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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 5, Iss. 4 — Apr. 1, 2014
  • pp: 1099–1113
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Motion-artifact-robust, polarization-resolved second-harmonic-generation microscopy based on rapid polarization switching with electro-optic Pockells cell and its application to in vivo visualization of collagen fiber orientation in human facial skin

Yuji Tanaka, Eiji Hase, Shuichiro Fukushima, Yuki Ogura, Toyonobu Yamashita, Tetsuji Hirao, Tsutomu Araki, and Takeshi Yasui  »View Author Affiliations


Biomedical Optics Express, Vol. 5, Issue 4, pp. 1099-1113 (2014)
http://dx.doi.org/10.1364/BOE.5.001099


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Abstract

Polarization-resolved second-harmonic-generation (PR-SHG) microscopy is a powerful tool for investigating collagen fiber orientation quantitatively with low invasiveness. However, the waiting time for the mechanical polarization rotation makes it too sensitive to motion artifacts and hence has hampered its use in various applications in vivo. In the work described in this article, we constructed a motion-artifact-robust, PR-SHG microscope based on rapid polarization switching at every pixel with an electro-optic Pockells cell (PC) in synchronization with step-wise raster scanning of the focus spot and alternate data acquisition of a vertical-polarization-resolved SHG signal and a horizontal-polarization-resolved one. The constructed PC-based PR-SHG microscope enabled us to visualize orientation mapping of dermal collagen fiber in human facial skin in vivo without the influence of motion artifacts. Furthermore, it implied the location and/or age dependence of the collagen fiber orientation in human facial skin. The robustness to motion artifacts in the collagen orientation measurement will expand the application scope of SHG microscopy in dermatology and collagen-related fields.

© 2014 Optical Society of America

1. Introduction

Second-harmonic-generation (SHG) is a nonlinear optical phenomenon resulting from the nonlinear interaction of a high-peak-power optical electric field with a material [1

1. A. Yariv, Introduction to Optical Electronics, (Holt McDougal, 1977).

] and has been widely used for wavelength conversion of laser light with a nonlinear optical crystal. On the other hand, when ultrashort pulse light is focused onto biological tissue instead of a crystal, SHG enables unique nonlinear optical microscopy having selectivity specific to non-centrosymmetric structures in biomolecules, such as collagen, myosin, and microtubules [2

2. S. Fine and W. P. Hansen, “Optical second harmonic generation in biological systems,” Appl. Opt. 10(10), 2350–2353 (1971). [CrossRef] [PubMed]

4

4. 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(1), 493–508 (2002). [CrossRef] [PubMed]

]. Furthermore, a contrast mechanism used in SHG microscopy is based on a naturally endogenous SHG process inherent in these molecules themselves, making it possible to visualize them in vivo freely from photobleaching, phototoxicity and additional staining with a fluorochrome. In particular, the collagen molecule is an important structural protein in the human body and forms structural aggregates successively, i.e., microfibrils, fibrils, fibers, and bundles of different sizes in biological tissues. Therefore, in vivo imaging of these collagen aggregates in tissues is an important biological application of SHG microscopy [5

5. K. König and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8(3), 432–439 (2003). [CrossRef] [PubMed]

12

12. R. Tanaka, S. Fukushima, K. Sasaki, Y. Tanaka, H. Murota, T. Matsumoto, T. Araki, and T. Yasui, “In vivo visualization of dermal collagen fiber in skin burn by collagen-sensitive second-harmonic-generation microscopy,” J. Biomed. Opt. 18(6), 061231 (2013). [CrossRef] [PubMed]

] because it is difficult to visualize collagen in vivo using conventional methods, including histological methods.

PR-SHG imaging has to be performed by acquiring successive SHG images while changing the polarization angle of the incident laser light. Usually, the polarization angle is adjusted by mechanical rotation of a half-wave plate [13

13. P. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da Silva, “Polarization-dependent optical second-harmonic imaging of a rat-tail tendon,” J. Biomed. Opt. 7(2), 205–214 (2002). [CrossRef] [PubMed]

, 15

15. T. Yasui, Y. Tohno, and T. Araki, “Characterization of collagen orientation in human dermis by two-dimensional second-harmonic-generation polarimetry,” J. Biomed. Opt. 9(2), 259–264 (2004). [CrossRef] [PubMed]

20

20. G. Latour, I. Gusachenko, L. Kowalczuk, I. Lamarre, and M.-C. Schanne-Klein, “In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy,” Biomed. Opt. Express 3(1), 1–15 (2012). [CrossRef] [PubMed]

