WO2017082357A1

WO2017082357A1 - Super-resolution microscope

Info

Publication number: WO2017082357A1
Application number: PCT/JP2016/083399
Authority: WO
Inventors: 池滝　慶記; 熊谷　寛; 大助岡田
Original assignee: オリンパス株式会社; 学校法人北里研究所
Priority date: 2015-11-12
Filing date: 2016-11-10
Publication date: 2017-05-18
Also published as: JPWO2017082357A1

Abstract

According to the present invention, a sample 10 is scanned using a plurality of fluorescent spots by: generating a plurality of illumination lights for scanning by transmitting an illumination light, which includes a first light that excites molecules and a second light that suppresses the excitation of molecules, from a plurality of pinholes 26a while rotating a pinhole array disk 26; and shining, on the sample 10 via a spatial modulation element 27 and a microscope objective lens 28 that have optical transparency for the first light and the second light, these illuminating lights for scanning as a plurality of fluorescent spots that exceed the diffraction limit.

Description

Super-resolution microscope

The present invention relates to a super-resolution microscope.

As a super-resolution microscope, for example, in Patent Documents 1 and 2, fluorescence capable of observing a sample including a molecule having at least two excited quantum states with a high spatial resolution exceeding the diffraction limit using a double resonance absorption process. A microscope is disclosed. The fluorescence microscopes disclosed in Patent Documents 1 and 2 are pump light for generating fluorescence by exciting a molecule in a sample from a stable state to a first quantum state, and further transitioning the molecule to another second quantum state. The sample surface is spatially scanned with a fluorescent spot (excitation light spot) contracted below the diffraction limit using erase light for suppressing the generation of fluorescence. A fluorescence image having a resolution exceeding the spatial resolution of the diffraction limit is obtained by two-dimensionally arranging the fluorescence signals at each measurement point on a computer and performing image processing.

Although the fluorescence microscopes disclosed in Patent Documents 1 and 2 have an excellent function, it takes time to measure one screen because the sample is scanned with one scanning beam including pump light and erase light as a set. It takes. For this reason, when observing a living biological sample, the spatial resolution may be reduced due to changes in the shape and position of the biological sample at the focal point of the laser beam, and a good super-resolution microscope image may not be obtained. is there.

Note that, as a method of obtaining an image in a short time with one scanning beam, for example, it is conceivable to narrow the observation region or shorten the integration time of the fluorescence signal for each observation point. However, in the former case, inconvenience that it is difficult to grasp the structure of the entire sample occurs, and in the latter case, the S / N of the image is reduced.

On the other hand, as another super-resolution microscope, for example, Patent Document 3 discloses a STED (stimulated emission depletion :) microscope that uses excitation light for exciting a sample and suppression light for suppressing excitation of the sample by stimulated emission. Yes. The STED microscope disclosed in Patent Document 3 is formed on a lens disk that holds a plurality of microlens arranged in an array, a pinhole disk that has a plurality of pinholes formed in the same pattern as the arrangement of microlenses, and a microlens. A phase adjustment unit. Then, while rotating the lens disk and the pinhole disk integrally, the excitation light and the suppression light superimposed on the same axis are simultaneously incident on a plurality of microlenses, and the sample is irradiated through a pair of pinholes. ing.

According to the STED microscope disclosed in Patent Document 3, since a sample is scanned with a plurality of fluorescent spots exceeding the diffraction limit, high-speed scanning is possible, and a super-resolution microscope image can be obtained in a short time.

JP 2001-100102 A JP 2010-15026 A JP 2012-226145 A

However, in the STED microscope disclosed in Patent Document 3, a suppression light transmission filter is formed in the phase adjustment unit of the microlens, and an excitation light transmission filter is formed in a region other than the phase adjustment unit of the microlens. Here, the suppression light transmission filter has a characteristic of transmitting the suppression light and not transmitting the excitation light. Further, the excitation light transmission filter has a characteristic of transmitting the excitation light and not transmitting the suppression light. Therefore, it is not easy to separately form the suppression light transmission filter and the excitation light transmission filter in the small-diameter microlens, and there is a concern that the cost may increase. Moreover, since the transmission region within the aperture of the excitation light and the suppression light is divided and shielded, the intensity of the excitation light and the suppression light is extremely reduced. In particular, since the suppression light requires high intensity, a high-power laser light source is required. Furthermore, since each pupil region is shielded, it is difficult to obtain a sufficient super-resolution function by spreading or deforming the focused beam shape.

Therefore, an object of the present invention made in view of such a viewpoint is to provide an improved super-resolution microscope capable of obtaining a good super-resolution microscope image in a short time.

