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

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/7751503
• 关键词: Antibodies; Biological; Biological Process; Blinking; Buffers; Caliber; Cell physiology; Cells; Characteristics; Color; Complex; Detection; Development; Diagnostic; Dyes; Encapsulated; Environment; Exhibits; Fluorescence; Fluorescence Microscopy; Fluorescent Probes; Goals; Grant; Heterogeneity; Image; In Vitro; Individual; Label; Lasers; Life; Medical Imaging; Microscopy; Oligonucleotides; Optics; Photobleaching; Probability; Process; Property; Protein Dynamics; Quantum Dots; Recovery; Resolution; Semiconductors; Signal Transduction; Solutions; Spectrum Analysis; Time; absorption; base; biological systems; cell fixing; design; fluorescence imaging; fluorophore; improved; interest; minimally invasive; nanomaterials; research study; residence; single molecule; success

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.

长期的项目目标是创建小型，高荧光和光学 可调节的纳米材料可实现一系列全新的高灵敏度成像和 在极高的背景生物学环境中的检测功能。精美 善于探测机械异质性，单分子（SM）实验很棒 揭示导致生物活性的复杂步骤的潜力。不幸的是，一切 SM实验在某种程度上受到可用特性的限制 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便捷观察所必需的。选择性 长期以来，在光谱法中使用了感兴趣的探针的调节来提取非常 来自高背景环境的信号较弱，但没有荧光团显示 可调节发射，也不同时调节背景发射， 排除在生物成像中相似的灵敏度。通过两个具体目标， 我们将通过 我们独特的Ag纳米射线可能会获得亮度增长和选择性的光学调制 发射器。在目标I中，我们将阐明我们的Ag纳米人的独特光体物理学 启用长波长，次生激光诱导的光学耗竭 深色状态可显着提高总体排放率。由于此次要激光是 比主要激光激发和荧光较低的能量更低，我们 可以选择性地调节明亮的Ag纳米发射，而不是背景 通过调节次级激光强度。详细的光物理 迄今为止创建的所有纳米模特的表征至少将识别5个 具有基于调制的灵敏度增长的频谱纯纳米模型。在AIM II中，我们将 采用这5种不同颜色可调节的纳米模型来提取真正的细胞内 SM荧光信号通过整个图像调制。允许的限制 SM信号提取的背景将直接针对每个发射器进行探测 设计的控制实验。调制也将用于信号提取 单分子的基于活细胞荧光相关光谱观测。 这种选择性的光学调制应使整个图像被调制，并且 与检测的检测同步> 10倍的灵敏度在SM或大量中提高 纳米成像。有了成功的可能性，我们将发展这些Ultrabright， 高光稳定的发射器，其目标是基于光学调制的细胞内SM 观察。