WO2017175258A1 - Microscope à ultra-haute résolution - Google Patents

Microscope à ultra-haute résolution Download PDF

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Publication number
WO2017175258A1
WO2017175258A1 PCT/JP2016/001948 JP2016001948W WO2017175258A1 WO 2017175258 A1 WO2017175258 A1 WO 2017175258A1 JP 2016001948 W JP2016001948 W JP 2016001948W WO 2017175258 A1 WO2017175258 A1 WO 2017175258A1
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WO
WIPO (PCT)
Prior art keywords
light
super
optical
phase plate
resolution microscope
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PCT/JP2016/001948
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English (en)
Japanese (ja)
Inventor
池滝　慶記
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オリンパス株式会社
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Priority to JP2018510015A priority Critical patent/JP6714689B2/ja
Priority to PCT/JP2016/001948 priority patent/WO2017175258A1/fr
Publication of WO2017175258A1 publication Critical patent/WO2017175258A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens

Definitions

  • the present invention relates to a super-resolution microscope.
  • a fluorescence microscope capable of observing a sample containing a molecule having at least two excited quantum states with a high spatial resolution exceeding the diffraction limit using a double resonance absorption process is known (for example, see Patent Documents 1 and 2).
  • the fluorescence microscopes disclosed in Patent Documents 1 and 2 include a first light for exciting molecules in a sample from a stable state to a first quantum state (hereinafter also referred to as pump light), and a molecule in another quantum state.
  • a second surface light (hereinafter also referred to as erase light) is used as a set, and the sample surface is spatially scanned with a fluorescent spot contracted below the diffraction limit.
  • a fluorescence image having a resolution exceeding the spatial resolution of the diffraction limit is obtained by processing the image by arranging the fluorescence signals at each measurement point in a two-dimensional or three-dimensional manner on a computer.
  • Non-Patent Document 1 discloses a phase plate in which two quartz substrates having anisotropic refractive index characteristics are joined in a ring-shaped manner. If this phase plate is used, the phase of the erase light can be inverted by ⁇ at the center, so that an erase light beam having a three-dimensional hollow shape can be formed.
  • the thickness of the substrate is adjusted by integrally polishing two quartz substrates joined in a ring shape.
  • the electric field that is, the phase is inverted in the central annular region for the erase light, and the phase is shifted by an integral multiple of 2 ⁇ for the pump light having a different wavelength.
  • the wave front of the pump light is not substantially affected, and only the erase light is converted into a beam having a three-dimensional hollow shape.
  • Pump light and erase light can be introduced into a commercially available laser scanning microscope using a single common mode fiber. Therefore, if the above phase plate is inserted into the optical path of the illumination optical system of the laser scanning microscope and the pump light and erase light are condensed on the sample surface by the microscope objective lens, the pump light is placed in the hollow center of the erase light. Can be automatically condensed without axis misalignment.
  • the super-resolution microscopy can be implemented by attaching a phase plate to a commercially available laser scanning microscope, and thus is expected as an epoch-making microscope.
  • the phase plate used in the super-resolution microscope is not limited to the one obtained by bonding the above-described anisotropic optical substrate.
  • the phase plate may be configured by coating an optical thin film on a substrate (for example, see Non-Patent Document 2).
  • This phase plate is configured by dividing a substrate surface into a plurality of regions and coating each region with an optical thin film (for example, SiO 2 ) with a different thickness.
  • each region on the substrate may be etched to a different depth to form a phase plate (see, for example, Non-Patent Document 3).
  • the phase plate disclosed in Non-Patent Document 2 and Non-Patent Document 3 optimizes the film thickness to be coated in each region and the etching depth, so that the wavefront of erase light is not affected without affecting the wavefront of pump light. Can be controlled to provide wavelength dispersion capable of condensing in a hollow shape.
  • a phase plate formed by joining two quartz substrates having anisotropic refractive index characteristics in a ring shape requires strict processing accuracy as described below.
  • the quartz substrate In order for a quartz substrate to function as a substrate having optical anisotropy, the quartz substrate is cut at an accurate angle with respect to the optical axis of the quartz substrate, and the refractive index between the phase advance axis and the delay axis is determined. Need to control.
  • phase plate produced by coating an optical thin film on the substrate or etching the substrate
  • a step occurs at the interface of each region, so that light that has passed through the substrate in the boundary region diffracts. For this reason, when the pump light or erase light transmitted through the phase plate is condensed, the diffraction pattern overlaps in addition to the normal condensing pattern, and the spatial resolution function as the super-resolution microscope may be deteriorated.
  • phase plate formed by joining two quartz substrates is hindered from spreading the technology because the quartz substrate is expensive unlike ordinary optical glass. Since a phase plate formed by coating an optical thin film or a phase plate formed by etching a substrate requires different processing steps in each region, it takes time to manufacture.
