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HIGH PERFORMANCE PHOTON-COUNTING IMAGER

高性能光子计数成像仪

基本信息

批准号：
6648393
负责人：
SHIMON WEISS
金额：
$ 13.9万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-09-01 至 2005-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6648393
关键词：
DNA directed RNA polymerase bioengineering /biomedical engineering biosensor device computer data analysis computer program /software computer system design /evaluation computer system hardware electromagnetic radiation fibroblasts fluorescence spectrometry method development monitoring device semiconduction

项目摘要

DESCRIPTION (provided by applicant): The objectives of this proposal are to develop a high performance photon counting imager optimized for ultrahigh sensitivity fluorescence spectroscopy and imaging and to develop new methodologies and protocols that take advantage of its unique capabilities. The detector will be capable of registering single photons with high-resolution in space and time at high-count rates and high quantum efficiency. It will be suitable for imaging and spectroscopy of single molecules, molecular complexes and macromolecules in living cells and tissues with increased sensitivity, signal to noise and signal to background, while providing multi-parametric, high information-content from each detected photon. They dub this detector H33D (pronounced `heed', for: high-spatial resolution, high-temporal resolution and high count-rate 3-dimensional detector). The targeted detector performance: (i) a spatial resolution of 100 microm x 100 microm per pixel (spatially resolvable units) with a detector dimension (25 mm diameter) allowing at least 256 x 256 resolution elements (pixels), (ii) 100 picoseconds (ps) temporal resolution, (iii) a maximum count rate of 10(5) Hertz (100 kHz) per single pixel and 5.10(7) Hertz (50 MHz) over the whole detector and (iv) a quantum efficiency (QE) of > 40 percent @ 600nm. The proposed work is "high risk, high impact" and fits well the structure of the R21/R33 phased innovation mechanism. They will develop 3 generations of H33D detectors. The specifications/milestones for the pilot phase are already superior to those of current commercial detectors. They anticipate that these milestones will be met within 12 months. In the expanded development phase (R33), they will build two more generations. To deliver on the milestones, they will need to develop high QE photocathode (feasibility unknown, high risk); low-gain, moderate-resolution spatial detection (proof of principle exists); time-stamping of simultaneous spatially-separable events (feasibility unknown, high risk); high-count rate at high timing resolution (feasibility good). The development of the H33D would open new windows in multi-parameters studies of single molecules (simultaneous acquisition of lifetime, fluorescence spectrum and polarization), in time-resolved wide-field imaging of in vivo cellular processes or in vitro enzymatic reactions and high-throughput fluorescence correlation spectroscopy (FCS). They will demonstrate proof-of-principle biological applications building on other funded projects in their laboratory: (i) Time-correlated imaging and spectroscopy of semiconductor nanocrystals (NCs); (ii) Digital time-gated imaging of NCs in live fibroblast cells; (iii) Fluorescence lifetime imaging (FLIM) of live cells; (iv) Line-confocal time-resolved spectral imaging of cells; (v) Single-pair FRET conformational dynamics of surface-immobilized RNA polymerase molecules; (vi) Microfluidic applications; (vii) Multiconfocal fluorescence correlation spectroscopy (FCS); (viii) High frame-rate cell imaging.

描述(由申请人提供)：本提案的目标是开发一种针对超高灵敏度荧光光谱和成像进行优化的高性能光子计数成像仪，并开发利用其独特功能的新方法和协议。该探测器将能够以高计数速率和高量子效率在空间和时间上以高分辨率记录单光子。它将适用于活细胞和组织中单分子、分子络合物和大分子的成像和光谱，具有更高的灵敏度、对噪声的信号和对背景的信号，同时提供来自每个检测到的光子的多参数、高信息含量。他们将这种探测器命名为H33D(发音为‘heed’，意为：高空间分辨率、高时间分辨率和高计数率的三维探测器)。目标探测器的性能：(I)空间分辨率为每像素100微米x 100微米(空间可分辨单位)，探测器尺寸(直径25 mm)，允许至少256 x 256分辨率元素(像素)，(Ii)100皮秒(Ps)时间分辨率，(Iii)每单个像素的最大计数速率为10(5)赫兹(100 KHz)，整个探测器的最大计数速率为5.10(7)赫兹(50 MHz)，(Iv)量子效率(QE)为&gt；40%@600 nm。拟开展的工作是“高风险、高影响”，与R21/R33阶段性创新机制的结构非常契合。他们将开发3代H33D探测器。试验阶段的规格/里程碑已经优于目前的商业探测器。他们预计这些里程碑将在12个月内实现。在扩展开发阶段(R33)，他们将再建造两代。为了实现这些里程碑，他们将需要开发高QE光电阴极(可行性未知，高风险)；低增益，中等分辨率的空间探测(存在原则证据)；同时空间可分离事件的时间戳(可行性未知，高风险)；高时间分辨率的高计数率(可行性好)。 H33D的开发将为单分子的多参数研究(同时获取寿命、荧光光谱和偏振)、体内细胞过程或体外酶反应的时间分辨广域成像以及高通量荧光相关光谱(FCS)打开新的窗口。他们将在实验室其他资助项目的基础上展示原理验证生物学应用：(I)半导体纳米晶体(NCS)的时间相关成像和光谱；(Ii)活成纤维细胞中NCS的数字时间门控成像；(Iii)活细胞的荧光寿命成像(FLIM)；(Iv)细胞的线共聚焦时间分辨光谱成像；(V)表面固定RNA聚合酶分子的单对FRET构象动力学；(Vi)微流控应用；(Vii)多聚焦荧光相关光谱(FCS)；(Viii)高帧速率细胞成像。