JP2020098257A - Microscope and observation method - Google Patents

Abstract

To provide a microscope that can acquire a super-resolution image even when a periodic direction of structured illumination is not changed.SOLUTION: A microscope comprises: a first irradiation section that irradiates a first area of a sample surface with first light in which intensity periodically changes in a first direction of the sample surface where a sample irradiated with the first light to generate fluorescent light is arranged; a second irradiation section that irradiates a second area of the sample surface with second light in which the intensity changes in a second direction intersecting with the first direction, and which suppresses generation of the fluorescent light; a scanning section that scans the sample surface in the second direction using the first light and the second light; and a detection section that detects the fluorescent light generated on the basis of the first light and the second light scanned.SELECTED DRAWING: Figure 1

Description

The present invention relates to a microscope and an observation method.

A microscope using structured illumination is known (for example, see JP-A-2014-109490 below). Structured illumination may be utilized in microscopes that acquire super-resolution images beyond the diffraction limit of the optical system. The microscope in this case uses, for example, a stripe pattern in which the intensity periodic direction is one direction, as structured illumination, and detects the sample by changing the periodic direction to a plurality of directions. In a microscope using such structured illumination, for example, it is desirable to reduce the processing for changing the periodic direction of the structured illumination in order to speed up the processing.

JP, 2014-109490, A

According to the first aspect of the present invention, the first light whose intensity is cyclically changed in the first direction of the sample surface on which the sample that is irradiated with the first light and emits fluorescence is arranged is changed to the first light of the sample surface. A first irradiation unit that irradiates a first region, and a second irradiation unit that irradiates a second region of the sample surface with second light whose intensity changes in a second direction intersecting the first direction and suppresses the generation of fluorescence. And a scanning unit that scans the sample surface in the second direction with the first light and the second light, and a detection unit that detects fluorescence generated based on the scanned first light and the second light. A microscope is provided.

According to the second aspect of the present invention, the first light whose intensity is cyclically changed in the first direction of the sample surface on which the sample, which is irradiated with the first light and emits fluorescence, is arranged, Irradiating one region, irradiating the second region of the sample surface with the second light whose intensity changes in the second direction intersecting the first direction and suppressing the generation of fluorescence, and An observation method is provided that includes scanning with first light and second light in two directions, and detecting fluorescence generated based on the scanned first light and second light.

It is a figure which shows the microscope of 1st Embodiment. (A) And (B) is a figure which shows the 1st irradiation part of 1st Embodiment. FIG. 9A is a diagram showing a region on the pupil plane of the first embodiment on which the first light is incident, and FIG. 9B is a diagram showing an intensity distribution of the first light on the sample surface of the first embodiment. (A) And (B) is a figure which shows the 2nd irradiation part of 1st Embodiment. (A) is a figure which shows the phase of the 2nd light in the pupil surface of 1st Embodiment, (B) is a figure which shows the 2nd area|region in the sample surface of 1st Embodiment, (C) is 1st. It is a figure which shows the intensity distribution of the 2nd light in the sample surface of embodiment. (A)-(C) is a figure which shows operation|movement of the microscope of 1st Embodiment. It is a flowchart which shows the observation method of 1st Embodiment. It is a figure which shows the microscope of 2nd Embodiment. (A) And (B) is a figure which shows the 1st irradiation part and 2nd irradiation part of 2nd Embodiment. (A) And (B) is a figure which shows the phase adjustment part of 2nd Embodiment. (A) to (C) is a figure which shows the mask of 2nd Embodiment. (A) And (B) is a figure which shows the optical path of the 2nd irradiation part of 2nd Embodiment. (A) And (B) is a figure which shows the 1st irradiation part of 3rd Embodiment. (A)-(C) is a figure which shows the branch part of 3rd Embodiment. (A) to (C) are diagrams showing processing of the image processing unit of the third embodiment. (A)-(C) is a figure which shows the 2nd irradiation part of 4th Embodiment. (A) to (C) are diagrams showing processing of the image processing unit of the fourth embodiment. It is a flowchart which shows operation|movement of the microscope of 5th Embodiment.

[First Embodiment]
The first embodiment will be described.
FIG. 1 is a diagram showing a microscope according to the first embodiment. The microscope MS is used for fluorescence observation of the sample S labeled with a fluorescent substance. The microscope MS detects the fluorescence emitted by the fluorescent substance contained in the sample S. The sample S may contain living cells, cells fixed with a tissue fixing solution such as a formaldehyde solution, or may contain tissues. The sample S may include a holding member that holds the observation target. The holding member is, for example, a cover glass or a petri dish.

In the present embodiment, the fluorescent material includes a photoswitchable probe (referred to as PP as appropriate). The fluorescent substance transitions from the inactive state to the active state by being irradiated with the activation light. The fluorescent substance transits from the ground state to the excited state by being irradiated with the excitation light in the active state. The fluorescent substance emits fluorescence in the excited state and transitions to the ground state. Further, the fluorescent substance transits from the activated state to the inactivated state by being irradiated with the deactivating light. In the inactive state, the fluorescent substance does not transition to the excited state even when irradiated with the excitation light, and does not emit fluorescence.

The fluorescent substance may be, for example, a fluorescent dye such as a cyanine dye or a fluorescent protein. The type of fluorescent substance contained in the sample S may be one type or two or more types. The wavelength of the activation light, the wavelength of the excitation light, and the wavelength of the deactivation light are determined by the type of fluorescent substance. The wavelength of the activating light is different from the wavelength of the deactivating light. The wavelength of the excitation light may be the same as the wavelength of the activating light or the wavelength of the inactivating light depending on the type of the fluorescent substance. The wavelength of the excitation light may differ from both the wavelength of the activation light and the wavelength of the deactivation light depending on the type of the fluorescent substance.

The sample S is irradiated with the first light L1 to generate fluorescence L. The first light L1 is fluorescence generation light that causes the fluorescent substance contained in the sample S to generate fluorescence L. In the present embodiment, the first light L1 serves as both activation light that activates the fluorescent substance contained in the sample S and excitation light that excites the fluorescent substance activated by the activation light. In this embodiment, the wavelength of the excitation light is the same as the wavelength of the activation light. The sample S is irradiated with the first light L1 as the activation light and the excitation light, and the fluorescence L is generated.

