螢光奈米鑽石之操控與生物影像應用

Manipulation and Bioimaging Applications of Fluorescent Nanodiamonds

曾彥凱(Yan-Kai Tzeng)

指導教授 ： 李遠哲

共同指導教授 ： 張煥正

國立臺灣大學/理學院/化學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.01590

摘要

鑽石是碳的同素異形體。不同於其它碳材料的獨特性質是，鑽石具有光學透明性，而常含有氮元素的缺陷，此缺陷可當作發光中心。帶負電荷的氮空位的缺陷，是在鑽石中最值得一提的發光中心，因為發出具有高穩定性的遠紅外螢光。這種獨特的光學特性結合良好的生物相容性，使奈米尺寸的鑽石在生物成像中，成為有潛力的螢光探針，尤其是細胞追蹤研究上。首先，我們利用小於20奈米的螢光奈米鑽石作為螢光共振能量轉移的供體和近紅外螢光染料作為受體。此螢光共振能量轉移的效率是大約7%。緊接著，針對螢光奈米鑽石，我們建立一套超高解析度的受激發射損耗顯微術系統（STED），展示出奈米等級的螢光成像精度。螢光奈米鑽石在受激發射損耗顯微術，是最佳的螢光標記，因為它不會光漂白（Photobleach）。相對之下，經由高功率的STED雷射光束照射，有機螢光染料或是螢光蛋白是很容易光損傷。螢光奈米鑽石的另一個顯著特徵是，螢光生命週期超過13奈秒，生命週期明顯長於普通的有機螢光染料或是綠色螢光蛋白（Green Fluorescent Protein; 簡稱GFP），以及細胞的自體螢光。藉由時間選通技術，成功地降低了細胞與組織的自體螢光背景訊號，我們應用螢光奈米鑽石作為細胞長時間的追蹤和證實移植的小鼠肺部幹細胞之歸巢和植入的能力。最後，在鑽石內部的氮空缺中心具有一個非常獨特的量子系統，並且可以通過光偵測磁共振技術進行操控，適用於測量環境的變化，例如是溫度改變。使用光偵測磁共振技術，我們實現了高靈敏度的溫度測量，在尺寸為100 奈米螢光鑽石的周圍溫度。以現代生物醫學和生物技術應用而言，所有的實驗結果證實，螢光奈米鑽石在奈米功能顯影劑技術和感測器探測，是最具有潛力而理想之選項。

關鍵字

螢光奈米鑽石 ； 螢光共振能量轉移 ； 受激發射損耗顯微術 ； 螢光生命週期影像顯微術 ； 幹細胞 ； 光偵測磁共振技術

並列摘要

Diamond is an allotrope of carbon. A unique property that distinguishes it from other carbon materials is that diamond is optically transparent and often contains point defects as color centers. Negatively charged nitrogen-vacancy (NV−) defects are the most noteworthy color centers in diamond because it emits far-red fluorescence with high photostability. This unique optical property combined with good biocompatibility makes nanoscale diamonds a promising fluorescent probe for bioimaging, particularly cell tracking studies. Firstly, we measured the efficiency of Forster resonance energy transfer (FRET) with sub-20-nm fluorescent nanodiamonds (FNDs) as the FRET donors and near-infrared dyes as the acceptors. A FRET efficiency of ~7% was found. Next, we built a super-resolution stimulated emission depletion (STED) microscopy system for FNDs and demonstrated the nanoscale precision for fluorescence imaging. FND is an ideal candidate for STED, since it does not photobleach. In contrast, organic dyes or fluorescent proteins are easily photodamaged by the high-power STED laser beam. Another distinct feature of FND is that its fluorescence lifetime is more than 13 ns, significantly longer than that of common organic dyes or green fluorescent proteins (GFPs) as well as cell auto-fluorescence. Using a time-gating technique, which successfully reduces cell and tissue auto-fluorescence background signals, we applied FNDs as long-term cell trackers and demonstrated the homing and engraftment capacity of lung stem cells transplanted in mice. Finally, the NV− center in diamond is a very unique quantum system, and can be manipulated by optical detected magnetic resonance (ODMR), a technique applicable to measure environmental changes such as temperature shifts. With the ODMR technique, we achieved high-sensitivity detection of the surrounding temperature of 100-nm FND particles at the nanoscale. All the experimental results demonstrate that FNDs are ideal candidates for potential applications in modern biomedical science and biotechnologies as nanotechnology-enabled imaging agents and sensors.

並列關鍵字

Fluorescent Nanodiamonds ； Fluorescence Resonance Energy Transfer ； Stimulated Emission Depletion Microscopy ； Fluorescence Lifetime Imaging Microscopy ； Stem Cell ； Optically Detected Magnetic Resonance

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