The super-resolution microscope according to the present invention that achieves the above object is as follows.
Illumination light including first light that excites molecules in the sample from the stable state to the first quantum state and second light that suppresses excitation of the molecules is used as the illumination light of the first light and the second light. An illumination optical system that irradiates the sample with at least a portion of the spatially superimposed;
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 scanning illumination light generation unit, a spatial modulation element, and a microscope objective lens,
The scanning illumination light generation unit generates a plurality of scanning illumination lights that are displaced from the incident illumination light in different trajectories,
The microscope objective lens condenses a plurality of the scanning illumination light from the scanning illumination light generation unit on the sample,
The spatial modulation element has optical transparency with respect to the first light and the second light, and each of the plurality of scanning illumination lights condensed by the microscope objective lens is converted into the first light. Spatially modulate at least a portion of either the first light or the second light so that the second light has a maximum light intensity and the second light has a minimum light intensity. ,
The detection optical system includes a two-dimensional optical sensor that photoelectrically converts the response light from the sample by the plurality of scanning illumination lights,
The sample is scanned with a plurality of scanning illumination lights, and the sample is observed based on the output of the two-dimensional sensor.

The spatial modulation element may modulate the polarization state or phase state of either the first light or the second light.

The scanning illumination light generator includes a rotatable pinhole array disk,
The pinhole array disk has a plurality of pinholes arranged in a plane perpendicular to the rotation axis, and transmits the illumination light from a plurality of the pinholes in a part of the plane, thereby a plurality of the scanning illumination lights Produces
The sample may be scanned with a plurality of scanning illumination lights while rotating the pinhole array disk.

The scanning illumination light generator includes a rotatable microlens array disk,
The microlens array disk has a plurality of microlenses arranged on a plane orthogonal to a rotation axis, and transmits the illumination light from a plurality of the microlenses that are part of the plane, thereby a plurality of illumination lights for scanning. Produces
The sample may be scanned with a plurality of scanning illumination lights while rotating the microlens array disk.

The spatial modulation element may be provided so as to be bonded to each microlens of the microlens array disk.

The illumination optical system further includes a spatial modulation element array disk that is disposed on the incident surface side or the exit surface side of the illumination light with respect to the microlens array disk and is rotatable integrally with the microlens array disk,
A plurality of the spatial modulation element array discs are arranged on the optical axis of each of the plurality of microlenses in the same arrangement pattern as the arrangement pattern of the microlenses in the microlens array disc on a plane orthogonal to the rotation axis. You may have the said spatial modulation element arranged.

The scanning illumination light generation unit includes a microlens array disk and a pinhole array disk that can rotate together,
The microlens array disk has a plurality of microlenses arranged in a plane perpendicular to the rotation axis,
The pinhole array disk has a plurality of pinholes arranged in the same arrangement pattern as the arrangement pattern of the microlens on a plane orthogonal to the rotation axis, and the pinhole is located at the image side focal position of the microlens. Arranged to
Passing the illumination light through the plurality of microlenses and the plurality of pinholes to generate the plurality of scanning illumination lights;
The sample may be scanned with a plurality of scanning illumination lights while rotating the microlens array disk and the pinhole array disk.

The illumination optical system includes a deflection unit that deflects the illumination light and moves a scanning region of the sample,
Based on the output of the two-dimensional sensor in the plurality of scanning regions, an image signal of the sample in which the plurality of scanning regions are combined may be generated.

The detection optical system includes:
The response from the sample that is arranged in the optical path of the illumination light or the scanning illumination light incident on the microscope objective lens, transmits the illumination light or the scanning illumination light, and enters the microscope objective lens through the microscope objective lens A dichroic mirror that reflects light,
An imaging lens for imaging the response light reflected by the dichroic mirror on the two-dimensional sensor;
A spectral filter disposed in an optical path between the imaging lens and the two-dimensional sensor and transmitting the response light may be further included.

The illumination optical system may further include a beam shaping unit that shapes the shape of the illumination area by the illumination light incident on the scanning illumination light generation unit.

According to the present invention, it is possible to realize an improved super-resolution microscope that can be easily configured without incurring an increase in cost and can obtain a good super-resolution microscope image in a short time.

It is a schematic block diagram of the super-resolution microscope which concerns on 1st Embodiment. It is a fragmentary sectional view of FIG. It is a top view which shows schematic structure of an example of a spatial modulation element. It is a figure for demonstrating the optical characteristic of the spatial modulation element of FIG. 3A. It is a top view which shows schematic structure of the other example of a spatial modulation element. It is a figure for demonstrating the optical characteristic of the spatial modulation element of FIG. 4A. It is a schematic block diagram of the super-resolution microscope which concerns on 2nd Embodiment. FIG. 6 is a partial bottom view of the microlens array disk of FIG. 5. FIG. 6 is a partial cross-sectional view of the microlens array disk of FIG. 5. It is a schematic block diagram of the super-resolution microscope which concerns on 3rd Embodiment. It is a schematic block diagram of the super-resolution microscope which concerns on 4th Embodiment. It is a schematic block diagram of the super-resolution microscope which concerns on 5th Embodiment. It is a schematic block diagram of the super-resolution microscope which concerns on 6th Embodiment. It is a figure explaining the irradiation area | region by the illumination light with respect to the micro lens array disk of FIG. It is a schematic block diagram of the super-resolution microscope which concerns on 7th Embodiment.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(First embodiment)
FIG. 1 is a schematic configuration diagram of a super-resolution microscope according to the first embodiment. The super-resolution microscope according to the present embodiment observes the sample 10 stained with a fluorescent dye having at least two or more excited quantum states by super-resolution using a double resonance absorption process. Therefore, in the super-resolution microscope shown in FIG. 1, the first light (pump light) for exciting the fluorescent dye from the stable state to the first quantum state and the fluorescent dye are further transitioned to another second quantum state. Second light (erase light) for suppressing excitation of fluorescent molecules is used as illumination light.

