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WO2010024235A1 - Microscope confocal - Google Patents

Microscope confocal Download PDF

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Publication number
WO2010024235A1
WO2010024235A1 PCT/JP2009/064763 JP2009064763W WO2010024235A1 WO 2010024235 A1 WO2010024235 A1 WO 2010024235A1 JP 2009064763 W JP2009064763 W JP 2009064763W WO 2010024235 A1 WO2010024235 A1 WO 2010024235A1
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WO
WIPO (PCT)
Prior art keywords
light
component
image
confocal
microscope apparatus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2009/064763
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English (en)
Japanese (ja)
Inventor
寺田　浩敏
小林　正典
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Hamamatsu Photonics KK
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Hamamatsu Photonics KK
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Filing date
Publication date
Application filed by Hamamatsu Photonics KK filed Critical Hamamatsu Photonics KK
Publication of WO2010024235A1 publication Critical patent/WO2010024235A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036—Scanning details, e.g. scanning stages
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B5/00—Optical elements other than lenses
    • G02B5/04—Prisms
    • G02B5/045—Prism arrays

Definitions

  • the present invention relates to a confocal microscope apparatus that generates a confocal image of an object.
  • a confocal microscope is used to obtain a high-resolution image of an object.
  • a confocal microscope has a pinhole plate in which a large number of minute pinholes are provided between the image sensor and the objective lens, and only the light imaged on the surface of the pinhole plate by the objective lens is used as illumination light. Is passed through the same pinhole as the pinhole that has passed through, and the scattered light that does not form an image on the surface of the pinhole plate is blocked by the pinhole plate. As a result, a clear image free from noise due to out-of-focus or flare light is generated (for example, see Patent Document 1 below).
  • JP 2007-310202 A Japanese translation of PCT publication No. 2002-535716
  • the present invention has been made in view of such problems, and provides a confocal microscope apparatus that facilitates adjustment work when obtaining a confocal image and that can reduce the size of the apparatus. Objective.
  • a confocal microscope apparatus of the present invention is a confocal microscope apparatus that generates a confocal image of an object, and includes a light source that emits illumination light, and an object that corresponds to the illumination light.
  • the illumination light emitted from the light source is transmitted through the first transmission part of the mask part, so that the object is irradiated with a pattern having a predetermined pitch.
  • the first component including the confocal imaging light among the light from the first light is received by the first light detection unit after passing through the first transmission unit.
  • the second component that does not include the confocal imaging light among the light from the object is transmitted / refracted by the second transmission unit and then received by the second light detection unit, and the first component is received.
  • a confocal image can be generated based on the image data obtained by the second light detection unit.
  • the light source can be unified and the two photodetecting portions can be arranged on one side of the mask portion, so that the adjustment of the mounting angle and position thereof is simplified, and the photodetector It is possible to easily reduce the size of the apparatus including the optical system by unitizing the optical system.
  • FIG. 2A is a plan view of the prism array mask of FIG. 1 viewed from the incident direction of illumination light
  • FIG. 2B is a side view of the prism array mask of FIG.
  • A) is the top view seen from the incident direction of the illumination light of the prism array mask concerning the modification of this invention
  • (b) is a side view of the prism array mask of (a).
  • (A) is the top view seen from the incident direction of the illumination light of the prism array mask concerning the modification of this invention
  • (b) is a side view of the prism array mask of (a).
  • (A) is the top view seen from the incident direction of the illumination light of the prism array mask concerning the modification of this invention
  • (b) is a side view of the prism array mask of (a).
  • (A) is the top view seen from the incident direction of the illumination light of the mask part concerning the modification of this invention
  • (b) is a side view of the mask part of (a).
  • (A) is the top view seen from the incident direction of the illumination light of the prism array mask concerning the modification of this invention
  • (b) is a side view of the prism array mask of (a).