]. However, such mechanical rotation requires a long waiting time and thus hinders achievement of in vivo PR-SHG imaging, because of unwanted motion artifacts caused by heart beats, breathing, and/or mechanical vibrations of the subjects. Although an electro-optic Pockells cell (PC) enables fast rotation of the laser light polarization in PR-SHG microscopy [14

14. P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J. 82(6), 3330–3342 (2002). [CrossRef] [PubMed]

], it is difficult to achieve rapid image acquisition for in vivo applications because the required lock-in amplifier cannot be used with a highly sensitive photon-counting photomultiplier. In the work described in this article, by rapidly switching the polarization angle between vertical and horizontal directions at every pixel with a PC, we successively demonstrated in vivo visualization of collagen fiber orientation in human skin without the influence of motion artifacts.

2. Experimental methods

2.1 PR-SHG imaging based on two orthogonal polarizations

PR-SHG microscopy has two operation modes, depending on the method of polarization change. The first mode provides a radar graph of SHG light intensity or corresponding intensity profile with respect to the polarization angle by rotating the linear polarization angle continuously [13

13. P. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da Silva, “Polarization-dependent optical second-harmonic imaging of a rat-tail tendon,” J. Biomed. Opt. 7(2), 205–214 (2002). [CrossRef] [PubMed]

18

18. J. C. Mansfield, C. P. Winlove, J. Moger, and S. J. Matcher, “Collagen fiber arrangement in normal and diseased cartilage studied by polarization sensitive nonlinear microscopy,” J. Biomed. Opt. 13(4), 044020 (2008). [CrossRef] [PubMed]

, 20

20. G. Latour, I. Gusachenko, L. Kowalczuk, I. Lamarre, and M.-C. Schanne-Klein, “In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy,” Biomed. Opt. Express 3(1), 1–15 (2012). [CrossRef] [PubMed]

]. The resulting SHG radar graph indicates detailed information of the dominant orientation angle and the degree of fiber alignment. On the other hand, the second mode is based on the contrast of two SHG light beams when switching the laser polarization between the vertical and horizontal directions [17

17. T. Yasui, K. Sasaki, Y. Tohno, and T. Araki, “Tomographic imaging of collagen fiber orientation in human tissue using depth-resolved polarimetry of second-harmonic-generation light,” Opt. Quantum Electron. 37(13-15), 1397–1408 (2005). [CrossRef]

, 19

19. T. Yasui, Y. Takahashi, S. Fukushima, Y. Ogura, T. Yamashita, T. Kuwahara, T. Hirao, and T. Araki, “Observation of dermal collagen fiber in wrinkled skin using polarization-resolved second-harmonic-generation microscopy,” Opt. Express 17(2), 912–923 (2009). [CrossRef] [PubMed]

]. Since the second mode involves simpler adjustment of the polarization angle than the first mode and is effective for a simple analysis of uniaxially oriented collagen fiber, we considered extending the second mode to in vivo applications in this work. To evaluate the collagen orientation quantitatively in the second mode, the polarization anisotropy of SHG light (α) was defined as follows
α=IVIHIV+IH
(1)
where IV and IH are the SHG intensities when the incident light is vertically and horizontally polarized, respectively. Then, the α image was calculated by substituting IV and IH values at each pixel of two PR-SHG images for Eq. (1). The α image reflects the distribution of collagen fiber orientation because strong SHG light is observed when laser polarization is parallel to the collagen orientation, and SHG light is considerably weaker if the laser polarization is perpendicular to the collagen orientation. The collagen orientation is uniaxial for α = ± 1 and random or biaxial for α = 0. The sign of α gives the dominant direction of the collagen orientation: positive for a vertical orientation and negative for a horizontal orientation.

To solve the above problem, we should consider speeding up the polarization switching and improving the data acquisition method. The polarization switching can be speeded up to a few tens MHz by using a PC. Regarding the improvement of the data acquisition method, a VPR-SHG signal and an HPR-SHG signal should be acquired alternately at every pixel, as shown in Fig. 1(b). In this case, the VPR-SHG image is composed of odd data acquisition numbers ( = 1, 3, 5, •••••, 2m-5, 2m-3, 2m-1), whereas the HPR-SHG image is composed of even data acquisition numbers ( = 2, 4, 6, •••••, 2m-4, 2m-2, 2m). Since the polarization-switching rate by the PC is much faster than the motion artifacts, it is easy to satisfy a pixel-to-pixel correspondence between the VPR-SHG and HPR-SHG images. Therefore, a combination of the rapid polarization switching by PC and step-wise raster scanning by galvanometer mirror (GM) will enable us to obtain an α image correctly without motion artifacts.

