發展新拉曼光譜與成像方法( I )

Abstract

目前交大分子科學研究所的究極光譜呈像研究室(USIL)正在進行兩個高度原創性的 研究─「真空紫外雙光子電子超拉曼(HR)光譜學研究」及「活細胞中絕對定量性的拉 曼光譜分子呈像」。在第一個研究項目中，我們引領全球以HR 光譜學研究電子轉移現 象。一般而言，電子轉移都是以紫外可視領域的吸收或發散(螢光/磷光)光譜學研究之。 紫外可視吸收光譜對液態或溶液態的樣本來說十分便利，不過並不適用於顯微鏡下對 微小晶體的研究。螢光/磷光光譜學則適用於顯微鏡下的研究，不過它僅能用於取得基 態電子的光譜情報。HR 則可取得與紫外可視吸收相當，帶有激發態電子情報的光譜。 再者，HR 的泛用性十分高，可輕易地應用在顯微鏡下以測量微小的樣本。於第二個研 究項目中，我們將開發針對活細胞的快速並帶絕對定量性的拉曼分光顯微鏡。本計畫 由二部組成─導入可同時觀測400 個焦點的共軛焦拉曼技術以及定出生物分子的絕對 拉曼截面積。近年來，拉曼分光及呈像技術的進步已令單一活體細胞中分子層級的研 究難度大為降低。然而，目前的拉曼影像僅能表現生物分子的相對濃度而無從得知細 胞中生物分子的正確數量。若細胞中生物分子的絕對數量能被測出，以脂質為例我們 便能探討一個活細胞中已脂肪型態儲存的生物能量有多少。除此之外，細胞中脂質的 數量亦能有效表示出其「肥胖」程度。拉曼分光與呈像絕對定量性的特質將能開啟許 多新的生物學研究方向。這些研究主題都是奠基於申請者過去三十年在東京打下的研 究基礎及過去五年在USIL 的研究成果。這些研究所需要大部份的儀器皆已經(如紅外 電子吸收及時差式紅外光譜儀、單模氬離子雷射、低頻拉曼分光用的多色分光儀及一 臺實驗室製作的拉曼分光顯微鏡)或即將(如一臺飛秒超拉曼光譜儀及一臺多波長拉曼 分光顯微鏡)自東京轉移過來。此次申請的預算將被用以補足申請者在東京研發中的新 型儀器所需的費用。此研究計畫的目標是將USIL 更加推向國際。此二項研究計畫皆具 高度原創性，不僅在基礎科學領域中可稱前所未見，並同時具有高度於工業及生醫領 域的應用潛力。若這些計畫能順利付諸實行，它們將能提高USIL 在全球的能見度並使 之成為一個世界頂尖的分子光譜呈像中心。

Two highly original research subjects in molecular spectroscopy and imaging, “Electronic hyper-Raman (HR) spectroscopy with two photon excitation in the vacuum ultraviolet” and “Absolutely quantitative molecular imaging of living cells by Raman microspectroscopy”, are intensively investigated at the Ultimate Spectroscopy and Imaging Laboratory (USIL), Institute of Molecular Science (IMS), National Chiao Tung University NCTU). In the first subject, we lead the world to challenge HR spectroscopy of electronic transitions. Electronic transitions are usually measured with UV-VIS absorption or emission (fluorescence/phosphorescence). UV-VIS absorption spectroscopy is very convenient for liquid/solution samples. However, it is not suitable for measuring very small crystalline samples under a microscope. Fluorescence/phosphorescence spectroscopy is highly sensitive and suitable for microspectroscopy but the spectral information is limited to the ground electronic state. HR can provide electronic spectra that exactly correspond to UV-VIS absorption, bearing spectral information on the excited electronic states. HR is highly versatile and can easily measure very small samples under a microscope. In the second subject, we develop fast and absolutely quantitative Raman microspectroscopy of living cells. The plan consists of two parts, the introduction of 400 foci confocal Raman technique and the determination of absolute Raman cross sections of bio-molecules. Recent developments in Raman spectroscopy and imaging have facilitated in vivo molecular-level studies of single living cells. At the present stage, however, the obtained Raman images represent only the relative concentrations of bio-molecules. We can not tell how many bio-molecules are there in a living cell. If the absolute number of biomolecules, say lipids, in a cell is known, we can discuss how much energy is stored and used in the form of lipids in a living cell. Furthermore, the absolute number of lipid molecules provides a reliable measure of the “fatness” of a cell. The absolute quantification will surely open up many new possibilities of Raman spectroscopy and imaging of living cells. These research subjects are based on the background that the applicant has built in the past thirty years at Tokyo as well as the achievements in the past five years at USIL. Major portion of the necessary equipments have already been transferred (IR electroabsorption and time-resolved IR systems, a single-mode Ar+ laser and a polychromator for low-frequency Raman spectroscopy and a laboratory constructed Raman microspectrometer) or will be transferred from Tokyo (a femtosecond hyper-Raman spectrometer and a multi-wavelength Raman micro-spectrometer). The requested budget will be spent complementarily for introducing new apparatus that the applicant is developing at Tokyo. The goal of the present project is to further establish USIL internationally. Two research subjects are highly original and unprecedented in fundamental science but with high potentials for industrial and biomedical applications. If successfully performed, they will attract much attention to make USIL a world center of excellence of molecular spectroscopy and imaging.