The pump light and the erase light are coaxially synthesized from the light source unit 20 as a linearly polarized continuous wave or pulse wave, respectively, and emitted as illumination light. The light source unit 20 includes, for example, a laser light source for pump light and a laser light source for erase light, and the pump light and the erase light emitted from these laser light sources directly or through a filter or the like are coaxially formed by a known beam combiner. Combined and emitted as illumination light. The light source unit 20 may be configured to use a fundamental wave and a predetermined harmonic wave from a single laser light source as pump light and erase light.

The illumination light emitted from the light source unit 20 is guided to the microscope main body 22 through the single mode fiber 21. The illumination light emitted from the light source unit 20 may be a continuous wave or a pulse wave of pump light and erase light. In the case of a pulse wave, both of the emission timings may be the same, or the erase light emission timing may be a maximum of the excitation lifetime of the fluorescent molecules of the sample 10 relative to the pump light emission timing (several nanoseconds to several microseconds). ) May be delayed.

In the microscope main body 22, the illumination light emitted from the single mode fiber 21 is condensed on the sample 10 by the microscope objective lens 28 through the collimator lens 23, the scanning illumination light generation unit 50, the dichroic mirror 25, and the spatial modulation element 27. Is done.

The collimator lens 23 converts the illumination light diffused and emitted from the exit end face of the single mode fiber 21 into parallel light, expands the illumination light, and causes the illumination light to enter the scanning illumination light generation unit 50.

The scanning illumination light generator 50 generates a plurality of scanning illumination lights that are displaced from the illumination light along different trajectories. In the present embodiment, the scanning illumination light generation unit 50 includes a microlens array disk 24 and a pinhole array disk 26. The microlens array disk 24 and the pinhole array disk 26 are configured in the same manner as the microlens array disk and the pinhole array disk in a Nipo type confocal microscope, for example, and are integrally formed coaxially by a disk drive unit 29 having a motor or the like. Driven by rotation. That is, the microlens array disk 24 includes a plurality of microlenses 24a arranged in a plane orthogonal to the rotation axis with different rotation trajectories.

Similarly, the pinhole array disk 26 includes a plurality of pinholes 26a arranged in the same arrangement pattern as the arrangement pattern of the microlenses 24a on a plane orthogonal to the rotation axis. The pinhole array disk 26 is arranged so that each pinhole 26a is positioned at the focal point of the corresponding microlens 24a of the microlens array disk 24, and the pinhole 24a functions as a confocal pinhole. In FIG. 1, for the sake of clarity, four corresponding microlenses 24a and four pinholes 26a are shown.

Illumination light emitted from the collimator lens 23 as parallel light is parallel to the rotation axis of the microlens array disk 24 in the scanning illumination light generation unit 50 and is planar on the plane of the microlens array disk 24 at a predetermined distance from the rotation axis. A part is irradiated. Thereby, the illumination light is simultaneously incident on the plurality of microlenses 24 a located in the irradiation region of the microlens array disk 24. As shown in the partial cross-sectional view of FIG. 1, the illumination light incident on the microlens array disk 24 is condensed by a plurality of microlenses 24a, and corresponding pinholes 26a as a plurality of scanning illumination lights. Transparent.

The dichroic mirror 25 is disposed in the optical path of the scanning illumination light between the microlens array disk 24 and the pinhole array disk 26. The dichroic mirror 25 is configured to transmit scanning illumination light and reflect fluorescence (response light) from the sample 10 described later.

The plurality of scanning illumination lights transmitted through the plurality of pinholes 26a are enlarged to the aperture size of the microscope objective lens 28 by the action of the corresponding microlenses 24a and are condensed at different positions of the sample 10 by the microscope objective lens 28. .

The spatial modulation element 27 is disposed in the optical path of the scanning illumination light between the pinhole array disk 26 and the microscope objective lens 28. The spatial modulation element 27 has optical transparency with respect to pump light and erase light. The spatial modulation element 27 pumps the pump light so that the pump light has the maximum value of the light intensity and the erase light has the minimum value of the light intensity for each of the scanning illumination lights condensed by the microscope objective lens 28. And at least a part of either one of the erase lights.

For example, the spatial modulation element 27 condenses the pump light by the microscope objective lens 28 without beam shaping as a normal Gaussian beam. Further, the erase light is modulated so as to be condensed after being shaped into a hollow shape having a minimum intensity at the focal point of the microscope objective lens 28. Thereby, a plurality of fluorescent spots exceeding the diffraction limit in which the fluorescence emission around the irradiation region of the pump light is suppressed are formed on the sample 10.

The spatial modulation element 27 uses, for example, (1) an optical multilayer film, (2) etching, (3) a wave plate, (4) a photonic material, or the like, and a polarization state of one of pump light and erase light. Or it is configured to modulate the phase state.