Further, the sample S is irradiated with the second light L2 by the second irradiation unit 2 described later, and the generation of the fluorescence L is suppressed. The second light is fluorescence suppression light that suppresses the generation of fluorescence L in the fluorescent substance contained in the sample S. In the present embodiment, the second light L2 includes inactivating light that inactivates the fluorescent substance contained in the sample S. The second light L2 suppresses the generation of fluorescence L in the fluorescent substance by deactivating the fluorescent substance activated by being irradiated with the first light as the activation light.

The microscope MS according to the embodiment is, for example, a line scanning fluorescence microscope that illuminates the sample S while scanning the linear first light L1 and detects the fluorescence L. Reference numeral SF in FIG. 1 denotes a sample surface on which the sample S is arranged when the sample S is observed. The microscope MS according to the embodiment is, for example, a confocal microscope that suppresses the detection of the fluorescence L generated at a position away from the sample surface SF. The microscope MS includes a first irradiation unit 1, a second irradiation unit 2, a scanning unit 3, a detection unit 4, and an image processing unit 5.

The first irradiation unit 1 irradiates the sample S with the first light. The first irradiation unit 1 irradiates the first region L1 on the sample surface SF with the first light L1. The intensity of the first light L1 changes periodically in the first direction D1 of the sample surface SF. In FIG. 1 and the like, reference numeral D2 is a second direction different from the first direction D1 on the sample surface SF. The second direction D2 is a direction intersecting the first direction D1 (eg, non-parallel direction, vertical direction). The first light L1 emitted to at least a part of the first area A1 (for example, the area A0 in FIG. 1) is a light flux that is longer in the first direction D1 than in the second direction D2. The sample surface SF includes a surface to be irradiated with light (eg, first light). For example, the first direction D1 includes a direction intersecting (eg, orthogonal to) the optical axis of the first light L1 or the vertical direction on the sample surface SF.

The second irradiation unit 2 irradiates the sample S with the second light L2. The intensity of the second light L2 changes in the second direction D2 that intersects the first direction D1. The second irradiation unit 2 irradiates the second area A2a and the second area A2b on the sample surface SF with the second light L2. The second area A2a is set on the first side in the second direction D2 with respect to at least a part of the first area A1 (for example, the area A0 in FIG. 1). The first side is, for example, the side opposite to the direction of the arrow in the second direction D2 of FIG. The second area A2b is set on the second side opposite to the first side in the second direction D2 with respect to at least a part of the first area A1 (for example, the area A0 in FIG. 1). The second side is, for example, the same side as the direction of the arrow in the second direction D2 of FIG. The second area A2b is arranged on the side opposite to the second area A2a with respect to the area A0.

In the following description, the second area A2a and the second area A2b are appropriately paired and referred to as a pair of second areas A2a and A2b, and the second area A2a and the second area A2b are collectively included in the second area A2. Express. The second area A2 is set at a position sandwiching at least a part of the area A0 of the first area A1 in the second direction D2. The area A0 is a target area for detecting the fluorescence L. The region A0 is, for example, a strip-shaped region that is longer in the first direction D1 than in the second direction D2. The reference numeral D3 is a third direction perpendicular to both the first direction D1 and the second direction D2. The third direction D3 is, for example, a direction perpendicular to the sample surface SF. The third direction D3 is, on the sample surface SF, a direction coaxial with the optical axis of the optical system of the microscope MS or a direction parallel to the optical axis of the optical system of the microscope MS.

The scanning unit 3 scans the sample surface SF in the second direction D2 with the first light L1 and the second light L2. The scanning unit 3 changes the position of the first area A1 on the sample surface SF where the first light L1 is incident and the position of the second area A2 on the sample surface SF where the second light L2 is incident. The detector 4 detects the fluorescence L generated in the sample S. The image processing unit 5 generates an image based on the detection result of the detection unit 4. Hereinafter, each part of the microscope MS will be described.

The first irradiation unit 1 irradiates the sample S with the first light L1. The first light L1 is a striped pattern in which bright portions and dark portions are alternately arranged on the sample surface SF. In the following description, the period of the intensity of the first light L1 in the first direction D1 of the sample surface SF is appropriately referred to as the stripe period of the first light L1. The first irradiation unit 1 includes a first light source 11 and a first optical system 12. The first light source 11 includes a solid-state light source such as a laser diode or a light emitting diode.

The first optical system 12 irradiates the sample S with the first light L1 emitted from the first light source 11. The first optical system 12 illuminates the sample S with a stripe pattern of the first light L1. The first optical system 12 splits the first light L1 emitted from the first light source 11 into a plurality of light fluxes, and forms a fringe pattern of the first light L1 by the interference of two or more light fluxes that are split. The first optical system 12 splits the first light L1 into a plurality of diffracted lights by diffraction and forms a fringe pattern of the first light L1 on the sample surface SF by interference of two or more diffracted lights. For example, the first optical system 12 forms the fringe pattern of the first light L1 on the sample surface SF by the two-beam interference of the +1st-order diffracted light and the −1st-order diffracted light.

The first optical system 12 includes a lens 13, an optical member 14, a branch portion 15, a lens 16, a mask 17, a lens 18, a lens 19, and a mirror 20 in the order from the first light source 11 to the sample S. A dichroic mirror 21, a lens 22, a dichroic mirror 23, a scanning mirror 24, a lens 25, a lens 26, and a lens 27.

In FIG. 1, reference numeral P0 is a pupil plane of the lens 27, and reference numerals P1 to P3 are pupil conjugate planes optically conjugate with the pupil plane P0. Reference numerals SF1 to SF5 are sample conjugate surfaces that are optically conjugate with the sample surface SF. The first optical system 12 forms a stripe pattern of the first light L1 at the position of the sample conjugate surface SF2, and projects the formed stripe pattern of the first light L1 on the sample surface SF. In FIG. 1, the optical system of the microscope MS is a catadioptric optical system. In the following description, at least a part of the optical system of the microscope MS will be replaced with a refraction optical system equivalent to that in FIG.

