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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 2, Iss. 10 — Oct. 31, 2007
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In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy

Che-Hang Yu, Shih-Peng Tai, Chun-Ta Kung, I-Jong Wang, Han-Chieh Yu, Hsiang-Ju Huang, Wen-Jeng Lee, Yi-Fan Chan, and Chi-Kuang Sun  »View Author Affiliations


Optics Express, Vol. 15, Issue 18, pp. 11167-11177 (2007)
http://dx.doi.org/10.1364/OE.15.011167


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Abstract

Elastin is an essential and widespread structural protein in charge of the integrity on tissues and organs. In this study, we demonstrate that elastin is a major origin of the third-harmonic-generation (THG) contrast under Cr:forsterite laser excitation operating at 1230nm, with selective visualization inside many tissues such as lung tissues and arteries. In vivo imaging of the nude mouse elastic cartilage beneath the hypodermis by epi-THG microscopy keeps the high resolution and contrast in all three dimensions. Combined with second-harmonic-generation microscopy, THG microscopy exhibits the ability to show the extraordinary proliferation of elastic fibers for the ophthalmic disease of pterygium and the capability of distinguishable visualization from collagen.

© 2007 Optical Society of America

1. Introduction

2. Material and Methods

2.1 Nonlinear scanning microscopy

Fig. 1. System setup. (a) Experimental diagram of THG and multiphoton microscopes. (b) Fixation of the nude mouse ear. (c) Acquire images of the test animal under the microscope with a thermal blanket.

2.2 Tissue preparation and animals

The human lung tissues were stored in liquid nitrogen and obtained from the tissue bank of National Taiwan University Hospital. The rat aortas were excised and also frozen by liquid nitrogen. They both were cut into 120um thin sections for intrinsic imaging and followed by eosin staining for extrinsic imaging. In the in vivo study, nude mouse is used as our animal model. The experimental protocols were approved by the National Taiwan University Institutional Animal Care and Use Committee (NTU-IACUC). After anaesthetization, the nude mouse ear is fixed as shown in Fig. 1(b). Fig. 1(c) is an example to rest the nude mouse in our microscope system with a thermal blanket maintaining the body temperature of the test animal. In the experiment of pterygium, surgical specimens of pterygium and normal conjunctiva specimens from patients were obtained from the Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan. The specimens were fixed in 3.7% formaldehyde in 0.1 mol/L of phosphate-buffered-saline (PH 7.4) at 4°C for storage before imaging.

3. Ex vivo THG and endogenous fluorescence imaging of elastic fibers

Fig. 2. Normalized emission spectra of elastin powder under 1230 nm nonlinear excitation. All emission spectra are with a central wavelength around 655nm. The emission spectra from elastin powder of aorta and lung are denoted in black and red colors respectively; while the emission spectra from elastin solution of aorta and lung are denoted in green and blue colors respectively.

Elastin is considered as one of the endogenous fluorescent sources for the two-photon fluorescence (2PF) microscopy under Ti:sapphire laser excitation operating at about 800nm. In the last few years, several articles have been devoted to study the structural and functional properties of elastic fibers in the vascular wall by using the endogenous 2PF microscopy [18

18. J.D. Ania-Castañón, I.O. Nasieva, N. Kurukitkoson, S.K. Turitsyn, C. Borsier, and E. Pincemin, “Nonlinearity management in fiber transmission systems with hybrid amplification,” Opt. Commun.233, 353(2004). [CrossRef]

20

20. C. Xu, X. Liu, and X. Wei, “DPSK for high spectral efficiency optical transmissions,” J. Selected Topics Quantum Electron. 10, 281–293 (2004). [CrossRef]

