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    High spatial resolution imaging of a structure of interest in a specimen
    1.
    发明授权
    High spatial resolution imaging of a structure of interest in a specimen 有权
    样本中感兴趣结构的高空间分辨率成像

    公开(公告)号：US08174692B2

    公开(公告)日：2012-05-08

    申请号：US12949159

    申请日：2010-11-18

    申请人： Stefan W. Hell ,  Jonas Fölling ,  Christian Eggeling ,  Alexander Egner ,  Andreas Schönle ,  Mariano Bossi

    发明人： Stefan W. Hell ,  Jonas Fölling ,  Christian Eggeling ,  Alexander Egner ,  Andreas Schönle ,  Mariano Bossi

    IPC分类号： G01J3/30

    CPC分类号： G01N21/6458 ,  G01N21/6428 ,  G02B21/16 ,  G02B21/367 ,  G02B27/58

    摘要： In high spatial resolution imaging, a structure in a specimen is marked with a substance which, in a first electronic state, is excited by light of one wavelength to emit fluorescent light, which is also converted from its first into a second electronic state by that light, and which returns from its second into its first electronic state. The specimen is imaged onto a sensor at a spatial resolution not resolving an average spacing between neighboring molecules of the substance, and exposed to the light at such an intensity that the molecules in the first state are alternately excited to emit fluorescent light and converted into their second state, and that at least 10% of the molecules presently in their first state lie at a distance from their closest neighboring molecules in their first state which is greater than the spatial resolution of the imaging onto the sensor.

    摘要翻译： 在高空间分辨率成像中，样本中的结构用物质标记，该物质在第一电子状态下被一个波长的光激发以发射荧光，其也从其第一个电子状态转换成第二电子状态 光，从第二个回到第一个电子状态。 将样本以不分辨物质的相邻分子之间的平均间隔的空间分辨率成像到传感器上，并以如此强度暴露于光，使得第一状态下的分子被交替地激发以发射荧光并转换成它们 并且目前处于其第一状态的至少10％的分子处于与它们的第一状态中最接近的相邻分子相距一定距离，其大于成像在传感器上的空间分辨率。

    Photoactivable fluorescent dyes for optical microscopy and imaging techniques
    4.
    发明授权
    Photoactivable fluorescent dyes for optical microscopy and imaging techniques 有权
    用于光学显微镜和成像技术的可光化荧光染料

    公开(公告)号：US08617827B2

    公开(公告)日：2013-12-31

    申请号：US13395323

    申请日：2009-09-10

    申请人： Stefan W. Hell ,  Vladimir N. Belov ,  Vadim P. Boyarskiy ,  Christian A. Wurm ,  Stefan Jakobs ,  Claudia Geisler

    发明人： Stefan W. Hell ,  Vladimir N. Belov ,  Vadim P. Boyarskiy ,  Christian A. Wurm ,  Stefan Jakobs ,  Claudia Geisler

    IPC分类号： G01N33/53 ,  C07D285/06 ,  C12Q1/02 ,  C09B46/00

    CPC分类号： C09B11/24

    摘要： The present invention relates to novel photoactivable rhodamine or carbopyronine derivatives of the following general formulae G1-G4 (I), G1: A1=O, A2=N, A3=C; G2: A1=S, A2=N, A3=C; G3: A1=O, A2=O, A3=N; G4: A1=S, A2=O, A3=N; comprising UV light absorbing chromophores which after photolysis by irradiation at 254-490 nm (preferably at 375-420 nm) generate fluorescent rhodamine or carbopyronine derivatives and small non-toxic fragments such as N2 or N2O. The invention also relates to methods for preparing such compounds and to the use of these compounds for optical microscopy and imaging techniques.

    摘要翻译： 本发明涉及以下通式G1-G4（I），G1：A1 = O，A2 = N，A3 = C的新型可光致发生罗丹明或卡吡龙衍生物。 G2：A1 = S，A2 = N，A3 = C; G3：A1 = O，A2 = O，A3 = N; G4：A1 = S，A2 = O，A3 = N; 包括紫外光吸收发色团，其在254-490nm（优选在375-420nm）下照射光解后产生荧光罗丹明或卡吡喃衍生物和小的无毒片段如N 2或N 2 O。 本发明还涉及制备这些化合物的方法以及这些化合物用于光学显微镜和成像技术的用途。

    Fluorescent light microscope for measuring a sample using red-shifted stokes lines
    5.
    发明授权
    Fluorescent light microscope for measuring a sample using red-shifted stokes lines 有权
    荧光光学显微镜用于使用红移斯托克斯线测量样品

    公开(公告)号：US08039815B2

    公开(公告)日：2011-10-18

    申请号：US12753349

    申请日：2010-04-02

    申请人： Stefan W. Hell ,  Brian Rankin ,  Robert Kellner ,  Jaydev Jethwa ,  Thorsten Staudt