2.2. Experimental setup

Figure 2(a)
Fig. 2 (a) Experimental setup. PC: Electro-optic Pockells cell; PMT: photon-counting photomultiplier with Peltier cooling. Inset is a photograph of the attachment ring. (b) Comparison of autocorrelation traces of the laser light before and after passing though the prism pair and PC. (c) Photograph of in vivo measurement of the human facial skin. (d) Timing chart of scanning signals of slow GM and fast GM, switching signal of PC, and timing signal of data acquisition.
shows the experimental setup. The developed PR-SHG microscope was equipped with a mode-locked Cr:Forsterite laser (Avesta Project Ltd., CrF-65P, center wavelength = 1250 nm, pulse duration = 70 fs, repetition rate = 73 MHz) for a laser source that reduces the laser-induced photodamage [21

21. 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 sources,” Opt. Quantum Electron. 34(12), 1251–1266 (2002). [CrossRef]

23

23. S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, “In vivo optical biopsy of hamster oral cavity with epi-third-harmonic-generation microscopy,” Opt. Express 14(13), 6178–6187 (2006). [CrossRef] [PubMed]

] and enhances the penetration depth [8

8. T. Yasui, Y. Takahashi, M. Ito, S. Fukushima, and T. Araki, “Ex vivo and in vivo second-harmonic-generation imaging of dermal collagen fiber in skin: comparison of imaging characteristics between mode-locked Cr:Forsterite and Ti:Sapphire lasers,” Appl. Opt. 48(10), D88–D95 (2009). [CrossRef] [PubMed]

]. We used a PC made of lithium tantalate crystal (Conoptics Inc., 360-120, thickness = 120 mm, half-wave voltage = 160 V at 1250 nm, contrast ratio = 100:1, refractive index = 2.13 at 1250 nm, group velocity dispersion = 144 fs2/mm), driven by a digital amplifier (Conoptics Inc., 25D, bandwidth < 30 MHz, maximum voltage = 175 V), for rapid switching of two orthogonal polarizations. To suppress the temporal broadening of the pulse duration in the PC, we prechirped the laser light using a pair of dispersion prisms made of S-NPH3 glass (Ohara Inc., refractive index = 1.90 at 1250 nm, group velocity dispersion = −30 fs2/nm) before passing through PC. Figure 2(b) compares the autocorrelation traces of the laser light before and after passing through the prism pair and PC. The adjusted negative dispersion in the prism pair cancels the positive dispersion in the PC, making it possible to maintain the original pulse duration of this laser source after passing through the prism pair and PC. A half-wave plate (Lattice Electro Optics Inc., CWO-1250-02-10, retardation tolerance = λ/500), set into a stepping-motor-driven rotatory mount (Suruga Seiki Co., Ltd., K491-30, angle resolution = 0.004 deg/pulse, driving pulse frequency = 1.875 kHz, rotating speed = 7.5 deg/s), after PC was used for the mechanical-rotation-based PR-SHG imaging described later. After switching the polarization between the vertical and horizontal directions with the PC, the focus spot of the laser beam is scanned two-dimensionally onto a sample by use of a pair of GMs, composed of a fast GM (scanning freq. = 64 Hz) and a slow GM (scanning freq. = 0.5 Hz), a pair of relay lenses, and an objective lens (OL; Nikon Instruments Inc., CFI Plan 50 × H, magnification = 50, NA = 0.9, working distance = 350 μm, oil-immersion type). To cancel the polarization dependence of the reflectivity in the GM, the vertical and horizontal polarizations of the laser light were respectively set at a direction of 45 degree and −45 degree from the p-polarization in the fast GM or the s-polarization in the slow GM. The average power of the laser light was set to be 28–35 mW on the sample. Backscattered SHG light was collected via the objective lens and then was separated from the laser light by a harmonic separator (Lattice Electro Optics Inc., LWP-45-Runp625-Tunp1250-B-1013, reflected wavelength = 625 nm) and an optical filter with a sharp pass-band (Semrock Inc., 625/26 nm BrightLine, pass wavelength = 612 to 638 nm). Finally, the SHG light was detected by a photon-counting photomultiplier with Peltier cooling (PMT; Hamamatsu, H7421-40) connected with a pulse counter. We used a customized attachment ring [see an inset of Fig. 2(a)] for in vivo imaging of human facial skin, which is attached to the subject’s skin by a double-stick tape and used to reduce the motion of its measured position. Figure 2(c) shows a photograph of in vivo measurement of the human facial skin.

