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1. 发明授权

US06661909B2 Calibration of fluorescence resonance energy transfer in microscopy 失效
公开(公告)号：US06661909B2
公开(公告)日：2003-12-09
申请号：US09912117
申请日：2001-07-23
申请人： Dougalas C. Youvan , Christopher M. Silva , Edward J. Bylina , William J. Coleman , Michael R. Dilworth , Mary M. Yang
发明人： Dougalas C. Youvan , Christopher M. Silva , Edward J. Bylina , William J. Coleman , Michael R. Dilworth , Mary M. Yang
IPC分类号： G06K900
CPC分类号： G01N21/6458 , G01N21/278 , G01N21/6428 , G01N2021/6441
摘要： Imaging hardware, software, calibrants, and methods are provided to visualize and quantitate the amount of Fluorescence Resonance Energy Transfer (FRET) occurring between donor and acceptor molecules in epifluorescence microscopy. The MicroFRET system compensates for overlap among donor, acceptor, and FRET spectra using well characterized fluorescent beads as standards in conjunction with radiometrically calibrated image processing techniques. The MicroFRET system also provides precisely machined epifluorescence cubes to maintain proper image registration as the sample is illuminated at the donor and acceptor excitation wavelengths. Algorithms are described that pseudocolor the image to display pixels exhibiting radiometrically-corrected fluorescence emission from the donor (blue), the acceptor (green) and FRET (red). The method is demonstrated on samples exhibiting FRET between genetically engineered derivatives of the Green Fluorescent Protein (GFP) bound to the surface of Ni chelating beads by histidine-tags.

2. 发明授权

US5914245A Solid phase enzyme kinetics screening in microcolonies 失效
标题翻译：微菌落中的固相酶动力学筛选
公开(公告)号：US5914245A
公开(公告)日：1999-06-22
申请号：US98202
申请日：1998-06-16
申请人： Edward J. Bylina , William J. Coleman , Michael R. Dilworth , Christopher M. Silva , Mary M. Yang , Douglas C. Youvan
发明人： Edward J. Bylina , William J. Coleman , Michael R. Dilworth , Christopher M. Silva , Mary M. Yang , Douglas C. Youvan
IPC分类号： C12Q1/04 , C12Q1/34 , C12Q1/44 , C12Q1/00 , C12Q1/37 , C12Q1/54
CPC分类号： C12Q1/04 , C12Q1/34 , Y10S435/808 , Y10S435/968
摘要： A MicroColonyImager instrument and solid phase methods to screen cells expressing mutagenized enzymes for enhanced activity. The MicroColonyImager instrument and methods permit high throughput screening of enzyme libraries by time course analyses of single-pixels, using either absorption, fluorescence or FRET. This high throughput assay can detect small differences in enzyme rates within microcolonies grown at a nearly confluent density on an assay disk. Each microcolony is analyzed simultaneously at single-pixel resolution, requiring less than 100 ml substrate/measurement. By simultaneously assaying different substrates tagged with spectrally distinct chromogenic or fluorogenic reporters, the substrate specificity of an enzyme can be changed.
摘要翻译： MicroColonyImager仪器和固相方法筛选表达诱变酶的细胞以增强活性。 MicroColonyImager仪器和方法允许通过使用吸收，荧光或FRET的单个像素的时程分析来酶库的高通量筛选。这种高通量测定可以检测在测定盘上以几乎汇合的密度生长的微菌落内的酶速度的小差异。每个微绒毛在单像素分辨率下同时进行分析，需要少于100毫升的底物/测量。通过同时测定用光谱不同的显色或荧光报告物标记的不同底物，可以改变酶的底物特异性。

3. 发明授权

US06456734B1 Calibration of fluorescence resonance energy transfer in microscopy 失效
公开(公告)号：US06456734B1
公开(公告)日：2002-09-24
申请号：US09092316
申请日：1998-06-05
申请人： Douglas C. Youvan , Christopher M. Silva , Edward J. Bylina , William J. Coleman , Michael R. Dilworth , Mary M. Yang
发明人： Douglas C. Youvan , Christopher M. Silva , Edward J. Bylina , William J. Coleman , Michael R. Dilworth , Mary M. Yang
IPC分类号： G06K900
CPC分类号： G01N21/6458 , G01N21/278 , G01N21/6428 , G01N2021/6441
摘要： Imaging hardware, software, calibrants, and methods are provided to visualize and quantitate the amount of Fluorescence Resonance Energy Transfer (FRET) occurring between donor and acceptor molecules in epifluorescence microscopy. The MicroFRET system compensates for overlap among donor, acceptor, and FRET spectra using well characterized fluorescent beads as standards in conjunction with radiometrically calibrated image processing techniques. The MicroFRET system also provides precisely machined epifluorescence cubes to maintain proper image registration as the sample is illuminated at the donor and acceptor excitation wavelengths. Algorithms are described that pseudocolor the image to display pixels exhibiting radiometrically-corrected fluorescence emission from the donor (blue), the acceptor (green) and FRET (red). The method is demonstrated on samples exhibiting FRET between genetically engineered derivatives of the Green Fluorescent Protein (GFP) bound to the surface of Ni chelating beads by histidine-tags.