In the case of (1) above, the spatial modulation element 27 is configured, for example, as shown in FIGS. 3A and 3B (see, for example, JP 2010-15026 A). 3A is a plan view showing a schematic configuration of the spatial modulation element 27, and FIG. 3B is a diagram for explaining optical characteristics of the spatial modulation element 27. FIG. The spatial modulation element 27 uses the entire region of the incident light beam on the glass substrate 27a as a modulation region, and has a plurality of regions divided around the optical axis in this modulation region, in this case, eight regions, in which eight regions are erased. with respect to the wavelength lambda ₂ of the optical phase by lambda _2/8 are differently multilayer film 27b is formed.

Here, as shown in FIG. 3B schematically showing the phase distribution of the pump light and the erase light, the multilayer film 27b increases in phase in a stepwise manner so that it becomes a Laguerre Gaussian beam. It is designed to go around 2π. On the other hand, for the pump light, the multilayer film 27b is designed such that a phase change of an integer multiple of 2π occurs with respect to the wavelength λ ₁ of the pump light in each region due to wavelength dispersion.

Further, the spatial modulation element 27 is configured so that, for example, a single layer film is independently coated on each region instead of the multilayer film 27b of FIG. 3A, and a phase difference (optical path difference) is given to the pump light and the erase light. (For example, refer to JP2014-182239A).

In the case of (2) above, the spatial modulation element 27 divides the glass substrate into a radial pattern, for example, and adjusts the etching depth of each region to give independent phase differences to the pump light and the erase light. Configured as follows.

In the case of (3) above, the spatial modulation element 27 is configured, for example, as shown in FIGS. 4A and 4B (see, for example, Opt. Lett. Vol. 40, Issue 6, pp. 1057-1060 (2015)). . 4A is a plan view showing a schematic configuration of the spatial modulation element 27, and FIG. 4B is a diagram for explaining optical characteristics of the spatial modulation element 27. FIG. The spatial modulation element 27 includes a cylindrical substrate 27c and an annular substrate 27d that are joined concentrically.

The cylindrical substrate 27c and the annular substrate 27d are made of, for example, a quartz substrate having a fast axis (indicated by a solid line arrow) and a slow axis (indicated by a broken line arrow) that are orthogonal to each other. It joins so that the fast axis of the board | substrate 27d may orthogonally cross. The cylindrical substrate 27c and the annular substrate 27d are adjusted in thickness so that they are half-wave plates for erase light and single-wave plates for pump light, and the polarization states of the pump light and erase light are adjusted. Modulate.

In the spatial modulation element 27 in FIG. 4A, the pump light is transmitted in the same phase, and the erase light is transmitted with the phase inverted, as shown in FIG. 4B. When the pump light transmitted through the spatial modulation element 27 is collected by the microscope objective lens 28, the pump light is collected in a form close to a normal Gaussian beam. On the other hand, when the erase light transmitted through the spatial modulation element 27 is condensed by the microscope objective lens 28, the electric field cancels out only by the focal point due to interference, so that it is condensed as a three-dimensional hollow beam. Therefore, in this case, since the fluorescent spot can be contracted also in the optical axis direction, a three-dimensional super-resolution effect can be obtained.

In the case of (4) above, the spatial modulation element 27 is configured by arranging minute holes or pit patterns of diffraction limit on a crystal substrate made of a photonic crystal material. The refractive index for a specific wavelength can be adjusted by adjusting the size and / or interval of the hole or pit pattern.

For example, if holes or pit patterns are arranged at different sizes and / or intervals with respect to the direction orthogonal to the plane of the crystal substrate, different refractive indexes can be given in the orthogonal direction. By utilizing this characteristic, different refractive indexes can be given independently to the pump light and the erase light. Thereby, the pump light and the erase light transmitted through the spatial modulation element 27 are similarly caused to have the maximum value of the light intensity by the microscope objective lens 28, and the erase light has the minimum value of the light intensity. The sample 10 can be condensed.

On the other hand, the fluorescence emitted from the sample 10 by the irradiation of the plurality of scanning illumination lights follows the optical path opposite to the optical path of the corresponding scanning illumination light, and the microscope objective lens 28, the spatial modulation element 27, and the corresponding pins. The light enters the dichroic mirror 25 through the hole 26a. Fluorescence incident on the dichroic mirror 25 is reflected by the dichroic mirror 25, then forms an image on the image sensor 32 through the imaging lens 30 and the spectral filter 31, and is output as a photoelectric conversion signal from the image sensor 32. Is done. The photoelectric conversion signal output from the image sensor 32 is input to the image processing unit 33. The image processing unit 33 processes the input photoelectric conversion signal and outputs a fluorescence image signal of the sample 10.

In order to improve the S / N ratio of the fluorescence image signal output from the image processing unit 33, the spectral filter 31 converts the wavelength component of the fluorescence emitted from the sample 10 from the wavelength component of the light reflected by the dichroic mirror 25. It is configured to transmit light. The spectral filter 31 may be omitted when the dichroic mirror 25 has a characteristic of reflecting only fluorescence of a desired wavelength component and transmitting other wavelength components. The image sensor 32 is configured by a two-dimensional sensor including a solid-state imaging device such as a CCD (Charge-Coupled Device), a BBD (Bucket-Brigade Device), and a CMOS (Complementary Metal-Oxide Semiconductor).