To correctly perform the data acquisition procedure in Fig. 1(b), scanning signals of the slow GM and fast GM, a switching signal of the PC, and a timing signal of the data acquisition should be synchronized. To this end, we used a timing chart for these four signals as shown in Fig. 2(d). First, the slow GM was scanned at 0.5 Hz with a step-wise waveform (number of steps = 128, time duration of one step = 15.6 ms). Second, the fast GM was scanned at 64 Hz with a step-wise waveform (number of steps = 128, time duration of one step = 122 µs). Third, the PC was driven by a 8,192-Hz square wave swinging between the half-wave voltage ( = 160 V, corresponding polarization direction = vertical) and the no-bias voltage ( = 0 V, corresponding polarization direction = horizontal) because the polarization angle has to be changed from the vertical to the horizontal direction during one step of the step-wise waveform for the fast GM in order to switch the polarization at the same pixel. Fourthly, the timing signal of the data acquisition was set to be twice as high frequency ( = 16,384 Hz) as the switching signal of the PC. Finally, the serial data train of SHG signals (data acquisition number = 1, 2, 3, •••••, 2m-2, 2m-1, 2m = 1, 2, 3, •••••, 65534, 65535, 65536) was acquired by a counter board in a computer and sorted into the VPR-SHG image (data acquisition number = 1, 3, 5, •••••, 2m-5, 2m-3, 2m-1 = 1, 3, 5, •••••, 65531, 65533, 65535) and the HPR-SHG image (data acquisition number = 2, 4, 6, •••••, 2m-4, 2m-2, 2m = 2, 4, 6, •••••, 65532, 65534, 65536). The two scanning signals of the fast GM and slow GM, the switching signal of the PC, and the timing signal of the data acquisition were provided from two signal generators (NF Corporation, WF1974), synchronizing to each other. We confirmed the synchronization of all signals by monitoring the two angle-sensor signals of GMs, the two optical signals separated by a polarization beamsplitter after passing through PC, and data acquisition signal from the signal generator simultaneously in another setup (not shown). In this way, one can obtain the α image, satisfying a pixel-to-pixel correspondence between VPR-SHG and HPR-SHG images even though the motion artifacts exist.

2.3 Sample preparation

We prepared the human Achilles tendon as the sliced specimen for ex vivo PR-SHG imaging. The human Achilles possesses the uniaxial orientation of collagen fiber along the axial direction. The human Achilles was sliced to 1 mm thick along the axial direction. After washing the sliced specimen with distilled water, the specimen was put between a cover slip and a glass slide to flatten the sample surface.

