens pitch).
shows the custom built horizontal multiphoton microscope used to characterize the GRIN lens systems. A mode-locked Ti:sapphire laser (Tsunami, Spectra Physics, Inc.) was used as the excitation source at 800 nm with 10 nm bandwidth. Dual axis (5 mm diameter) galvo based scan mirrors (GVSM002, Thorlabs Inc.), and two scan lenses of 10 cm and 30 cm focal length (respectively, AC508-100-B-ML and AC508-300-B-ML, Thorlabs Inc.) were used to scan the beam angle at the overfilled back aperture of a 0.1 NA microscope objective. The focus of the objective was raster scanned by the galvo mirrors across the proximal face of the GRIN lens systems. The GRIN lens systems were mounted on a three axis manual linear translational stage close to the focal plane of the objective to aid alignment. All optical characterizations were conducted by moving the sample mounted on a 3D stage (MP-285, Sutter Instruments) allowing axial scanning of the sample while maintaining the GRIN lens system in a fixed position. The fluorescent signal from the sample was epi-collected through the GRIN lenses and the microscope objective. Collected light is reflected by a dichroic beam splitter (FF-665-Di01, Semrock Inc.). After passing through two 575/250 bandpass filters (HQ575_250 2p, Chroma Technology Corp.) separated by a colored glass (FGS900, Thorlabs Inc.), the fluorescence is detected by a photo-multiplier tube (PMT) (HC125-02, Hamamatsu Photonics). Data acquisition and motion control were implemented using a DAQ card (PCI-6115, National Instruments Corp.) and MPScan software [25

25. Q. T. Nguyen, P. S. Tsai, and D. Kleinfeld, “MPScope: a versatile software suite for multiphoton microscopy,” J. Neurosci. Methods 156(1-2), 351–359 (2006). [CrossRef] [PubMed]

]. The axial resolution of the GRIN lens systems was characterized in air using the full width at half maximum (FWHM) two-photon excited fluorescence signal from a 500 nm thin film of Rhodamine B (RhB) dye, while the lateral resolution was characterized using FWHM two-photon excited fluorescence from subresolution (0.2 µm) fluorescent beads. The field of view (FOV) was characterized by raster-scanning the proximal face of the GRIN lens system and measuring the one-photon transmission using a photodiode (SM05PD1A, Thorlabs Inc.), and was defined as the FWHM of the resulting intensity profile.

4. GRIN endoscope characterization

The on-axis lateral and axial resolutions and FOV of the systems are summarized in Table 1. Figure 2
Fig. 2 Two-photon lateral and axial resolution of GRIN system 2C (285 mm length). (a) Lateral intensity line profile across a subresolution fluorescent bead with a Gaussian fit in black. (b) Axial intensity profile across a thin film rhodamine slide with a Lorentzian fit in black.
shows the beam profile along the axial and lateral directions of the longest GRIN system (2C). Increasing the length of the relay lens results in only a small deterioration of the lateral resolution. The axial resolution declines more significantly for the longer systems. We believe that this is mainly due to increased accumulation of spherical aberrations, as can be seen by the asymmetric thin film response curve in Fig. 2(b). These characterizations were done in air; we would expect the axial resolution scaled by a factor of ~1.3 in tissue. Nevertheless, axial resolution remains on the order of one layer of mammalian cells for all of the GRIN lenses tested. The FOV for all systems (195-370 μm) is relatively large for the small OD systems. Figure 3
Fig. 3 Normalized one photon transmission intensity across the field of view for (a) GRIN system 1B and (b) GRIN system 2C.
shows the most and least uniform intensity curves obtained from the transmission imaging (GRIN system 1B and 2C respectively).

The off-axis axial resolutions of the different systems were measured by acquiring a through-focus z-series of the RhB thin film and fitting a Lorentzian function to individual off-axis areas of ~6 by 6 µm in size. The resulting FWHM of each area is plotted in Fig. 4
Fig. 4 Off-axis performance. Axial FWHM of GRIN system 2C in µm plotted (a) Across the FOV and (b) Across a line (dashed blue in (a)) through the center of the FOV. Scale bar is 50µm.
. The lateral off-axis performance was also measured for system 1B using sub-resolution fluorescent beads, and the FWHM of the obtained PSF remained below 1.2 µm up to the edge of the FOV. These results indicate that the off-axis resolution of these GRIN systems remains within ~20% of the on-axis resolution for most of the FOV (~80% of the area). The ability to deliver ultrashort pulses to the sample is critically important for multiphoton imaging. The effect of the longest GRIN lens system (285 mm in length, system 2C) on the excitation laser pulse was characterized. Without dispersion compensation, the initial 80 fs pulse width was broadened to 740 fs. Using precompensation with a rotating cylindrical lens and grating [26

26. M. E. Durst, D. Kobat, and C. Xu, “Tunable dispersion compensation by a rotating cylindrical lens,” Opt. Lett. 34(8), 1195–1197 (2009). [CrossRef] [PubMed]

], we were able to achieve an 85 fs pulse width at the sample, indicating that a simple dispersion compensation setup that accounts for the second order dispersion is sufficient for delivering pulses on the order of 80 fs.

