- 學位論文
螢光生命週期顯微術之原理及應用
Fluorescence lifetime imaging microscopy principles and applications
摘要
在本篇論文中，我們成功架設了多光子螢光生命週期顯微系統(fluorescence lifetime imaging microscopy, FLIM)，提供螢光波長(或頻率)以外另一維度，使得各種不同的螢光可以藉由系統展現不同的生命週期特性，尤其對於生物體內本身的自發螢光，在同樣的雷射光源激發下常常擁有類似發射波段，這時螢光生命週期顯微系統尤其發揮了重要的功能，提供時域上的資訊，作為生物研究應用上，資訊交叉分析比對的重要基礎，而在實際應用時，我們採用的Cr:forsterite 近紅外光雷射光源，有著相對於採用Ti:Sapphire雷射更為簡單明確的結果，因為在Ti:Sapphire 雷射所激發的生物體自發螢光種類多而繁複，作為特定問題的分析反而形成阻礙；另外Cr:forsterite雷射還有著較深的穿透深度和相對低侵入的光破壞(photodamage)，更適合進行生物樣本的觀察，而且利用Cr:forsterite近紅外光1230奈米波段的特性，我們還整合了實驗室發展的非線性二倍頻及三倍頻顯微系統，觀察進行時就可以配合高數值孔徑的物鏡，同時地取得高解析度的螢光強度、螢光生命週期、二倍頻以及三倍頻的豐富影像訊息，形成一個深入分子層級的生物研究平台，可用來解決某些特定相對複雜的生物議題。 螢光生命週期是每個分子的專有特性，因此螢光生命週期顯微術是研究分子影像很適合的工具，作為分子影像領域的開端，我們測量了多種細胞胞器中代表性的分子，得到其螢光光譜以及生命週期，作為分辨次細胞結構的基本依據。 同時比對實際組織中的螢光，對可能的分子來源提供非常有利的證據，甚或作為某些胞器的指標，對於次細胞等級的資訊分析有著相當的影響，對未來分子層級的生物醫學影像觀察具有相當大的幫助。 癌症長達25年佔據國人十大死因的首位，而肺癌和肝癌則佔據第一和第二大癌症死因，長期以來一直持續威脅人類的生命，因此我們利用前段所提及的多模態光學資訊，針對正常以及癌症的樣本進行觀察，藉由這些資訊我們進行交叉比對分析，藉著各種定量的指標，我們可以有相當高的機率成功判別生物的樣本是否有癌症的發生，而且先天的光學切片能力相較於傳統的切片染色更為迅速而方便，接下來可以持續評估其作為切除手術引導之可行性，做為判別癌症的臨床依據之一。 螢光生命週期顯微系統，結合實驗室已發展的倍頻顯微術，提供了多維度的光學資訊，更直接或間接地激發不同維度的分析方式，可以有效率地進行某些特定的生物研究，進一步分析各種複雜的醫學議題。
並列摘要
In this thesis, we successfully implement the fluorescence lifetime imaging microscopy (FLIM), which maps the fluorescence lifetime distribution and thus provides a new dimension for observation in addition to frequency or wavelength. Disparate fluorescent molecules can be inherently discriminated with different fluorescence lifetime; with the help of additional time-resolved information, biological issues can be analyzed accurately. The inherent contrast in fluorescence lifetime is especially significant for the endogenous fluorescence, where different molecules often have highly overlapping spectra under the same excitation source. According to many published studies, the Ti:sapphire laser is known to excite fluorescence from numerous kinds of molecules, which is advantageous in some applications but may form an obstacle for specific biological studies since unwanted fluorescence shows up as well. So an infrared Cr:forsterite laser is used instead and the endogenous fluorescence appears simpler and more definite in contrast with that from a Ti:sapphire laser. Besides, the Cr:forsterite provides good penetration depth and the least invasive observation due to low absorption at the wavelength by biological tissues. Its infrared working wavelength also enables us to integrate FLIM with our developed second and third harmonic generation microscopy; multi-modality information, including fluorescence, SHG and THG, are thus obtained simultaneously. With abundant optical information, the FLIM-integrated multi-modality laser scanning system has become a powerful tool for biological studies to solve certain relatively complicated issues Fluorescence lifetime is a specific property of a fluorescent molecule, and therefore, molecular imaging can be realized with fluorescence lifetime imaging. As an initial study, several typical and essential molecules are selected to measure their fluorescence spectra and lifetime, which could be taken as a data base for molecular imaging. We then compare them with the autofluorescence in real human tissues, and possible fluorescent origins are found with the help of the fluorescence lifetime and spectra. Cancer has been the leading cause of death for 25 years, and lung cancer and liver cancer are the first and the second place, respectively, in all kinds of cancer. Using the FLIM-integrated multi-modality system, we perform quantitative analysis on normal and abnormal samples and diagnose the occurrence of liver cancer with a high accuracy. In the case of lung cancer, the specific kind of adenocarcinoma is identified using fluorescence lifetime. In contrast to conventional tedious biopsy, the technique is very potential in clinical diagnosis of cancer and in serving as guidance for tumor removal surgery. FLIM, combined with the developed harmonic generation microscopy in our group, provides multi-dimensional information, and creates a solid basis for cross analysis for biological studies. More complicated medical issues can be further studied using the FLIM-integrated multi-modality optical system, which could also be modified depending on different demands.
參考文獻
延伸閱讀
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