3. Results

3.1 Ex vivo PR-SHG imaging of human Achilles tendon with uniaxial collagen orientation

3.2 In vivo PR-SHG imaging of dermal collagen fiber in human facial skin

To evaluate the robustness to motion artifacts, we compared α images of the cheek skin of a male subject in his 24 years old continuously acquired with the PC-based PR-SHG microscopy and the mechanical-rotation-based PR-SHG microscopy. Figure 5
Fig. 5 A series of α images (image size = 400 µm by 400 µm region, pixel size = 128 pixels × 128 pixels) acquired every 2 s using the PC-based PR-SHG microscopy (Media 1). The same region in the cheek skin for a male subject in his 24 years old was continuously measured.
shows a series of α images (image size = 400 µm by 400 µm region, pixel size = 128 pixels × 128 pixels) acquired every 2 s (1 s for VPR-SHG imaging and 1 s for HPR-SHG imaging) at the same region in the cheek skin using the PC-based PR-SHG microscopy. Also, Media 1 shows a corresponding movie of a series of continuously acquired VPR-SHG images, HPR-SHG images, and α images. Pores in the skin appeared as a black circle in those images because of no collagen content. Although the captured images fluctuated due to the motion artifacts in the subject, a little bluish tone and its distribution were maintained in the same way for all α images. This result is evidence that there is a pixel-to-pixel correspondence between the VPR-SHG image and the HPR-SHG image under the motion artifacts. On the other hand, α images obtained with the mechanical-rotation-based PR-SHG microscopy made a striking contrast to those obtained with the PC-based PR-SHG microscopy. Figure 6
Fig. 6 A series of α images (image size = 400 µm by 400 µm region, pixel size = 256 pixels × 256 pixels) acquired every 10 s using the mechanical-rotation-based PR-SHG microscopy (Media 2). The same region in the cheek skin for a male subject in his 24 years old was continuously measured.
shows a series of α images (image size = 400 µm by 400 µm region, pixel size = 256 pixels × 256 pixels) acquired every 10 s (2 s for VPR-SHG imaging, 6 s for the mechanical rotation, and 2 s for HPR-SHG imaging) at the same region in the cheek skin of the same subject using the mechanical-rotation-based PR-SHG microscopy, and Media 2 shows a corresponding movie for a series of continuously acquired VPR-SHG images, HPR-SHG images, and α images. The captured images in Fig. 6 and Media 2 also fluctuated due to the motion artifacts in the same manner as that in Fig. 5 and Media 1. However, the behavior of α images is quite different from that in the PC-based PR-SHG microscopy. That is to say, the tone of the α image largely varies between bluish and reddish colors every frame of the α image. This is because the waiting time for the mechanical rotation ( = 6 s) makes it difficult to keep the pixel-to-pixel correspondence between the VPR-SHG image and the HPR-SHG image, resulting in incorrect α images due to pixel mismatching between them. Considering the comparison between Figs. 5 and 6, it should be noted that the data from two different methods were compared with different time acquisition. However, since the SHG microscopy was constructed firmly and the customized attachment ring was used to suppress the motion artifacts of the measured position in the human facial skin, the effect of microscope drift is negligible. Furthermore, although the total image acquisition time ( = 10 s) can be reduced by the fast motor or liquid crystal retarder, we consider that it is still difficult even for these methods to satisfy a pixel-to-pixel correspondence between two PR-SHG images due to the motion artifacts. A comparison of α images between Figs. 5 and 6, or Media 1 and Media 2, underlines the clear advantage of the PC-based PR-SHG microscopy over the mechanical-rotation-based PR-SHG microscopy regarding the motion artifacts.

Finally, to evaluate the potential for in vivo applications in the dermatology, we applied the PC-based PR-SHG microscopy to visualize the distribution of dermal collagen fiber orientation in human facial skin in vivo. We selected two portions of the facial skin for measurement: cheek skin and eye corner skin. To enlarge the imaging region, we acquired an SHG image of a 400 µm by 400 µm regions by using the GM and successively acquired the neighboring regions after scanning the sample position by 400 µm by using a stepping-motor-driven translation stage as shown in Fig. 2(a) [11

11. T. Yasui, M. Yonetsu, R. Tanaka, Y. Tanaka, S. Fukushima, T. Yamashita, Y. Ogura, T. Hirao, H. Murota, and T. Araki, “In vivo observation of age-related structural changes of dermal collagen in human facial skin using collagen-sensitive second harmonic generation microscope equipped with 1250-nm mode-locked Cr:Forsterite laser,” J. Biomed. Opt. 18(3), 031108 (2013). [CrossRef] [PubMed]

, 12

12. R. Tanaka, S. Fukushima, K. Sasaki, Y. Tanaka, H. Murota, T. Matsumoto, T. Araki, and T. Yasui, “In vivo visualization of dermal collagen fiber in skin burn by collagen-sensitive second-harmonic-generation microscopy,” J. Biomed. Opt. 18(6), 061231 (2013). [CrossRef] [PubMed]

]. We obtained a large SHG image with a size of 1.6 mm by 1.6 mm by arranging the 16 acquired SHG images in a matrix of four rows and four lines. Figures 7(a)
Fig. 7 Comparison of α images in human facial skin measured by PC-based PR-SHG microscopy (image size = 1.6 mm by 1.6 mm region). (a) Cheek skin and (b) eye corner skin for a male subject in his 24 years old, and (c) cheek skin and (d) eye corner skin for a male subject in his 57 years old.
and 7(b) respectively show α images in the cheek skin and the eye corner skin for a male subject in his 24 years old. A mixture of bluish and reddish colors in Fig. 7(a) indicated that the cheek skin has a neutral distribution of the vertical and horizontal orientation of the collagen fibers. By contrast, the α image in Fig. 7(b) became more reddish than that in Fig. 7(a), indicating that the horizontal orientation of the collagen fiber is dominant in the eye corner skin. We also performed a similar measurement for a male subject in his 57 years old, as shown in Figs. 7(c) and 7(d). The resulting α images are similar to those in the male subject in his 24 years old, indicating the mixed distribution of vertical and horizontal collagen orientation in his cheek skin and the predominant distribution of horizontal orientation in his eye corner skin. However, bluish color in Fig. 7(a) and reddish color in Fig. 7(b) were enhanced in Figs. 7(c) and 7(d). We will discuss the location and/or age dependence of the collagen fiber orientation in human facial skin later.

