組織內三倍頻之分子對比與外加顯影劑

這篇論文的目的是利用了以中心波長為1230nm的鉻貴橄欖石雷射做為光源的非線性雷射掃瞄顯微鏡系統去探索生物體中其他仍未被發現的三倍頻對比來源以及尚未被使用的三倍頻外加顯影劑，並初步的應用於醫學研究上。在這個論文所討論的範圍包含了，偵測到來自彈性蛋白纖維的三倍頻對比並加以證實，且驗證了醋酸與蘇木素作為三倍頻外加顯影劑的可行性。彈性蛋白是生物體中一個不可或缺且廣泛分布的構造性蛋白，負責了組織以及器官的完整性。在這篇論文中，在使用中心波長為1230nm的鉻貴橄欖石雷射光源激發之下，證實了彈性蛋白是三倍頻的主要來源之ㄧ，且在許多生物體中如肺組織和血管中有著選擇性的影像。在血管中，彈性蛋白纖維產生了比雙光子螢光信號大上一到兩個等級強度的三倍頻信號，顯示出來自彈性蛋白纖維很強的三倍頻信號對比提供了彈性蛋白絕佳的選擇性影像。利用三倍頻顯微術取得在活體裸鼠耳朵中的彈性軟骨影像，深於皮下組織之下仍保持著三度空間上的高解析度和高對比度，這展現出三倍頻顯微術用於觀察活體內彈性軟骨的優越性。結合了二倍與三倍頻顯微術之後，這項技術有希望可以用來作為結膜翼狀贅片的纖維化檢測且能夠區分出彈性纖維和膠原纖維的影像。欲發展一套三倍頻分子影像顯微術應用於臨床癌症疾病的診斷上，使用外加三倍頻顯影劑加強在細胞核處的三倍頻對比度是必需的。在醋酸暫時性的作用之下，敏感於局部線性與三階非線性係數變化的三倍頻信號在細胞核處被選擇性的加強。這個現象在穿刺出的人類和老鼠肺組織中或是活體的中國倉鼠口腔中皆被觀察到且不影響到後續病理切片的製作。因此，利用醋酸作為三倍頻顯影劑的三倍頻分子影像顯微術提供了非侵入式的疾病檢查技術，特別針對了如癌症等與細胞型態有關的疾病。由於癌化的過程與膠原蛋白的變化有關，而膠原蛋白是二倍頻信號的對比來源之一，所以二倍頻顯微術在協助癌症診斷上是一個有用的工具。包含了高空間解析度、非侵入式、光學切片特性的倍頻顯微術利用醋酸作為三倍頻顯影劑擁有諸多的好處，對協助快速的癌症診斷提供了新的工具且對型態組織學提供了新的觀點。蘇木素染色是一個最廣泛使用的染色方法只需要幾分鐘的時間，組織學上主要是染色細胞核以供醫學診斷。在蘇木素的使用下，利用三倍頻顯微術觀察到在細胞核處很強的三倍頻信號增加提供了證據證實蘇木素是一個出色的三倍頻細胞核顯影劑。憑藉著光學切片的能力，在不需進一步切片的情況下，增強的三倍頻信號立即地提供了極需的三度空間細胞核資訊，且根據特定的細胞核排列與較大的細胞核大小等癌化特徵能夠區分出正常與癌化的人類肺組織。結合現有存在的診斷工具，這項技術可應用於協助與細胞核型態有關的大部分醫學診斷，且應該立即廣泛地應用到其他的器官或是組織上，做為在癌化過程中評估細胞核變化的一個方法。

關鍵字

倍頻顯微術；非線性光學顯微術；醫學生物影像

並列摘要

The purpose of this thesis is to explore other yet undiscovered contrast origins and unused contrast agents of third-harmonic-generation (THG) inside biological tissues, and apply to the preliminary studies of medicine based on a nonlinear laser scanning microscopy system using a femtosecond Cr:forsterite laser centered at 1230-nm. The scope of this thesis contains that THG contrast from elastic fibers is detected and demonstrated, and using acetic acid or hematoxylin as THG contrast agents is verified to be feasible. Elastin is an essential and widespread structural protein in charge of the integrity on tissues and organs. In this thesis, we demonstrate that elastin is one of major origins of the THG contrast under Cr:forsterite laser excitation operating at 1230nm, with selective visualization inside many tissues such as lung tissues and arteries. In arteries, elastic fibers produce a THG signal 1-2 orders of magnitude larger than two-photon fluorescence (2PF) signal, revealing that the strong THG emissions from elastin could be a promising contrast to provide selective imaging. In vivo imaging of the nude mouse elastic cartilage beneath the hypodermis by THG microscopy keeps the high resolution and contrast in all three dimensions, which exhibits the superiority of intra-vital observations on elastic cartilage by THG microscopy. Combined with second-harmonic-generation (SHG) microscopy, THG microscopy exhibits the promise for examination on elastosis of the ophthalmic disease, pterygium, and the capability of distinguishable visualization from collagen. To develop a molecular THG microscopy for future clinical cancer diagnosis, a contrast agent to enhance THG at nuclear site is necessary. With the transitory effect of the acetic acid, THG process, sensitive to local differences in both linear and third-order nonlinear susceptibility, is selectively enhanced and observed in cell nuclei either in the biopsied human and mouse lung tissues without interference with the followed pathological sections or in the live hamster mouse oral cavity. Therefore, our developed molecular THG microscopy using acetic acid as a contrast agent shows promise for noninvasive diseases examination especially for those diseases associated with nuclear morphology such as cancer. Because neoplastic process is associated with changes of collagen fiber, which is a contrast origin of SHG signal, SHG microscopy is a useful tool for assisting cancer diagnosis. The highly spatially resolved, noninvasive, and intrinsic optical sectioning characteristics of harmonics-optical microscopy (HOM) with the acetic acid as the THG contrast agent have the advantages of offering new tool for assisting immediate cancer diagnosis and new insights into the morphological histology. Hematoxylin stain, which is the most widely used stain method and takes a few minutes, mainly stains the cell nuclei in histology for medical diagnosis. With the use of hematoxylin, the strong enhancement at cell nuclei taken from THG microscopy provides the evidences that hematoxylin is an excellent cell nucleus contrast agent in THG microscopy. Enhanced THG signals provide immediate much-required cell nuclei information in all three dimensions without further sectioning due to the intrinsic optical sectioning capability, and allow separating the normal and cancerous human lung tissues, underlying neoplastic characteristic features of particular nuclear arrangement and larger nuclear size. Integrated with existing diagnosis tool, this technique is easily applicable to assist most medical diagnosis associated with cell morphology, and should be readily generalized to other organs or tissues, as an immediate method to assess cell nuclear changes during neoplsia.

並列關鍵字

Harmonics optics microscopy ； Nonlinear optics microscopy ； Medical and biological imaging

參考文獻

Anderson RR, Parish JA (1981) The optics of human skin. J Invest Dermat 77:13

Armstrong JA, Bloembergen N, Ducuing J, Pershan PS (1962) Interactions between light waves in a nonlinear dielectric. Phys Rev 127:1918

Azzopardi JG, Zayid I (1972) Elastic tissue in tumours of salivary glands. J Pathol. 107:149-156

Azzopardi JG, Laurini RN (1974) Elastosis in breast cancer. Cancer 33:174

Barad Y, Eisenberg H, Horowitz M, Silberberg Y (1997) Nonlinear scanning laser microscopy by third harmonic generation. Appl Phys Lett 70:922

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組織內三倍頻之分子對比與外加顯影劑

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