公开(公告)号：US07534576B2

公开(公告)日：2009-05-19

申请号：US11730931

申请日：2007-04-04

申请人： Rudolf Rigler ,  Manfred Eigen ,  Karsten Henco ,  Ulo Mets ,  Jerker Widengren ,  Michael Stuke ,  Michael Brinkmeyer ,  Wolfgang Simm ,  Olaf Lehman

发明人： Rudolf Rigler ,  Manfred Eigen ,  Karsten Henco ,  Ulo Mets ,  Jerker Widengren ,  Michael Stuke ,  Michael Brinkmeyer ,  Wolfgang Simm ,  Olaf Lehman

IPC分类号： G01N33/569

CPC分类号： G01N21/6452 ,  C12Q1/04 ,  C12Q1/6827 ,  G01N21/64 ,  G01N21/6402 ,  G01N21/6408 ,  G01N21/6428 ,  G01N21/6458 ,  G02B21/24 ,  Y10S435/808 ,  Y10S436/805 ,  Y10T436/11 ,  C12Q2565/107 ,  C12Q2561/113 ,  C12Q2535/131

摘要： A Method for identifying one or a small number of molecules, especially in a dilution of ≦1 μM, using laser excited FCS with measuring times ≦500 ms and short diffusion paths of the molecules to be analyzed, wherein the measurement is performed in small volume units of preferably ≦10−14 l, by determining material-specific parameters which are determined by luminescence measurements of molecules to be examined.The device which can be preferably used for performing the method according to the invention is a per se known system of microscope optics for laser focusing for fluorescence excitation in a small measuring compartment of a very diluted solution and for imaging the emitted light in the subsequent measurement through confocal imaging wherein at least one system of optics with high numerical aperture of preferably ≧1.2 N.A. is employed, the light quantity is limited by a confocally arranged pinhole aperture in the object plane behind the microscope objective, and the measuring compartment is positioned at a distance of between 0 and 1000 μm from the observation objective.

摘要翻译： 使用测量时间<= 500ms的激光激发FCS和要分析的分子的短扩散路径来识别一个或少数分子，特别是稀释度为<=1μM的分子的方法，其中测量在 通过确定由待分析的分子的发光测量确定的材料特异性参数，优选<=10-14μl的小体积单位。 可以优选用于执行根据本发明的方法的装置是本身已知的用于在非常稀释的溶液的小测量室中进行荧光激发的激光聚焦的显微镜光学系统，并且用于在后续测量中对发射的光进行成像 通过共焦成像，其中使用具有优选> = 1.2NA的高数值孔径的至少一个光学系统，光量受到在显微镜物镜后面的物平面中的共同排列的针孔的限制，并且测量室位于 与观察目标之间的距离为0至1000μm。

公开(公告)号：US07839500B2

公开(公告)日：2010-11-23

申请号：US11920789

申请日：2006-05-29

申请人： Jerker Widengren

发明人： Jerker Widengren

IPC分类号： G01N21/64

CPC分类号： G01N21/00 ,  G01N21/6408 ,  G01N21/6428 ,  G01N21/6452 ,  G01N2021/6432 ,  G01N2021/6441

摘要： The present invention relates to a spectroscopic method and associated apparatus and computer program for measuring and analysing intensities of fluorescent molecules excited by an energy pulse. The method includes the steps of: a) generating a transient state build-up in the fluorescent molecules by means of an excitation pulse, within which pulse repetitive excitation-emission cycles are induced in the fluorescent molecules between their ground, typically singlet (So) and excited, typically singlet (SO states, resulting also in transition from S] to the transient state, b) relaxation of population of the transient state by transition back to the ground state in a time period following directly after the excitation pulse, c) determination of the transient state population by recording the fluorescence. The invention is characterised by varying pulse characteristics from one sequence of pulses to the next so as to circumvent the need of time-resolution in the detection.