5. Portable endoscope design and system characterization

To demonstrate that these GRIN lens systems have potential clinical applications, a compact, fiber-coupled multiphoton GRIN lens endoscope was constructed for in vivo image acquisition (Fig. 5
Fig. 5 Portable GRIN endoscope. (a) Optical drawing and (b) Solidworks drawing of the GRIN based endoscope system. Total system length of the portable device is 10.6” (including GRIN system).
). The 800 nm femtosecond pulse from the Ti:sapphire laser is delivered by a 2 meter hollow core PCF fiber (HC-800B, Thorlabs Inc.), and collimated to a beam of about 2 mm diameter using an aspheric lens. A small aperture (3 mm) galvo scanning mirror system (6210H, Cambridge Technology) was selected for a fast imaging rate (up to 4 frames/s at 512 by 512 pixels). The beam is then expanded by two scan lenses of 18 and 36 mm focal length (respectively, LSM02-BB and LSM03-BB, Thorlabs Inc.) to underfill a 0.3 NA microscope objective (RMS10X-PF, Thorlabs Inc.) to achieve an effective NA of ~0.1. The microscope objective couples the excitation beam into the proximal side of the GRIN lens system. We selected the longest 1 mm diameter system (1B) for this demonstration. The fluorescence signal from the sample is epi-collected through the GRIN lenses and the objective, and is reflected by a dichroic beam splitter (FF705-Di01, Semrock Inc.). After passing through two short pass filters (FF01-720/SP, Semrock Inc.) separated by colored glass (FGS900, Thorlabs Inc.), a second dichroic (Di01-R405, Semrock Inc.) separated the signal into the second harmonic and the autofluorescence channel. The housing of the GRIN lens endoscope was constructed from custom machined aluminum components using a milling machine with a fabrication tolerance of 0.001”. Optical elements in the GRIN endoscope were aligned in the z-axis to specification using calipers. We verified that illumination light projected through the system was centered as it passed through each optical element. The GRIN lens position was verified by confirming the focal length of the lens met specification (i.e. verified GRIN lens working distance by locating peak two-photon excited signal of a fluorescent sample ~130µm from the lens using a three-axis micrometer). The hollow core fiber has anomalous dispersion at 800 nm. This allowed us to precompensate the dispersion of the fiber and the GRIN lens by using a 10 cm rod of dispersive SF10 glass in the beam path before coupling into the fiber, resulting in a ~120 fs pulse width at the sample. The axial resolution of the GRIN lens endoscope system is 6.5 µm FWHM in air and 7.4 µm FWHM in water, measured using the RhB thin film as described above. The measured lateral resolution in both air and water is 0.85 µm FWHM using sub-resolution beads.

6. In vivo imaging

The ultimate goal of multiphoton endoscopy is imaging human patients in a clinical environment. A critical intermediate step is imaging unstained, live animals. Here, we demonstrate the capability of the portable GRIN endoscope in in vivo imaging of unstained tissues in live rats. A male rat (250-350 grams, Sprague-Dawley, Charles River Laboratories International, Inc.) was anesthetized using a gas anesthetic (~5% isoflurane-oxygen mixture) in an induction chamber. After reaching the appropriate level of sedation for surgery, the animal was fitted with a nose cone to maintain sedation (~2-3% isoflurane-oxygen mixture). Rat body temperature was maintained with a feedback-controlled heat blanket set to 36 °C. A ventral-midline abdominal incision exposed the internal organs to the GRIN endoscope. The kidney and liver were isolated and elevated using tongue depressors, and the surfaces of these organs were imaged using the portable GRIN endoscope. Another incision was made into the colon to expose and subsequently image the inner lining of this organ.