FIG. 2A and FIG. 2B are views showing the first irradiation unit of the first embodiment. In the following description, the XYZ orthogonal coordinate system shown in FIG. The Z direction of this XYZ orthogonal coordinate system is the direction of the optical axis at each position on the optical axis of the optical system of the microscope MS. The X direction is a direction that is orthogonal to the Z direction at each position on the optical axis of the optical system of the microscope MS and corresponds to the second direction D2 in FIG. The Y direction is a direction that is orthogonal to the Z direction at each position on the optical axis of the optical system of the microscope MS and corresponds to the first direction D1 in FIG.

FIG. 2A is a view of the optical path from the first light source 11 to the sample conjugate plane SF2 in the first irradiation section 1 as seen from the X direction. Further, FIG. 2B is a view of the optical path from the first light source 11 to the sample conjugate plane SF2 in the first irradiation section 1 as seen from the Y direction.

The lens 13 is, for example, a collimator. The lens 13 converts the first light L1 emitted from the first light source 11 into parallel light. In the optical member 14, the power corresponding to the second direction D2 is stronger than the power corresponding to the first direction D1. In this specification, the power is the reciprocal of the focal length, and is also called the refractive power in the case of a transmissive optical member such as a lens. The power corresponding to the second direction D2 is the reciprocal of the focal length in the plane including the direction (here, the X direction) corresponding to the second direction D2 and the optical axis direction of the optical system. The power corresponding to the second direction D2 is a refracting power that deflects a light beam in the X direction corresponding to the second direction D2. The power corresponding to the first direction D1 is the reciprocal of the focal length in a plane including the direction corresponding to the first direction D1 (here, the Y direction) and the optical axis direction of the optical system (here, the Z direction). .. The power corresponding to the first direction D1 is a refracting power that deflects a light beam in the Y direction corresponding to the first direction D1. In this specification, the optical member having power may be a transmissive optical member such as a lens or a reflective optical member such as a concave mirror. The optical member 14 is an astigmatism member that acts on the first light L1 to generate astigmatism. The optical member 14 is, for example, a cylindrical lens. Instead of the optical member 14, the first optical system 12 may include an astigmatism optical system that acts on the first light L1 to generate astigmatism.

As shown in FIG. 2A, the optical member 14 has a refractive power of almost 0 for deflecting the light beam in the Y direction. The first light L1 that has passed through the optical member 14 enters the branch portion 15 as substantially parallel light on the YZ plane. As shown in FIG. 2B, the optical member 14 has a stronger refracting power for deflecting the light beam in the X direction than that for deflecting the light beam in the Y direction. The first light L1 that has passed through the optical member 14 is condensed on the branch portion 15 on the XZ plane.

The branching unit 15 branches the first light L1 into a plurality of light beams. The branching unit 15 branches the first light L1 in the Y direction corresponding to the first direction D1 of the sample surface SF. The branching unit 15 branches the first light L1 into a plurality of light beams (eg, diffracted light) by diffraction. In the present embodiment, the branch portion 15 is described as a diffraction grating, and the diffraction grating as the branch portion 15 is represented by reference numeral 15. The branching unit 15 may use a spatial light modulator (SLM, as appropriate) instead of the diffraction grating.

The structure of the diffraction grating 15 changes periodically in the Y direction corresponding to the first direction D1. The diffraction grating 15 has, for example, a one-dimensional periodic structure in a plane that intersects the optical axis of the first optical system 12. This periodic structure is a structure in which one or both of the transmittance and the phase difference are periodically changed. The diffraction grating 15 is arranged at a position of the sample conjugate plane SF1 optically conjugate with the sample plane SF or a position in the vicinity thereof. The “near position” is a position deviated from the target within a range where optical characteristics are allowed. For example, in the case of the diffraction grating 15, when the “optical characteristic” is the contrast of the stripe pattern on the sample surface SF and the “target” is the sample conjugate surface SF1, the position near the diffraction grating 15 is the position of the sample surface SF. It is a position deviated from the sample conjugate plane SF1 within a range where the contrast of the stripe pattern is allowed. In the following description, "nearby position" is used with the same meaning as appropriate.

The first irradiation unit 1 can change the phase of the stripe pattern of the first light L1 on the sample surface SF by controlling the branching unit 15. For example, when the branching portion 15 is a diffraction grating, the first irradiation unit 1 moves the diffraction grating 15 in the Y direction corresponding to the first direction D1 so that the first light L1 on the sample surface SF is first. The phase of the stripe pattern in the direction D1 is changed. When the branching unit 15 is an SLM, the first irradiation unit 1 may change the phase of the stripe pattern of the first light L1 by changing the distribution of the refractive index in the SLM.

The lens 16 is arranged so that the focus on the light source side is at the position of SF1 or a position in the vicinity thereof. In the following description, the focus on the light source side of the lens is referred to as the light source side focus, and the focus on the sample side of the lens is referred to as the sample side focus. The lens 16 converts the first light L1 into parallel light on the XZ plane.

The mask 17 is arranged at the position of the pupil conjugate plane P1 or in the vicinity thereof. The mask 17 is arranged at the position of the focal point of the lens 16 on the sample side or in the vicinity thereof. The mask 17 has an opening 17a through which the first light L1 passes. A portion around the opening 17a is a light blocking portion 17b that blocks the first light L1. The opening 17a is arranged at a position where the diffracted light forming the fringe pattern of the plurality of diffracted lights generated by the diffraction grating 15 is incident.

In the present embodiment, the fringe pattern of the first light L1 is formed by the interference between the +1st-order diffracted light and the -1st-order diffracted light, and the opening 17a has a position where the +1st-order diffracted light generated by the diffraction grating 15 enters and the diffraction grating. It is arranged at a position where the −1st order diffracted light generated in 15 is incident. The light shielding portion 17b is arranged at a position where the 0th order diffracted light generated by the diffraction grating 15 is incident. As shown in FIG. 2B, the first light L1 is incident on the mask 17 as parallel light when viewed from the Y direction. The size of the opening 17a in the X direction corresponding to the second direction D2 is larger than that in the Y direction corresponding to the first direction D1.

The first light L1 that has passed through the mask 17 enters the lens 18. The lens 18 is arranged so that the focus on the light source side is at the position of the pupil conjugate plane P1 or a position in the vicinity thereof. The lens 18 is arranged so that its sample-side focus is at the position of the sample conjugate plane SF2 or in the vicinity thereof. On the sample conjugate plane SF2, the intensity distribution of the first light L1 becomes an intensity distribution corresponding to the pattern of the diffraction grating 15. In this way, the stripe pattern of the first light L1 is formed on the sample conjugate plane SF2.

