3D OPTICAL IMAGING OF AN INTAC TYMPANIC MEMBRANE BY USING HARMONIC GENERATION MICROSCOPY

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3D OPTICAL IMAGING OF AN INTAC TYMPANIC MEMBRANE BY USING HARMONIC GENERATION MICROSCOPY
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  3D OPTICAL IMAGING OF AN INTAC TYMPANIC MEMBRANE BY USING HARMONIC GENERATION MICROSCOPY Chi-Kuang Sun a , Szu-Yu Chen b , and Wen-Jeng Lee c a Graduate Institute of Electro-Optical Engineering and Department of Electrical Engineering, National Taiwan University, and Research Center for Applied Sciences, Academia Sinica. b Graduate Institute of Electro-Optical Engineering and Department of Electrical Engineering, National Taiwan University. c Graduate Institute of Electrical Engineering, National Taiwan University. No.1 Sec.4 Roosevelt Road, Taipei, 10617 TAIWAN, R.O.C Email: sun@cc.ee.ntu.edu.tw KEY WORDS: Tympanic membrane, 3D imaging, virtual sectioning, harmonic generation microscopy, noninvasive, diagnostic tool. The tympanic membrane (TM) is a thin membrane that separates the ear canal and the middle ear. The TM can transfer the vibration of sound waves to attached auditory ossicles, and is important for protecting the middle ear. Perforations of TM often occur and lead to hearing loss. To know how perforations affect TM’s work, the vibration model of TM is needed. To  build a vibration model, thickness distribution of an intact TM is needed. Traditionally, an excised and flatten TM is used for analyzing [1]. However, distortions may result from sample preparation. Harmonic generation microscopy (HGM), combining second- and third-harmonic generation (SHG and THG), obeys energy conservation, and least-invasive nature and high penetrability can be achieved by using an infrared laser source. Based on intrinsic sectioning ability, HGM has high 3D spatial resolution. For an average thickness of 100 μ m, HGM is thus ideal for in vivo analyzing 3D thickness distribution of the TM. THG can reveal interfaces between materials with different refractive index, while SHG has contrast on non-centrosymmetric and highly organized structures [2]. Figure 1 shows the epi-SHG and epi-THG images of the TM. The middle layer, composed of collagen fibers, was revealed by strong epi-SHG (Fig. 1(a)) while two epithelium layers (Fig. 1(b)) were revealed  by epi-THG. Based on virtual sectioning capability, the thickness distribution of an intact TM can be successfully obtained by using HGM. Due to the noninvasive nature, HGM can also be developed to be a in vivo diagnostic tool. Due to high spatial resolution, extremely fine  perforations and changes can be diagnosed. Moreover, using strong SHG from the collagen, the healing process of TM, which relaying on collagen, can be studied in vivo. This project is sponsored by National Health Research Institute of Taiwan. [1] W. F. S. Decraemer et al .  “Measurement of the thickness of the tympanic membrane in cat using a heterodyne interferometer,” P. Proceedings of the SPIE  , 5503,  106-115 (2004). [2] S.-W. Chu   et al. “Nonlinear bio-photonic crystal effects revealed with multi-modal nonlinear microscopy,”  J. Microscopy 208 , 190-200   (2002). Figure 1: SHG (a) and THG (b) images of TMinaxialdirection.Scalebar:200m.   (a)(b)
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