  • an object of the present invention made in view of such a viewpoint is to provide a super-resolution microscope that can easily produce a phase plate and can be inexpensive as a whole.
  • a super-resolution microscope for observing a sample containing a molecule having at least two excited quantum states, Illumination light having a plurality of wavelengths including a first light for exciting the molecule from a stable state to a first quantum state and a second light for causing the molecule to transition to another quantum state,
  • An illumination optical system for condensing and irradiating at least a part of the first light and the second light on the sample in a spatially overlapping manner;
  • a scanning unit that scans the sample by relatively displacing the illumination light and the sample;
  • a detection optical system for detecting response light from the sample by irradiation of the illumination light to the sample, and
  • the illumination optical system includes a phase plate that spatially modulates the first light and the second light,
  • the phase plate has a flat space shape with a predetermined thickness having a plurality of divided regions through which the illumination light passes.
  • the plurality of divided regions are made of at least two kinds of
  • the phase plate is composed of a plurality of optical substrates having different refractive indexes in the plurality of divided regions, Each of the optical substrates may have a refractive index that is spatially isotropic and is different with respect to the first light and the second light.
  • the thickness of the divided region made of the optical medium material is d, and the refractive index of light at the wavelength ⁇ of the i-th divided region among the plurality of divided regions is n i ( ⁇ ), j-th
  • the refractive index of light at the wavelength ⁇ of the divided region is n j ( ⁇ )
  • the wavelength of the first light is ⁇ p
  • the wavelength of the second light is ⁇ e
  • the thickness of the divided region made of the optical medium material is d, and the refractive index of light at the wavelength ⁇ of the i-th divided region among the plurality of divided regions is n i ( ⁇ ), j-th
  • the refractive index of light at the wavelength ⁇ of the divided region is n j ( ⁇ )
  • the wavelength of the first light is ⁇ p
  • the wavelength of the second light is ⁇ e
  • an arbitrary integer is m, Satisfy (2).
  • the phase plate has two optical substrates, one of the optical substrates may have a ring shape, and the other optical substrate may have a cylindrical shape bonded to the inner peripheral surface of the one optical substrate. .
  • the plurality of optical substrates of the phase plate may be configured by adding impurities at different densities.
  • FIG. 2 is a perspective view conceptually showing a first configuration example of a phase plate in FIG. 1. It is explanatory drawing of one manufacturing method of the phase plate of FIG. It is a perspective view which shows notionally the 2nd structural example of the phase plate of FIG.
  • FIG. 6 is a plan view conceptually showing a third configuration example of the phase plate in FIG. 1.
  • FIG. 1 is a diagram showing a conceptual configuration of a super-resolution microscope according to an embodiment of the present invention.
  • Pump light and erase light are introduced from one common single mode fiber 21 to the super-resolution microscope 20 according to the present embodiment.
  • the pump light for example, light having a wavelength of 532 nm using an Nd: YVO4 laser as a light source is used.
  • the erase light for example, light having a wavelength of 633 nm using a He—Ne laser as a light source is used.
  • Pump light and erase light emitted from each light source are coaxially combined by a known beam combiner and incident on the single mode fiber 21.
  • the pump light and erase light introduced from the single mode fiber 21 are collimated in common by the collimator lens 22, and then pass through the iris 23, the phase plate 10, and the bandpass filter 24 to be galvanomirrors. Introduced into the optical system 25.
  • the pump light and erase light introduced into the galvano mirror optical system 25 are deflected and scanned in the two-dimensional direction by the galvano mirror optical system 25, and are condensed on the sample S by the microscope objective lens 27 through the pupil projection lens 26.
  • the beam diameter of the erase light incident on the phase plate 10 is adjusted together with the pump light so as to satisfy the interference condition that forms a hollow shape on the focal plane of the microscope objective lens 27.
  • the band pass filter 24 is configured to transmit the pump light and the erase light and reflect the fluorescence from the sample S.
  • the fluorescence generated from the sample S by the irradiation of the pump light traces the optical path of the illumination light of the pump light and erase light, enters the band pass filter 24, is reflected by the band pass filter 24, and is reflected by the illumination optical system. Separated from the optical path.
  • the fluorescence reflected by the band pass filter 24 is collected only by the block filter 31 and then condensed by the condenser lens 32 and received by the photodetector 34 such as a photomultiplier through the pinhole 33.
  • the illumination optical system includes the single mode fiber 21, the collimator lens 22, the phase plate 10, the pupil projection lens 26, and the microscope objective lens 27.
  • the scanning unit includes a galvanometer mirror optical system 25.
  • the detection optical system includes a bandpass filter 24, a block filter 31, a condenser lens 32, a pinhole 33, and a photodetector 34.
  • a phase plate 10 is inserted into the optical path of an illumination optical system of a commercially available laser scanning microscope, and a pump is used using a single common mode fiber in the illumination optical path. It can be easily configured by introducing light and erase light.
  • phase plate 10 Next, a configuration example of the phase plate 10 will be described.
