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Multiharmonic-generation biopsy of skin Chi-Kuang Sun, Cheng-Chi Chen, Shi-Wei Chu, and Tsung-Han Tsai Department of Electrical Engineering and Graduate Institute of Electro-Optical Engineering, National Taiwan University, Taipei 10617, Taiwan
Yung-Chih Chen and Bai-Ling Lin Molecular and Cell Biology Division, Development Center for Biotechnology, Taipei 10672, Taiwan Received July 8, 2003 Because it avoids the in-focus photodamage and phototoxicity problem of two-photon-f luorescence excitation, multiharmonic-generation biopsy based on a 1200 – 1300-nm light source could provide a truly noninvasive and highly penetrative optical sectioning of skin. We study multiharmonic-generation biopsy of fixed mouse skin. Our preliminary study suggests that this technique could provide submicrometer-resolution deep-tissue noninvasive biopsy images in skin without the use of f luorescence and exogenous markers. © 2003 Optical Society of America OCIS codes: 170.1870, 180.6900, 180.5810, 190.4160.
Traditional biopsy requires the removal, f ixation, and staining of tissues, cells, or f luids from a living body. The histological procedure is invasive and painful. Noninvasive in vivo optical biopsy is thus required, which should provide highly penetrative, threedimensional (3D) imaging with submicrometer spatial resolution. Optical coherence tomography1 – 3 (OCT) is one potential method for optical biopsy of skin that can provide high penetration by use of 1.3-mm-wavelength light1 or submicrometer axial resolution with a 725-nm broadband light source.3 Optical biopsy based on scanning two-photon f luorescence microscopy is another choice.4,5 Compared with OCT, laser-scanning nonlinear microscopy provides much higher lateral resolution while the sectioned two-dimensional images are in the plane parallel to the surface, in contrast with the cross-sectional (tangential) images provided by OCT. Previous two-photon f luorescence biopsy of skin4,5 based on 780-nm femtosecond light provided high-resolution imaging from the skin surface through the epidermal –dermal junction. However, for future clinical applications without surgery, current 700– 850-nm-based laser scanning technology still presents several limitations, including low penetration depth, in-focus cell damage, and multiphoton phototoxicity as a result of high optical intensity in the 800-nm wavelength region and toxicity as a result of required exogenous f luorescence markers. Several mechanisms that cause nonlinear photon damage with 740– 800-nm excitation were recently identified, including oxidative photodamage caused by two-photon excitation of endogenous and exogenous f luorophores6 or multiphoton-induced plasma generation.7,8 These reports on the multiphotonabsorption-induced photodamage by Ti:sapphire lasers in two-photon f luorescence microscopy indicate the importance of reducing possible tissue damage while performing optical biopsy of skin with a high-intensity light source. These photodamage phenomena also limit the maximum optical intensity applicable in a two-photon f luorescence microscopy–based biopsy system, which limits the signal intensity and thus the penetration depth. Previous studies suggested that, with an 80-MHz Ti:sapphire laser and an objective 0146-9592/03/242488-03$15.00/0
with a N.A. of ⬃1, less than 7-mW average power could be applied to a live subject, which would prevent possible optical damage.6 A previous study also indicated that increasing the excitation laser wavelength to 1047 nm will allow long-term two-photon f luorescence imaging of mammalian embryos without compromising viability; however, the average illumination power was still limited to 13 mW, and a total exposure of only 2 J could be applied to one embryo within a 24-h imaging period.9 In this Letter we report a study of multiharmonicgeneration biopsy of excised mouse skin, using a femtosecond Cr:forsterite laser centered at 1230 nm.10 The structures in the human dermis observed by in vivo two-photon imaging were found to be similar to those of excised mouse skin.5 According to a previous study, light attenuation (including both absorption and scattering) in human skin11 reaches a minimum near the 1200– 1300-nm wavelength because of the combination of diminishing