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1. 发明申请

WO2009115108A1 A METHOD AND AN APPARATUS FOR LOCALIZATION OF SINGLE DYE MOLECULES IN THE FLUORESCENT MICROSCOPY 审中-公开
标题翻译：用于荧光显微镜中单分子分子定位的方法和装置
公开(公告)号：WO2009115108A1
公开(公告)日：2009-09-24
申请号：PCT/EP2008/009671
申请日：2008-11-14
申请人： RUPRECHT-KARLS-UNIVERSITÄT HEIDELBERG , LEMMER, Paul , CREMER, Christoph , BADDELEY, David , EIPEL, Heinz
发明人： LEMMER, Paul , CREMER, Christoph , BADDELEY, David , EIPEL, Heinz
IPC分类号： G01N21/64
CPC分类号： G01N21/6458 , G01N21/6428 , G01N2021/6441 , G02B21/0076 , G02B21/16 , G02B21/367 , G02B27/58
摘要： The invention concerns a method and a corresponding apparatus for obtaining a sub-resolution spatial information of a sample labeled with at least one type fluorescent label, said sub-resolution spatial information comprising localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction. The method comprises: - acquiring localization image data by employing fluorescence localization microscopy, wherein said localization image data comprises a series of images obtained by - illuminating a region of interest of the sample with illumination light having intensity in the range of approximately 1 kW/cm2 to approximately 1 MW/cm2, thereby transferring at least a portion of the fluorescent molecules of the at least one type fluorescent label from a first state to a second state and by means of emission of fluorescent light to a third inactive state, - detecting at least a portion of the emitted fluorescent light by an information acquiring sensor, thereby obtaining an image of the region of interest; - repeating the steps of illuminating and detecting of the emitted fluorescent light a plurality of times, thereby obtaining the series of images, each image being taken at a different time step; and - processing the acquired localization image data to thereby obtain said localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction, wherein the step of processing comprises determining in each of the detected images of the series the positions of the ban/centers of the detected fluorescence emission distributions from the single fluorescent molecules of the one or more fluorescent labels in at least one spatial direction.
摘要翻译：本发明涉及一种用于获得标记有至少一种类型荧光标签的样本的亚分辨率空间信息的方法和相应装置，所述子分辨率空间信息包括关于至少一种类型的荧光分子的位置的定位信息荧光标记在至少一个空间方向。该方法包括： - 采用荧光定位显微镜获取定位图像数据，其中所述定位图像数据包括通过以强度在约1kW / cm 2范围内的照明光照射样品的感兴趣区域而获得的一系列图像至约1MW / cm 2，从而将至少一种类型荧光标记物的荧光分子的至少一部分从第一状态转移到第二状态，并且通过将荧光发射到第三无效状态， - 在通过信息获取传感器发射的荧光的至少一部分，从而获得感兴趣区域的图像; - 重复多次发光荧光的照明和检测步骤，从而获得一系列图像，每个图像以不同的时间步长拍摄; 以及 - 处理所获取的定位图像数据，从而在至少一个空间方向上获得关于所述至少一种类型荧光标签的荧光分子的位置的所述定位信息，其中所述处理步骤包括在每个检测到的图像中确定将至少一个空间方向上的一个或多个荧光标记的单个荧光分子的检测到的荧光发射分布的禁止/中心的位置串联起来。