All animal procedures were reviewed and approved by the Cornell Institutional Animal Care and Use Committee. Throughout imaging sessions, the portable GRIN endoscope was mounted on a flexible mechanical arm (18041-XL-Special, Flexbar Machine Corporation), which allowed for rapid coarse movement of the endoscope from organ to organ. The flexible arm was in turn mounted on a 3D stage (MP-285, Sutter Instruments) for fine movement control. Images were acquired at a rate of 4 frames/second without averaging and with approximately 75mW at the sample. Example images are shown in Fig. 6
Fig. 6 Unaveraged in vivo images of unstained rat tissue. The pseudo-color images show red SHG signal (<405 nm) and green intrinsic fluorescent emission (405-700 nm). (a) Image of the superficial kidney renal cortex shows dark renal interstitium (RI), dark cellular nuclei (N) and bright intrinsic fluorescent cytoplasm (CY) that form the epithelial cells in the renal tubules (RT), SHG signal from the tough fibrous layer that forms the renal capsule (RC), and the dark blood filled lumen (L) inside the renal tubules. (b) Image of the inner colon wall shows bright intrinsic fluorescent signal from entrocytes (E) surrounding dark circular crypts (C). (c) Image of the rat liver showing ~20 µm diameter hapatocytes (coarse dashed line) with dark nuclei (N, solid line) chained together to form hepatic chords (HC), a dark bile duct (BD, fine dashed line) and bright intrinsically fluorescent bile salts (BS), as well as SHG emission from the septa (S) a fibrous tissue bands that separates hapatocyte nodules. (d) Image of the rat liver without labels shown for clarity. In these images, scale bars are 20 µm.
, pseudocolored with autofluorescence in green and second harmonic signal in red. Motion artifacts present a major challenge for in vivo imaging experiments. Most of the observed artifacts were due to respiratory motion, and seemed to increase the closer the imaged organ was to the diaphragm. Most of the images of the colon (>90%) are of comparable quality to those shown in Fig. 6, and this was true for ~70% of the kidney images. During imaging of both these organs, respiratory rate of the animal was at around 45 breaths per minute. The liver images suffered the most from motion artifacts, with only ~65% of similar quality as Fig. 6 at 45 breaths per minute. Once respiratory rate was decreased temporarily to ~25 breaths per minute by increasing the isofluorane:oxygen ratio in the gas anesthetic, the amount of high-quality images increased to ~80%. In comparison, we note that the respiratory rate for a healthy human adult is approximately 12-20 breaths per minute.

7. Discussion

While long GRIN lens systems are limited to rigid endoscope applications, they offer several advantages over flexible multiphoton endoscopes. A significant advantage is the diameter of the endoscope probe. GRIN lenses are commercially available in diameters as small as 350 μm, which allows them to be inserted into needles as small as 22 gauge (inner diameter of 413 μm). Using the doublet design, we would expect similar imaging performance with such a lens except with a smaller FOV of about 70 μm diameter. Furthermore, the use of externally mounted galvo based scanning mirrors solves several challenges faced by other endoscope designs when considering clinical implementation such as uniformity of the scan and durability of the device. Because GRIN lenses are inexpensive, they could ultimately be used as disposable devices, eliminating the need for sterilizing the endoscope probe between procedures. The GRIN lens approach would also allow for an external focus adjustment in the depth of the sample without movement of the endoscope probe that has penetrated the tissue. By axially translating the scan objective, a z-scan in the sample of 0 to 95 μm from the surface of the lens has previously been shown [20

20. 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] [PubMed]

].

8. Conclusions

We have demonstrated that TPF and SHG imaging are possible through long GRIN lens systems up to 28.5 cm in length. Long GRIN lenses can be integrated with a compact and portable two-photon microscope suitable for a clinical environment. The device presented can acquire TPF and SHG images at a rate of 4 frames/s with a field of view of ~200 μm diameter and with subcellular resolution. The presented in vivo results of unstained organs of live rats show great promise for using GRIN endoscopy for optical biopsy.

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 Number R01-EB006736, “Development of Medical Multiphoton Microscopic Endoscopy.” We thank members of the Xu and Webb research groups as well as Dr. Douglas Scherr and Dr. Sushmita Mukherjee of Weill Cornell Medical College for discussions and technical suggestions. We also thank Dr. Wendy Williams of the Cornell Center for Animal Resources and Education for her assistance with the in vivo imaging experiments and Mr. Herbert Stürmer of GRINTECH GmbH 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.

S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, “Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging,” Opt. Lett. 31(18), 2756–2758 (2006). [CrossRef] [PubMed]

3.

S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton microscopy,” Proc. SPIE 7161, 716117, 716117-10 (2009). [CrossRef]

4.

I. Pavlova, K. R. Hume, S. A. Yazinski, J. Flanders, T. L. Southard, R. S. Weiss, and W. W. Webb, “Multiphoton microscopy and microspectroscopy for diagnostics of inflammatory and neoplastic lung,” J. Biomed. Opt. 17(3), 036014 (2012). [CrossRef]

5.

M. C. Skala, J. M. Squirrell, K. M. Vrotsos, J. C. Eickhoff, A. Gendron-Fitzpatrick, K. W. Eliceiri, and N. Ramanujam, “Multiphoton microscopy of endogenous fluorescence differentiates normal, precancerous, and cancerous squamous epithelial tissues,” Cancer Res. 65(4), 1180–1186 (2005). [CrossRef] [PubMed]

6.

