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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 2, Iss. 8 — Aug. 1, 2011
  • pp: 2317–2328

In vivo optical virtual biopsy of human oral mucosa with harmonic generation microscopy

Ming-Rung Tsai, Szu-Yu Chen, Dar-Bin Shieh, Pei-Jen Lou, and Chi-Kuang Sun  »View Author Affiliations

Biomedical Optics Express, Vol. 2, Issue 8, pp. 2317-2328 (2011)

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Recent clinical studies on human skin indicated that in vivo multi-harmonic generation microscopy (HGM) can achieve sub-micron resolution for histopathological analysis with a high penetration depth and leave no energy or photodamages in the interacted tissues. It is thus highly desired to apply HGM for in vivo mucosa histopathological diagnosis. In this paper, the first in vivo optical virtual biopsy of human oral mucosa by using epi-HGM is demonstrated. We modified an upright microscope to rotate the angle of objective for in vivo observation. Our clinical study reveals the capability of HGM to in vivo image cell distributions in human oral mucosa, including epithelium and lamina propria with a high penetration depth greater than 280 μm and a high spatial resolution better than 500 nm. We also found that the third-harmonic-generation (THG) contrast on nucleus depends strongly on its thicknesses, in agreement with a numerical simulation. Besides, 4% acetic acid was found to be able to enhance the THG contrast of nucleus in oral mucosa, while such enhancement was found to decay due to the metabolic clearance of the contrast enhancer by the oral mucosa. Our clinical study indicated that, the combined epi-THG and epi-second-harmonic-generation (SHG) microscopy is a promising imaging tool for in vivo noninvasive optical virtual biopsy and disease diagnosis in human mucosa.

© 2011 OSA

OCIS Codes
(180.5810) Microscopy : Scanning microscopy
(180.6900) Microscopy : Three-dimensional microscopy
(190.4160) Nonlinear optics : Multiharmonic generation

ToC Category:

Original Manuscript: April 7, 2011
Revised Manuscript: July 16, 2011
Manuscript Accepted: July 20, 2011
Published: July 21, 2011

Ming-Rung Tsai, Szu-Yu Chen, Dar-Bin Shieh, Pei-Jen Lou, and Chi-Kuang Sun, "In vivo optical virtual biopsy of human oral mucosa with harmonic generation microscopy," Biomed. Opt. Express 2, 2317-2328 (2011)