摘要翻译： 本发明涉及一种用于测量和分析由能量脉冲激发的荧光分子的强度的光谱方法及相关装置和计算机程序。 该方法包括以下步骤：a）通过激发脉冲在荧光分子中产生瞬时状态积聚，其中脉冲重复激发 - 发射循环在它们的地面之间的荧光分子中被诱导，通常是单重态（So） 并且激发，通常为单态（SO状态，也从S转变为瞬态），b）通过在激励脉冲之后的时间段内转变回基态来缓和瞬态的群体，c） 通过记录荧光测定瞬态群体。 本发明的特征在于从脉冲序列到下一个序列的脉冲特性变化，从而避免在检测中需要时间分辨率。

公开(公告)号：US20150132775A1

公开(公告)日：2015-05-14

申请号：US14117347

申请日：2012-05-11

申请人： Johan Strömqvist ,  Sofia Johansson ,  Jerker Widengren

发明人： Johan Strömqvist ,  Sofia Johansson ,  Jerker Widengren

IPC分类号： G01N21/64 ,  C12Q1/68 ,  G01N33/58

CPC分类号： G01N21/6428 ,  C12Q1/6818 ,  G01N21/6408 ,  G01N33/542 ,  G01N33/581 ,  G01N33/582 ,  G01N2021/6417 ,  G01N2021/6432 ,  G01N2021/6441

摘要： The present invention provides a FCCS method for determining the concentration and/or the diffusion coefficient of at least a first labeled species, a second labeled species and/or a complex between said first and second labeled species, in a system, wherein the method comprises the steps of determining a cross-talk parameter K, wherein K is the ratio between the brightness of the first labeled species and the second labeled species at the centre of each focus, as detected for both species in the channel for detecting the second labeled species; using the cross talk parameter K for determining a displacement parameter ro and using K, ro, or both K and ro for determining the concentration and/or the diffusion coefficient of said first and/or a second labeled species and/or a complex between said first and second labeled species.

摘要翻译： 本发明提供了一种用于在系统中确定至少第一标记物质，第二标记物质和/或所述第一和第二标记物质之间的复合物的浓度和/或扩散系数的FCCS方法，其中所述方法包括 确定串扰参数K的步骤，其中K是在用于检测第二标记物质的通道中检测到的针对每个焦点中心的第一标记物质的亮度和第二标记物质之间的比率 ; 使用串扰参数K来确定位移参数ro并且使用K，ro或K和ro两者来确定所述第一和/或第二标记物质的浓度和/或扩散系数和/或所述第二和/ 第一和第二标记物种。

公开(公告)号：US20090040518A1

公开(公告)日：2009-02-12

申请号：US11920789

申请日：2006-05-29

申请人： Jerker Widengren

发明人： Jerker Widengren

IPC分类号： G01N21/64

CPC分类号： G01N21/00 ,  G01N21/6408 ,  G01N21/6428 ,  G01N21/6452 ,  G01N2021/6432 ,  G01N2021/6441

摘要： The present invention relates to a spectroscopic method and associated apparatus and computer program for measuring and analysing intensities of fluorescent molecules excited by an energy pulse. The method includes the steps of: a) generating a transient state build-up in the fluorescent molecules by means of an excitation pulse, within which pulse repetitive excitation-emission cycles are induced in the fluorescent molecules between their ground, typically singlet (So) and excited, typically singlet (SO states, resulting also in transition from S] to the transient state, b) relaxation of population of the transient state by transition back to the ground state in a time period following directly after the excitation pulse, c) determination of the transient state population by recording the fluorescence. The invention is characterised by varying pulse characteristics from one sequence of pulses to the next so as to circumvent the need of time-resolution in the detection.

摘要翻译： 本发明涉及一种用于测量和分析由能量脉冲激发的荧光分子的强度的光谱方法及相关装置和计算机程序。 该方法包括以下步骤：a）通过激发脉冲在荧光分子中产生瞬时状态积聚，其中脉冲重复激发 - 发射循环在它们的地面之间的荧光分子中被诱导，通常是单重态（So） 并且激发，通常为单态（SO状态，也从S转变为瞬态），b）通过在激励脉冲之后的时间段内转变回基态来缓和瞬态的群体，c） 通过记录荧光测定瞬态群体。 本发明的特征在于从脉冲序列到下一个序列的脉冲特性变化，从而避免在检测中需要时间分辨率。

公开(公告)号：US07241569B2

公开(公告)日：2007-07-10

申请号：US10435674

申请日：2003-05-12

申请人： Rudolf Rigler ,  Manfred Eigen ,  Karsten Henco ,  Ulo Mets ,  Jerker Widengren ,  Michael Stuke ,  Michael Brinkmeyer ,  Wolfgang Simm ,  Olaf Lehman