Returning to the description of FIG. 1, the first optical system 12 relays the stripe pattern formed on the sample conjugate plane SF2 to the sample plane SF. The first light L1 that has passed through the sample conjugate surface SF2 enters the lens 19, is refracted by the lens 19, and enters the mirror 20. The mirror 20 is a folding mirror. The first light L1 reflected by the mirror 20 enters the dichroic mirror 21.

Reference numeral 21A in FIG. 1 represents the optical characteristics of the dichroic mirror 21 with respect to the wavelength. In FIG. 1, the reflectance with respect to the wavelength and the transmittance with respect to the wavelength are shown as the optical characteristic 21A. In the optical characteristic 21A, λ1 is the wavelength of the first light L1 and λ2 is the wavelength of the second light L2. The wavelength λ1 may be the center wavelength of the first light L1 or the peak wavelength at which the intensity of the first light L1 is maximized. The wavelength λ2 may be the center wavelength of the second light L2 or the peak wavelength at which the intensity of the second light L2 is maximized.

The reflectance of the dichroic mirror 21 for the wavelength λ1 of the first light L1 is lower than the reflectance of the second light L2 for the wavelength λ2. Further, the dichroic mirror 21 has a higher transmittance for the wavelength λ1 of the first light L1 than for the wavelength λ2 of the second light L2. At least part of the first light L1 that has entered the dichroic mirror 21 passes through the dichroic mirror 21 and enters the lens 22.

A pupil conjugate plane P2 is arranged in the optical path between the lens 19 and the lens 22. The lens 22 is a relay optical system, and relays the intensity distribution of the first light L1 on the pupil conjugate plane P2 to the scanning unit 3. The first light L1 that has passed through the lens 22 enters the dichroic mirror 23. Reference numeral 23A in FIG. 1 represents the optical characteristics of the dichroic mirror 23 with respect to the wavelength. In FIG. 1, the reflectance with respect to the wavelength and the transmittance with respect to the wavelength are shown as the optical characteristic 23A.

In the optical characteristic 23A, λ3 is the wavelength of the fluorescence L. The wavelength λ3 may be the center wavelength of the fluorescence L or the peak wavelength at which the intensity of the fluorescence L is maximized. The reflectance of the dichroic mirror 23 with respect to the wavelength λ1 of the first light L1 is higher than the reflectance of the fluorescence L with respect to the wavelength λ3. Further, the dichroic mirror 23 has a lower transmittance for the wavelength λ1 of the first light L1 than that for the wavelength λ3 of the fluorescence L.

At least part of the first light L1 that has entered the dichroic mirror 23 is reflected by the dichroic mirror 23 and enters the scanning mirror 24. The scanning unit 3 includes, for example, a galvanometer mirror, a MEMS mirror, or a polygon mirror. The scanning unit 3 deflects the first light L1 and adjusts the position where the first light L1 is incident on the sample surface SF. The scanning unit 3 causes the scanning mirror 24 to move the first region A1 where the first light L1 is incident on the sample surface SF in the second direction D2 on the sample surface SF.

The scanning unit 3 includes a scanning mirror 24 and a driving unit 29. The scanning mirror 24 is arranged at the position of the pupil conjugate plane or in the vicinity thereof. The first light L1 reflected by the dichroic mirror 23 enters the scanning mirror 24 and is reflected by the scanning mirror 24. The drive unit 29 drives the scanning mirror 24 and changes the angle of the scanning mirror 24 with respect to the optical axis of the first optical system 12. The position where the first light L1 is incident on the sample surface SF changes based on the angle of the scanning mirror 24 with respect to the optical axis of the first optical system 12.

The first light L1 reflected by the scanning mirror 24 enters the lens 25, is refracted by the lens 25, and enters the lens 26. The sample conjugate plane SF3 is arranged in the optical path between the lens 25 and the lens 26. The first light L1 that has entered the lens 26 is refracted by the lens 26 and enters the lens 27. The lens 27 is an objective lens. The sample side focus of the lens 27 corresponds to the position of the sample surface SF. The light source side focus of the lens 27 corresponds to the pupil plane P0. The first light that has entered the lens 27 is refracted by the lens 27 and is applied to the sample surface SF.

FIG. 3A is a diagram showing a region where the first light is incident on the pupil plane of the first embodiment. The first light L1 is incident on the area P0a and the area P0b on the pupil plane P0. The region P0a and the region P0b are regions corresponding to the openings 17a of the mask 17 shown in FIG. The regions P0a and P0b are strip-shaped regions that are longer in the X direction corresponding to the second direction D2 than in the Y direction corresponding to the first direction D1. The area P0a and the area P0b are arranged in the Y direction.

FIG. 3B is a diagram showing the intensity distribution of the first light on the sample surface of the first embodiment. Reference numeral ΔD1 in FIG. 3B is the interval between positions where the intensity of the first light L1 is maximized in the first direction D1. The interval ΔD1 is the same as the interval at which the intensity of the first light L1 is minimized in the first direction D1. The fringe period of the first light L1 is expressed using the interval ΔD1. The position of the end of the first region A1 in the second direction D2 is represented by the position of the boundary at which the intensity of the first light L1 is equal to or more than the intensity required to activate and excite the fluorescent material.

Returning to the description of FIG. 1, the second irradiation unit 2 irradiates the sample S with the second light L2. The second irradiation unit 2 irradiates the second region A2 on the sample surface SF with the second light L2. The second region A2a is arranged on one side of the second direction D2 with respect to at least a part of the first region A1 (for example, a target region for detecting fluorescence) A0. The second area A2b is arranged on the other side in the second direction D2 with respect to at least a part of the area A0 of the first area A1. The second area A2b is arranged on the opposite side of the second area A2a in the second direction D2 with respect to at least a part of the area A0 of the first area A1. For example, the plurality of second areas A2 (second area A2a, second area A2b, etc.) are arranged on both sides of the area A0 along the second direction D2 with reference to at least a part of the area A0 of the first area A1. Adjacent to each other.