  • the phase plate 10 is an optically isotropic optical medium material having the same thickness that does not affect the wavefront accuracy without using a special anisotropic optical medium material, that is, a standard having an isotropic refractive index. A plurality of typical optical medium materials are used.
  • the phase plate 10 has a surface that is geometrically flat and has an optically sufficient flatness.
  • an optical medium material for example, standard glass BK7 or quartz glass can be used.
  • optical thin film materials such as TiO 2 can be used.
  • FIG. 2 is a perspective view conceptually showing a first configuration example of the phase plate 10.
  • the phase plate 10 shown in FIG. 2 includes a first optical glass 11-1 and a second optical glass 11-2.
  • the first optical glass 11-1 is formed in an annular shape.
  • the second optical glass 11-2 is formed in a cylindrical shape and joined to the inner peripheral surface of the first optical glass 11-1. That is, the phase plate 10 has two divided regions in which the first optical glass 11-1 and the second optical glass 11-2 are bonded in a ring shape.
  • the first optical glass 11-1 and the second optical glass 11-2 have the same thickness d in the optical axis direction of the transmitted pump light and erase light, and have different refractive indexes.
  • the refractive index at the wavelength ⁇ of the first optical glass 11-1 is n 1 ( ⁇ )
  • the refractive index at the wavelength ⁇ of the second optical glass 11-2 is n 2 ( ⁇ ).
  • the wavelength of the pump light and lambda p, the wavelength of the erase light and lambda e are examples of the wavelength of the erase light and lambda e.
  • phase difference ⁇ of the erase light when transmitted through the first optical glass 11-1 and the second optical glass 11-2 is given by the following expression (3).
  • phase difference ⁇ of the pump light when transmitted through the first optical glass 11-1 and the second optical glass 11-2 is given by the following equation (4).
  • the erase light transmitted through the phase plate 10 in order for the erase light transmitted through the phase plate 10 to be condensed as a beam spot of the hollow hole by the microscope objective lens 27, the erase light transmitted through the outer first optical glass 11-1 and the inner light It is desirable that the phase of the erase light transmitted through the second optical glass 11-2 is reversed. In this case, it is only necessary to satisfy the following expression (5), where m is an arbitrary integer.
  • the pump light needs to be condensed as a normal Gaussian beam.
  • the phase disturbance of the beam surface of the pump light needs to be at least ⁇ / 4 or less. That is, it is necessary to satisfy the condition of the following formula (6). Under this condition, the pump light can be condensed in a circular shape without reversing the polarity of the phase in all wavefronts.
  • the first optical glass 11-1 and the second optical glass 11-2 having different refractive indexes are selected, and the supplied pump light and What is necessary is just to choose thickness d of the conditions which satisfy
  • Table 1 shows parameters in this design example.
  • NB-Bak1 manufactured by Schott
  • NF2 manufactured by Schott
  • phase difference ⁇ of the pump light is ⁇ p / 210 when converted to the wavelength of the pump light. This value is negligible because the planar accuracy of a normal optical polishing glass substrate is ⁇ p / 10. Therefore, even if the pump light is transmitted through the phase plate 10, the wavefront is not affected at all.
  • the phase plate 10 shown in FIG. 2 can be manufactured by a very standard processing technique.
  • the NB-Bak1 substrate constituting the first optical glass 11-1 is cut into an annular shape.
  • the N—F 2 substrate constituting the second optical glass 11-2 is cut into a columnar shape with the same thickness as the first optical glass 11-1.
  • the columnar second optical glass 11-2 is bonded to the inner peripheral surface of the ring-shaped first optical glass 11-1 with, for example, an ultraviolet curable resin. Thereafter, the bonded first optical glass 11-1 and second optical glass 11-2 are optically polished integrally until optical flatness is satisfied.
  • the first optical glass 11-1 and the second optical glass 11-2 were used as commercially available optical glass.
  • the first optical glass 11-1 and the second optical glass 11-2 are made of, for example, ordinary fused silica glass so as to have optimum refractive indexes according to the respective wavelengths of the pump light and erase light to be used. Further, a small amount of impurities such as Ti may be added at different densities. Further, the phase plate 10 can be coated with an antireflection film on the surface to suppress generation of scattered light of illumination light.
  • the manufacturing method described above since the first optical glass 11-1 and the second optical glass 11-2 having isotropic refractive indexes are used, a conventional optical substrate having anisotropic refractive index characteristics is obtained. Compared with the case of using, the two optical substrates can be cut without worrying about the direction of the optical axis. In addition, when joining two substrates, there is no need to worry about the direction of the optical axis, so the work process becomes much simpler. In addition, since optical glass with an isotropic refractive index is used, advanced cutting technology is not required, and an expensive quartz substrate is not required. There are also advantages in terms of production and cost.
  • the phases of the erase light transmitted through the first optical glass 11-1 and the erase light transmitted through the second optical glass 11-2 were inverted. However, even if the two are not completely reversed, the electric field of at least the erase light transmitted through the outer first optical glass 11-1 and the erase light transmitted through the inner second optical glass 11-2. If the polarities are different, the electric field at the focal point of the microscope objective lens 27 can be canceled.