  • (A) is the top view seen from the incident direction of the illumination light of the mask part concerning the modification of this invention
  • (b) is a side view of the mask part of (a).
  • FIG. 1 is a diagram showing a schematic configuration of a confocal microscope apparatus 1 according to the first embodiment of the present invention.
  • a confocal microscope apparatus 1 shown in FIG. 1 is an apparatus for generating a confocal image of a sample (object) A, and is used in particular for observing a fluorescence image accompanying fluorescence emitted from the sample A.
  • the confocal microscope apparatus 1 is provided with a fiber light source 2 that irradiates illumination light L 1 and an excitation filter 3 positioned in front of the irradiation direction of the fiber light source 2, and is emitted from the fiber light source 2 by the excitation filter 3.
  • the illumination light L1 a specific range of wavelength components is selectively transmitted.
  • a dichroic mirror 4 is disposed in front of the fiber light source 2 through the excitation filter 3, and the illumination light L 1 transmitted through the excitation filter 3 by the dichroic mirror 4 is reflected toward the sample A in the microscope 9. .
  • the large condensing lens 5 the prism array mask (mask part) 6, the imaging lens 7, and the objective lens 8 is arranged.
  • the prism array mask 6 is located on the primary imaging plane of the microscope 9 including the imaging lens 7 and the objective lens 8.
  • the illumination light L1 is uniformly irradiated on the surface of the prism array mask 6 by the large condenser lens 5.
  • the illumination light L ⁇ b> 1 in which a bright and dark pattern with a predetermined pitch is formed by passing straight through the prism array mask 6 is incident on the microscope 9 including the imaging lens 7 and the objective lens 8.
  • the sample A in front of the objective lens 8 is illuminated by the illumination light L1.
  • the illumination light L1 refracted when passing through the prism array mask 6 is removed from the microscope 9 and absorbed by the light trap 10.
  • Fluorescence emitted from the sample A according to the illumination light L1 having such a bright and dark pattern is captured by the objective lens 8 and imaged on the surface of the prism array mask 6 via the imaging lens 7. Is done.
  • the first component (confocal imaging light) L2 that is in focus with respect to the sample A is transmitted straight through the prism array mask 6, and out of this fluorescence with respect to the sample A.
  • Most of the second component (non-confocal imaging light) L3 excluding the first component that is not in focus is transmitted and refracted by the prism array mask 6 (details will be described later).
  • a large condensing lens 5, a fluorescent filter 12, small condensing lenses 13A and 13B, and image sensors (light detection units) 11A and 11B are provided in this order. ing.
  • the imaging light L2 of the first component passes through the large condensing lens 5, and then is collimated into parallel light and passes through the dichroic mirror 4 having a property of transmitting the fluorescent wavelength component.
  • the fluorescent filter 12 provided between the two image pickup devices 11A and 11B.
  • the imaging light L2 of the first component from which the fluorescence wavelength component is extracted is condensed by the small condenser lens 13A disposed on the optical axis, and provided on the optical axis of the imaging light L2. An image is formed and received on the image pickup device 11A.
  • the imaging light L3 of the second component travels obliquely with respect to the optical axis of the imaging light L2, passes through the large condenser lens 5, is collimated into parallel light, and passes through the fluorescent filter 12. Thus, the fluorescence wavelength component is extracted.
  • the imaging light L3 of the second component is condensed by a small condenser lens 13B disposed on the optical axis, and provided adjacent to the image sensor 11A on the optical axis of the imaging light L3. An image is formed and received on the image sensor 11B.
  • a TDI camera incorporating a CCD (Charge Coupled Devices) capable of TDI (Time Delay Integration) operation is preferably used as the image pickup devices 11A and 11B.
  • CCD Charge Coupled Devices
  • TDI Time Delay Integration
  • the difference between the image data corresponding to the imaging light L2 generated by the imaging element 11A and the image data corresponding to the imaging light L3 generated by the imaging element 11B as described above is obtained.
  • a confocal image of the sample A is generated.
  • FIG. 2A is a plan view of the prism array mask 6 viewed from the incident direction of the illumination light L1
  • FIG. 2B is a side view of the prism array mask 6.
  • the prism array mask 6 is illuminated from the central portion on the surface 14a on which the illumination light L1 is incident to the central portion on the surface 14b opposite to the incident surface 14a.