4. Discussion

It will be interesting to compare the PR-SHG imaging to another SHG imaging without the need for the polarization rotation because both methods are robust to the motion artifacts in the imaging of the collagen fiber orientation. Recently, the analysis of the collagen fiber orientation in tissues has been achieved based on two-dimensional discrete Fourier transform (2D-FT) of SHG image [24

24. R. A. Rao, M. R. Mehta, and K. C. Toussaint Jr., “Fourier transform-second-harmonic generation imaging of biological tissues,” Opt. Express 17(17), 14534–14542 (2009). [CrossRef] [PubMed]

26

26. A. Ghazaryan, H. F. Tsai, G. Hayrapetyan, W.-L. Chen, Y.-F. Chen, M. Y. Jeong, C.-S. Kim, S.-J. Chen, and C.-Y. Dong, “Analysis of collagen fiber domain organization by Fourier second harmonic generation microscopy,” J. Biomed. Opt. 18(3), 031105 (2013). [CrossRef] [PubMed]

]. In this method, under incidence of a circularly polarized laser light, a single SHG image was obtained, and then 2D-FT analysis of the image was effectively performed for evaluation of collagen fiber orientation. Due to 2D-FT analysis, the calculation of the collagen fiber orientation has been done from a single SHG image without performing PR-SHG imaging. Although this method does not need the polarization rotation and hence can be performed by the simpler setup, SHG image has to be visualized at sufficiently high spatial resolution for correct image analysis with 2D-FT. In other word, this method cannot provide the information at the molecular level due to the insufficient spatial resolution. On the other hand, because the polarization dependence of SHG efficiency is arisen from the molecular axis, PR-SHG imaging will be useful for investigating the information at the molecular level, for example, the calculation of the ratio of the χ(2) tensor elements [27

27. S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90(2), 693–703 (2006). [CrossRef] [PubMed]

].

5. Conclusion

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research Nos. 22300154, 23240069, 23650260, and 25560200 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Japan. We are grateful to Dr. Mamoru Hashimoto of Osaka University for fruitful discussions.

References and links

1.

A. Yariv, Introduction to Optical Electronics, (Holt McDougal, 1977).

2.

S. Fine and W. P. Hansen, “Optical second harmonic generation in biological systems,” Appl. Opt. 10(10), 2350–2353 (1971). [CrossRef] [PubMed]

3.

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4.

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5.

K. König and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8(3), 432–439 (2003). [CrossRef] [PubMed]

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T. Yasui, Y. Takahashi, M. Ito, S. Fukushima, and T. Araki, “Ex vivo and in vivo second-harmonic-generation imaging of dermal collagen fiber in skin: comparison of imaging characteristics between mode-locked Cr:Forsterite and Ti:Sapphire lasers,” Appl. Opt. 48(10), D88–D95 (2009). [CrossRef] [PubMed]

9.

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T. Yasui, M. Yonetsu, R. Tanaka, Y. Tanaka, S. Fukushima, T. Yamashita, Y. Ogura, T. Hirao, H. Murota, and T. Araki, “In vivo observation of age-related structural changes of dermal collagen in human facial skin using collagen-sensitive second harmonic generation microscope equipped with 1250-nm mode-locked Cr:Forsterite laser,” J. Biomed. Opt. 18(3), 031108 (2013). [CrossRef] [PubMed]

12.

R. Tanaka, S. Fukushima, K. Sasaki, Y. Tanaka, H. Murota, T. Matsumoto, T. Araki, and T. Yasui, “In vivo visualization of dermal collagen fiber in skin burn by collagen-sensitive second-harmonic-generation microscopy,” J. Biomed. Opt. 18(6), 061231 (2013). [CrossRef] [PubMed]

13.

P. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da Silva, “Polarization-dependent optical second-harmonic imaging of a rat-tail tendon,” J. Biomed. Opt. 7(2), 205–214 (2002). [CrossRef] [PubMed]

14.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J. 82(6), 3330–3342 (2002). [CrossRef] [PubMed]

15.

T. Yasui, Y. Tohno, and T. Araki, “Characterization of collagen orientation in human dermis by two-dimensional second-harmonic-generation polarimetry,” J. Biomed. Opt. 9(2), 259–264 (2004). [CrossRef] [PubMed]

16.