  • phase difference between the erase light transmitted through the first optical glass 11-1 and the erase light transmitted through the second optical glass 11-2 is expressed by the following formula (6) It is only necessary to satisfy 7).
  • the area and transmittance of the optical glass 11-1 and the second optical glass 11-2 may satisfy the following formula (8).
  • the integration region “in” indicates the region of the inner second optical glass 11-2
  • the integration region “out” indicates the region of the outer first optical glass 11-1.
  • T in indicates transmittance distribution of the erase light in the second optical glass 11-2
  • T out denotes the transmittance distribution of the erase light of the first optical glass 11-1.
  • ⁇ in indicates the phase distribution of the erase light that has passed through the second optical glass 11-2
  • ⁇ out indicates the phase distribution of the erase light of the first optical glass 11-1.
  • X and y indicate the coordinates of the surface of the phase plate 10.
  • FIG. 4 is a perspective view conceptually showing a second configuration example of the phase plate 10.
  • the phase plate 10 shown in FIG. 4 has a large number of divided regions having a multi-annular zone structure having optical glasses 12-1 to 12-n having the same thickness joined together in a concentric manner.
  • the polarities of the electric fields of erase light that pass through adjacent optical glasses are different.
  • the area and transmittance of each optical glass having a multiple ring zone structure may be different.
  • the equation (8) is expressed as the following equation (9).
  • j represents the jth optical glass.
  • FIG. 5 is a plan view conceptually showing a third configuration example of the phase plate 10.
  • the phase plate 10 shown in FIG. 5 has four divided regions in which four optical glasses 13-1 to 13-4 having different refractive index characteristics and the same thickness are joined in a circular shape by ⁇ / 2 rads.
  • the four optical glasses 13-1 to 13-4 change the phase of the transmitted erase light step by step by ⁇ / 4.
  • phase plate 10 shown in FIG. 5 it is possible to form a very tight erase light hollow beam on the focal plane of the microscope objective lens 27.
  • the phase disturbance of the beam surface of the pump light passing through the four optical glasses 13-1 to 13-4 needs to be at least ⁇ / 4 or less.
  • phase plate 10 In the first to third configuration examples of the phase plate described above, optical glasses having the same thickness are used as a plurality of optical medium materials having different isotropic refractive indexes.
  • the phase plate 10 is configured by coating a plurality of optical thin films having the same film thickness and different refractive indexes on the same optical substrate based on the concept of using optical medium materials having the same thickness. .
  • a glass substrate having an optically isotropic and uniform thickness is used, and the surface thereof is, for example, as shown in FIG. 2, FIG. 4 or FIG. It is divided into a plurality of areas of a band or 4 divisions. Then, each divided region is coated with an optical thin film having the same film thickness and different refractive index that satisfies the conditions described in the first to third configuration examples. That is, as in the first to third configuration examples, instead of joining a plurality of optical glasses with different isotropic refractive indexes and the same thickness, a plurality of optical fibers with the same thickness having different isotropic refractive indexes.
  • the phase plate 10 is formed by forming a thin film on the surface of a glass substrate having an optically isotropic and uniform thickness.
  • the optical thin film can be formed using an optical thin film material such as TiO 2 .
  • the optical thin film may be a single layer film or a multilayer film.
  • the number of layers and the optical medium material are appropriately selected under the condition that the entire film thickness is constant, and more precise independent phase control is performed for pump light and erase light. It is possible to guide the super-resolution microscope function.
  • the optical medium material to be coated is an organic material, for example, an ultraviolet curable resin, unlike an inorganic material, not only a technique such as vapor deposition and sputtering, but also a spin coating method, printing, etc. Can also be used. Therefore, there is an advantage that the manufacturing process of the phase plate 10 is simplified.
  • the phase plate 10 is composed of any one of the first to fourth configuration examples described above. Therefore, since the phase plate 10 can be easily manufactured, high-precision super-resolution microscope observation is possible with a low-cost configuration as a whole.
  • the phase plate 10 is not limited to the configuration example described above, and may be configured by bonding a plurality of optical substrates having different refractive index characteristics and the same thickness and various shapes.
  • the phase plate 10 may be formed by coating a plurality of regions divided on the same optical substrate with a plurality of optical thin films having various refractive indexes and the same thickness.
  • the configuration of the illumination optical system, the scanning unit, the detection unit, etc. of the super-resolution microscope 20 shown in FIG. 1 is an example, and can be changed to an appropriate configuration having the same function.
  • Phase plate 11-1, 11-2, 12-1 to 12-n, 13-1 to 13-4 Optical glass 20
  • Super-resolution microscope 21 Single mode fiber 22
  • Collimator lens 23 Iris 24
  • Bandpass filter 25 Galvanometer mirror optics System 26
  • Pupil projection lens 27 Microscope objective lens S
  • Block filter 32 Condensing lens 33 Pinhole 34 Photodetector