  • a rectangular transmission part 15 that transmits the light L1 and the imaging light L2 and L3 is formed, and a shielding part 16 that shields the illumination light L1 and the imaging light L2 and L3 is formed around the transmission part 15 on the surface 14a. Is formed.
  • the transmission part 15 is formed with a first transmission part 15a and a second transmission part 15b that are alternately arranged in a strip shape.
  • the first transmission portion 15a has a flat surface formed on the surfaces 14a and 14b, and is entirely made of a material having optical transparency such as glass.
  • the second transmission portion 15b is formed with a so-called triangular prism-like prism so as to have a surface inclined in a direction perpendicular to the longitudinal direction on the surface 14a, while a flat surface is formed on the surface 14b side.
  • the whole is made of a material having optical transparency such as glass.
  • the arrangement interval and the width along the arrangement direction of the second transmission parts 15b may be selected depending on the magnification and NA of the objective lens of the microscope and the target sample. It is preferable that the ratio of the width along the arrangement direction of the first transmission parts 15a to the width along the arrangement direction of the second transmission parts 15b is 1: 1. In this case, it is not necessary to correct between the two image data when calculating the image data generated by the image sensors 11A and 11B, and the processing efficiency is improved.
  • the illumination light L1 that is perpendicularly incident on the surface 14a of the first transmission portion 15a is transmitted straight through the first transmission portion 15a and is directed vertically to the sample A from the surface 14b. It is emitted toward.
  • the illumination light L1 incident on the second transmission part 15b is refracted by the prism, passes through the second transmission part 15b, is emitted in an oblique direction from the surface 14b, and the light trap 10 (FIG. 1) absorbed.
  • the sample A is irradiated with the illumination light L1 having a strip pattern reflecting the shape and arrangement pitch of the first transmission parts 15a.
  • the first component L2 that is, the confocal imaging light
  • the prism array mask 6 is transmitted through the prism array mask 6 along the incident direction of the illumination light L1.
  • the first fluorescent component L2 passes straight through the first transmission portion 15a from the surface 14b toward the surface 14a.
  • the imaging light L2 received by the image sensor 11A forms a band-like image reflecting the pattern of the first transmission part 15a.
  • the second component L3, that is, most of the non-confocal imaging light returns to the second transmission portion 15b of the prism array mask 6.
  • the second fluorescent component L3 is transmitted from the surface 14b toward the surface 14a through the second transmission portion 15b, and is refracted in a direction different from the first component L2 by the prism on the surface 14a side.
  • the imaging light L3 received by the image sensor 11B forms a band-like image reflecting the pattern of the second transmission part 15b.
  • the arrangement interval of the second transmission parts 15b can be appropriately selected according to the pupil diameter of the objective lens 8, the required resolution, and contrast.
  • the arrangement interval is reduced.
  • the arrangement interval is reduced.
  • the arrangement interval is increased.
  • MTF ModulationModTransfer Function
  • the arrangement interval may be determined so as to be on the low frequency side, and on the high frequency side when the resolution is important.
  • the illumination light L1 irradiated from the fiber light source 2 is transmitted through the first transmission portion 15a of the prism array mask 6 so that the sample A is irradiated with a pattern having a predetermined pitch.
  • the first component L2 including the confocal imaging light among the fluorescence generated from the sample A is transmitted through the first transmission part 15a and then received by the image sensor 11A.
  • the second component L3 that does not include the confocal imaging light among the fluorescence generated from the sample A is transmitted / refracted by the second transmission unit 15b and then received by the image sensor 11B.
  • a confocal image can be generated based on the image data obtained by the imaging elements 11A and 11B.
  • the light source can be unified and the two imaging elements can be arranged on one side of the mask portion, so that the adjustment of the mounting angle and position thereof is simplified.
  • the optical system can be shared by integrating the two image pickup devices into one unit, it is possible to easily reduce the size of the device including the optical system.
  • the image sensors 11A and 11B are configured by a TDI camera, the image of the sample A is moved in accordance with the vertical transfer timing of the pixel lines of the image sensors 11A and 11B without moving the mask portion. An averaged high-sensitivity confocal image can be obtained.
