| Summary: | 博士 === 國立臺灣大學 === 電機工程學研究所 === 92 === In this thesis, a harmonics optical microscope is built based on a modified optical scanning microscope system and a Cr:forsterite laser. Both second harmonic generation (SHG) and third harmonic generation (THG) have been used as nonlinear imaging modalities. Due to their nonlinear natures, the generated SHG and THG intensities depend on square and cubic of the incident light intensity, respectively, providing an intrinsic optical sectioning capability without the need of a power-consuming pinhole. The energy-conservation and intrinsic-emission characteristics of these optical harmonics provide the optical noninvasive nature desirable for biomedical imaging applications. Based on a near-infrared excitation source at 1230-nm, the millimeter penetration and sub-micrometer resolution of our developed imaging system has been demonstrated. Both SHG and THG modalities provide unprecedented structure contrast. SHG, in biological samples, is generated from orderly arranged nano-scaled structures, which is termed bio-photonic crystal and thus exhibit a highly specific imaging contrast. THG, on the other hand, is interface-sensitive due to the Gouy phase shift effect, and can be used as a general structural imaging tool.
Such a microscopic tool would provide a significant impact on developmental biology and histology researches, as presented in the following chapters. As novel biopsy instrumentation for noninvasive deep tissue observation, it is of vital importance to characterize harmonic generations from collagen and muscle tissues. The full second-order nonlinear susceptibility tensor with 27 elements in muscle tissue can be determined through the polarization dependency of optical harmonics. Based on a cylindrical fiber theory, the third-order nonlinear susceptibility tensor elements can also be estimated with reasonable accuracy. The resolved nonlinear tensor can provide information on the molecular origin of the biological nonlinearities. For thick tissue in vivo biopsy, it is only practical to collect the backward SHG (B-SHG). We have demonstrated and discussed in detail all the mechanisms contributing to B-SHG. As a result, an asymmetric bipolar emission profile from thin collagen fibrils is revealed, for the first time. Moreover, the elaborate explanations of the complex B-SHG power dependency on the thickness and the local arrangement of collagen fibrils have been presented. The contribution of backscattering in thick tissues is also be characterized. These characterizations are of fundamental importance for the realization of epi-detected harmonics optical microscopy, which is a pragmatic solution for in vivo deep tissue inspection.
To improve the performance of our microscopy technique, several technical advancements, such as incorporating more channels and increasing the frame rate (signal intensity), are demonstrated. We have presented the combination of harmonics optical microscope with other nonlinear imaging modalities, such as three- and four-photon excited fluorescence. The first four-photon excited fluorescence is observed from a bulk gallium nitride sample, with simultaneously emitted strong SHG and THG. The superb spatial resolution of the four-photon fluorescence is manifested. The combined multimodal nonlinear microscope can provide more complete information about the structural and functional properties of both biological and semiconductor samples. We have also demonstrated that the frame rate of the nonlinear scanning microscopy system can be significantly increased with the aid of a high-repetition rate femtosecond laser. Most important of all, the nonlinear photodamage is greatly reduced by increasing the repetition rate. This concept is able to integrate with other fast-scanning scheme to further improve the frame rate.
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