2. 发明申请

WO2007110126A1 VERFAHREN ZUR MIKROSKOPISCHEN ORTSBESTIMMUNG EINES AUSGEWÄHLTEN, INTRAZELLULÄREN DNA-ABSCHNITTS BEKANNTER NUKLEOTIDSEQUENZ 审中-公开
标题翻译：方法选择的，细胞内的DNA SECTION已知核苷酸的微观位置确定
公开(公告)号：WO2007110126A1
公开(公告)日：2007-10-04
申请号：PCT/EP2007/001467
申请日：2007-02-21
申请人： RUPRECHT-KARLS-UNIVERSITÄT HEIDELBERG , HAUSMANN, Michael , CREMER, Christoph
发明人： HAUSMANN, Michael , CREMER, Christoph
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6841 , C12Q2563/107 , C12Q2543/10 , C12Q2537/119
摘要： Das Verfahren zur mikroskopischen Ortsbestimmung eines ausgewählten intrazellulären, nativen Genomabschnitts mit bekannter Nukleotidsequenz in situ ist durch die Art und Reihenfolge der folgenden Maßnahmen gekennzeichnet: (1.) Die Target-DNA wird anhand von Genomdatenbanken hinsichtlich Teilsequenzen analysiert, die ein einmaliges Muster innerhalb des Genoms darstellen. (2) Es werden einzelsträngige Sondensequenzen bereitgestellt, die mit diesen Teilsequenzen übereinstimmen oder komplementär dazu sind, und die über eine Watson-Crick-Bindung zur Hybridisierung an die Einzelstränge dieser Teilsequenzen geeignet sind. (3.) Die Sondensequenzen werden mit Markermolekülen gekoppelt sind, wobei alle Einheiten aus Sondensequenz und Markermolekül(en) das gleiche Bindungsverhalten oder den gleichen Schmelzpunkt mit dem zu ihr komplementären Einzelstrang der Target-DNA aufweisen. (4.) Die Sondensequenzen werden in die Zelle eingebracht und mit der Target-DNA zusammengebracht, so daß sie mit den entsprechenden, temporär als zwei Einzelstränge vorliegenden Teilsequenzen der Target-DNA hybridisieren. (5.) Die emittierten Markersignale werden detektiert und (6.) anhand des Vorhandenseins und/oder der Intensität und/oder des gleichzeitigen Auftretens von verschiedenen Markersignalen wird der Ort der Target-DNA auf dem Genom identifiziert.
摘要翻译：对于具有原位已知核苷酸序列的选择的细胞内天然基因组节段的微观本地化的方法的特征在于，类型和以下的顺序：（1）目标DNA为部分序列分析的基因组中的独特模式的基因组数据库的基础上代表。（2）是对应于这些部分序列或是与其互补，并且其适于通过沃森 - 克里克结合杂交到这些部分序列的单链中提供单链探针序列。（3）探针序列加上标志物分子，其中探针序列和具有相同的结合行为或与单链相同的熔点，与靶DNA互补的标记分子（多个）的所有单元。（4）所述的探针序列引入所述细胞，并与靶DNA，使得它们与杂交作为靶DNA的两条单链存在的部分序列的暂时对应的汇集。（5）所发射的标记物信号被检测，和（6）的基础上的存在和/或强度和/或不同的标记的同时发生的信号上的基因组中的靶DNA的位置被识别。

3. 发明申请

WO2003031951A1 FAR YIELD LIGHT MICROSCOPICAL METHOD, SYSTEM AND COMPUTER PROGRAM PRODUCT FOR ANALYSING AT LEAST ONE OBJECT HAVING A SUBWAVELENGTH SIZE 审中-公开
标题翻译：用于分析至少一个具有亚水深尺寸的对象的微光微观方法，系统和计算机程序产品
公开(公告)号：WO2003031951A1
公开(公告)日：2003-04-17
申请号：PCT/EP2002/011343
申请日：2002-10-09
申请人： RUPRECHT-KARLS-UNIVERSITÄT HEIDELBERG , CREMER, Christoph , FAILLA, Antonio, Virgilio , ALBRECHT, Benno
发明人： CREMER, Christoph , FAILLA, Antonio, Virgilio , ALBRECHT, Benno
IPC分类号： G01N21/64
CPC分类号： G01N21/6458 , G01N21/6428 , G01N2021/6421 , G01N2021/6439 , G01N2021/6441 , G02B21/00 , G02B21/002 , G02B21/0076 , G02B21/16
摘要： The present invention relates to a far field light microscopical method, respectively a system and a computer program product for analysing at least one object having a subwavelength size in at least one spatial direction to obtain spatial information of the object, in particular size and topology thereof, comprising the steps of: - labelling the object(s) with one or more suitable optical markers; - providing suitably structured illumination light to at least partially illuminate the object(s); - subjecting the object(s) to the structured illumination light; - detecting an optical response of the object(s); - obtaining the spatial information of the object(s) by comparing the obtained response with simulation data of an optical response of object(s) having known spatial information.
摘要翻译：本发明涉及远场光显微镜方法，分别是用于在至少一个空间方向上分析具有亚波长尺寸的至少一个物体的系统和计算机程序产品，以获得物体的空间信息，特别是其尺寸和拓扑结构包括以下步骤： - 用一个或多个合适的光学标记对物体进行标记; - 提供适当结构的照明光以至少部分照亮所述物体; - 对物体施加结构照明光; - 检测物体的光学响应; - 通过将获得的响应与具有已知空间信息的对象的光学响应的仿真数据进行比较来获得对象的空间信息。