In FIG. 1, the second area A2a and the second area A2b are set so as to overlap a part of the first area A1. For example, the second area A2a overlaps with a partial area A1a of the first area A1. Further, the second area A2b overlaps with a partial area A1b of the first area A1. Note that one or both of the second area A2a and the second area A2b may not overlap the first area A1.

The second irradiation unit 2 includes a second light source 31 and a second optical system 32. The second light source 31 includes a solid-state light source such as a laser diode or a light emitting diode. The second optical system 32 irradiates the sample S with the second light L2 emitted from the second light source 31. The second optical system 32 illuminates the sample S with the second light L2 whose intensity changes in the second direction D2. The second optical system 32 relatively changes the phases of a plurality of light beams included in the second light L2 emitted from the second light source 31, thereby forming an intensity distribution (for example, intensity pattern) of the second light. To do. For example, the second optical system 32 forms the pattern light (for example, structured light) of the second light L2 by giving a phase difference between the first light flux and the second light flux of the second light. The intensity distribution of the second light L2 will be described later with reference to FIG.

The second optical system 32 includes a lens 33, a phase adjustment unit 34, an optical member 35, a lens 36, a dichroic mirror 21, a lens 22, and a dichroic mirror 23 in order from the second light source 31 toward the sample S. , A scanning mirror 24, a lens 25, a lens 26, and a lens 27. The second optical system 32 shares the configuration from the dichroic mirror 21 to the lens 27 with the first optical system 12. Each component (for example, an optical member such as a lens) from the dichroic mirror 21 to the lens 27 is arranged in a region including a region where the first light L1 is incident and a region where the second light L2 is incident. The optical path of the second light L2 may be common to at least a part of the optical path of the first light L1 between the dichroic mirror 21 and the lens 27. The optical axis of the second optical system 32 is coaxial with the optical axis of the first optical system 12 between the dichroic mirror 21 and the lens 27.

4A and 4B are views showing the second irradiation unit of the first embodiment. FIG. 4A is a view of the optical path from the second light source 31 to the lens 22 in the second irradiation section 2 as seen from the X direction. Further, FIG. 4B is a view of the optical path from the second light source 31 to the lens 22 in the second irradiation section 2 as viewed from the Y direction. The optical axis of the second optical system 32 is bent by the dichroic mirror 21 (see FIG. 1) between the lens 36 and the lens 22, but the dichroic mirror 21 is not shown in FIGS. 4(A) and 4(B). , Is shown as an equivalent refracting optical system so that the optical axis becomes a straight line.

The phase adjustment unit 34 is an intensity distribution adjustment unit that adjusts the intensity distribution of the second light L2 on the sample surface SF. The phase adjuster 34 is arranged at the position of the pupil conjugate plane P3 or in the vicinity thereof. As shown in FIG. 4B, the phase adjustment unit 34 includes a region 34a and a region 34b on which the second light L2 is incident. The boundary between the regions 34a and 34b is a plane including the optical axis 32a of the second optical system 32 and parallel to the Y direction corresponding to the first direction D1 in FIG.

The phase adjuster 34 gives a phase difference between the second light L2 incident on the area 34a and the second light L2 incident on the area 34b. In the following description, the second light L2 incident on the region 34a is represented by reference symbol L2a, and the second light L2 incident on the region 34b is represented by reference symbol L2b, as appropriate. The phase adjustment unit 34 adjusts the intensity distribution of the second light L2 on the sample surface SF by adjusting the phase difference between the second light L2a and the second light L2b. The intensity distribution of the second light L2 on the sample surface SF corresponds to the interference fringe formed by the interference between the second light L2a and the second light L2b. In this interference fringe, the position D2a where the intensity is maximum (shown later in FIG. 5C) and the position D2c where the intensity is minimum (shown later in FIG. 5C) are the second light L2a and the second light. It depends on the phase difference from L2b. The phase adjusting unit 34 adjusts the phase difference between the second light L2a and the second light L2b so that the first area A1 and the second area A2 shown in FIG. 1 have a predetermined positional relationship.

In FIG. 1, the optical axis of the first optical system 12 on the light emitting side and the optical axis of the second optical system 32 on the light emitting side are coaxial. The pair of second areas A2a and A2b are arranged symmetrically with respect to the first area A1 when the phase difference between the second light L2a and the second light L2b is an odd multiple of π. In the present embodiment, the phase difference between the second light L2a and the second light L2b is set to an odd multiple of π. For example, the phase adjustment unit 34 adjusts the phase difference between the second light L2a and the second light L2b to be an odd multiple of π.

In the phase adjustment unit 34, the optical distance in the area 34a is different from the optical distance in the area 34b. The phase adjustment unit 34 includes a first portion 37 and a second portion 38. The first portion 37 is a member that transmits at least part of the second light L2. In the first portion 37, the dimension (eg, thickness) of the second optical system 32 in the optical axis direction is different between the region 34a and the region 34b. The thickness of the first portion 37 in the region 34a changes stepwise with respect to the thickness of the first portion 37 in the region 34b. The first portion 37 of FIG. 4B is thinner in the region 34a than in the region 34b.

The second portion 38 has a refractive index different from that of the first portion 37. In FIG. 4, the second portion 38 is a void or space occupied by atmospheric gas or the like. The second portion 38 may include a member having a refractive index different from that of the first portion 37. The second portion 38 is arranged in the region 34a. The optical distance of the second light L2a in the region 34a differs from the optical distance of the second light L2b in the region 34b by an odd multiple of half the wavelength of the second light L2. The phase difference of the second light L2a transmitted through the region 34a from the second light L2b transmitted through the region 34b is an odd multiple of π. The second light L2 transmitted through the phase adjustment unit 34 enters the optical member 35.

The optical member 35 has stronger power corresponding to the second direction D2 than power corresponding to the first direction D1. The power corresponding to the second direction D2 is a refracting power for deflecting the light beam in the X direction corresponding to the second direction D2. The power corresponding to the first direction D1 is a refracting power that deflects a light beam in the Y direction corresponding to the first direction D1. The optical member 35 is an astigmatism member that acts on the first light L1 to generate astigmatism. The optical member 35 is, for example, a cylindrical lens. The second optical system 32 may include, instead of the optical member 35, an astigmatism optical system that acts on the second light L2 to generate astigmatism.