    发明人： Stefan W. Hell ,  Brian Rankin ,  Robert Kellner ,  Jaydev Jethwa ,  Thorsten Staudt

    IPC分类号： G01N21/64

    CPC分类号： G02B21/16 ,  G01N21/6458 ,  G01N21/65 ,  G01N2021/655 ,  H01S3/302

    摘要： A fluorescent light microscope for measuring a sample comprises a light source providing transfer light having a transfer wavelength for transferring a fluorescent dye in the sample from one state into another state, and a detector which measures fluorescent light from the sample with spatial resolution. The light source comprises a laser, an optical wave guide connected to the laser, and a wavelength-selective device connected to the optical wave guide. The laser emits pump light of a pump wavelength other than the transfer wavelength and injects the pump light into the optical wave guide. The pump light, due to Raman scattering being stimulated in the optical wave guide, generates a light spectrum emerging from the optical wave guide which has, besides the pump wavelength, at least one red-shifted Stokes line whose full width at half maximum is smaller than half of its distance to its next neighbor line on the blue side of the spectrum; and the wavelength-selective device singles out the transfer light by its transfer wavelength from the red-shifted Stokes lines of the light spectrum.

    摘要翻译： 用于测量样品的荧光显微镜包括提供具有用于将样品中的荧光染料从一种状态转移到另一状态的转印波长的转移光，以及以空间分辨率测量来自样品的荧光的检测器。 光源包括激光器，连接到激光器的光波导和连接到光波导的波长选择器件。 激光器发射除了转移波长之外的泵波长的泵浦光，并将泵浦光注入到光波导中。 由于在光波导中被拉曼散射的泵浦光产生从光波导出射的光谱，除了泵浦波长之外，还有至少一个红移斯托克斯线，其半峰全宽较小 超过其在光谱蓝色边上的下一个相邻线的距离的一半; 并且波长选择装置通过其来自光谱的红移斯托克斯线的转移波长来分离转移光。

    STED-fluorescent light microscopy with two-photon excitation
    6.
    发明授权
    STED-fluorescent light microscopy with two-photon excitation 有权
    具有双光子激发的STED-荧光光学显微镜

    公开(公告)号：US07863585B2

    公开(公告)日：2011-01-04

    申请号：US12706388

    申请日：2010-02-16

    申请人： Stefan W. Hell ,  Katrin Willig

    发明人： Stefan W. Hell ,  Katrin Willig

    IPC分类号： G01N21/64

    CPC分类号： G01N21/6458 ,  G01N21/636 ,  G02B21/0076

    摘要： A method of high spatial resolution imaging a structure in a sample comprises: marking the structure with molecules of a fluorescent dye; selecting a first wavelength for excitation light which excites the molecules of the fluorescent dye via a multi photon process for spontaneous emission of fluorescent light; focusing pulses of the excitation light into the sample to excite those molecules of the fluorescent dye present in a focal area of the focused excitation light; selecting a second wavelength shorter than the first wavelength for de-excitation light which de-excites excited molecules of the fluorescent dye prior to their spontaneous emission; during a plurality of the pulses of the excitation light, continuously directing the de-excitation light onto the sample to de-excite excited molecules of the fluorescent dye, which are located outside an measurement area which is a fraction of the focal area; and recording the fluorescent light spontaneously emitted by the molecules of the fluorescent dye in the sample.

    摘要翻译： 对样品中的结构进行高空间分辨率成像的方法包括：用荧光染料的分子标记结构; 选择激发光的第一波长，其通过用于自发发射荧光的多光子过程激发荧光染料的分子; 将激发光的脉冲聚焦到样品中以激发存在于聚焦激发光的焦点区域中的荧光染料的那些分子; 选择比第一波长短的第二波长用于去激发荧光染料在其自发发射之前激发分子的去激发光; 在激发光的多个脉冲期间，将去激发光连续地引导到样品上以去激发位于焦点区域的一部分的测量区域外的荧光染料的激发分子; 并记录由荧光染料分子自发发射的荧光。

    Three-dimensionally localizing light emitting molecules of unknown orientation and unknown Z-position
    7.
    发明授权
    Three-dimensionally localizing light emitting molecules of unknown orientation and unknown Z-position 有权
    三维定位未知取向和未知Z位的发光分子

    公开(公告)号：US08559021B2

    公开(公告)日：2013-10-15

    申请号：US13441043

    申请日：2012-04-06

    申请人： Johann Engelhardt ,  Stefan W. Hell ,  Jan Keller-Findeisen

    发明人： Johann Engelhardt ,  Stefan W. Hell ,  Jan Keller-Findeisen

    IPC分类号： G01B11/14 ,  G01J3/30 ,  G06K9/00

    CPC分类号： G02B21/0076 ,  G01N21/6456 ,  G01N2021/6463

    摘要： To the end of three-dimensionally localizing light emitting marker entities of unknown orientation and unknown position in a sample, the light emitted by each single marker entity is imaged in at least two different ways onto at least one detection plane which corresponds to a focal plane (13) in the sample resulting in at least two images of the marker entity. Virtual x- and y-positions of the marker entity in parallel to the focal plane (13) are separately determined from the emitted light intensity distribution over each image of the marker entity. Further, the z-position of the marker entity normal to the focal plane is determined from the emitted light intensity distributions over the images of the marker entity. The real x- and y-positions of the marker entity in parallel to the focal plane (13) are determined based on its virtual x- and y-positions and on its z-position.

