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Three dimensional, position observation method and apparatus

外国特許コード F110005367
整理番号 K01707WO
掲載日 2011年9月5日
出願国 アメリカ合衆国
出願番号 98818706
公報番号 20090219549
公報番号 8203720
出願日 平成18年6月29日(2006.6.29)
公報発行日 平成21年9月3日(2009.9.3)
公報発行日 平成24年6月19日(2012.6.19)
国際出願番号 JP2006312958
国際公開番号 WO2007004497
国際出願日 平成18年6月29日(2006.6.29)
国際公開日 平成19年1月11日(2007.1.11)
優先権データ
  • 特願2005-197049 (2005.7.6) JP
  • 2006JP312958 (2006.6.29) WO
発明の名称 (英語) Three dimensional, position observation method and apparatus
発明の概要(英語) A three-dimensional position observation apparatus provided with a lens system having focusing and diaphragm mechanisms, for forming an image on an imaging plane by light from an observation object includes a beam steering member disposed in a light path extending from the observation object to the imaging plane, for changing a traveling direction of observation light into a plurality of different directions, and an image analyzing unit for analyzing a position of the observation object based on a positional relation between a plurality of images on the imaging plane formed by light passing through the beam steering member.
従来技術、競合技術の概要(英語) BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a three-dimensional position observation method and apparatus for detecting with high precision a three-dimensional position of an observation object, in particular, three-dimensional motion of a microscopic particle under a microscope.
2. Description of the Related Art
In recent years, there has been tremendous progress in optical microscopy, which now reaches a stage where a single protein molecule in an aqueous solution can be studied as an object.
This progress has been achieved with the aid of new optical technologies such as total reflection illuminations, development of various types of highly sensitive cameras, improvement in properties of optical filters, and the like.
A number of experimental techniques have been developed, and thus a new field called "a single molecular physiology" has emerged.
For example, in a molecular motor or a proteolytic enzyme, substrate binding involves a dynamic structural change, which is considered to be closely linked to a function.
A demanded technique is to make such a structural change occurring inside a single biomolecule visible in a molecular level in a viable condition under a microscope.
Innovative techniques from a new perspective are required to advance this growing field to a next new step.
One of techniques for observing a single protein molecule is that a protein is specifically labeled with a fluorescence dye to catch a signal from a single fluorophore.
A fluorescence microscope has a structure incorporating an optical system for emitting an excitation light for brightening the fluorescence dye when receiving a light with a specific wavelength, with use of a dye emitting a light with a longer wavelength than the specific wavelength, and an optical microscope for observing thus generated fluorescence.
Where a reagent bound to a fluorescence dye is bound to a structure inside a cell as an observation object and then the fluorophore is irradiated with a light with a predetermined wavelength, the structure inside the cell as the object generates fluorescence in a black background.
Since the number of fluorophores observable with a general fluorescence microscope is several dozen or more, it is impossible to discern the single fluorophore.
This is because an optical signal strength of a noise, i.e., a background, is greater than that obtained from the single fluorophore.
In this regard, fluorescence microscopes improved for performance upgrade, with which the single fluorophore is visible, have been developed by improving a property of filters, a quality of objective lenses, and the like.
The single fluorophore is observed through the use of such a property of the fluorophore as generating fluorescence by evanescent field illumination.
Specifically, the fluorophore is made to generate fluorescence by illumination of an object sample with use of the evanescent field as a non-propagating light, which is generated around a boundary surface between an aqueous solution containing the object sample and a glass by irradiating the boundary surface with a laser beam at a total reflection angle or greater by means of total reflection from the side of the glass.
Since the evanescent field is exponentially-attenuated with respect to a direction perpendicular to the boundary surface, only a local field near the boundary surface is irradiated, thereby providing the advantage that the intensity of background light is extremely low compared with that of illumination with normal light.
Even under a condition that a number of fluorophores are present in the aqueous solution containing the object sample, there is a low probability that the fluorophore is present on the side of the aqueous solution near the boundary surface, thereby resulting in a low fluorescence from fluorophores other than the single target fluorophore secured to a top of the boundary surface.
Therefore, the noise due to the fluorescence from the background light or other fluorophores is extremely low, which enables observation of the fluorescence from the single target fluorophore.