]. In a similar way, to make sure whether elastin still fluoresces under 1230nm excitation, we measured the nonlinear emission spectra of the purified elastin powders extracted from the human lung and aorta, either in the solid form or being dissolved in liquid. Under 1230 nm excitation, as shown in Fig. 2, all the nonlinear emission peaks of the elastin powder spectra are with the same central wavelength around 655nm. Besides, the measured fluorescence power was found to be in quadratic dependence on the excitation power, confirming its two-photon nature. In addition, the two-photon excitation action cross-section (σTPE, defined as the product of the two-photon absorption cross-section and the fluorescence quantum yield) [21

21. X. Wei and X. Liu, “Analysis of intrachannel four-wave mixing in differential phase-shift keying transmission with large dispersion,” Opt. Lett. 28, 2300–2302 (2003). [CrossRef] [PubMed]

] of purified elastin in the solution was measured. The measurement of the σTPE followed the method performed by Blab et al. previously [21

21. X. Wei and X. Liu, “Analysis of intrachannel four-wave mixing in differential phase-shift keying transmission with large dispersion,” Opt. Lett. 28, 2300–2302 (2003). [CrossRef] [PubMed]

]. We used the reported value of σTPE of HcRed excited with 1230nm [22

22. R. I. Killey, H. J. Thiele, V. Mikhailov, and P. Bayvel, “Reduction of intrachannel nonlinear distortion in 40-Gb/s-based WDM transmission over standard fiber,” IEEE Photon. Technol. Lett. 12, 1624–1626 (2000). [CrossRef]

] as a reference and considered the difference of transmission of optics in different wavelengths to calibrate the value of σTPE of elastin based on the measured power of two-photon fluorescence of elastin excited with 1230nm laser beam. The value of the σTPE is 0.125±0.0044GM (1GM=10-50 cm4 s/photon) with an excitation wavelength of 1230nm, indicating that elastin is a low efficiency two-photon fluorescence protein.

Figure 3 is the intra-tissue emission spectrum measured from the area of the elastic fiber in a rat aorta. From Fig. 3, the THG signal emitted from the elastic fiber is about two orders of magnitude larger than the 2PF signal with the central wavelength around 655nm, which is the same as the spectral measurement of elastin powders. This result implies that the strong THG emissions from elastin could be a promising contrast to provide selective imaging.

Fig. 3. Intra-tissue emission spectrum measured from the area of the elastic fiber in a rat aorta. SHG signal comes from the collagen closely adjacent to the elastic fiber in the artery media.
Fig. 4. THG and multi-photon fluorescence imaging of human lung tissue and rat aorta. (a) and (b) are simultaneous THG (blue) and endogenous 2PF (magenta) images of a human lung tissue and a rat aorta, respectively. (c) and (d) are simultaneous THG (blue) and eosin-stained exogenous fluorescence (orange) images of a human lung tissue and a rat aorta, respectively. The co-localization of blue and orange colors shows white color. All yellow arrows within four images indicate the location of elastic fibers. Scale bar: 20µm.

Then, we recorded the endogenous 2PF and THG images simultaneously in the intact human lung tissues and the rat aorta samples. In the image of human lung tissue [indicated by yellow arrows in Fig. 4(a)], some THG signals (blue) excellently correlating with endogenous 2PF (magenta) are the sites of elastic fibers, revealing the alveolar structure. In the image of rat aorta, what has to be noticed is that the parallel THG signals is filled with the endogenous 2PF signals from elastic fibers [indicated by yellow arrows in Fig. 4(b)]; in other words, it should be the exact location of elastic fibers between the parallel THG signals, characterizing the lamellar structure in the artery media. These experiments highly suggest that it is the elastic fibers responsible for some of the dominating THG signals.

4. Ex vivo THG and exogenous fluorescence imaging of elastic fibers

To further prove the origin of THG signals from the elastin fibers, we stained the human lung tissue and the rat aorta with added probe, eosin, to exactly label the elastin. Eosin, a specific and strongly fluorescent elastin marker, has the ability to selectively visualize elastin fibers in different tissues for the fluorescence microscope, and the elastin endogenous fluorescence contributes no or only little comparatively [14

14. C. Rasmussen, S. Dey, F. Liu, J. Bennike, B. Mikkelsen, P. Mamyshev, M. Kimmitt, K. Springer, D. Gapontsev, and V. Ivshin, “Transmission of 40×42.7 Gb/s over 5200 km ultraWave fiber with terrestrial 100 km spans using turn-key ETDM transmitter and receiver,” in proceedings ECOC’2002, DK, Copenhaguen, paper PD4.4.