4. 发明申请

WO2014180566A1 METHOD AND APPARATUS FOR COMBINATION OF LOCALIZATION MICROSCOPY AND STRUCTURED ILLUMINATION MICROSCOPY 审中-公开
标题翻译：本地化显微镜和结构化照明显微镜的组合方法与装置
公开(公告)号：WO2014180566A1
公开(公告)日：2014-11-13
申请号：PCT/EP2014/001228
申请日：2014-05-07
申请人： RUPRECHT-KARLS-UNIVERSITÄT HEIDELBERG
发明人： BEST, Gerrit , CREMER, Christoph , ROSSBERGER, Sabrina , DITHMAR, Stefan
IPC分类号： G02B21/00 , G01N21/64 , G02B21/16 , G02B21/36 , G02B27/58
CPC分类号： G02B21/367 , G01N21/6445 , G01N21/6458 , G01N2201/0675 , G02B21/0076 , G02B21/16 , G02B21/361 , G02B27/58
摘要： The invention relates to a fluorescent microscope and a respective method for obtaining super-resolution images of a sample labelled with at least one type fluorescent label by combining localization microscopy and structured illumination microscopy. In an aspect, the fluorescent microscope comprises one or more light sources and an illumination system having a structured illumination path, in which a pattern generation system is positioned, for illuminating the sample with structured illumination light and a localization illumination path for illuminating the sample with localization illumination light. A switching mechanism is configured to switch between a first, a second and/or a third mode, wherein in the first mode at least a portion of the light emitted from the one or more light sources propagates through one of the illumination paths; in the second mode at least a portion of the light emitted from the one or more light sources propagates through the other one of the illumination paths; and in the third mode at least a portion of the light emitted from one or more of the light sources propagates through one illumination path while simultaneously at least another portion of the light emitted from one or more of the light sources propagates through the other illumination path. At least one image detector positioned in an optical detection path, configured to detect at least a portion of fluorescent light emitted from fluorescent molecules of the illuminated sample. Another aspect concerns a method for obtaining super-resolution image data of a sample labeled with at least one type of fluorescent label comprising illuminating the sample with localization illumination light and with structured illumination light; detecting at least a portion of the fluorescent light emitted from the excited fluorescent molecules of the at least one fluorescent label, thereby obtaining at least one image of the illuminated sample; and processing the obtained at least one image of the sample image to obtain super-resolution image data.
摘要翻译：本发明涉及通过组合定位显微镜和结构化照明显微镜获得用至少一种类型荧光标记物标记的样品的超分辨率图像的荧光显微镜和相应方法。在一方面，荧光显微镜包括一个或多个光源和具有结构化照明路径的照明系统，其中定位图案生成系统，用于以结构化的照明光照射样本，并且具有用于照亮样品的定位照明路径，定位照明灯。切换机构被配置为在第一模式，第二模式和/或第三模式之间切换，其中在第一模式中，从一个或多个光源发射的光的至少一部分传播通过照明路径之一; 在第二模式中，从一个或多个光源发射的光的至少一部分传播通过另一个照明路径; 并且在第三模式中，从一个或多个光源发射的光的至少一部分传播通过一个照明路径，同时，从一个或多个光源发射的光的至少另一部分传播通过另一个照明路径。位于光学检测路径中的至少一个图像检测器，被配置为检测从被照射样品的荧光分子发射的荧光的至少一部分。另一方面涉及一种用于获得用至少一种类型的荧光标签标记的样品的超分辨率图像数据的方法，包括用定位照明光和结构化照明光照射样品; 检测从所述至少一个荧光标记的激发的荧光分子发射的荧光的至少一部分，从而获得所述被照射样品的至少一个图像; 以及处理获得的样本图像的至少一个图像以获得超分辨率图像数据。

5. 发明公开

EP0946855A1 VERFAHREN UND VORRICHTUNGEN ZUR DISTANZMESSUNG ZWISCHEN OBJEKTSTRUKTUREN 失效
标题翻译：方法和距离测量之间的对象结构DEVICES
公开(公告)号：EP0946855A1
公开(公告)日：1999-10-06
申请号：EP97953660.0
申请日：1997-12-20
申请人： Ruprecht-Karls-Universität Heidelberg
发明人： CREMER, Christoph , HAUSMANN, Michael , BRADL, Joachim , RINKE, Bernd
IPC分类号： G01Q60 , G01Q80 , G01N21 , G02B21
CPC分类号： B82Y15/00 , G02B21/16 , G02B21/26 , G02B21/34
摘要： The invention relates to a method and devices for far field microscopy and flow fluorometry for geometrical telemetry between fluorchrome-marked object structures, which are the structures to be measured, wherein distances can be smaller than the half width of the principal maximum of the effective point image function. According to this method, the measuring structures are marked with fluorescent colorants having different or the same spectral signature depending on the distance between said structures. Calibration targets of defined sizes and spatial arrangements are marked with the same fluorescent colorants. Calibration targets and measuring structures are prepared together or separately on an object holder and submitted to microscopic or flow-fluorometric examination. In each case, two defined calibration targets of different spectral signature are measured according to wavelength-dependent imaging and locational behavior of the corresponding optical system. The measuring values thus obtained are compared with the previously known actual distances, and the difference is used as calibrating value for correcting the misalignment determined by the optical system in the detection of measuring structures. The devices comprise calibrating targets and an axial tomograph for implementing the inventive method.