    摘要翻译： 在三维定位样品中未知取向和未知位置的发光标记实体的三维定位结束时，由每个单个标记实体发射的光以至少两种不同的方式成像到至少一个对应于焦平面的检测平面 （13），导致标记实体的至少两个图像。 标记实体与焦平面（13）平行的虚拟x和y位置根据标记实体的每个图像上的发射光强度分布分别确定。 此外，从标记实体的图像上的发射光强度分布确定垂直于焦平面的标记实体的z位置。 标记实体与焦平面（13）平行的真实x和y位置基于其虚拟x和y位置及其z位置来确定。

    Method of exciting molecules out of a first state into a second states using an optical signal
    8.
    发明授权
    Method of exciting molecules out of a first state into a second states using an optical signal 有权

    公开(公告)号：US07224452B2

    公开(公告)日：2007-05-29

    申请号：US11525505

    申请日：2006-09-22

    申请人： Stefan W. Hell

    发明人： Stefan W. Hell

    IPC分类号： G01N21/64

    CPC分类号： G01N21/6458 ,  G02B21/16

    摘要： In a fluorescence-microscopic method of examining a sample with high spatial resolution, the sample is first cooled to a temperature of below 5° C.; next the cooled sample is transferred out of a ground state into a fluorescent state within an area captured by a detector using an excitation beam of light; next the cooled sample is subject to de-excitation of excited molecules by stimulated emission in the area captured by the detector, except at desired measuring points, using an de-exciting beam of light, the de-exciting beam of light having a spatial intensity distribution comprising a zero point located at the desired measuring points, and the excited sample being transferred back into its ground state by the de-exciting beam of light; and then fluorescence light spontaneously emitted by the cooled sample is measured with the detector, the detector detecting fluorescence light that is emitted only from the measuring points.

    NOVEL PHOTOACTIVABLE FLUORESCENT DYES FOR OPTICAL MICROSCOPY AND IMAGING TECHNIQUES
    10.
    发明申请
    NOVEL PHOTOACTIVABLE FLUORESCENT DYES FOR OPTICAL MICROSCOPY AND IMAGING TECHNIQUES 有权
    用于光学显微镜和成像技术的新型可光化荧光染料

    公开(公告)号：US20120202216A1

    公开(公告)日：2012-08-09

    申请号：US13395323

    申请日：2009-09-10

    申请人： Stefan W. Hell ,  Vladimir N. Belov ,  Vadim P. Boyarskiy ,  Christian A. Wurm ,  Stefan Jakobs ,  Claudia Geisler

    发明人： Stefan W. Hell ,  Vladimir N. Belov ,  Vadim P. Boyarskiy ,  Christian A. Wurm ,  Stefan Jakobs ,  Claudia Geisler

    IPC分类号： G01N33/53 ,  C07D285/06 ,  C12Q1/02 ,  C09B46/00

    CPC分类号： C09B11/24

    摘要： The present invention relates to novel photoactivable rhodamine or carbopyronine derivatives of the following general formulae G1-G4 (I), G1: A1=O, A2=N, A3=C; G2: A1=S, A2=N, A3=C; G3: A1=O, A2=O, A3=N; G4: A1=S, A2=O, A3=N; comprising UV light absorbing chromophores which after photolysis by irradiation at 254-490 nm (preferably at 375-420 nm) generate fluorescent rhodamine or carbopyronine derivatives and small non-toxic fragments such as N2 or N2O. The invention also relates to methods for preparing such compounds and to the use of these compounds for optical microscopy and imaging techniques.

    摘要翻译： 本发明涉及以下通式G1-G4（I），G1：A1 = O，A2 = N，A3 = C的新型可光致发生罗丹明或卡吡龙衍生物。 G2：A1 = S，A2 = N，A3 = C; G3：A1 = O，A2 = O，A3 = N; G4：A1 = S，A2 = O，A3 = N; 包括紫外光吸收发色团，其在254-490nm（优选在375-420nm）下照射光解后产生荧光罗丹明或卡吡喃衍生物和小的无毒片段如N 2或N 2 O。 本发明还涉及制备这些化合物的方法以及这些化合物用于光学显微镜和成像技术的用途。