孫啟光臺灣大學：電機工程學研究所朱士維2007-11-262018-07-062007-11-262018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/53122在這篇論文中，我們提出並實現了倍頻顯微影像術，更進一步將其應用在生物和材料科學領域。由研究倍頻產生的機制，我們提出了生物光子晶體的概念，並在許多生物樣本上得到證實。此外，更利用了倍頻的極化關係，求得生物組織中的非線性張量，並由此成功的解釋生物體中引發非線性效應的分子級結構特性。我們也是首次提出在生物體中存在著反向二倍頻強過順向二倍頻的材料（膠原蛋白纖維），並且經由實驗證實了這個違反直覺的現象。同時，我們首次在半導體材料中觀測到四光子螢光，並將此螢光信號配合倍頻顯微術在氮化鎵中取得了世界上第一張四光子螢光影像，我們也說明了如何將這些不同的顯微術模態對應到不同的物理特性上而能對氮化鎵材料有更深入的瞭解。 倍頻顯微影像術所用的信號是來自生物體或材料本身發出的二倍頻和三倍頻，無須外加任何染劑或額外處理。同時由於倍頻產生時的能量守恆特性，不會有多餘能量累積在樣本中，因此不會對樣本造成傷害。結合上述兩個特性，倍頻顯微影像術可說是提供了一個真正的非侵入式顯微檢測系統。此外，二倍頻強度和入射光強度平方成正比，三倍頻強度則和入射光強度三次方成正比，這樣的非線性效應提供了倍頻顯微影像術非常好的光學切片能力，且不需使用會讓信號衰減很多的共焦孔隙。這個特性配合上我們所使用的鉻貴橄欖石雷射，使得我們取像深度可以超過一毫米，還可以有次微米的解析度，而不會像一般光學顯微鏡的模糊不清。此外，倍頻顯微影像術具有非常高的對比，二倍頻信號主要是在生物體內整齊排列的奈米級構造產生，稱之為生物光子晶體效應。而三倍頻則是在介面不連續處產生，可以提供一個完整的細胞型態資訊。兩者結合才能夠對生物體中複雜的結構提供進一步的資訊。 我們驗證了把倍頻顯微影像術應用在胚胎發展生物學和皮膚組織學上，都能在不傷害樣本的條件下，提供傳統顯微鏡無法取得的深層組織高解析度影像，對於組織研究提供了良好的非侵入式觀測工具。另外，由於倍頻是由分子級的構造所引起，因此解出倍頻的非線性張量將可對其中的分子結構或排列有更進一步的瞭解。在此論文中，我們也成功利用了倍頻的極化關係求出了肌肉二階非線性張量中的每一項，同時也在合理的誤差範圍內對三階非線性張量做了相當精確的估計。 為了使我們的顯微技術具有更多功能，我們也試著將螢光信號整合進來。由於雷射波長的限制，要觀察到一般的染劑或生物體螢光分子的螢光，三或四光子激發是必須的。我們在染色的肝細胞上實現了世界上第一個結合了倍頻和三光子螢光的顯微術。更在氮化鎵中觀測到四光子激發螢光，同時將此信號和倍頻顯微術結合，取得第一張四光子激發螢光影像。而為了要實現即時倍頻影像觀測系統，必須要讓非線性的倍頻信號增加又不會對脆弱的生物樣本造成傷害，我們提出了利用提高雷射重複率的方法來達成這個要求，並且成功的以此原理做出即時倍頻顯微術。最後，由於動量守恆的緣故，倍頻信號在理論上應該是大部分都順著入射光方向向前傳播，但若要將倍頻顯微影像術發展為醫療用途，則必須要收集反向傳播的倍頻信號。我們深入探討了反向倍頻產生的機制，並首次證實了在膠原蛋白纖維中反向倍頻會比順向倍頻還要強，更提出了利用反向和順向倍頻的比例來估計膠原蛋白纖維的粗細的可能性。為未來將此技術應用在生物醫學領域奠定了良好的基礎。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.Contents 誌謝 I Abstract IV 摘要 VI Contents VIII Publication list XI Chapter 1. Introduction 1 1.1 Historical overview 1 1.2 Scope of this thesis 7 Chapter 2. Basic principles 10 2.1 Basic concepts of microscopy 10 2.1.1 Diffraction and resolution 10 2.1.2 Aberration 13 2.2 Confocal laser scanning microscopy 15 2.3 Nonlinear optics (at focal region) 18 2.3.1 Second harmonic generation (SHG) 20 2.3.2 Third harmonic generation (THG) 26 2.3.3 Two-photon fluorescence (2PF) 32 2.4 Nonlinear laser scanning microscopy 35 2.4.1 Optical sectioning capability 36 2.4.2 Reduced photodamage and photobleach effects 38 2.4.3 Increased penetration depth 40 Chapter 3. Home-built harmonics optical microscope 44 3.1 Laser source selection 44 3.2 HOM setup 51 3.2.1 PMT-based HOM 51 3.2.2 Spectrum-based HOM 53 Chapter 4. Applications of HOM 56 4.1 Bio-photonic crystalline structure probing 57 4.2 In vivo observation of zebrafish embryogenesis 72 4.2.1 HOM imaging in a live embryo (0-24 hpf) 74 4.2.2 HOM imaging in a larva (after 24 hpf) 82 4.3 Dermatology studied by harmonics optical biopsy (HOB) 86 4.4 Nonlinear susceptibility determination in bio-tissue by polarization HOM (P-HOM) 94 4.4.1 Theoretical analysis of harmonic generations in muscle 96 4.4.2 Experimental results and discussion 102 Chapter 5. Advancement of HOM 112 5.1 Multiphoton multiharmonic microscopy 112 5.1.1 Three-photon fluorescence microscopy + HOM 112 5.1.2 Four-photon fluorescence (4PF) microscopy + HOM in GaN 116 5.2 Real-time HOM based on a 2-GHz repetition rate laser 125 5.2.1 Laser source 126 5.2.2 Enhanced SHG in muscle tissue 128 5.3 Epi-HOM (SHG modality) 137 5.3.1 Artificial single collagen experiment 142 5.3.2 B-SHG in collagen tissue 147 5.3.3 Epi-HOM in thick tissues 153 Chapter 6. Summary 163 Reference 1688170504 bytesapplication/pdfen-US飛秒脈衝非線性效應二倍頻多光子螢光共焦顯微術三倍頻nonlinear effectsfemtosecond lasersecond harmonic generationthird harmonic generationconfocal microscopymultiphoton fluorescencethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/53122/1/ntu-93-D90941004-1.pdf