scattering cross section with increasing wavelength and avoiding the resonant molecular absorption of common tissue constituents such as water, melanin, and hemoglobin. Previous studies comparing contrast and penetration depth between 800- and 1300-nm light sources for OCT in live tissues suggested superior performance at 1200–1300 nm.1 Moving the operating wavelength of a nonlinear optical biopsy to the 1200– 1300-nm spectral region can not only increase the penetration depth in skin but also reduce the multiphoton absorption cross section9 and thus reduce the potential photodamage and phototoxicity. Our recent study using a femtosecond Cr:forsterite (1230-nm) laser indicated that zebraf ish embryos can be imaged repeatedly over the course of hours to days without observable damage and that the embryos all developed into normal healthy larvae even when exposed to 100-mW average illumination power, for a corresponding total exposure of 1 –10 kJ per embryo.12 Even though the long excitation wavelength suppresses autof luorescence in skin, moving the excitation wavelength to longer than 1200 nm makes third-harmonic generation (THG) and second-harmonic generation (SHG) microscopy © 2003 Optical Society of America
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possible because with the visible THG and SHG wavelengths one can avoid strong UV absorption in biotissues. Harmonic generation is known to deposit no energy in the matter with which it interacts because of the energy-conservation characteristic of the generation. This characteristic provides the noninvasive nature desirable for clinical imaging. Because of their nonlinear nature, the generated SHG and THG intensities depend on the square and the cube of the incident light intensity, respectively. Similarly to the multiphoton-induced f luorescence process, this nonlinear dependence allows localized excitation and is ideal for intrinsic optical sectioning in scanning laser microscopy. A study of multiharmonic-generation biopsy of excised mouse skin was performed with a femtosecond Cr:forsterite laser centered at 1230 nm with a 140-fs pulse width at a 110-MHz repetition rate. Although the whole nonlinear spectrum in the visible and near-IR region was acquired,13 no autof luorescence except strong SHG and THG signals could be found from the mouse skin and ear samples under our experimental conditions. The IR laser beam was f irst shaped by a telescope and then directed into a modified beam scanning system (Olympus Fluoview 300) and a microscope (Olympus BX-51) with an IR waterimmersion objective (LUMplanFL/IR 603 兾N.A. 0.9). The scattered and ref lected backward SHG signals were collected in the ref lection geometry by use of the same objective; the signals passed through the same scanner, f iltered by a 615-nm narrowband interference filter, and detected by a photomultiplier tube. The transmitted forward SHG and THG were collected by a 1.2-N.A. achromatic condenser, divided by a chromatic beam splitter, and detected by two separate photomultiplier tubes with 410- and 615-nm narrowband interference f ilters in front. We did not detect backward THG in this study because of the limitation of the available chromatic beam splitters. Average illumination power of 50– 100 mW was applied to the sample during the study. The diffractionlimited spatial resolution was 1000 nm in the axial and 400 nm in the radial direction for transmission THG microscopy. The fixed integument samples were removed from the caudal dorsal plane of B6 mice and preserved in formalin at 4 ±C. Most of the hair of the B6 mice was removed before they were euthanized in a CO2 chamber. The thickness of the samples under observation was of the order of 0.5 –1 mm. Figure 1 shows the backward- (B-) SHG, forward (F-) SHG, F-THG, and combined cross-sectional (x z) as well as the paradermal (x y) scanning sectioned images. Because of momentum conservation, the generated harmonic signals tend toward forward emission and can thus be collected with the present transmission geometry. However, for future optical biopsy, ref lection geometry will be preferred for signal collection because human bodies are optically thick. The images in Fig. 1 were log processed to overcome the finite dynamic range in the 12-bit system. From the cross-sectional images, the general histological structures in both the epidermis and dermis layer can be identified with THG because of its sensitivity to local
