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1. WO2006123967 - FLUORESCENT NANOSCOPY METHOD

Publication Number WO/2006/123967
Publication Date 23.11.2006
International Application No. PCT/RU2006/000231
International Filing Date 05.05.2006
IPC
G01N 21/00 2006.1
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
21Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
G01N 21/64 2006.1
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
21Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
63optically excited
64Fluorescence; Phosphorescence
CPC
G01N 21/6428
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
21Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
63optically excited
64Fluorescence; Phosphorescence
6428Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
G01N 21/6458
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
21Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
63optically excited
64Fluorescence; Phosphorescence
645Specially adapted constructive features of fluorimeters
6456Spatial resolved fluorescence measurements; Imaging
6458Fluorescence microscopy
G01N 21/6486
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
21Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
63optically excited
64Fluorescence; Phosphorescence
6486Measuring fluorescence of biological material, e.g. DNA, RNA, cells
H04N 13/275
HELECTRICITY
04ELECTRIC COMMUNICATION TECHNIQUE
NPICTORIAL COMMUNICATION, e.g. TELEVISION
13Stereoscopic video systems; Multi-view video systems; Details thereof
20Image signal generators
275from 3D object models, e.g. computer-generated stereoscopic image signals
H04N 7/18
HELECTRICITY
04ELECTRIC COMMUNICATION TECHNIQUE
NPICTORIAL COMMUNICATION, e.g. TELEVISION
7Television systems
18Closed circuit television systems, i.e. systems in which the signal is not broadcast
Y10S 977/84
YSECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
10TECHNICAL SUBJECTS COVERED BY FORMER USPC
STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
977Nanotechnology
84Manufacture, treatment, or detection of nanostructure
Applicants
  • КЛИМОВ Андрей Алексеевич KLIMOV, Andrey Alexeevich [RU]/[RU]
  • КЛИМОВ Дмитрий Андреевич KLIMOV, Dmitry Andreevich [RU]/[RU] (UsOnly)
  • КЛИМОВ Евгений Андреевич KLIMOV, Evgeniy Andreevich [RU]/[RU] (UsOnly)
  • КЛИМОВА Татьяна Витальевна KLIMOVA, Tatiana Vitalyevna [RU]/[RU] (UsOnly)
Inventors
  • КЛИМОВ Андрей Алексеевич KLIMOV, Andrey Alexeevich
  • КЛИМОВ Дмитрий Андреевич KLIMOV, Dmitry Andreevich
  • КЛИМОВ Евгений Андреевич KLIMOV, Evgeniy Andreevich
  • КЛИМОВА Татьяна Витальевна KLIMOVA, Tatiana Vitalyevna
Common Representative
  • КЛИМОВ Дмитрий Андреевич KLIMOV, Dmitry Andreevich
Priority Data
200511505218.05.2005RU
Publication Language Russian (ru)
Filing Language Russian (RU)
Designated States
Title
(EN) FLUORESCENT NANOSCOPY METHOD
(FR) PROCEDE DE NANOSCOPIE PAR FLUORESCENCE
(RU) СПОСОБ ФЛУОРЕСЦЕНТНОЙ НАНОСКОПИИ
Abstract
(EN) The invention relates to analysis of object dyed with fluorescent colouring agents carried out with the aid of a fluorescent microscope. The microscope comprises an optical system for visualising and projecting a sample image to a video camera, a computer for recording and processing images, a sample holder arranged in front of an object lens and a fluorescent radiation exciting source. The separately fluorescent visible molecules and nanoparticles are periodically formed in different object part. A laser produces the oscillation thereof which is sufficient for recording the non-overlapping images of said molecules and nanoparticles and for decolouring already recorded fluorescent molecules. The pictures of recorded individual molecule and nanoparticle images are processed by a computer for searching the co-ordinates of the stain centres and building the object image according to the calculated co-ordinates of the stain centres corresponding to the co-ordinates of the individual fluorescent molecules and nanoparticles. Said invention makes it possible to obtain a two- and three-dimensional image with a resoling power better than 20 nm and to record a colour image by dyeing proteins, nucleic acids and lipids with different colouring agents.
(FR) Le procédé se rapporte à l'analyse d'objets qui sont peints avec des colorants fluorescents, ladite analyse s'effectuant au moyen d'un microscope à fluorescence amélioré qui, grâce à sa résolution améliorée, est parfois appelé 'nanoscope'. L'invention proposé un procédé pour obtenir une image au moyen d'un microscope qui contient: un système optique d'observation visuelle et de projection de l'image d'un échantillon dans une caméscope capable d'enregistrer et de numériser avec un faible niveau de bruit les images de molécules et de nanoparticules fluorescentes isolées, un ordinateur destiné à l'enregistrement et au traitement d'images, un support d'échantillons, disposé en face de l'objectif, une source d'excitation de rayonnement fluorescent, un ensemble de filtres bloquants interchangeables destinés à créer une lumière fluorescente de l'échantillon, qui se distingue en ce que dans de différentes zones de l'objet on crée périodiquement des molécules et des nanoparticules fluorescentes visibles isolément. Le laser assurer leur excitation qui est suffisante non seulement pour l'enregistrement de leurs images non superposées mais aussi pour la décoloration des molécules fluorescentes non encore enregistrées. Des dizaines de milliers de poses comprenant les images enregistrées de molécules et de nanoparticules isolées (ayant la forme de taches possédant un diamètre de l'ordre de la longueur d'onde de la lumière fluorescente multipliée par l'agrandissement du microscope) sont traitées sur un ordinateur à des fins de détermination des coordonnées du centre des taches et de construction d'une image de l'objet sur la base des millions de coordonnées calculées de centres des taches qui correspondent aux coordonnées des molécules et des nanoparticules fluorescentes individuelles. L'invention permet d'obtenir des images en deux et trois dimensions avec une résolution supérieure à 20 nm (à comparer avec la formule I), d'enregistrer des images couleur en cas de coloration avec des colorants différents des protéines, des acides nucléiques, des lipides, etc.
(RU) Изобретение относится к анализу объектов, покрашенных флуоресцентными красителями, осуществляемому с помощью модифицированного флуоресцентного микроскопа. Микроскоп содержит: оптическую систему для визуального наблюдения и проецирования изображения образца на видеокамеру, компьютер для записи и обработки изображений, держатель образца, расположенный напротив объектива, источник возбуждения флуоресцентного излучения. В разных участках объекта периодически создаются видимые раздельно флуоресцирующие молекулы и наночастицы. Лазер обеспечивает их возбуждение, достаточное не только для регистрации не перекрывающихся изображений, но и для обесцвечивания уже зарегистрированных флуоресцирующих молекул. Кадры с зарегистрированными изображениями одиночных молекул и наночастиц обрабатываются на компьютере с целью поиска координат центров пятен и постройки изображения объекта по вычисленным координатам центров пятен, соответствующих координатам индивидуальных флуоресцирующих молекул и наночастиц. Технический результат-получение двух- и трехмерного изображения с разрешением лучше 20 нм, возможность регистрации цветного изображения при прокраске различными красителями белков, нуклеиновых кислот, липидов.
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