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Abstract

Selon l'invention, une lumière de pompage et une lumière d'effacement sont modulées spatialement par une lame de phase (10) présentant une forme spatiale plate qui a une épaisseur prédéfinie et qui possède une pluralité de régions de division, et elles sont collectées avec une ultra-haute résolution sur un échantillon (S). La pluralité de régions de division de la lame de phase (10) comprend au moins deux types de matériaux de support optique spatialement isotropes, et elle commande en phase de façon indépendante la lumière de pompage et la lumière d'effacement.
PCT/JP2016/001948 2016-04-07 2016-04-07 Microscope à ultra-haute résolution WO2017175258A1 (fr)

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JP2018510015A JP6714689B2 (ja) 2016-04-07 2016-04-07 超解像顕微鏡
PCT/JP2016/001948 WO2017175258A1 (fr) 2016-04-07 2016-04-07 Microscope à ultra-haute résolution

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PCT/JP2016/001948 WO2017175258A1 (fr) 2016-04-07 2016-04-07 Microscope à ultra-haute résolution

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010529486A (ja) * 2007-06-01 2010-08-26 マックス プランク ゲゼルシャフト ツール フェルデルング デル ヴィッセンシャフテン エー．ヴェー． 波長又は偏光を感知する光学装置及びこの光学装置の使用
JP2015031882A (ja) * 2013-08-05 2015-02-16 オリンパス株式会社 超解像顕微鏡及び変調光学素子

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010529486A (ja) * 2007-06-01 2010-08-26 マックス プランク ゲゼルシャフト ツール フェルデルング デル ヴィッセンシャフテン エー．ヴェー． 波長又は偏光を感知する光学装置及びこの光学装置の使用
JP2015031882A (ja) * 2013-08-05 2015-02-16 オリンパス株式会社 超解像顕微鏡及び変調光学素子

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