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  1. N. Houssami, S. Ciatto, I. Ellis, and D. Ambrogetti, “Underestimation of malignancy of breast core-needle biopsy: concepts and precise overall and category-specific estimates,” Cancer 109(3), 487–495 (2007). [CrossRef] [PubMed]
  2. G. C. Zografos, F. Zagouri, T. N. Sergentanis, A. Nonni, D. Koulocheri, M. Fotou, E. Panopoulou, N. Pararas, C. Fotiadis, and J. Bramis, “Minimizing underestimation rate of microcalcifications excised via vacuum-assisted breast biopsy: a blind study,” Breast Cancer Res. Treat. 109(2), 397–402 (2008). [CrossRef] [PubMed]
  3. F. Feldchtein, V. Gelikonov, R. Iksanov, G. Gelikonov, R. Kuranov, A. Sergeev, N. Gladkova, M. Ourutina, D. Reitze, and J. Warren, “In vivo OCT imaging of hard and soft tissue of the oral cavity,” Opt. Express 3(6), 239–250 (1998). [CrossRef] [PubMed]
  4. J. M. Ridgway, W. B. Armstrong, S. Guo, U. Mahmood, J. Su, R. P. Jackson, T. Shibuya, R. L. Crumley, M. Gu, Z. Chen, and B. J. Wong, “In vivo optical coherence tomography of the human oral cavity and oropharynx,” Arch. Otolaryngol. Head Neck Surg. 132(10), 1074–1081 (2006). [CrossRef] [PubMed]
  5. N. Ozawa, Y. Sumi, K. Shimozato, C. Chong, and T. Kurabayashi, “In vivo imaging of human labial glands using advanced optical coherence tomography,” Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 108(3), 425–429 (2009). [CrossRef] [PubMed]
  6. W. M. White, M. Rajadhyaksha, S. González, R. L. Fabian, and R. R. Anderson, “Noninvasive imaging of human oral mucosa in vivo by confocal reflectance microscopy,” Laryngoscope 109(10), 1709–1717 (1999). [CrossRef] [PubMed]
  7. K.-B. Sung, C. Liang, M. Descour, T. Collier, M. Follen, and R. Richards-Kortum, “Fiber-optic confocal reflectance microscope with miniature objective for in vivo imaging of human tissues,” IEEE Trans. Biomed. Eng. 49(10), 1168–1172 (2002). [CrossRef] [PubMed]
  8. K. C. Maitland, A. M. Gillenwater, M. D. Williams, A. K. El-Naggar, M. R. Descour, and R. R. Richards-Kortum, “In vivo imaging of oral neoplasia using a miniaturized fiber optic confocal reflectance microscope,” Oral Oncol. 44(11), 1059–1066 (2008). [CrossRef] [PubMed]
  9. P. Wilder-Smith, K. Osann, N. Hanna, N. E. 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]
  10. J. Sun, T. Shilagard, B. Bell, M. Motamedi, and G. Vargas, “In vivo multimodal nonlinear optical imaging of mucosal tissue,” Opt. Express 12(11), 2478–2486 (2004). [CrossRef] [PubMed]
  11. 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]
  12. A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80(4), 2029–2036 (2001). [CrossRef] [PubMed]
  13. 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]
  14. C.-K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, “Higher harmonic generation microscopy for developmental biology,” J. Struct. Biol. 147(1), 19–30 (2004). [CrossRef] [PubMed]
  15. S.-Y. Chen, C.-S. Hsieh, S.-W. Chu, C.-Y. Lin, C.-Y. Ko, Y.-C. Chen, H.-J. Tsai, C.-H. Hu, and C.-K. Sun, “Noninvasive harmonics optical microscopy for long-term observation of embryonic nervous system development in vivo,” J. Biomed. Opt. 11(5), 054022 (2006). [CrossRef] [PubMed]
  16. C.-S. Hsieh, S.-U. Chen, Y.-W. Lee, Y.-S. Yang, and C.-K. Sun, “Higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos,” Opt. Express 16(15), 11574–11588 (2008). [PubMed]
  17. C.-S. Hsieh, C.-Y. Ko, S.-Y. Chen, T.-M. Liu, J.-S. Wu, C.-H. Hu, and C.-K. Sun, “In vivo long term continuous observation of gene expression in zebrafish embryos nerve systems by using harmonic generation microscopy and morphant technology,” J. Biomed. Opt. 13(6), 064041 (2008). [CrossRef] [PubMed]
  18. S.-Y. Chen, S.-U. Chen, H.-Y. Wu, W.-J. Lee, Y.-H. Liao, and C.-K. Sun, “In vivo virtual biopsy of human skin by using noninvasive higher harmonic generation microscopy,” IEEE J. Sel. Top. Quantum Electron. 16(3), 478–492 (2010). [CrossRef]
  19. S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, “In vivo harmonic generation biopsy of human skin,” J. Biomed. Opt. 14(6), 060505 (2009). [CrossRef] [PubMed]
  20. J. M. Schins, T. Schrama, J. Squier, G. J. Brakenhoff, and M. Muller, “Determination of material properties by use of third-harmonic generation microscopy,” J. Opt. Soc. Am. B 19(7), 1627–1634 (2002). [CrossRef]
  21. S.-W. Chu, I.-H. Chen, T.-M. Liu, P. C. Chen, C.-K. Sun, and B.-L. Lin, “Multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser,” Opt. Lett. 26(23), 1909–1911 (2001). [CrossRef] [PubMed]
  22. S.-H. Chia, T.-M. Liu, A. A. Ivanov, A. B. Fedotov, A. M. Zheltikov, M.-R. Tsai, M.-C. Chan, C.-H. Yu, and C.-K. Sun, “A sub-100 fs self-starting Cr:forsterite laser generating 1.4 W output power,” Opt. Express 18(23), 24085–24091 (2010). [CrossRef] [PubMed]
