Optically modulated, sequence- and color-tunable single Ag nanodot biolabels

光调制、序列和颜色可调的单银纳米点生物标记

基本信息

批准号：
7751503
负责人：
ROBERT M DICKSON
金额：
$ 27.89万
依托单位：
GEORGIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-01 至 2011-08-31
项目状态：
已结题

项目摘要

The long-term project goal is to create small, highly fluorescent, and optically modulatable nanomaterials to enable a whole new array of high sensitivity imaging and detection capabilities in extremely high background biological environments. Beautifully adept at probing mechanistic heterogeneity, single molecule (SM) experiments hold great potential to unravel the complex steps leading to biological activity. Unfortunately, all SM experiments are at some level limited by the disadvantageous properties of available fluorescent labels, with at least 10-fold improvements in brightness and photostability being necessary for facile observation in the high background cellular milieu. Selective modulation of the probe of interest has long been utilized in spectroscopy to extract very weak signals from high background environments, but no fluorophores exhibit modulatable emission without also simultaneously modulating background emission, precluding similar sensitivity gains in biological imaging. Through two Specific Aims, we will demonstrate intracellular observability of individual fluorophores through brightness gains and selective optical modulation possible with our unique Ag nanodot emitters. In Aim I, we will elucidate the unique photophysics of our Ag nanodots that enable long wavelength, secondary laser-induced optical depletion of a photoaccessible dark state to significantly increase overall emission rate. Since this secondary laser is of lower energy than both the primary laser excitation and the fluorescence it enhances, we can selectively modulate the bright Ag nanodot emission independent of the background through modulation of the secondary laser intensity. Detailed photophysical characterizations of all nanodots created to date are expected to identify at least 5 spectrally pure nanodots exhibiting modulation-based sensitivity gains. In Aim II we will employ these 5 different color modulatable nanodots for extraction of true intracellular SM fluorescence signals through whole image modulation. Limits of permissible background for SM signal extraction will be directly probed for each emitter in well- designed control experiments. Modulation will also be utilized for signal extraction in live cell fluorescence correlation spectroscopy-based observations of single molecules. This selective optical modulation should enable entire images to be modulated and synchronized with detection for potential >10-fold sensitivity increases in SM or bulk nanodot imaging. With a high probability of success, we will develop these ultrabright, highly photostable emitters with the goal of optical modulation-based intracellular SM observation.

长期项目目标是创造小型、高荧光、光学可调节纳米材料，可实现一系列全新的高灵敏度成像和极高本底生物环境中的检测能力。漂亮地擅长探索机制异质性，单分子 (SM) 实验效果很好揭示导致生物活性的复杂步骤的潜力。不幸的是，所有 SM 实验在某种程度上受到现有材料的不利特性的限制。荧光标签，亮度和光稳定性至少提高10倍对于在高背景细胞环境中进行轻松观察是必要的。选择性感兴趣的探针的调制长期以来一直用于光谱学中以提取非常来自高背景环境的微弱信号，但没有荧光团表现出来可调制发射，无需同时调制背景发射，排除生物成像中类似的灵敏度增益。通过两个具体目标，我们将通过以下方式展示单个荧光团的细胞内可观察性我们独特的银纳米点可以实现亮度增益和选择性光学调制发射器。在目标 I 中，我们将阐明银纳米点的独特光物理学实现长波长、二次激光诱导光损耗的光可达暗状态显着提高整体发射率。由于该次级激光器是能量低于初级激光激发及其增强的荧光，我们可以独立于背景选择性地调节明亮的银纳米点发射通过调制二次激光强度。详细光物理迄今为止创建的所有纳米点的特征预计将识别至少 5 光谱纯的纳米点表现出基于调制的灵敏度增益。在目标 II 中，我们将使用这 5 种不同颜色的可调节纳米点来提取真正的细胞内 SM 荧光信号通过整个图像调制。允许的限度 SM信号提取的背景将直接探测井中的每个发射器设计了对照实验。调制也将用于信号提取基于活细胞荧光相关光谱的单分子观察。这种选择性光学调制应该能够调制整个图像并与 SM 或散装中潜在的 >10 倍灵敏度增加的检测同步纳米点成像。我们将开发这些超亮、高度光稳定的发射器，目标是基于光调制的细胞内 SM 观察。