In the present embodiment, the illumination optical system includes a collimator lens 23, a microlens array disk 24, a dichroic mirror 25, a pinhole array disk 26, a spatial modulation element 27, and a microscope objective lens 28. The detection optical system includes a microscope objective lens 28, a spatial modulation element 27, a pinhole array disk 26, a dichroic mirror 25, an imaging lens 30, a spectral filter 31, and an image sensor 32.

The light source unit 20, the disk drive unit 29, and the image processing unit 33 described above are controlled by the control unit 35. The control unit 35 may be realized by software using a computer or the like, or may be configured by a dedicated processor such as a DSP (digital signal processor). The image processing unit 33 may be built in the control unit 35.

In the present embodiment, the microlens array disk 24 and the pinhole array disk 26 are coaxially connected by the disk drive unit 29 while driving the light source unit 20 to emit illumination light having pump light and erase light from the single mode fiber 21. Rotate together at a predetermined rotational speed. When the microlens array disk 24 rotates, the plurality of microlenses 24a move along the irradiation locus of the illumination light irradiated on the microlens array disk 24 with different rotation trajectories. As a result, the sample 10 is raster-scanned by, for example, a plurality of fluorescent spots that exceed the diffraction limit due to the plurality of scanning illumination lights displaced along different trajectories, and images of the plurality of fluorescent spots are formed on the image sensor 32.

The image processing unit 33 converts the photoelectric conversion signal read from the pixel position of the image sensor 32 to the rotational speed of the microlens array disk 24 and the irradiation area of the illumination light irradiated to the microlens array disk 24. Processing is performed based on the position information, and a fluorescent image signal of the sample 10 is output. This fluorescent image signal can be input to a monitor to display a fluorescent image, or can be input to a recording unit to be recorded on a recording medium.

According to the present embodiment, since the sample 10 is scanned with a plurality of fluorescent spots exceeding the diffraction limit, the sample is not reduced without narrowing the observation area of the sample 10 or shortening the integration time of the fluorescence signal at each observation point. Ten three-dimensional observations are possible. Therefore, the observation area is not reduced and the S / N reduction of the microscope image is not caused. Further, since the spatial modulation element 27 is light transmissive with respect to the pump light and the erase light, it can be easily configured without increasing the cost. As described above, according to the present embodiment, it is possible to realize an improved super-resolution microscope that can obtain a good super-resolution microscope image in a short time. In the present embodiment, since the scanning illumination light generation unit 50 includes the microlens array disk 24, the illumination light from the light source unit 20 can be effectively used by the plurality of microlenses 24a. Therefore, the light source unit 10 can be easily configured using a relatively low power laser light source.

(Second Embodiment)
FIG. 5 is a schematic configuration diagram of a super-resolution microscope according to the second embodiment. In the super-resolution microscope according to the present embodiment, the pinhole array disk 26 and the spatial modulation element 27 are omitted from the configuration shown in FIG. The scanning illumination light generation unit 50 includes a microlens array disk 24, and a spatial modulation element 37 is bonded to each microlens 24 a of the microlens array disk 24.

For example, as shown in FIGS. 6A and 6B, a partial bottom view and a partial cross-sectional view of the microlens array disk 24, the spatial modulation element 37 is directly on the image side surface of each microlens 24a, for example, an optical multilayer film or a single layer film. It is constructed by joining. As a result, a plurality of scanning illumination light in which one of the pump light and the erase light is spatially modulated by the plurality of microlenses 24 a to which the illumination light from the collimator lens 23 is incident and the corresponding plurality of spatial modulation elements 37. Generated. The plurality of scanning illumination lights are beam-formed by the microscope objective lens 28 and condensed on the sample 10. Other configurations are the same as those in FIG.

According to the present embodiment, the fluorescent spot condensed by the microscope objective lens 28 through each microlens 24a contracts in the optical axis direction as in the case of using the spatial modulation element 27 shown in FIGS. 4A and 4B. It has a 3D super-resolution function. Therefore, since three-dimensional super-resolution observation can be performed without using the pinhole array disk 26 as shown in FIG. 1, the same effect can be obtained with a simpler configuration than in the case of FIG. Is possible.

(Third embodiment)
FIG. 7 is a schematic configuration diagram of a super-resolution microscope according to the third embodiment. The super-resolution microscope according to the present embodiment is rotated between the collimator lens 23 and the microlens array disk 24 in the configuration shown in FIG. 5 instead of providing the spatial modulation element 37 bonded to the microlens 24a. A spatial modulation element array disk 38 is provided as possible.

The spatial modulation element array disk 38 includes a plurality of spatial modulation elements 37 arranged in the same arrangement pattern as the arrangement pattern of the microlenses 24a on a plane orthogonal to the rotation axis. The spatial modulation element array disk 38 is arranged so that the spatial modulation element 28 is positioned on the optical axis of each microlens 24 a, and is rotationally driven integrally with the microlens array disk 24 by the disk drive unit 29. Other configurations are the same as those in FIG.