T. Yasui, Y. Tohno, and T. Araki, “Determination of collagen fiber orientation in human tissue by use of polarization measurement of molecular second-harmonic-generation light,” Appl. Opt. 43(14), 2861–2867 (2004). [CrossRef] [PubMed]

17.

T. Yasui, K. Sasaki, Y. Tohno, and T. Araki, “Tomographic imaging of collagen fiber orientation in human tissue using depth-resolved polarimetry of second-harmonic-generation light,” Opt. Quantum Electron. 37(13-15), 1397–1408 (2005). [CrossRef]

18.

J. C. Mansfield, C. P. Winlove, J. Moger, and S. J. Matcher, “Collagen fiber arrangement in normal and diseased cartilage studied by polarization sensitive nonlinear microscopy,” J. Biomed. Opt. 13(4), 044020 (2008). [CrossRef] [PubMed]

19.

T. Yasui, Y. Takahashi, S. Fukushima, Y. Ogura, T. Yamashita, T. Kuwahara, T. Hirao, and T. Araki, “Observation of dermal collagen fiber in wrinkled skin using polarization-resolved second-harmonic-generation microscopy,” Opt. Express 17(2), 912–923 (2009). [CrossRef] [PubMed]

20.

G. Latour, I. Gusachenko, L. Kowalczuk, I. Lamarre, and M.-C. Schanne-Klein, “In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy,” Biomed. Opt. Express 3(1), 1–15 (2012). [CrossRef] [PubMed]

21.

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 sources,” Opt. Quantum Electron. 34(12), 1251–1266 (2002). [CrossRef]

22.

S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, H.-J. Tsai, and C.-K. Sun, “In vivo developmental biology study using noninvasive multi-harmonic generation microscopy,” Opt. Express 11(23), 3093–3099 (2003). [CrossRef] [PubMed]

23.

S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, “In vivo optical biopsy of hamster oral cavity with epi-third-harmonic-generation microscopy,” Opt. Express 14(13), 6178–6187 (2006). [CrossRef] [PubMed]

24.

R. A. Rao, M. R. Mehta, and K. C. Toussaint Jr., “Fourier transform-second-harmonic generation imaging of biological tissues,” Opt. Express 17(17), 14534–14542 (2009). [CrossRef] [PubMed]

25.

P. Matteini, F. Ratto, F. Rossi, R. Cicchi, C. Stringari, D. Kapsokalyvas, F. S. Pavone, and R. Pini, “Photothermally-induced disordered patterns of corneal collagen revealed by SHG imaging,” Opt. Express 17(6), 4868–4878 (2009). [CrossRef] [PubMed]

26.

A. Ghazaryan, H. F. Tsai, G. Hayrapetyan, W.-L. Chen, Y.-F. Chen, M. Y. Jeong, C.-S. Kim, S.-J. Chen, and C.-Y. Dong, “Analysis of collagen fiber domain organization by Fourier second harmonic generation microscopy,” J. Biomed. Opt. 18(3), 031105 (2013). [CrossRef] [PubMed]

27.

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90(2), 693–703 (2006). [CrossRef] [PubMed]

28.

P. J. Campagnola and C.-Y. Dong, “Second harmonic generation microscopy: principles and applications to disease diagnosis,” Laser Photon. Rev. 5(1), 13–26 (2011). [CrossRef]

OCIS Codes
(170.1870) Medical optics and biotechnology : Dermatology
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(190.4160) Nonlinear optics : Multiharmonic generation
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Microscopy

History
Original Manuscript: January 13, 2014
Revised Manuscript: February 16, 2014
Manuscript Accepted: March 2, 2014
Published: March 7, 2014

Citation
Yuji Tanaka, Eiji Hase, Shuichiro Fukushima, Yuki Ogura, Toyonobu Yamashita, Tetsuji Hirao, Tsutomu Araki, and Takeshi Yasui, "Motion-artifact-robust, polarization-resolved second-harmonic-generation microscopy based on rapid polarization switching with electro-optic Pockells cell and its application to in vivo visualization of collagen fiber orientation in human facial skin," Biomed. Opt. Express 5, 1099-1113 (2014)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-5-4-1099