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optical inhomogeneity.13 – 15 The cover glass – formalin interface was also picked up by THG in the top part of the cross-sectional image. F-SHG, on the other hand, ref lects the distribution of connected tissues in the dermis layer.16 – 18 An abrupt disappearance of SHG signals close to the bottom of the cross-sectional image suggests the reach of the dermis –hypodermis junction, while the loosely structured hypodermis layer does not provide significant SHG and THG signals. This was confirmed with histological sectioning of the same sample. It is interesting to compare the B-SHG images with the F-SHG images. It can be found that B-SHG not only displays the distribution of the generation sources (such as connective tissues) but also the scattering and ref lection properties of the tissues. Our detected B-SHG intensity is of the order of 1兾10 of the F-SHG intensity, indicating the importance of scattering in the collection of B-SHG. The rete ridges that are due to the dermal papillae interdigitated with the downward projections of the epidermis can clearly be seen.19 Taking a paradermal sectioning in the surface region (Fig. 1, bottom row), we find that the observed THG images were dominated by the top contour prof ile of the stratum corneum because of the large index difference between the stratum corneum –formalin interface, while weak THG signals pick up f ine structures, including wavy collagen f ibers in connected tissues in both the epidermal and the dermal layers. Figure 2 shows paradermal images taken in a smoother region so that detailed tissue structures picked up by the THG can be easily revealed. These images are shown on their original linear scales. Figure 2(a) shows a sectioned image in the stratum basale layer on top of the epidermis– dermis junction. The cell membranes of densely packed basal cells can be clearly resolved by THG signals. Connective tissue in the dermis layer that generates SHG can already be seen on the right-hand side of the image because of the unf lat
Fig. 1. (a) B-SHG, (b) F-SHG, (c) F-THG, and (d) the combined cross-sectional (x z, into the skin, top row) as well as the paradermal (x y, parallel to the surface, bottom row) scanning images of mouse skin. B-SHG, F-SHG, and F-THG signals are denoted by green, red, and blue, respectively. The dotted lines in the top (bottom) row mark the corresponding z ( y) position for the bottom (top) row. Image size: 235 mm 3 310 mm for x z images and 235 mm 3 235 mm for x y images. i, cover glass – formalin interface; e, epidermis layer; de, dermis layer.
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Fig. 2. Paradermal multiharmonic-generation biopsy images of mouse skin. (a) Sectioned image in the stratum basale layer. Image size: 59 mm 3 59 mm. (b) Sectioned image of hairs above skin. (c) Sectioned image deep in the dermis. Image size for (b) and (c): 235 mm 3 235 mm. h, hair; s, sebaceous gland. B-SHG, F-SHG, and F-THG signals are denoted by green, red, and blue, respectively.
noninvasive biopsy tool with excellent 3D spatial resolution because of its virtual transition and nonlinear characteristics. Our preliminary study suggests that the multiharmonic generation technique, combined with a 1200–1300-nm light source, could provide submicrometer-resolution deep-tissue noninvasive biopsy images of animal skin without the use of f luorescence and exogenous markers. By improving detection sensitivity, this technique could be useful for noninvasive biopsy of skin diseases and even for early diagnosis of skin cancer symptoms such as angiogenesis. This work was sponsored by the National Health Research Institute of Taiwan, Republic of China, through grant NHRI-EX92-9201EI. C.-K. Sun’s e-mail address is
[email protected]. References
Fig. 3. Multiharmonic-generation biopsy images of a mouse ear. (a) Paradermal sectioned F-SHG image inside the mouse’s external ear showing the elastic cartilage. Image size: 235 mm 3 235 mm. (b) and (c) are the 3D reconstructed transverse and longitudinal F-THG images of capillaries inside the mouse’s external ear, respectively. Image size: 80 mm 3 80 mm. p, capillary.