As shown in FIG. 4(A), the optical member 35 has a refractive power of almost 0 for deflecting the light beam in the Y direction. The second light L2 that has passed through the optical member 35 enters the branch portion 15 as substantially parallel light on the YZ plane. As shown in FIG. 4B, the optical member 35 has a stronger refracting power for deflecting the light beam in the X direction than that for deflecting the light beam in the Y direction. The second light L2 that has passed through the optical member 35 is condensed on the sample conjugate plane SF4 on the XZ plane.

The second light L2a transmitted through the region 34a of the phase adjustment unit 34 and the second light L2b transmitted through the region 34b of the phase adjustment unit 34 are condensed on the sample conjugate plane SF4 and interfere with each other to generate the second light L2. Pattern light is formed. The second optical system 32 projects the pattern light of the second light L2 formed on the sample conjugate surface SF4 onto the sample surface SF (see FIG. 1). The second light L2 that has passed through the sample conjugate plane SF2 enters the lens 36. The lens 36 is arranged so that the focus on the light source side is at the position of the sample conjugate plane SF4 or in the vicinity thereof. The lens 36 is arranged so that its sample-side focal point is located at or near the position of the pupil conjugate plane P2.

Returning to the description of FIG. 1, the second light L2 that has entered the lens 36 is refracted by the lens 36 and enters the dichroic mirror 21. As indicated by the optical characteristic 21A, the dichroic mirror 21 has a reflectance for the wavelength λ2 of the second light L2 higher than that for the wavelength λ1 of the first light L1. Further, the dichroic mirror 21 has a lower transmittance for the wavelength λ2 of the second light L2 than that for the wavelength λ1 of the first light L1. At least part of the second light L2 that has entered the dichroic mirror 21 is reflected by the dichroic mirror 21 and enters the lens 22. A pupil conjugate plane P2 is arranged in the optical path between the lens 36 and the lens 22.

The second light L2 emitted from the lens 22 enters the dichroic mirror 23. As indicated by the optical characteristic 23A, the dichroic mirror 23 has a higher reflectance for the wavelength λ2 of the second light L2 than the reflectance for the wavelength λ3 of the fluorescence L. Further, the dichroic mirror 23 has a lower transmittance for the wavelength λ2 of the second light L2 than that for the wavelength λ3 of the fluorescence L.

At least part of the second light L2 that has entered the dichroic mirror 23 is reflected by the dichroic mirror 23 and enters the scanning mirror 24. The scanning unit 3 deflects the second light L2 and adjusts the position where the second light L2 is incident on the sample surface SF. The scanning unit 3 moves the second area A2 on the sample surface SF on which the second light L2 is incident in the second direction D2 on the sample surface SF. The scanning unit 3 moves the first area A1 and the second area A2 in synchronization with each other on the sample surface SF.

In this way, the second light L2 reflected by the dichroic mirror 23 enters the scanning mirror 24 and is reflected by the scanning mirror 24. The drive unit 29 drives the scanning mirror 24 and changes the angle of the scanning mirror 24 with respect to the optical axis of the second optical system 32. The position where the second light L2 enters the sample surface SF changes based on the angle of the scanning mirror 24 with respect to the optical axis of the second optical system 32.

Then, the second light L2 reflected by the scanning mirror 24 enters the lens 25, is refracted by the lens 25, and enters the lens 26. The sample conjugate plane SF3 is arranged in the optical path between the lens 25 and the lens 26. The second light L2 that has entered the lens 26 is refracted by the lens 26 and enters the lens 27. The second light L2 incident on the lens 27 is refracted by the lens 27 and is irradiated on the sample surface SF.

FIG. 5A is a diagram showing the phase of the second light on the pupil plane of the first embodiment. In FIG. 5A, reference numeral FP is a plane that includes the optical axis 32A of the second optical system 32 and that is parallel to the Y direction (parallel to the YZ plane) corresponding to the first direction D1. Reference numerals P0c and P0d are regions where the second light L2 is incident, respectively. The region P0c is arranged on one side of the plane FP, and the region P0d is arranged on the opposite side of the region P0c with respect to the plane FP. The second light L2 incident on the region P0c and the second light L2 incident on the region P0d are out of phase with each other. The phase difference between the second light L2 entering the region P0c and the second light L2 entering the region P0d is, for example, π.

FIG. 5B is a diagram showing the second region on the sample surface of the first embodiment, and FIG. 5C is a diagram showing the intensity distribution of the second light on the sample surface of the first embodiment. The center position of the second region A2a in the second direction D2 is a position D2a where the intensity of the second light L2 has a maximum value (appropriately referred to as a first maximum position). Further, the center position of the second area A2b is a position D2b at which the intensity of the second light L2 is maximized (which is appropriately referred to as a second maximum position). The position D2a and the position D2b at which the intensity of the second light becomes maximum in the second direction D2 are set outside the region A0 of at least a part of the first region A1 shown in FIG.

Further, reference numeral D2c is a position between the position D2a and the position D2b where the intensity of the second light L2 in the second direction D2 is minimized (appropriately referred to as a minimum position). At least a partial area A0 of the first area A1 shown in FIG. 1 is an area including a position D2c at which the intensity of the second light L2 is minimized in the second direction D2. The position D2c at which the intensity of the second light L2 is minimal is set, for example, at the center position of the first area A1 (see FIG. 1) in the second direction D2. In FIG. 5C, the symbol Ith is the intensity required to inactivate the fluorescent substance. The position of the end of the second area A2a and the position of the end of the second area A2b in the second direction D2 are respectively represented by the positions of the boundaries where the intensity of the second light L2 is equal to or higher than the intensity Ith.

As described in FIG. 4B, the position D2a where the intensity of the second light L2 is maximum and the position D2c where the intensity of the second light L2 is minimum on the sample surface SF are the second light L2a and the second light L2a. It depends on the phase difference between the two lights L2b. This phase difference is set so that at least a part of the first area A1 (for example, an area where fluorescence L is detected) is arranged between the pair of second areas A2a and A2b. This phase difference is set to an odd multiple of π, for example. The phase difference between the second light L2a and the second light L2b is set to a value different from an even multiple of π and may be an odd multiple of π or a different value from an odd multiple of π.