4. 发明授权

US06834122B2 Visualization and processing of multidimensional data using prefiltering and sorting criteria 失效
标题翻译：使用预过滤和排序标准可视化和处理多维数据
公开(公告)号：US06834122B2
公开(公告)日：2004-12-21
申请号：US09767595
申请日：2001-01-22
申请人： Mary M. Yang , Edward J. Bylina , William J. Coleman , Michael R. Dilworth , Steven J. Robles , Douglas C. Youvan
发明人： Mary M. Yang , Edward J. Bylina , William J. Coleman , Michael R. Dilworth , Steven J. Robles , Douglas C. Youvan
IPC分类号： G06K968
CPC分类号： G06K9/00127 , G06K9/0014 , G06K9/0063 , G06K9/6253 , G06K2009/00644 , G06K2009/4657 , G06T7/0012 , G06T2200/24 , G06T2207/10016 , G06T2207/10056 , G06T2207/30024 , Y10S128/922 , Y10S707/99937
摘要： Complex multidimensional datasets generated by digital imaging spectroscopy can be organized and analyzed by applying software and computer-based methods comprising sorting algorithms. Combinations of these algorithms to images and graphical data, allow pixels or features to be rapidly and efficiently classified into meaningful groups according to defined criteria. Multiple rounds of pixel or feature selection may be performed based on independent sorting criteria. In one embodiment sorting by spectral criteria (e.g., intensity at a given wavelength) is combined with sorting by temporal criteria (e.g., absorbance at a given time) to identify microcolonies of recombinant organisms harboring mutated genes encoding enzymes having desirable kinetic attributes and substrate specificity. Restriction of the set of pixels analyzed in a subsequent sort based on criteria applied in an earlier sort (“sort and lock” analyses) minimize computational and storage resources. User-defined criteria can also be incorporated into the sorting process by means of a graphical user interface that comprises a visualization tools including a contour plot, a sorting bar and a grouping bar, an image window, and a plot window that allow run-time interactive identification of pixels or features meeting one or more criteria, and display of their associated spectral or kinetic data. These methods are useful for extracting information from imaging data in applications ranging from biology and medicine to remote sensing.
摘要翻译：通过数字成像光谱法生成的复杂多维数据集可以通过应用软件和基于计算机的方法进行组织和分析，包括排序算法。这些算法与图像和图形数据的组合允许像素或特征根据定义的标准快速有效地分类为有意义的组。可以基于独立排序标准来执行多轮像素或特征选择。在一个实施方案中，通过光谱标准（例如，给定波长处的强度）的排序与通过时间标准（例如，给定时间的吸光度）排序相结合，以鉴定携带突变基因的重组体的微菌落，所述突变基因编码具有期望的动力学属性和底物特异性的酶。基于先前排序（“排序和锁定”分析）中应用的标准，在后续排序中分析的像素集合的限制最小化了计算和存储资源。用户定义的标准也可以通过包括可视化工具的图形用户界面结合到分类过程中，该可视化工具包括轮廓图，分类栏和分组条，图像窗口和允许运行时间的绘图窗口满足一个或多个标准的像素或特征的交互式识别，以及它们相关联的光谱或动力学数据的显示。这些方法可用于从生物学和医学到遥感等应用中从成像数据中提取信息。

5. 发明授权

US06738502B1 Multispectral taxonomic identification 失效
标题翻译：多光谱分类鉴定
公开(公告)号：US06738502B1
公开(公告)日：2004-05-18
申请号：US09585951
申请日：2000-06-02
申请人： William Coleman , Michael A. Tanner , Christopher M. Silva , Edward Bylina , Steven J. Robles , Michael R. Dilworth , Douglas C. Youvan , Mary M. Yang
发明人： William Coleman , Michael A. Tanner , Christopher M. Silva , Edward Bylina , Steven J. Robles , Michael R. Dilworth , Douglas C. Youvan , Mary M. Yang
IPC分类号： G06K900
CPC分类号： G01N21/274 , G01N21/6458 , G01N2201/127
摘要： The present invention provides an instrument and methods for a multispectral optical technique that can simultaneously classify individual biological cells within mixed populations. This invention, known as Multispectral Taxonomic Identification (MTID), shows that microscopy can be combined with a software analysis program to measure and categorize the fluorescence and other spectroscopically identifiable signals from complex populations of cells in situ, without cultivation. The invention thus enables high-throughput screening of cells for taxonomic classification.
摘要翻译：本发明提供了一种用于多光谱光学技术的仪器和方法，其可以在混合群体内同时分类各个生物细胞。称为多光谱分类鉴定（MTID）的本发明表明，显微镜可以与软件分析程序相结合，以便对来自复杂原位细胞群的荧光和其他可光谱鉴定的信号进行原位测量和分类，无需培养。因此，本发明使得能够进行用于分类分类的细胞的高通量筛选。

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