Therefore, also in the present embodiment, three-dimensional super-resolution observation is possible as in the second embodiment. Further, according to the present embodiment, since the spatial modulation element array disk 38 is used, the spatial modulation element array is changed with respect to the change of the wavelengths of the illumination light of the pump light and the erase light as compared with the case of the second embodiment. It is possible to respond immediately by exchanging the disk 38.

(Fourth embodiment)
FIG. 8 is a schematic configuration diagram of a super-resolution microscope according to the fourth embodiment. In the super-resolution microscope according to the present embodiment, in the configuration shown in FIG. 1, the microlens array disk 24 is omitted and the scanning illumination light generation unit 50 is configured by the pinhole array disk 26.

In the present embodiment, the illumination light emitted as collimated light from the collimator lens 23 is transmitted through the dichroic mirror 25 and is parallel to the rotation axis of the pinhole array disk 26 and at a predetermined distance from the rotation axis. A part of the plane of the array disk 26 is irradiated. As a result, the illumination light is simultaneously transmitted through the plurality of pinholes 26a located in the illumination light irradiation region, and a plurality of scanning illumination lights are generated.

The scanning illumination light generated through each of the plurality of pinholes 26a is enlarged to the aperture size of the microscope objective lens 28, condensed through the spatial modulator 27, and condensed at different positions on the sample 10 by the microscope objective lens 28. Is done. Other configurations are the same as those in FIG.

According to the present embodiment, the sample 10 can be scanned with a plurality of fluorescent spots exceeding the diffraction limit by rotating the pinhole array disk 26 without using a microlens array disk. Therefore, it is possible to obtain a good super-resolution microscope image in a short time with a simpler configuration than in the case of FIG.

(Fifth embodiment)
FIG. 9 is a schematic configuration diagram of a super-resolution microscope according to the fifth embodiment. The super-resolution microscope according to the present embodiment has the configuration shown in FIG. 1 and a deflection that deflects the incident position of the illumination light to the microlens array disk 24 between the collimator lens 23 and the microlens array disk 24. A portion 40 is provided.

The deflecting unit 40 includes, for example, a galvano mirror 41 and a pupil projection lens 42, deflects illumination light from the collimator lens 23 in a two-dimensional direction by the galvano mirror 41, passes through the pupil projection lens 42, and passes through the microlens array disk. 24 is irradiated. The galvanometer mirror 41 is driven by the control unit 35 via the mirror driving unit 43. When the illumination light is deflected by the galvanometer mirror 41, the incident position of the illumination light on the microlens array disk 24 is displaced, and the scanning region of the sample 10 by the plurality of scanning illumination lights is moved.

In the present embodiment, the driving of the galvanometer mirror 41 by the mirror driving unit 43 is controlled by the control unit 35 to move the scanning area of the sample 10, and the microlens array disk 24 and the pinhole array are each in the plurality of scanning areas. By rotating the disk 26, the sample 10 is scanned with a plurality of fluorescent spots exceeding the diffraction limit. The galvanometer mirror 41 preferably deflects the illumination light so that a plurality of scanning regions in the sample 10 are continuous.

Under the control of the control unit 35, the image processing unit 33 combines the fluorescence image signals obtained in the respective scanning regions based on the illumination light deflection information by the galvano mirror 41, that is, the movement information of the scanning region of the sample 10. Thus, a fluorescence image signal of the sample 10 obtained by combining a plurality of scanning regions is generated. Other configurations are the same as those in FIG.

According to the present embodiment, the scanning region of the sample 10 is moved by the deflecting unit 40, the sample 10 is scanned with a plurality of fluorescent spots exceeding the diffraction limit in each scanning region, and the plurality of scanning regions are combined. An image signal is obtained. Therefore, it is possible to observe a super-resolution microscope image over a wide range of the sample 10 in a short time without causing a decrease in S / N.

(Sixth embodiment)
FIG. 10 is a schematic configuration diagram of a super-resolution microscope according to the sixth embodiment. In the super-resolution microscope according to the present embodiment, a beam shaping unit 60 is provided between the pupil projection lens 42 and the microlens array disk 24 in the configuration shown in FIG. The beam shaping unit 60 includes, for example, a cylindrical lens 61 and a telescope 62.

The cylindrical lens 61 condenses the cross-sectional illumination light from the pupil projection lens 42 in a cross-section band shape. The telescope 62 converts the illumination light collected in a cross-sectional band shape by the cylindrical lens 61 into parallel light. As a result, the beam shaping unit 60 shapes the illumination light having a circular cross section from the pupil projection lens 42 into illumination light having a cross section and irradiates the microlens array disk 24. Other configurations are the same as those in FIG.

FIG. 11 is a diagram for explaining an irradiation region of the microlens array disk 24 by illumination light from the beam shaping unit 60. FIG. The microlens array disk 24 shown in FIG. 11 has a plurality of spiral arrangement patterns 63 in which a plurality of microlenses 24a are arranged in a spiral like the microlens array disk in the Nipo type confocal microscope. In this embodiment, according to the deflection of the illumination light by the deflection unit 40, for example, the illumination light that is beam-shaped by the beam shaping unit 60 in a cross-sectional band shape is selected on the inner peripheral side and the outer peripheral side of the microlens array disk 24. The scanning region of the sample 10 is moved.