In the single-molecule observation by means of the total reflection, proteins, or biomolecule such as DNA or ATP as a substrate, which are labeled with fluorescence dyes, are bound to a glass surface to detect respective molecules as an independent bright point.
For two-dimensional imaging of a weak signal from the single molecule, a highly sensitive camera is used, such as an image intensifier or a cooled CCD camera.
The present inventors manufactured a total reflection fluorescence microscope to thereby detect a structural change of a specific part of the single biomolecule in real time by observation.
For example, "Total Reflection Fluorescence Microscope" described in Japanese Patent No. 3,577,514 relates to the basic concept and the optical system of this technique and discloses a structure of the total reflection fluorescence microscope which enables observation of a dye molecule having a vibrating surface oriented in an arbitrary direction.
"Total Reflection Type Fluorescence Microscope and Illumination Optical System" described in Japanese Patent No. 3,671,227 by the present inventors discloses a total reflection fluorescence microscope which enables observation of an target dye molecule regardless of a direction of shaking moment of a sample bound to a fluorophore.
As described above, although observation of a single biomolecule has become possible, a positional information obtainable according to the prior art is two-dimensional information.
That is, the information on a vertical direction in which an objective lens moves cannot be obtained.
Observation of the three-dimensional positional information of an atomic molecule moving under a microscope achieves quantum leaps such as precise detection of displacement of the single protein molecule.
For example, the following documents also relate to the fluorescence microscope in the prior art: Japanese Patent Application Laid-Open No. 2005-37572, "Illumination Device for Fluorescence Microscope and Fluorescence Microscope"; Japanese Patent Application Laid-Open No. 2000-56233, "Device for Focusing with Adjustments Wavelength or Wavelength Region in Light Irradiation Path in Microscope"; and PCT National Publication No. 11-513145, "Confocal Microscope with Doublet System".
There has been tremendous improvement in optical microscopy such as a bright field microscopy, a dark field microscopy, a phase difference microscopy, a differential interference microscopy, and a laser confocal microscopy.
However, the positional information obtained by conventional microscopic observation is two-dimensional information in a surface (an x-y plane) parallel to a slide glass corresponding to a viewing plane, and positional information on a direction (a z-axis) perpendicular to the aforementioned surface cannot be obtained.

特許請求の範囲(英語) [claim1]
1. A three-dimensional position observation apparatus provided with a lens system having focusing and diaphragm mechanisms, for forming an image on an imaging plane by light from an observation object, the three-dimensional position observation apparatus comprising: a beam steering member disposed in a light path extending from the observation object to the imaging plane, for changing a part of a traveling direction of observation light; and
an image analyzing unit for analyzing a position of the observation object based on a positional relation between an image on the imaging plane formed by light passing through the beam steering member and an image on the imaging plane formed by light not passing through the beam steering member.
[claim2]
2. The three-dimensional position observation apparatus according to claim 1, wherein the beam steering member is a wedge prism.
[claim3]
3. The three-dimensional position observation apparatus according to claim 2, wherein the beam steering member is two wedge prisms each having the same inclination angle, and wherein the two wedge prisms are disposed in a combined form such that inclination directions of inclination surfaces are reversely oriented.
[claim4]
4. The three-dimensional position observation apparatus according to claim 3, wherein at least one of the wedge prisms is disposed such that the inclination surface faces a side of the imaging plane.
[claim5]
5. The three-dimensional position observation apparatus according to claim 2, wherein at least one of the wedge prisms is disposed such that the inclination surface faces a side of the imaging plane.
[claim6]
6. A three-dimensional position observation method using a three-dimensional position observation apparatus provided with a lens system having focusing and diaphragm mechanisms, for forming an image on an imaging plane by light from an observation object, the three-dimensional position observation method comprising: disposing a beam steering member in a light path extending from the observation object to the imaging plane, for changing a part of a traveling direction of observation light; and analyzing a position of the observation object based on a positional relation between an image on the imaging plane formed by light passing through the beam steering member and an image on the imaging plane formed by light not passing through the beam steering member with an image analyzing unit.
  • 発明者/出願人(英語)
  • NISHIZAKA TAKAYUKI
  • MIZUTANI KANA
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
国際特許分類(IPC)
米国特許分類/主・副
  • 356/614
  • 356/620
  • 356/623
参考情報 (研究プロジェクト等) PRESTO Structure and Function of Biomolecules AREA
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