]. For the reason given above, we also measured the nonlinear fluorescence spectrum of eosin under 1230 nm excitation and found the emission peak value is 555nm (as shown in Fig. 5). According to the measured eosin spectrum, we recorded exogenous three-photon fluorescence and THG images [Fig. 4(c) and 4(d)] at the same time within tissues. The stained images (orange) are perfectly correlated with the endogenous fluorescence images and have the identical relation to the THG images (The overlapping area shows white color). This result further verifies that one significant contrast origin of THG in the biological tissues comes from elastin.

Fig. 5. Normalized spectrum of eosin. The emission maximum is around 555nm.

5. In vivo THG and endogenous fluorescence imaging of elastic cartilage

One principal advantage of THG microscopy is its minimal invasiveness for the study of live samples with a sub-micron resolution [3

3. “Lambdaxtreme Transport successfully completes field trial in Deutsche Telekom network,” Lucent press release, July 2002, www.lucent.com.

,4

4. “CoreStream Agility Optical Transport system data sheet,” January 2007, www.ciena.com.

]. In our ex vivo experiments we demonstrate that one significant contrast origin of THG in the biological tissues comes from elastin. It is pertinent to consider whether this technique could be feasible for imaging the elastin in vivo. Therefore, we recorded the in vivo images in the unstained elastic cartilage of the live nude mouse ear combining THG microscopy and endogenous 2PF microscopy simultaneously. As shown in Fig. 6, deep into the skin the interconnecting sheets of elastin material can be identified by means of the overlap of the weak 2PF and the strong epi-THG images. Besides, it must be noted that the cell membrane contributes the distinct rounded images probably on account of the great refractive index difference between the chondrocyte and the lacuna, which is the space the chondrocyte occupies. Figure 7 shows an example movie of a sequential set of horizontally sectioned 2PF and epi-THG images taken from the elastic cartilage of the nude mouse ear beneath the hypodermis.

Fig. 6. In vivo horizontal sections of nude mouse elastic cartilage using THG (blue) and endogenous 2PF (magenta) microscopes. (a) Endogenous 2PF image of elastic cartilage adjusted at the same contrast level with Fig. 6(c). PMT acquisition voltage: 3000V. (b)Endogenous 2PF image adjusted at a very low contrast level. PMT acquisition voltage: 3000V. (c) THG image. PMT acquisition voltage: 1200V. (d) Simultaneous THG and endogenous 2PF image. Yellow arrows indicate one of the locations of the elastic fibers. Elastic fibers in the elastic cartilage shows high contrast, high spatial resolution, and distinguishable intensity using in vivo THG microscopy. Scale bar: 20µm.
Fig. 7. (690 KB) A movie of in vivo depth-resolved horizontal sections in elastic cartilage of the nude mouse ear. This movie is composed of 15 horizontal images. The optical depth difference between adjacent images is 2.1 µm. Image size: 80µm×80µm. [Media 1]

Figure 8 is a movie of 3-D reconstruction of a sequential set of horizontally sectioned images provided by the in vivo epi-THG microscopy. The 3-D reconstruction comprises 25 horizontal sections and has 2.1-µm interval between two adjacent images. The network of elastic fibers can be found to be arranged in a 3-D honeycomb configuration. It could be concluded that epi-THG microscopy is feasible to offer high resolution 3-D intra-tissue histological information of the unstained elastic cartilage in vivo with least invasiveness to provide an ideal new platform for studies of supporting tissues in the future.