发明人： Rudolf Rigler ,  Manfred Eigen ,  Karsten Henco ,  Ulo Mets ,  Jerker Widengren ,  Michael Stuke ,  Michael Brinkmeyer ,  Wolfgang Simm ,  Olaf Lehman

IPC分类号： G01N33/543

CPC分类号： G01N21/6452 ,  C12Q1/04 ,  C12Q1/6827 ,  G01N21/64 ,  G01N21/6402 ,  G01N21/6408 ,  G01N21/6428 ,  G01N21/6458 ,  G02B21/24 ,  Y10S435/808 ,  Y10S436/805 ,  Y10T436/11 ,  C12Q2565/107 ,  C12Q2561/113 ,  C12Q2535/131

摘要： A Method for identifying one or a small number of molecules, especially in a dilution of ≦1 μM, using laser excited FCS with measuring times ≦500 ms and short diffusion paths of the molecules to be analyzed, wherein the measurement is performed in small volume units of preferably ≦10−14 l, by determining material-specific parameters which are determined by luminescence measurements of molecules to be examined.The device which can be preferably used for performing the method according to the invention is a per se known system of microscope optics for laser focusing for fluorescence excitation in a small measuring compartment of a very diluted solution and for imaging the emitted light in the subsequent measurement through confocal imaging wherein at least one system of optics with high numerical aperture of preferably ≧1.2 N.A. is employed, the light quantity is limited by a confocally arranged pinhole aperture in the object plane behind the microscope objective, and the measuring compartment is positioned at a distance of between 0 and 1000 μm from the observation objective.

摘要翻译： 使用测量时间<= 500ms的激光激发FCS和要分析的分子的短扩散路径来识别一个或少数分子，特别是稀释度为<=1μM的分子的方法，其中测量在 通过确定通过待检测分子的发光测量确定的材料特异性参数，优选<= 10 -14 / 1的小体积单位。 可以优选用于执行根据本发明的方法的装置是本身已知的用于在非常稀释的溶液的小测量室中进行荧光激发的激光聚焦的显微镜光学系统，并且用于在后续测量中对发射的光进行成像 通过共焦成像，其中使用具有优选> = 1.2NA的高数值孔径的至少一个光学系统，光量受到在显微镜物镜后面的物平面中的共同排列的针孔的限制，并且测量室位于 与观察目标之间的距离为0至1000μm。

公开(公告)号：US20120050734A1

公开(公告)日：2012-03-01

申请号：US13264651

申请日：2010-04-15

申请人： Stefan Wennmalm ,  Jerker Widengren

发明人： Stefan Wennmalm ,  Jerker Widengren

IPC分类号： G01J3/44 ,  G01N21/64 ,  G01J3/30

CPC分类号： G01N21/6408 ,  G01N15/0205 ,  G01N21/6458 ,  G01N21/65 ,  G01N21/658 ,  G01N2015/0038

摘要： A method is disclosed for analyzing particles or biomolecules in a liquid sample, including: detecting a signal and fluctuations in the signal from a detection volume in the sample; wherein the signal is generated from signal-generating molecules in the medium surrounding the particles or biomolecules and the fluctuations are transient reductions in the signal as the particles or biomolecules transit through the detection volume; and analyzing the detected fluctuations to obtain information about the particles or biomolecules in the liquid sample. At least one example embodiment of the present invention relates to a fluorescence correlation spectroscopy system including a laser, a zero-mode waveguide, guiding device for guiding the laser into the zero-mode waveguide, device for collecting fluorescence emission from excited molecules within the waveguide, a detector for detecting the fluorescence emission and means for autocorrelating the detected fluorescence signal, wherein the detector comprises a photomultiplier tube. Moreover, at least one embodiment relates to the use of a fluorescence correlation spectroscopy system for analyzing molecules of interest in a sample by detecting and analyzing fluctuations in a fluorescence signal that is generated from sample molecules surrounding the molecules of interest, wherein the fluctuations are transient reductions in the detected fluorescence signal.