  • FIG. 3 is a diagram showing a schematic configuration of a confocal microscope apparatus 101 according to the second embodiment of the present invention.
  • the confocal microscope apparatus 1 according to the first embodiment generates a fluorescent image from the sample A according to the irradiation of the illumination light L1 with respect to the sample A having a fluorescence property.
  • the microscope apparatus 101 is for generating a reflection image from the sample A.
  • the confocal microscope apparatus 101 differs from the confocal microscope apparatus 1 in that a polarizer 103 is provided in place of the excitation filter 3 and a polarization beam splitter 104 is provided in place of the dichroic mirror 4.
  • a quarter-wave plate 114 is newly provided between the prism array mask 6 and the imaging lens 7, and an analyzer 112 is newly provided between the large condenser lens 5 and the small condenser lenses 13A and 13B. It is a point.
  • the illumination light L11 emitted from the fiber light source 2 is extracted from the polarization component in one direction by the polarizer 103, and the polarization beam splitter 104 having a property of reflecting the polarization component in the direction is applied to the microscope 9 by the polarization beam splitter 104. Reflected towards.
  • the illumination light L11 transmitted straight through the large condensing lens 5 and the prism array mask 6 is converted into a polarization state from linearly polarized light to circularly polarized light by the quarter wavelength plate 114, and then advances straight into the microscope 9, and the sample.
  • An illumination pattern reflecting the pattern of the first transmitting portion 15a of the prism array mask 6 is connected to A.
  • the reflected light and scattered light from the sample A form an image on the prism array mask 6 via the objective lens 8 and the imaging lens 7.
  • the polarization state is converted from circularly polarized light to linearly polarized light by the quarter wavelength plate 114 immediately before entering the prism array mask 6.
  • the direction of the linearly polarized light at this time is different from the direction of the linearly polarized light of the illumination light L11 by about 90 degrees.
  • the first component L12 focused on the sample A straightens the prism array mask 6 along the incident direction of the illumination light L11.
  • the first component L12 is converted into parallel light by the large condensing lens 5 and then transmitted through the polarizing beam splitter 104. Further, the polarization purity is increased by the analyzer 112, and the first component L12 passes through the small condensing lens 13A.
  • Light is received by the image sensor 11A.
  • the polarization beam splitter 104 has a property of transmitting linearly polarized light whose polarization direction is 90 degrees different from that of the illumination light L11, and the analyzer 112 is for blocking the wraparound of the illumination light causing noise. is there.
  • the imaging light L12 received by the image sensor 11A forms a reflected image (an image including a confocal image) reflecting the pattern of the first transmission portion 15a of the prism array mask 6.
  • the second component L13 not focused on the sample A is transmitted and refracted by the prism array mask 6 in a direction different from that of the first component L12.
  • the second component L13 is collimated by the large condensing lens 5 and then the polarization purity is increased by the analyzer 112 and is received by the image sensor 11B via the small condensing lens 13B.
  • the imaging light L13 received by the image sensor 11B forms an image (an image including a non-confocal image) reflecting the pattern of the second transmission portion 15b of the prism array mask 6.
  • the arrangement direction of the first and second transmission parts 15 a and 15 b of the prism array mask 6 is not limited to the short direction of the transmission part 15, but is oblique with respect to the longitudinal direction or the boundary of the transmission part 15. It does not matter.
  • the arrangement in the short direction is preferable in terms of the image quality of the confocal image because the separation angle can be reduced and the angle of the prism of the second transmission portion 15b can be reduced.
  • the configuration of the prism array mask 6 used as the mask portion can take various modifications.
  • 4 to 9 are diagrams showing modified examples of the mask portion.
  • the second transmission part 115b arranged in a band shape in the transmission part 115 has a surface inclined in the opposite direction to the second transmission part 15b on the surface 14a.