6. 发明公开

EP1436597A1 FAR YIELD LIGHT MICROSCOPICAL METHOD, SYSTEM AND COMPUTER PROGRAM PRODUCT FOR ANALYSING AT LEAST ONE OBJECT HAVING A SUBWAVELENGTH SIZE 有权
标题翻译：用于确定至少一个对象FERNFELD光镜的方法和设备中所使用小于波长
公开(公告)号：EP1436597A1
公开(公告)日：2004-07-14
申请号：EP02785192.2
申请日：2002-10-09
申请人： Ruprecht-Karls-Universität Heidelberg
发明人： CREMER, Christoph , FAILLA, Antonio, Virgilio , ALBRECHT, Benno
IPC分类号： G01N21/64 , G02B21/00
CPC分类号： G01N21/6458 , G01N21/6428 , G01N2021/6421 , G01N2021/6439 , G01N2021/6441 , G02B21/00 , G02B21/002 , G02B21/0076 , G02B21/16
摘要： The present invention relates to a far field light microscopical method, respectively a system and a computer program product for analysing at least one object having a subwavelength size in at least one spatial direction to obtain spatial information of the object, in particular size and topology thereof, comprising the steps of: - labelling the object(s) with one or more suitable optical markers; - providing suitably structured illumination light to at least partially illuminate the object(s); - subjecting the object(s) to the structured illumination light; - detecting an optical response of the object(s); - obtaining the spatial information of the object(s) by comparing the obtained response with simulation data of an optical response of object(s) having known spatial information.

7. 发明授权

EP0946855B1 VERFAHREN UND VORRICHTUNGEN ZUR DISTANZMESSUNG ZWISCHEN OBJEKTSTRUKTUREN 失效
标题翻译：方法和距离测量之间的对象结构DEVICES
公开(公告)号：EP0946855B1
公开(公告)日：2001-10-17
申请号：EP97953660.4
申请日：1997-12-20
申请人： Ruprecht-Karls-Universität Heidelberg
发明人： CREMER, Christoph , HAUSMANN, Michael , BRADL, Joachim , RINKE, Bernd
IPC分类号： G01B9/04 , G02B21/16 , G02B21/34
CPC分类号： B82Y15/00 , G02B21/16 , G02B21/26 , G02B21/34

8. 发明授权

EP1436597B1 FAR YIELD LIGHT MICROSCOPICAL METHOD AND SYSTEM FOR ANALYSING AT LEAST ONE OBJECT HAVING A SUBWAVELENGTH SIZE 有权
标题翻译：用于确定至少一个对象FERNFELD光镜的方法和设备中所使用小于波长
公开(公告)号：EP1436597B1
公开(公告)日：2008-07-02
申请号：EP02785192.2
申请日：2002-10-09
申请人： Ruprecht-Karls-Universität Heidelberg
发明人： CREMER, Christoph , FAILLA, Antonio, Virgilio , ALBRECHT, Benno
IPC分类号： G01N21/64 , G02B21/00
CPC分类号： G01N21/6458 , G01N21/6428 , G01N2021/6421 , G01N2021/6439 , G01N2021/6441 , G02B21/00 , G02B21/002 , G02B21/0076 , G02B21/16
摘要： The present invention relates to a far field light microscopical method, respectively a system and a computer program product for analysing at least one object having a subwavelength size in at least one spatial direction to obtain spatial information of the object, in particular size and topology thereof, comprising the steps of: - labelling the object(s) with one or more suitable optical markers; - providing suitably structured illumination light to at least partially illuminate the object(s); - subjecting the object(s) to the structured illumination light; - detecting an optical response of the object(s); - obtaining the spatial information of the object(s) by comparing the obtained response with simulation data of an optical response of object(s) having known spatial information.

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