As a result, the illumination light is incident on the plurality of microlenses 24 a located on the inner peripheral side of each spiral arrangement pattern 63 in synchronization with the rotation of the microlens array disk 24 in the belt-shaped irradiation region 64 a on the inner peripheral side. The sample 10 is scanned. Similarly, in the belt-shaped irradiation region 64b on the outer peripheral side, in synchronization with the rotation of the microlens array disk 24, the illumination light is incident on the plurality of microlenses 24a positioned on the outer peripheral side of each spiral arrangement pattern 63, and the sample is irradiated. 10 is scanned. In this manner, the sample 10 is scanned with a plurality of fluorescent spots exceeding the diffraction limit in the band-shaped scanning regions respectively corresponding to the

irradiation regions

64a and 64b, and a fluorescent image signal in which the plurality of scanning regions are combined is obtained.

According to the present embodiment, since the selected region of the microlens array disk 24 is intensively illuminated by the beam shaping unit 60, a strong fluorescence suppression effect can be efficiently induced. As a result, the spatial resolution can be further improved. That is, the spatial resolution of a super-resolution fluorescence microscope using a double resonance absorption process depends on how quickly a fluorescent molecule transitions from the first quantum state to the second quantum state. That is, the size of the fluorescent spot is determined depending on how many fluorescent molecules have transitioned from the first quantum state to the second quantum state by irradiation with the erase light. In general, the higher the intensity of the erase light, the more the transition from the first quantum state to the second quantum state proceeds, the fluorescent spot size becomes smaller, and the spatial resolution is improved. In particular, when dealing with fluorescent molecules having a small cross-sectional area, it is desirable that the intensity of erase light be as high as possible. Therefore, as in this embodiment, if the illumination light is beam shaped by the beam shaping unit 60 and the selected area of the microlens array disk 24 is intensively illuminated, the intensity of the erase light can be increased. The resolution can be further improved.

(Seventh embodiment)
FIG. 12 is a schematic configuration diagram of a super-resolution microscope according to the seventh embodiment. In the super-resolution microscope according to the present embodiment, in the configuration shown in FIG. 1, the pinhole array disk 26 is omitted, and the spatial modulation element 27 includes the cylindrical substrate 27c and the wheel shown in FIGS. 4A and 4B. A belt substrate 27d is provided. Other configurations are the same as those in FIG.

According to this embodiment, an extremely simple super-resolution microscope can be configured. That is, in FIG. 1, a pinhole array disk 26 that functions as a confocal pinhole is provided so that a three-dimensional stereoscopic image can be acquired with improved vertical resolution. However, if the spatial modulation element 27 shown in FIGS. 4A and 4B is used, vertical spatial resolution can be improved. Therefore, an extremely simple super-resolution microscope with improved vertical spatial resolution can be obtained by omitting the pinhole array disk 26 and using the annular spatial modulator 27 shown in FIGS. 4A and 4B. Can be configured.

It should be noted that the present invention is not limited to the above embodiment, and many variations or modifications are possible. For example, in the third embodiment, the spatial modulation element array disk 38 is disposed on the incident surface side of the microlens array disk 24, but the spatial modulation element array disk 38 is disposed on the emission surface side of the microlens array disk 24. May be. Further, the deflection unit 40 in the fifth embodiment may be applied to the second to fourth and seventh embodiments. Further, in the second embodiment and the third embodiment, a pinhole array disk may be provided as in the first embodiment. In this case, the pinhole array disk may be displaceable in the rotation axis direction according to the observation depth position of the sample 10. In each of the above embodiments, the scanning illumination light generation unit 50 may be configured using, for example, a digital micromirror device (DMD).

Further, the beam shaping unit 60 shown in the sixth embodiment can be applied to the second to fourth and seventh embodiments. The beam shaping unit 60 is not limited to the configuration using the cylindrical lens 61 and the telescope 62, and may be configured using a liquid crystal type phase space element, for example. If a liquid crystal type phase space element is used, an arbitrary focused beam pattern can be generated by controlling the phase distribution on the beam surface. For example, a spiral pattern that matches the lens arrangement pattern of the microlens array disk 24 is also used. It can be formed easily.

The present invention is not limited to a super-resolution microscope using a double resonance absorption process, but also to known super-resolution microscopes such as a STED microscope, a RESOLFT (reversible / saturable / optical / fluorescence / transitions) microscope, and a GSD (ground / state depletion) microscope. It can be applied effectively. That is, the super-resolution microscope according to the present invention can be applied to a spectroscopic process involving a change in the amount of fluorescence or the fluorescence wavelength by a photochemical reaction involving a quantum state of two or more levels, for example, a triplet state or a photoisomerization state. it can.