  • the prism-shaped recessed part of the triangular prism which has may be formed. Even with such a prism array mask 106, the illumination light L1 transmitted through the second transmission portion 115 is emitted obliquely from the surface 14b, and most of the second component L3 out of the fluorescence emitted from the sample A. Is refracted in a direction different from that of the first component L2 when passing through the second transmission portion 115b.
  • the first component L2 including the confocal imaging light includes the confocal imaging light by the imaging element 11A.
  • the second component L3 that is not present can be received by the image sensor 11B.
  • the second transmission part 215b in the transmission part 215 may be one in which band-like prisms are formed on both sides of the surfaces 14a and 14b. According to such a prism array mask 206, the deflection angle in the second transmission part 215b can be increased, so that the illumination light L1 transmitted through the two transmission parts 15a and 215b can be separated, and the first and second fluorescent lights can be separated. The components L2 and L3 are reliably separated.
  • the first and second transmissions having a surface inclined in opposite directions on the surface 14a in the transmission part 315 and forming a prism having an isosceles triangle section.
  • the portions 315a and 315b may be included. According to such a prism array mask 306, the separation angle between the light beam including the illumination light L1 and the fluorescent first component L2 and the light beam including the fluorescent second component L3 is further increased. Noise in the image is reduced.
  • a mask portion 406 as shown in FIG. 7 may be used instead of the prism array mask 6, a mask portion 406 as shown in FIG. 7 may be used.
  • the mask portion 406 has an entire surface 14a that is continuously inclined with respect to the surface 14b, and the transmission portion 15 includes first and second band-like transmission portions 415a and 415 formed of materials having different refractive indexes. Transmissive portions 415b are alternately arranged.
  • Such a first transmission part 415a has, for example, a band-shaped slit formed from the surface 14a to the surface 14b, and the second transmission part 415b is refracted from the first transmission part 415a such as glass. It is formed in a strip shape with materials having different rates.
  • Such a mask unit 406 also enables separation of the illumination light L1 transmitted through the two transmission units 415a and 415b and separation of the first and second components L2 and L3 of fluorescence.
  • the entire second transmission part 415b is made of glass, and the first transmission part 415a is a cavity.
  • the first and second transmission portions 515a and 515b may be arranged in a zigzag manner along the surface 14a in the transmission portion 515 in a two-dimensional manner.
  • the directionality of the contrast of the generated confocal image can be suppressed as compared with the band-shaped prism.
  • the configuration example of such a staggered arrangement is not limited to the form shown in FIG. 8A and can take various forms.
  • a mask portion 606 as shown in FIG.
  • the mask portion 606 is provided with a rectangular second transmissive portion 615b made of a light-transmitting material such as glass in the transmissive portion 615, and along the surface 14a of the second transmissive portion 615b,
  • the first transmission parts 615a that are through holes are arranged two-dimensionally.
  • the first transmission part 615a may be formed of a material having a refractive index different from that of the second transmission part 615b.
  • Such a mask unit 606 can also suppress the directionality of the contrast of the confocal image generated.
  • TDI cameras are used as the image sensors 11A and 11B for generating image data
  • the prism array mask 6 is used as the optical axis of the illumination light L1.
  • a drive mechanism for making the illumination light uniform by shaking in the vertical direction may be provided.
  • a confocal image in which the sensitivity is averaged can also be obtained by acquiring the image data of the sample A while shaking the mask portion by such a driving mechanism.
  • At least one of the first transmission part and the second transmission part includes a prism.
  • the first component light and the second component light generated from the object in the mask portion can be reliably separated.
  • the first and second light detection units are arranged on the opposite side of the object with the mask portion interposed therebetween.
  • the first and second light detection units are configured by an image sensor capable of TDI (Time Delay Integration) operation.
  • TDI Time Delay Integration
  • the TDI operation of the image sensor is synchronized with the movement of the object. In this way, a highly sensitive confocal image can be obtained without moving the mask portion.
  • the present invention uses a confocal microscope apparatus that generates a confocal image of a target object, facilitates adjustment work when obtaining a confocal image, and can reduce the size of the apparatus.

