- 學位論文
超高解析度──受激放射耗乏顯微術及螢光生命期影像顯微術於活體內外以螢光奈米鑽石為生物標記之應用
In vitro and In vivo Applications of Stimulated Emission Depletion and Fluorescence Lifetime Imaging Microscopy with Fluorescent Nanodiamonds as Biomarkers
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摘要
遠場螢光顯微術（far-field fluorescence microscopy）因高靈敏度、高訊噪比、非侵入性、高專一性、擷取影像快速、架設簡易等優點，成為近年來研究生物系統的重要工具。然而，遠場光學顯微鏡受光學繞射極限限制，使用可見光為激發光源時，解析度約200到400奈米，限制其生物應用，因此開發超高解析度（super resolution）顯微術，突破繞射極限成為重要的研究方向。近年來，受激放射耗乏（stimulated emission depletion, STED）顯微術是諸多超高解析度顯微術中，影像擷取速度最快且唯一完全利用光學原理而開發的技術，原理為利用一道甜甜圈型態且波長與螢光波長相符的STED光束與激發光重疊，使其周圍的受激分子放射耗乏回到基態而不放光，每次僅取得接近STED光束中點、光束強度接近零處的數個分子的螢光，而達到超高解析度。本論文工作為架設STED顯微鏡，並觀察以電穿孔法（electroporation）或經由胞吞作用（endocytosis）進入HeLa細胞內，包覆有牛血清白蛋白（bovine serum albumin, BSA）的螢光奈米鑽石粒子，單一35奈米的螢光奈米鑽石二維解析度達到40奈米。 螢光顯微鏡應用於生物系統的另一大挑戰為：生物自體螢光，尤其活體應用時，訊噪比常因生物自體螢光干擾而下降，甚至無法辨別訊號真假，為解決此一問題，利用螢光奈米鑽石中(N-V)0及(N-V)-中心螢光生命期較生物自體螢光生命期長的優勢，搭配螢光生命期顯微術（fluorescence lifetime imaging microscopy, FLIM），於秀麗隱桿線蟲（Caenorhabditis elegans）活體中，追蹤單一奈米鑽石粒子的即時運動影像；同時搭配微注射技術（microinjection）將各種表面修飾的螢光奈米鑽石，送入秀麗隱桿線蟲的性腺（gonad）、腸道細胞（intestinal cell）、假體腔（pseudocoelom）、子宮精囊（spermatica）前、清道夫細胞（scavenger cell）coelomocyte附近，觀察螢光奈米鑽石在活體內的移動及命運。
並列摘要
Far-field fluorescence microscopy has become a powerful tool for imaging biological systems because of its high sensitivity, high signal-to-noise ratio, high specificity and non-invasiveness. The spatial resolution, however, is limited by light diffraction to ~200 nm, which is larger than most sub-cellular organelles. Therefore, developing sub-diffraction microscopy is an important task to image delicate biological structures. Among many superresolution imaging techniques, stimulated emission depletion (STED) microscopy is purely lens-based and has the shortest image acquisition time. In this thesis, a home-built STED microscope has been constructed and applied to image 35-nm albumin-coated fluorescent nanodiamonds (FNDs) taken up by HeLa cells through endocytosis or electroporation. A resolution of ~40 nm has been achieved for a single FND in cells. In bioimaging, cell and tissue autofluorescence is another big obstacle which decreases the signal-to-noise ratio and restricts in vivo applications of fluorescence microscopy. FNDs with nitrogen-vacancy (N-V) centers as fluorophores have longer fluorescence lifetimes than autofluorescence. Using fluorescence lifetime imaging microscopy (FLIM) and a model organism, Caenorhabditis elegans (C. elegans), we studied the fate of carboxylated and albumin-coated FNDs microinjected into the worm and performed real-time tracking of single FNDs in vivo over an extended period of time.
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