Single Particle Optical Characterization and In-Vivo Imaging application of Fluorescent Nanodiamonds In C.elegans

螢光奈米鑽石之單粒子光學特性與秀麗隱桿線蟲活體 影像應用

尼丁(Nitin Mohan)

指導教授 ： 張煥正 ； 曾繁根

國立清華大學/原子科學院/工程與系統科學系/博士(2011年)

https://doi.org/10.6843/NTHU.2011.00140

摘要

The negatively charged nitrogen-vacancy defect center, (N-V)–, in type Ib diamond has drawn much attention in recent years, as the center exhibits some distinct fluorescence features, such as extended red emission at ~700 nm and high photostability, i.e. no photoblinking and photobleaching. These properties, along with the diverse surface functionalizability and non-toxic nature of the nanomaterial, have made fluorescent nanodiamond (FND) a promising candidate among other conventional markers, i.e. organic dyes, fluorescent proteins, and quantum dots, for biological applications as a fluorescent probe. Along with these excellent photophysical and biochemical features, the size of FND is also an important parameter to characterize.

Here we report procedures for production and isolation of 10-nm FND particles utilizing ion irradiation and differential centrifugation methods respectively. Particle size analysis of the isolated 10-nm FNDs was performed by Dynamic Light Scattering. Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) measurements were done to further confirm the size distributions at single particle level. Optical characterization of single 10-nm FNDs by confocal microscopy revealed the photostability of (N-V)- within the diamond lattice. The defect centers were quantified to be 3 per crystal on an average, by photon correlation technique. Dynamics of the single 10-nm FNDs in solution was studied by Fluorescence correlation spectroscopy (FCS), where the particle size and fluorescence intensity could be measured simultaneously. Also we compared the normalized autocorrelation functions of the intensity fluctuations of DsRed-Monomer (a 4-nm monomeric red fluorescent protein) and 10-nm FND particles excited under the same experimental conditions (λex = 532 nm and Iex = 80 kW/cm2), and the average fluorescence intensity of single FND is found to be ~8-fold higher than that of a single DsRed-Monomer and smaller FNDs could be used as FRET donors with suitable acceptors.

Moving on, we explored the possibilities of using 100-nm FNDs for long term bio-imaging in vivo. We choose Caenorhabditis-elegans as our in vivo system. Fluorescent nanodiamonds were incorporated into wild type C. elegans by feeding them directly with FND solution. In our primary observations with bright field and epi-fluorescence microscopy, the FND particles remain with in the lumen of the C- elegans and could image the whole digestive system of the organism for several days. With proper surface fictionalization with BSA protein and Dextran we observed FNDs absorbed into the intestinal cells from lumen by endocytosis. The second choice of incorporating FND particles into the organism is by microinjection. The FND is micro-injected into the syncytial gonads of gravid hermaphrodites and could be seen embedded in the embryos and the newly hatched larvae of the organism. Toxicity studies and stress response gene analysis additionally proved that FNDs were non toxic to the organism.

關鍵字

單粒子光學特性 ； 螢光奈米鑽石 ； 活體影像

並列摘要

近年來，Type Ib 鑽石中含有的（氮－空缺）缺陷中心(nitrogen-vacancy defect center)成為熱門的研究主題，因其特殊的光學性質，如：近紅外光螢光、高螢光穩定性、無光閃爍及無光致漂白等，除此之外，奈米鑽石具有多元化的表面官能基、無毒性等性質，使螢光奈米鑽石凌駕於其他商業化有機染料、螢光蛋白、量子點等螢光探針，更適合應用於生物領域。 除光物理性質及生化性質外，螢光奈米鑽石的尺寸亦是重要的參數。本論文以離子束佈植製造螢光奈米鑽石並用差速離心法分離直徑10nm之螢光奈米鑽石。以動態光散射儀、原子力顯微鏡及穿透式電子顯微鏡檢測粒子尺寸；以共軛焦顯微鏡檢驗（氮－空缺）缺陷中心之光學穩定性；以fluorescence correlation spectroscopy (FCS)檢測10nm螢光奈米鑽石在水溶液中的動態性質，並同時量測螢光強度及尺寸。本實驗在相同條件下(λex = 532 nm and Iex = 80 kW/cm2)，比較4nm紅色螢光蛋白DsRed-Monomer與10nm螢光奈米鑽石螢光波動的歸一化自相關函數(autocorrelation function)，發現螢光奈米鑽石之螢光強度約為紅色螢光蛋白的八倍。此小尺寸螢光奈米鑽石十分適合做為螢光共振轉移之捐贈子(donor)。 另外，本實驗使用100nm螢光奈米鑽石觀察標準模型生物──秀麗隱桿線蟲(Caenorhabditis-elegans)的活體影像。餵食含有螢光奈米鑽石水溶液之線蟲，可用明視野及螢光顯微鏡觀察其消化系統管道達數天之久；餵食表面接有牛血清白蛋白(BSA)或葡萄聚醣(dextran)的100nm螢光奈米鑽石，可觀察到螢光奈米鑽石被腸道細胞以胞吞作用吸收。微注射100nm螢光奈米鑽石進入妊娠雌雄同體(gravid hermaphrodites)線蟲之合胞性腺(syncytial gonads)，螢光奈米鑽石可隨著細胞質流動進入胚胎中，利於觀察胚胎發育及幼蟲時期。進一步的毒性測試及逆境反應(stress response)下的基因分析，證明螢光奈米鑽石對線蟲不具毒性。

並列關鍵字

Single Particle Optical Characterization ； Fluorescent Nanodiamonds ； In-Vivo Imaging

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