摘要翻译： 公开了一种用于分析液体样品中的颗粒或生物分子的方法，包括：检测来自样品中检测体积的信号和信号波动; 其中所述信号由围绕所述颗粒或生物分子的介质中的信号产生分子产生，并且所述波动是当所述颗粒或生物分子通过所述检测体积传递时所述信号的瞬时减少; 并分析检测到的波动以获得关于液体样品中的颗粒或生物分子的信息。 本发明的至少一个示例实施例涉及一种荧光相关光谱系统，其包括激光器，零模式波导，用于将激光器引导到零模式波导中的引导装置，用于从波导内的激发分子收集荧光发射的装置 ，用于检测荧光发射的检测器和用于自动相关所检测的荧光信号的装置，其中所述检测器包括光电倍增管。 此外，至少一个实施方案涉及通过检测和分析由感兴趣分子周围的样品分子产生的荧光信号的波动来分析样品中感兴趣的分子的荧光相关光谱系统的用途，其中波动是瞬时的 检测到的荧光信号减少。

公开(公告)号：US20070243525A1

公开(公告)日：2007-10-18

申请号：US11730931

申请日：2007-04-04

申请人： Rudolf Rigler ,  Manfred Eigen ,  Karsten Henco ,  Ulo Mets ,  Jerker Widengren ,  Michael Stuke ,  Michael Brinkmeyer ,  Wolfgang Simm ,  Olaf Lehmann

发明人： Rudolf Rigler ,  Manfred Eigen ,  Karsten Henco ,  Ulo Mets ,  Jerker Widengren ,  Michael Stuke ,  Michael Brinkmeyer ,  Wolfgang Simm ,  Olaf Lehmann

IPC分类号： C12Q1/02 ,  C12Q1/70 ,  G01J3/30

CPC分类号： G01N21/6452 ,  C12Q1/04 ,  C12Q1/6827 ,  G01N21/64 ,  G01N21/6402 ,  G01N21/6408 ,  G01N21/6428 ,  G01N21/6458 ,  G02B21/24 ,  Y10S435/808 ,  Y10S436/805 ,  Y10T436/11 ,  C12Q2565/107 ,  C12Q2561/113 ,  C12Q2535/131

摘要： A Method for identifying one or a small number of molecules, especially in a dilution of ≦1 μM, using laser excited FCS with measuring times ≦500 ms and short diffusion paths of the molecules to be analyzed, wherein the measurement is performed in small volume units of preferably ≦10−14 l, by determining material-specific parameters which are determined by luminescence measurements of molecules to be examined. The device which can be preferably used for performing the method according to the invention is a per se known system of microscope optics for laser focusing for fluorescence excitation in a small measuring compartment of a very diluted solution and for imaging the emitted light in the subsequent measurement through confocal imaging wherein at least one system of optics with high numerical aperture of preferably ≧1.2 N.A. is employed, the light quantity is limited by a confocally arranged pinhole aperture in the object plane behind the microscope objective, and the measuring compartment is positioned at a distance of between 0 and 1000 μm from the observation objective.

公开(公告)号：US06582903B1

公开(公告)日：2003-06-24

申请号：US09021410

申请日：1998-02-10

申请人： Rudolf Rigler ,  Manfred Eigen ,  Karsten Henco ,  Ulo Mets ,  Jerker Widengren ,  Michael Stuke ,  Michael Brinkmeyer ,  Wolfgang Simm ,  Olaf Lehmann

发明人： Rudolf Rigler ,  Manfred Eigen ,  Karsten Henco ,  Ulo Mets ,  Jerker Widengren ,  Michael Stuke ,  Michael Brinkmeyer ,  Wolfgang Simm ,  Olaf Lehmann

IPC分类号： G01N33543

CPC分类号： G01N21/6402 ,  G01N21/6428 ,  G01N21/6458 ,  G01N2021/6417 ,  Y10S435/808 ,  Y10S436/805 ,  Y10T436/11

摘要： A method for identifying one or a small number of molecules, especially in a dilution of ≦1 &mgr;M, using laser excited FCS with measuring times ≦500 ms and short diffusion paths of the molecules to be analyzed, wherein the measurement is performed in small volume units of preferably ≦10−14 l, by determining material-specific parameters which are determined by luminescence measurements of molecules to be examined.

摘要翻译： 使用测量时间<= 500ms的激光激发FCS和要分析的分子的短扩散路径来识别一个或少数分子，特别是稀释度为<=1μM的分子的方法，其中测量在 通过确定由待分析的分子的发光测量确定的材料特异性参数，优选<=10-14μl的小体积单位。