rete ridge structure. Figure 2(b) shows a clear image of hair taken from the region above the epidermis. With the focal plane moved deep into the dermal layer, Fig. 2(c) shows a typical sectioned image in which the SHG picks up the connective tissues consisting of collagen f ibers and the THG ref lects the hairs and the surrounding sebaceous glands (S). To take advantage of the forward-emission characteristic of harmonic-generation biopsy, we also performed harmonic-generation biopsy in the transmission geometry with a f ixed external ear sample from a mouse. Figure 3(a) shows the F-SHG sectioning images inside the mouse’s external ear. With a 500-mm sample thickness, we found that 1230-nm light could penetrate the whole sample, while the THG picked up the prof iles of the epithelial cells on both sides of the mouse ear (not shown). SHG ref lects the elastic cartilage in the center of the mouse ear, which is composed of collagen fibrils [Fig. 3(a)]. Figures 3(b) and 3(c) are 3D reconstructed transverse and longitudinal THG images of capillaries with an ⬃10-mm diameter inside the mouse’s external ear, showing the excellent 3D resolution of the THG biopsy technique. In summary, current 800-nm-based two-photon optical biopsy technology still presents several limitations, including f inite penetration depth, on-focus cell damage, and phototoxicity as a result of the required high optical intensity and multiphoton absorption processes. Harmonic generation, however, can provide a truly
1. G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Boppart, C. Pitris, J. F. Southern, and J. G. Fujimoto, Science 276, 2037 (1997). 2. Y. Yasuno, S. Makita, Y. Sutoh, M. Itoh, and T. Yatagai, Opt. Lett. 27, 1803 (2002). 3. B. Povazay, K. Bizheva, A. Unterhuber, B. Hermann, H. Sattmann, A. F. Fercher, W. Drexler, A. Apolonski, W. J. Wadsworth, J. C. Knight, P. St. J. Russell, M. Vetterlein, and E. Scherzer, Opt. Lett. 27, 1800 (2002). 4. P. T. C. So, H. Kim, and I. E. Kochevar, Opt. Express 3, 339 (1998), http://www.opticsexpress.org. 5. B. R. Masters and P. T. C. So, Opt. Express 8, 2 (2001), http://www.opticsexpress.org. 6. U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and J.-J. Halbhuber, Exp. Cell Res. 263, 88 (2001). 7. K. König, P. T. C. So, W. W. Mantulin, and E. Gratton, Opt. Lett. 22, 135 (1997). 8. I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, Opt. Quantum Electron. 34, 1251 (2002). 9. J. M. Squirell, D. L. Wokosin, J. G. White, and B. D. Bavister, Nature Biotechnol. 17, 763 (1999). 10. T.-M. Liu, S.-W. Chu, C.-K. Sun, B.-L. Lin, P. C. Cheng, and I. Johnson, Scanning 23, 249 (2001). 11. R. R. Anderson and J. A. Parish, J. Investig. Dermatol. 77, 13 (1981). 12. C.-K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, “Harmonic generation microscopy for developmental biology,” J. Struct. Biol. (to be published). 13. S.-W. Chu, I.-H. Chen, T.-M. Liu, P. C. Cheng, C.-K. Sun, and B.-L. Lin, Opt. Lett. 26, 1909 (2001). 14. D. Yelin and Y. Silberberg, Opt. Express 5, 169 (1999), http://www.opticsexpress.org. 15. J. A. Squier, M. Muller, G. J. Brakenhoff, and K. R. Wilson, Opt. Express 3, 315 (1999), http:// www.opticsexpress.org. 16. Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, Opt. Lett. 22, 1323 (1997). 17. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002). 18. S.-W. Chu, I.-S. Chen, T.-M. Liu, C.-K. Sun, S.-P. Lee, B.-L. Lin, P.-C. Cheng, M.-X. Kuo, D.-J. Lin, and H.-L. Liu, J. Microsc. 208, 190 (2002). 19. H. Elias, J. E. Pauly, and E. R. Burns, Histology and Human Microanatomy, 4th ed. (Piccin, Padova, Italy, 1978).