Returning to the description of FIG. 1, in the first region A1 of the sample surface SF, the fluorescent substance in the region A0 that does not overlap the second region A2 is illuminated by the first light L1 and is not illuminated by the second light L2. L occurs. The fluorescent substance in the second region A2 is inactivated by the irradiation of the second light L2, so that the generation of the fluorescence L is suppressed. The detection unit 4 detects the fluorescence L generated in the sample S in the area A0. The sample surface SF is formed by at least the first area A1 and the second area A2.

The detector 4 includes a third optical system 41 and a light receiver 42. The third optical system 41 includes a lens 27, a lens 26, a lens 25, a scanning mirror 24, a dichroic mirror 23, a lens 43, and a mask 44 in order from the sample S toward the light receiving section 42. The configuration of the third optical system 41 from the lens 27 to the dichroic mirror 23 is shared by the first optical system 12 and the second optical system 32.

The fluorescence L generated from the sample S enters the scanning mirror 24 via the lens 27, the lens 26, and the lens 25. The fluorescence L is reflected by the scanning mirror 24 and enters the dichroic mirror 23. As shown by the optical characteristic 23A, the dichroic mirror 23 has a reflectance of the fluorescence L with respect to the wavelength λ3 that is lower than the reflectance with respect to the wavelength λ1 of the first light L1 and the wavelength λ2 of the second light L2. Further, the dichroic mirror 23 has a higher transmittance for the wavelength λ3 of the fluorescence L than for the wavelength λ1 of the first light L1 and the wavelength λ2 of the second light L2.

At least a part of the fluorescence L that has entered the dichroic mirror 23 passes through the dichroic mirror 23 and enters the lens 43. The fluorescence L that has entered the lens 43 is condensed on the sample conjugate plane SF5 which is optically conjugate with the sample plane SF. The mask 44 is arranged at the position of the sample conjugate plane SF5 or in the vicinity thereof. The mask 44 has an opening 44a through which the fluorescent light L passes. The mask 44 is arranged so that the fluorescence L generated on the sample surface SF passes through the opening 44a. The mask 44 is arranged so as to shield the fluorescence generated on the surface deviated from the sample surface SF in the third direction D3. The opening 44a has the same shape as the area A0 where the fluorescence L is generated on the sample surface SF. The opening 44a has, for example, a slit shape, and has a strip shape that is longer in the direction corresponding to the first direction D1 than in the direction corresponding to the second direction D2.

The light receiving section 42 is arranged at the position of the sample conjugate plane SF5 or a position in the vicinity thereof. The fluorescence L that has passed through the opening 44 a of the mask 44 enters the light receiving unit 42. The light receiving unit 42 detects the fluorescence L that has passed through the opening 44 a of the mask 44. The light receiving unit 42 includes a plurality of photodiodes arranged in a direction corresponding to the first direction D1. The light receiving unit 42 outputs the detection result of detecting the fluorescence L to the image processing unit 5. The third optical system 41 may include a relay optical system that relays the sample conjugate surface SF5, and the light receiving unit 42 is arranged at a position of the sample conjugate surface where the sample conjugate surface SF5 is relayed or a position in the vicinity thereof. May be done.

The image processing unit 5 generates an image based on the detection result of the light receiving unit 42. In FIG. 1, the image processing unit 5 is provided in the control device 6. The control device 6 includes an image processing unit 5, a control unit 7, and a storage unit 8. The control unit 7 controls each unit of the microscope MS. The control unit 7 controls the first irradiation unit 1 to irradiate the sample S with the first light L1 at a predetermined timing and intensity. The control unit 7 controls the first irradiation unit 1 to control the period during which the first light L1 is incident on the sample S (for example, the sample surface SF). The control unit 7 controls the second irradiation unit 2 to irradiate the sample S with the second light L2 at a predetermined timing and intensity. The control unit 7 controls the second irradiation unit 2 to control the period during which the second light L2 is incident on the sample S (for example, the sample surface SF). The control unit 7 controls the first irradiation unit 1 and the second irradiation unit 2 in synchronization with each other. The control unit 7 overlaps at least a part of the period during which the second light L2 is incident on the sample S (for example, the sample surface SF) and the period during which the first light L1 is incident on the sample S (for example, the sample surface SF). The 1st irradiation part 1 and the 2nd irradiation part 2 are controlled so that it may do. The control unit 7 controls the scanning unit 3 to control the position of the first area A1 and the position of the second area A2 on the sample surface SF. The controller 7 controls the detector 4 to detect the fluorescence L. The control unit 7 causes the control device 6 to output the detection result of the light receiving unit 42.

The storage unit 8 is, for example, a non-volatile memory, a hard disk drive (referred to as HDD as appropriate), a solid state drive (referred to as SSD as appropriate), or the like. The storage unit 8 stores, for example, setting information that defines the operation of each unit of the microscope MS. The operation of each of the above-mentioned parts includes an operation of irradiating the sample S with the first light L1 by the first irradiation unit 1, an operation of irradiating the sample S with the second light L2 by the second irradiation unit 2, and an operation of scanning the sample surface SF by the scanning unit 3. At least one of the operation of scanning with the first light L1 and the second light L2 and the operation of detecting the fluorescence L by the detection unit 4 is included. When the operation of each unit is an operation of irradiating the sample S with the first light L1 by the first irradiation unit 1, the setting information is, for example, that the first light L1 is irradiated on the sample surface SF by the first irradiation unit 1. And the intensity of the first light L1 with which the sample surface SF is irradiated. When the operation of each unit is an operation of irradiating the sample S with the second light L2 by the second irradiation unit 2, the setting information is, for example, that the second light L2 is irradiated on the sample surface SF by the second irradiation unit 2. And the intensity of the second light L2 with which the sample surface SF is irradiated. When the operation of each unit is an operation of scanning the sample surface SF with the first light L1 and the second light L2 by the scanning unit 3, the setting information is, for example, the angle of the scanning mirror 24 in the scanning unit 3 at each time. Contains information that specifies When the operation of each unit is an operation of detecting the fluorescence L by the detection unit 4, the setting information includes, for example, a period during which the detection unit 4 detects the fluorescence L (for example, timing of starting exposure, exposure time, exposure It includes information that defines the end timing).

