Endogenous and Exogenous Contrast Agents of Third-harmonic-generation Microscopy
Date Issued
2007
Date
2007
Author(s)
Yu, Che-Hang
DOI
en-US
Abstract
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.
Subjects
倍頻顯微術
非線性光學顯微術
醫學生物影像
Harmonics optics microscopy
Nonlinear optics microscopy
Medical and biological imaging
SDGs
Type
thesis
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