  23. B. E. Bouma, G. J. Tearney, I. P. Bilinsky, B. Golubovic, and J. G. Fujimoto, “Self-phase-modulated Kerr-lens mode-locked Cr:forsterite laser source for optical coherence tomography,” Opt. Lett. 21(22), 1839–1841 (1996). [CrossRef] [PubMed]
  24. C.-K. Sun, “Higher harmonic generation microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 17–56 (2005). [PubMed]
  25. A. N. S. I. Standard, Z136.1–2007, American National Standard for the Safe Use of Lasers (2007).
  26. S.-W. Chu, S.-P. Tai, C.-L. Ho, C.-H. Lin, and C.-K. Sun, “High-resolution simultaneous three-photon fluorescence and third-harmonic-generation microscopy,” Microsc. Res. Tech. 66(4), 193–197 (2005). [CrossRef] [PubMed]
  27. V. Barzda, C. Greenhalgh, J. Aus der Au, S. Elmore, J. van Beek, and J. Squier, “Visualization of mitochondria in cardiomyocytes by simultaneous harmonic generation and fluorescence microscopy,” Opt. Express 13(20), 8263–8276 (2005). [CrossRef] [PubMed]
  28. A. C. Millard, D. N. Fittinghoff, P. W. Wiseman, M. Muller, G. J. Brakenhoff, J. A. Squier, and K. R. Wilson, “Three dimensional, third harmonic microscopy of living systems,” Biophys. J. 78, 800 (2000). [PubMed]
  29. D. W. Fawcett, “Oral cavity and associated glands,” in A Textbook of Histology (Saunders, 1986), pp. 579–601.
  30. H. G. Burkitt, B. Young, and J. W. Heath, Wheater's Functional Histology: A Text and Colour Atlas, (Churchill-Livingstone, 1993), pp. 235–246.
  31. S.-W. Chu, I.-H. 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, “Nonlinear bio-photonic crystal effects revealed with multimodal nonlinear microscopy,” J. Microsc. 208(3), 190–200 (2002). [CrossRef] [PubMed]
  32. S.-W. Chu, S.-P. Tai, M.-C. Chan, C.-K. Sun, I.-C. Hsiao, C.-H. Lin, Y.-C. Chen, and B.-L. Lin, “Thickness dependence of optical second harmonic generation in collagen fibrils,” Opt. Express 15(19), 12005–12010 (2007). [CrossRef] [PubMed]
  33. C.-K. Sun, C.-H. Yu, S.-P. Tai, C.-T. Kung, I.-J. Wang, H.-C. Yu, H.-J. Huang, W.-J. Lee, Y.-F. Chan, and C.-K. Sun, “In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy,” Opt. Express 15(18), 11167–11177 (2007). [CrossRef] [PubMed]
  34. C.-F. Chang, C.-H. Yu, and C.-K. Sun, “Multi-photon resonance enhancement of third harmonic generation in human oxyhemoglobin and deoxyhemoglobin,” J Biophotonics 3(10-11), 678–685 (2010). [CrossRef] [PubMed]
  35. J. G. Cowpe, R. B. Longmore, and M. W. Green, “Quantitative exfoliative cytology of abnormal oral mucosal smears,” J. R. Soc. Med. 81(9), 509–513 (1988). [PubMed]
  36. A. H. Hande and M. S. Chaudhary, “Cytomorphometric analysis of buccal mucosa of tobacco chewers,” Rom. J. Morphol. Embryol. 51(3), 527–532 (2010). [PubMed]
  37. M. A. Landay and H. E. Schroeder, “Quantitative electron microscopic analysis of the stratified epithelium of normal human buccal mucosa,” Cell Tissue Res. 177(3), 383–405 (1977). [CrossRef] [PubMed]
  38. S.-P. Tai, T.-H. Tsai, W.-J. Lee, D.-B. Shieh, Y.-H. Liao, H.-Y. Huang, K. Y.-J. Zhang, H.-L. Liu, and C.-K. Sun, “Optical biopsy of fixed human skin with backward-collected optical harmonics signals,” Opt. Express 13(20), 8231–8242 (2005). [CrossRef] [PubMed]
  39. T.-H. Tsai, S.-P. Tai, W.-J. Lee, H.-Y. Huang, Y.-H. Liao, and C.-K. Sun, “Optical signal degradation study in fixed human skin using confocal microscopy and higher-harmonic optical microscopy,” Opt. Express 14(2), 749–758 (2006). [CrossRef] [PubMed]
  40. J. Sato, M. Yanai, T. Hirao, and M. Denda, “Water content and thickness of the stratum corneum contribute to skin surface morphology,” Arch. Dermatol. Res. 292(8), 412–417 (2000). [CrossRef] [PubMed]
  41. A. Myakov, L. Nieman, L. Wicky, U. Utzinger, R. Richards-Kortum, and K. Sokolov, “Fiber optic probe for polarized reflectance spectroscopy in vivo: design and performance,” J. Biomed. Opt. 7(3), 388–397 (2002). [CrossRef] [PubMed]
  42. C. J. Balas, G. C. Themelis, E. P. Prokopakis, I. Orfanudaki, E. Koumantakis, and E. S. Helidonis, “In vivo detection and staging of epithelial dysplasias and malignancies based on the quantitative assessment of acetic acid-tissue interaction kinetics,” J. Photochem. Photobiol. B 53(1-3), 153–157 (1999). [CrossRef] [PubMed]
  43. W.-J. Lee, C.-H. Yu, S.-P. Tai, H.-Y. Huang, and C.-K. Sun, “Acetic acid as a cell nucleus contrast agent in third-harmonic generation microscopy,” J. Med. Biol. Eng. 27, 161–164 (2007).
  44. A. B. MacLean, “Acetowhite epithelium,” Gynecol. Oncol. 95(3), 691–694 (2004). [CrossRef] [PubMed]

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