開發「受激拉曼散射顯微影像技術」應用於三維化學成像

Abstract

由於肉眼無法直接觀察微米尺度的變化，光學顯微影像技術提供了一個好的工具來觀察與探討微米或次微米尺度的物質的型態、結構與化學性質。相較於螢光顯微影像技術，拉曼顯微影像在無需外加螢光染劑的優勢下即可產生化學專一影像。然而，受限於自發性拉曼散射的低效率（低拉曼散射截面,~10-30 cm2/molecule），傳統拉曼散射的二維成像相當耗時間，因而限制了樣品的選擇，也不適合進行三維化學成像（以200  200像素的影像為例，若每一個像素積分時間為0.1秒，完成一張二維影像則需要1.1小時）。近幾年同調拉曼散射（coherent Raman scattering）顯微影像技術，如1982年由華盛頓海軍研究實驗室M.D. Duncan團隊所開發的同調反史托克拉曼散射顯微影像技術（coherent anti-Stokes Raman scattering）與2008年由哈佛大學X.S. Xie團隊所開發的受激拉曼散射顯微影像技術（stimulated Raman scattering）提供了新穎、高速、高靈敏且化學專一的光學影像技術。相對於同調反史托克拉曼散射顯微影像技術受限於不具化學專一性的背景訊號（non-resonant background）而降低了靈敏度，新興的受激拉曼散射顯微影像技術不但免於該背景訊號的干擾，且訊號與樣品濃度呈線性關係的特性更提供了具化學特性的影像定量能力。 此碩士論文中，我將介紹受激拉曼散射顯微影像系統的原理與其架設過程，並測試其特性。在自行架設的系統中，我們選擇皮秒脈衝雷射與光參共振波長可調雷射光源（optical parameter oscillator）作為激發光源以提供高光譜選擇性。由於需要直接偵測高強度的雷射，我們選擇設有反向偏壓（bias-voltage）的矽晶片光電二極管（Si photodiode），以提供在高雷射強度下該光電二極管依然能線性地輸出光電流，並擁有夠短的反應時間。此外，我將展示該顯微影像系統的生物應用，包括脂肪肝組織中脂肪球的體外成像，與具有高脂血症的活體斑馬魚體內的血管脂肪性病變成像。在最後，我也觀察觀察經由電噴灑方式自製的聚苯乙烯 （polystyrene）高分子纖維在聚甲基丙烯酸甲酯（poly(methyl methacrylate)）高分子膜上經由熱退火（thermal annealing）處理時，高分子材料的形貌變化。在此我們展現出在不同的加熱程度下，聚苯乙烯纖維在聚甲基丙烯酸甲酯膜上融化、波動（undulation）與形成水滴狀高分子的過程。我的碩士成果顯示了受激拉曼散射顯微影像技術應用在各種研究領域的高度潛力。

Optical microscopy has long been a powerful method to explore morphological, structural and chemical information of materials of varied types at a sub-micrometer scale. Comparing with fluorescence microscopy, Raman microscopy allows chemically specific imaging without labeling. Recently, the stimulated Raman scattering (SRS) has been employed by the Xie group at Harvard to develop a new modality of optical microscopy that allows chemical imaging at high-frame rate. Unlike the coherent anti-Stokes Raman scattering (CARS) microscopy that suffers from non-resonant background, the SRS signal depends linearly on the concentration of samples further allowing quantitative imaging with chemical specificity. Herein, I report the construction and characterization of our home-built SRS microscope. In our setup, a picosecond pulse laser and a coupled optical parameter oscillator are employed as the excitation source. Due to the detection of intense laser, a back-bias silicon photodiode is chosen for high linearity to high laser intensity and fast responding speed. To extract signal from intense laser, a lock-in amplifier coupled with an electro-optic modulator is chosen. Further, I illustrate the biological application of SRS microscopy by imaging lipid droplets in fatty liver tissues ex vivo and vascular fatty lesions of hyperlipidemic zebrafish in vivo. At the end, I investigate the dynamic morphological change of composite polymeric materials that comprise polystyrene (PS) fibers lying on a poly(methyl methacrylate) (PMMA) film during thermal annealing. I show that the melting and undulating processes of electrospun PS fiber and formation of PS droplets on the PMMA film in different heating stages. My work demonstrates a great of potential of SRS microscopy to different research fields.