Fig. 8. (2.19 MB) A movie of 3-D reconstruction of a sequential set of horizontally sectioned in vivo images from the elastic cartilage of the nude mouse ear. Green arrows indicate one of the locations of the elastic fibers. This movie is composed of 25 horizontal images. The optical depth difference between adjacent images is 2.1 µm. Image size: 120µm×120µm. [Media 2]

Similar to the endogenous multiphoton fluorescence microscopy and SHG microscopy, THG microscopy is with contrasts contributing from several major origins and is thus with a possibility of false positive examination of elastin. As shown in Fig. 8, there are some THG signals from chondrocytes instead of elastic fibers. We attempted to solve this problem by examining the polarization of the generated THG radiation from elastic fibers under linearly polarized incident radiation excitation. With a fixed polarized incident light, we examined the polarization of the THG signals with a polarizer in front of the detection. Figure 9 shows the measured THG intensity by rotating the angle θ of the THG polarizer to that of the excitation polarization for different elastin fiber orientations. Our polarization study indicated that the generated THG polarization is in parallel to the polarization of the excitation light, independent of the fiber orientations, similar to the case of an isotropic medium. It is thus unable to differentiate the elastic fiber from other isotropic media by means of polarization management. Our study also suggests that the THG enhancement in elastin fibers could be due to the two-photon resonance [23

23. E. Pincemin, D. Grot, C. Borsier, J.D. Ania-Castañòn, and S.K. Turitsyn, “Impact of the fiber type and dispersion management on the performance of an NRZ 16x40 Gb/s DWDM transmission system,” IEEE Photon. Technol. Lett. 16, 2362–2364 (2004). [CrossRef]

] of the elastin molecules rather than the fibril structures [24

24. J. P. Gordon and L. F. Mollenauer, “Phase noise in photonics communications systems using linear amplifier,” Opt. Lett. 15, 1351–1355 (1990). [CrossRef] [PubMed]

].

Fig. 9. Normalized THG intensity after a linear polarization as a function of the polarizer angle θ relative to the linear polarization angle of the incident excitation. The square data points represent the case that the excitation polarization is parallel (0°) to the elastic fiber. The triangular data points represent the case that the excitation polarization is 45° to the elastic fiber.

6. Imaging elastosis of the conjunctiva by using epi-THG microscopy

Fig. 10. Simultaneous THG (blue) and SHG (green) images of human conjunctiva (a) Image of normal conjunctiva shows distribution of collagen without elastosis. (b) Image of diseased conjunctiva, pterygium, shows elastin proliferation revealed by THG signals. Scale bar: 20µm.

7. Conclusion

Our study concludes that elastin is a major origin of THG contrast in varieties of tissues such as arteries and lung tissues, under Cr:forsterite laser excitation operating at 1230nm. Met the noninvasive requirement for in vivo studies, THG microscopy is a 3-D morphological imaging tool not only providing the submicron resolution for detailed histological information due to its interface-sensitive nature but also leaving no energy deposition to examined tissues due to the virtual-state-transition characteristic. Easily integrated with other nonlinear optical microscopy such as SHG and 2PF signals with the same laser system, THG microscopy could provide a new way to distinguish elastin and collagen fibers without staining.

Acknowledgments

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OCIS Codes
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(180.6900) Microscopy : Three-dimensional microscopy
(190.4160) Nonlinear optics : Multiharmonic generation
(190.4180) Nonlinear optics : Multiphoton processes

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: June 15, 2007
Revised Manuscript: July 24, 2007
Manuscript Accepted: July 24, 2007
Published: August 21, 2007

Virtual Issues
Vol. 2, Iss. 10 Virtual Journal for Biomedical Optics

Citation
Chi-Kuang Sun, Che-Hang Yu, Shih-Peng Tai, Chun-Ta Kung, I-Jong Wang, Han-Chieh Yu, Hsiang-Ju Huang, Wen-Jeng Lee, and Yi-Fan Chan, "In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy," Opt. Express 15, 11167-11177 (2007)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-15-18-11167


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