C. C. Wang, F. C. Li, R. J. Wu, V. A. Hovhannisyan, W. C. Lin, S. J. Lin, P. T. So, and C. Y. Dong, “Differentiation of normal and cancerous lung tissues by multiphoton imaging,” J. Biomed. Opt. 14(4), 044034 (2009). [CrossRef] [PubMed]

7.

P. Wilder-Smith, K. Osann, N. Hanna, N. El Abbadi, M. Brenner, D. Messadi, and T. Krasieva, “In vivo multiphoton fluorescence imaging: a novel approach to oral malignancy,” Lasers Surg. Med. 35(2), 96–103 (2004). [CrossRef] [PubMed]

8.

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]

9.

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]

10.

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]

11.

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]

12.

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]

13.

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]

14.

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]

15.

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

16.

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]

17.

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]

18.

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]

19.

B. A. Flusberg, J. C. Jung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, “In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope,” Opt. Lett. 30(17), 2272–2274 (2005). [CrossRef] [PubMed]

20.

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] [PubMed]

21.

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. Neurophysiol. 92(5), 3121–3133 (2004). [CrossRef] [PubMed]

22.

J. C. Jung and M. J. Schnitzer, “Multiphoton endoscopy,” Opt. Lett. 28(11), 902–904 (2003). [CrossRef] [PubMed]

23.

K. König, A. Ehlers, I. Riemann, S. Schenkl, R. Bückle, and M. Kaatz, “Clinical two-photon microendoscopy,” Microsc. Res. Tech. 70(5), 398–402 (2007). [CrossRef] [PubMed]

24.

R. S. Pillai, D. Lorenser, and D. D. Sampson, “Deep-tissue access with confocal fluorescence microendoscopy through hypodermic needles,” Opt. Express 19(8), 7213–7221 (2011). [CrossRef] [PubMed]

25.

Q. T. Nguyen, P. S. Tsai, and D. Kleinfeld, “MPScope: a versatile software suite for multiphoton microscopy,” J. Neurosci. Methods 156(1-2), 351–359 (2006). [CrossRef] [PubMed]

26.

M. E. Durst, D. Kobat, and C. Xu, “Tunable dispersion compensation by a rotating cylindrical lens,” Opt. Lett. 34(8), 1195–1197 (2009). [CrossRef] [PubMed]

27.

L. P. Gartner and J. L. Hiatt, Color Textbook of Histology (W.B. Saunders, Philadelphia, 2001).

28.

J. M. Dela Cruz, J. D. McMullen, R. M. Williams, and W. R. Zipfel, “Feasibility of using multiphoton excited tissue autofluorescence for in vivo human histopathology,” Biomed. Opt. Express 1(5), 1320–1330 (2010). [CrossRef] [PubMed]

29.

R. Ramasamy, J. Sterling, E. S. Fisher, P. S. Li, M. Jain, B. D. Robinson, M. Shevchuck, D. Huland, C. Xu, S. Mukherjee, and P. N. Schlegel, “Identification of spermatogenesis with multiphoton microscopy: an evaluation in a rodent model,” J. Urol. 186(6), 2487–2492 (2011). [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: February 9, 2012
Revised Manuscript: April 12, 2012
Manuscript Accepted: April 14, 2012
Published: April 19, 2012

Citation
David M. Huland, Christopher M. Brown, Scott S. Howard, Dimitre G. Ouzounov, Ina Pavlova, Ke Wang, David R. Rivera, Watt W. Webb, and Chris Xu, "In vivo imaging of unstained tissues using long gradient index lens multiphoton endoscopic systems," Biomed. Opt. Express 3, 1077-1085 (2012)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-3-5-1077


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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. S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, “Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging,” Opt. Lett.31(18), 2756–2758 (2006). [CrossRef] [PubMed]
  3. S. Mukherjee, J. S. Wysock, C. K. Ng, M. Akhtar, S. Perner, M. M. Lee, M. A. Rubin, F. R. Maxfield, W. W. Webb, and D. S. Scherr, “Human bladder cancer diagnosis using Multiphoton microscopy,” Proc. SPIE7161, 716117, 716117-10 (2009). [CrossRef]
  4. I. Pavlova, K. R. Hume, S. A. Yazinski, J. Flanders, T. L. Southard, R. S. Weiss, and W. W. Webb, “Multiphoton microscopy and microspectroscopy for diagnostics of inflammatory and neoplastic lung,” J. Biomed. Opt.17(3), 036014 (2012). [CrossRef]
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