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References

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  11. T. Yasui, M. Yonetsu, R. Tanaka, Y. Tanaka, S. Fukushima, T. Yamashita, Y. Ogura, T. Hirao, H. Murota, and T. Araki, “In vivo observation of age-related structural changes of dermal collagen in human facial skin using collagen-sensitive second harmonic generation microscope equipped with 1250-nm mode-locked Cr:Forsterite laser,” J. Biomed. Opt.18(3), 031108 (2013). [CrossRef] [PubMed]
  12. R. Tanaka, S. Fukushima, K. Sasaki, Y. Tanaka, H. Murota, T. Matsumoto, T. Araki, and T. Yasui, “In vivo visualization of dermal collagen fiber in skin burn by collagen-sensitive second-harmonic-generation microscopy,” J. Biomed. Opt.18(6), 061231 (2013). [CrossRef] [PubMed]
  13. P. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da Silva, “Polarization-dependent optical second-harmonic imaging of a rat-tail tendon,” J. Biomed. Opt.7(2), 205–214 (2002). [CrossRef] [PubMed]
  14. P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J.82(6), 3330–3342 (2002). [CrossRef] [PubMed]
  15. T. Yasui, Y. Tohno, and T. Araki, “Characterization of collagen orientation in human dermis by two-dimensional second-harmonic-generation polarimetry,” J. Biomed. Opt.9(2), 259–264 (2004). [CrossRef] [PubMed]
  16. T. Yasui, Y. Tohno, and T. Araki, “Determination of collagen fiber orientation in human tissue by use of polarization measurement of molecular second-harmonic-generation light,” Appl. Opt.43(14), 2861–2867 (2004). [CrossRef] [PubMed]
  17. T. Yasui, K. Sasaki, Y. Tohno, and T. Araki, “Tomographic imaging of collagen fiber orientation in human tissue using depth-resolved polarimetry of second-harmonic-generation light,” Opt. Quantum Electron.37(13-15), 1397–1408 (2005). [CrossRef]
  18. J. C. Mansfield, C. P. Winlove, J. Moger, and S. J. Matcher, “Collagen fiber arrangement in normal and diseased cartilage studied by polarization sensitive nonlinear microscopy,” J. Biomed. Opt.13(4), 044020 (2008). [CrossRef] [PubMed]
  19. T. Yasui, Y. Takahashi, S. Fukushima, Y. Ogura, T. Yamashita, T. Kuwahara, T. Hirao, and T. Araki, “Observation of dermal collagen fiber in wrinkled skin using polarization-resolved second-harmonic-generation microscopy,” Opt. Express17(2), 912–923 (2009). [CrossRef] [PubMed]
  20. G. Latour, I. Gusachenko, L. Kowalczuk, I. Lamarre, and M.-C. Schanne-Klein, “In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy,” Biomed. Opt. Express3(1), 1–15 (2012). [CrossRef] [PubMed]
  21. 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 sources,” Opt. Quantum Electron.34(12), 1251–1266 (2002). [CrossRef]
  22. S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, H.-J. Tsai, and C.-K. Sun, “In vivo developmental biology study using noninvasive multi-harmonic generation microscopy,” Opt. Express11(23), 3093–3099 (2003). [CrossRef] [PubMed]
  23. S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, “In vivo optical biopsy of hamster oral cavity with epi-third-harmonic-generation microscopy,” Opt. Express14(13), 6178–6187 (2006). [CrossRef] [PubMed]
  24. R. A. Rao, M. R. Mehta, and K. C. Toussaint., “Fourier transform-second-harmonic generation imaging of biological tissues,” Opt. Express17(17), 14534–14542 (2009). [CrossRef] [PubMed]
  25. P. Matteini, F. Ratto, F. Rossi, R. Cicchi, C. Stringari, D. Kapsokalyvas, F. S. Pavone, and R. Pini, “Photothermally-induced disordered patterns of corneal collagen revealed by SHG imaging,” Opt. Express17(6), 4868–4878 (2009). [CrossRef] [PubMed]
  26. A. Ghazaryan, H. F. Tsai, G. Hayrapetyan, W.-L. Chen, Y.-F. Chen, M. Y. Jeong, C.-S. Kim, S.-J. Chen, and C.-Y. Dong, “Analysis of collagen fiber domain organization by Fourier second harmonic generation microscopy,” J. Biomed. Opt.18(3), 031105 (2013). [CrossRef] [PubMed]
  27. S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J.90(2), 693–703 (2006). [CrossRef] [PubMed]
  28. P. J. Campagnola and C.-Y. Dong, “Second harmonic generation microscopy: principles and applications to disease diagnosis,” Laser Photon. Rev.5(1), 13–26 (2011). [CrossRef]

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