DESCRIPTION OF SYMBOLS 10 Sample 20 Light source part 21 Single mode fiber 22 Microscope main body part 23 Collimator lens 24 Microlens array disk 24a Microlens 25 Dichroic mirror 26 Pinhole array disk 26a Pinhole 27 Spatial modulation element 28 Microscope objective lens 29 Disk drive part 30 Imaging Lens 31 Spectral filter 32 Image sensor 33 Image processing unit 35 Control unit 37 Spatial modulation element 38 Spatial modulation element array disk 40 Deflection unit 41 Galvano mirror 42 Pupil projection lens 43 Mirror drive unit 50 Scanning illumination light generation unit 60 Beam shaping unit

Claims

Illumination light including first light that excites molecules in the sample and second light that suppresses excitation of the molecules is spatially overlapped with at least part of the first light and the second light. An illumination optical system for irradiating the sample together;
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 scanning illumination light generation unit, a spatial modulation element, and a microscope objective lens,
The scanning illumination light generation unit generates a plurality of scanning illumination lights that are displaced from the incident illumination light in different trajectories,
The microscope objective lens condenses a plurality of the scanning illumination light from the scanning illumination light generation unit on the sample,
The spatial modulation element has optical transparency with respect to the first light and the second light, and each of the plurality of scanning illumination lights condensed by the microscope objective lens is converted into the first light. Spatially modulate at least a portion of either the first light or the second light so that the second light has a maximum light intensity and the second light has a minimum light intensity. ,
The detection optical system includes a two-dimensional optical sensor that photoelectrically converts the response light from the sample by the plurality of scanning illumination lights,
A super-resolution microscope that scans the sample with a plurality of illumination lights for scanning and observes the sample based on the output of the two-dimensional sensor.
The super-resolution microscope according to claim 1,
The super-resolution microscope, wherein the spatial modulation element modulates a polarization state or a phase state of one of the first light and the second light.
The super-resolution microscope according to claim 1 or 2,
The scanning illumination light generator includes a rotatable pinhole array disk,
The pinhole array disk has a plurality of pinholes arranged in a plane perpendicular to the rotation axis, and transmits the illumination light from a plurality of the pinholes in a part of the plane, thereby a plurality of the scanning illumination lights Produces
A super-resolution microscope that scans the sample with the plurality of scanning illumination lights while rotating the pinhole array disk.
The super-resolution microscope according to claim 1 or 2,
The scanning illumination light generator includes a rotatable microlens array disk,
The microlens array disk has a plurality of microlenses arranged on a plane orthogonal to a rotation axis, and transmits the illumination light from a plurality of the microlenses that are part of the plane, thereby a plurality of illumination lights for scanning. Produces
A super-resolution microscope that scans the sample with the plurality of scanning illumination lights while rotating the microlens array disk.
The super-resolution microscope according to claim 4,
The super-resolution microscope, wherein the spatial modulation element is provided so as to be bonded to the microlens of each of the microlens array disks.
The super-resolution microscope according to claim 4,
The illumination optical system further includes a spatial modulation element array disk that is disposed on the incident surface side or the exit surface side of the illumination light with respect to the microlens array disk and is rotatable integrally with the microlens array disk,
A plurality of the spatial modulation element array discs are arranged on the optical axis of each of the plurality of microlenses in the same arrangement pattern as the arrangement pattern of the microlenses in the microlens array disc on a plane orthogonal to the rotation axis. A super-resolution microscope having the spatial modulation elements arranged.
The super-resolution microscope according to claim 1 or 2,
The scanning illumination light generation unit includes a microlens array disk and a pinhole array disk that can rotate together,
The microlens array disk has a plurality of microlenses arranged in a plane perpendicular to the rotation axis,
The pinhole array disk has a plurality of pinholes arranged in the same arrangement pattern as the arrangement pattern of the microlens on a plane orthogonal to the rotation axis, and the pinhole is located at the image side focal position of the microlens. Arranged to
Passing the illumination light through the plurality of microlenses and the plurality of pinholes to generate the plurality of scanning illumination lights;
A super-resolution microscope that scans the sample with a plurality of illumination lights for scanning while rotating the microlens array disk and the pinhole array disk.
The super-resolution microscope according to any one of claims 1 to 7,
The illumination optical system includes a deflection unit that deflects the illumination light and moves a scanning region of the sample,
A super-resolution microscope that generates an image signal of the sample in which a plurality of scanning regions are combined based on outputs of the two-dimensional sensors in the plurality of scanning regions.
The super-resolution microscope according to any one of claims 1 to 8,
The detection optical system includes:
The response from the sample that is arranged in the optical path of the illumination light or the scanning illumination light incident on the microscope objective lens, transmits the illumination light or the scanning illumination light, and enters the microscope objective lens through the microscope objective lens A dichroic mirror that reflects light,
An imaging lens for imaging the response light reflected by the dichroic mirror on the two-dimensional sensor;
A super-resolution microscope, further comprising: a spectral filter that is disposed in an optical path between the imaging lens and the two-dimensional sensor and transmits the response light.
The super-resolution microscope according to any one of claims 1 to 9,
The illumination optical system is a super-resolution microscope, further comprising a beam shaping unit that shapes the shape of an irradiation region by the illumination light incident on the scanning illumination light generation unit.