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Microscoopes, Condenser (AREA)
  • Optical Elements Other Than Lenses (AREA)

Abstract

L'invention concerne un microscope confocal qui facilite la procédure d'ajustement, lors de l'obtention d'images confocales, le dispositif étant plus petit. Le microscope confocal (1) est doté d'une source optique de fibres (2) qui irradie de la lumière (L1), un masque en réseau de prismes (6), dans lequel des premières parties de transmission (15a) qui transmettent un premier composant (L2) de la fluorescence de l'échantillon (A) qui retourne dans le sens d'incidence de la lumière et la lumière (L1), et des secondes parties de transmission (15b) qui transmettent et diffractent un second composant (L3) différent du premier composant (L2), de la fluorescence dans un sens différent du premier composant (L2), sont placés de manière alternative. Le microscope comprend également un élément d'imagerie (11A) qui reçoit un premier composant (L2) transmis par les premières partie de transmission (15a) et un élément d'imagerie (11B) qui reçoit le second composant (L3) transmis par les secondes parties (15b). Une image confocale de l'objet est formée sur la base des données d'images obtenues par les éléments d'imagerie (11A) et (11B).
PCT/JP2009/064763 2008-08-29 2009-08-25 Microscope confocal Ceased WO2010024235A1 (fr)

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JP2008-221974 2008-08-29
JP2008221974A JP2010054981A (ja) 2008-08-29 2008-08-29 共焦点顕微鏡装置

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104067158A (zh) * 2013-01-22 2014-09-24 株式会社高岳制作所 共焦扫描仪及使用它的光学测量装置
EP2758812B1 (fr) * 2011-09-23 2018-04-25 Carl Zeiss Microscopy GmbH Dispositif et procédé d'éclairage par transmission pour des microscopes optiques et système de microscope

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002535716A (ja) * 1999-01-22 2002-10-22 アイシス イノヴェイション リミテッド 共焦点顕微鏡検査装置および方法
WO2007010697A1 (fr) * 2005-07-21 2007-01-25 Nikon Corporation Dispositif de microscopes cofocaux
JP2007310202A (ja) * 2006-05-19 2007-11-29 Tokyo Seimitsu Co Ltd 共焦点顕微鏡

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002535716A (ja) * 1999-01-22 2002-10-22 アイシス イノヴェイション リミテッド 共焦点顕微鏡検査装置および方法
WO2007010697A1 (fr) * 2005-07-21 2007-01-25 Nikon Corporation Dispositif de microscopes cofocaux
JP2007310202A (ja) * 2006-05-19 2007-11-29 Tokyo Seimitsu Co Ltd 共焦点顕微鏡

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2758812B1 (fr) * 2011-09-23 2018-04-25 Carl Zeiss Microscopy GmbH Dispositif et procédé d'éclairage par transmission pour des microscopes optiques et système de microscope
CN104067158A (zh) * 2013-01-22 2014-09-24 株式会社高岳制作所 共焦扫描仪及使用它的光学测量装置
CN104067158B (zh) * 2013-01-22 2016-03-16 株式会社东光高岳 共焦扫描仪及使用它的光学测量装置

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