The control unit 7 controls each unit of the microscope MS based on the setting information stored in the storage unit 8. The storage unit 8 also stores the detection result of the light receiving unit 42 in association with the detection timing at which the light receiving unit 42 executes the detection. The image processing unit 5 reads the detection result of the light receiving unit 42 stored in the storage unit 8 and generates an image. The storage unit 8 stores the image data generated by the image processing unit 5. The control device 6 outputs the data of the image generated by the image processing unit 5 to the display device and displays the image on the display device. The display device may be a part of the microscope MS or a device external to the microscope MS (eg, desktop personal computer, portable terminal, etc.).

6A to 6C are diagrams showing the operation of the microscope of the first embodiment. For each part of the microscope MS, refer to FIG. 1 and the like as appropriate. The control unit 7 controls the first irradiation unit 1 to set the phase of the stripe pattern of the first light L1 to the first phase. In the state where the stripe pattern of the first light L1 is set to the first phase, the control unit 7 controls the scanning unit 3 to cause the first light L1 and the second light L2 to move the sample surface SF in a predetermined direction (this In that case, scanning is performed in the second direction D2) (see FIG. 6A). For example, the control unit 7 continuously changes the position of the first area A1 and the position of the second area A2 on the sample surface SF. Further, the control unit 7 controls the detection unit 4 to cause the light receiving unit 42 to perform detection at a predetermined sampling frequency while scanning the sample surface SF with the first light L1 and the second light L2.

The image processing unit 5 identifies the position of the first area A1 at each detection timing of the light receiving unit 42 based on the setting information stored in the storage unit 8. The image processing unit 5 associates the specified position of the first area A1 with the detection result of the light receiving unit 42 to represent the detection result obtained by the light receiving unit 42 while the scanning unit 3 executes a series of scans. An image (hereinafter referred to as the first image) is generated. In FIG. 6B, reference numeral Im1 is a first image representing the detection result of the light receiving unit 42 in the state where the stripe pattern of the first light L1 is set to the first phase. In FIG. 6B, a dotted arrow indicates a change in phase.

The control unit 7 controls the first irradiation unit 1 to control the first light L1 after the light receiving unit 42 completes the scheduled detection in the state where the stripe pattern of the first light L1 is set to the first phase. The phase of the stripe pattern is set to a second phase different from the first phase. The phase difference between the first phase and the second phase is, for example, 2π/3. The control unit 7 controls the scanning unit 3 to scan the sample surface SF in the predetermined direction with the first light L1 and the second light L2 while the stripe pattern of the first light L1 is set to the second phase. Let Further, the control unit 7 controls the detection unit 4 to cause the light receiving unit 42 to perform detection at a predetermined sampling frequency while scanning the sample surface SF with the first light L1 and the second light L2. Reference numeral Im2 is a first image representing the detection result of the light receiving unit 42 in the state where the stripe pattern of the first light L1 is set to the second phase.

The control unit 7 controls the first irradiation unit 1 to control the first irradiation unit 1 after the light receiving unit 42 completes the scheduled detection in the state where the stripe pattern of the first light L1 is set to the second phase. The phase of the stripe pattern is set to a third phase that is different from both the first phase and the second phase. The phase difference between the second phase and the third phase is, for example, 2π/3. The control unit 7 controls the scanning unit 3 to scan the sample surface SF in the predetermined direction with the first light L1 and the second light L2 in a state where the stripe pattern of the first light L1 is set to the third phase. Let Further, the control unit 7 controls the detection unit 4 to cause the light receiving unit 42 to perform detection at a predetermined sampling frequency while scanning the sample surface SF with the first light L1 and the second light L2. Reference numeral Im3 is a first image representing the detection result of the light receiving unit 42 in the state where the stripe pattern of the first light L1 is set to the second phase.

The image processing unit 5 generates a second image having a resolution different from that of the first image based on the plurality of first images (eg, the first image Im1, the first image Im2, the first image Im3) obtained above. .. The second image is, for example, a super-resolution image whose resolution is higher than that of the first image. For example, the image processing unit 5 generates a second image by separating the components for each region in the frequency space using the plurality of first images, reconstructing the separated components, and then returning the components to those in the real space. To do. In FIG. 6C, the symbol ky is the first axis direction in the frequency space corresponding to the first direction D1. The symbol kx is the second axis direction in the frequency space corresponding to the second direction D2.

Reference numeral F1 is a region corresponding to an optical transfer function (optical transfer function, appropriately referred to as OTF) of the optical system of the microscope MS. The spatial frequency component outside the area F1 is detected by the optical system because the spatial frequency shifts due to the illumination of the striped pattern and appears in the area F1 mixed with other spatial frequency components. The microscope MS according to the embodiment detects the fluorescence L emitted from the sample S under a plurality of conditions in which the phase of the first light L1 is different. The image processing unit 5 separates the spatial frequency component having the shifted spatial frequency using the plurality of detection results obtained by the light receiving unit 42 under the plurality of conditions in which the phase of the first light L1 is different. The image processing unit 5 arranges the separated spatial frequency components as the spatial frequency components of the spatial frequency before shifting, and reconstructs the distribution of the spatial frequency components. Then, the image processing unit 5 generates the second image by converting the reconstructed distribution of the spatial frequency components into the distribution of the components (eg, intensity) in the real space by the inverse Fourier transform.

Reference numeral F2 in FIG. 6C is an area in which the separated spatial frequency components are arranged. The area F2 is an area where the spatial frequency component exists. In the region F2, since the intensity of the first light L1 changes in the first direction D1, the region F2 spreads to a higher frequency side than the region F1 corresponding to the OTF in the first axial direction ky corresponding to the first direction D1. (Eg, high cutoff frequency). In addition, the region F2 is set to sandwich the first region A1 in the second direction D2, and thus the region F2 corresponds to the OTF in the second axial direction kx corresponding to the second direction D2. It extends to higher frequencies than F1 (eg, high cutoff frequency). The image processing unit 5 generates a super-resolution image with high resolution in both the first direction D1 and the second direction D2, for example, by performing an inverse Fourier transform on the spatial frequency component in the region F2. The second image may have the same resolution as that of the first image or a resolution lower than that of the first image in at least one direction.

Next, the observation method according to the embodiment will be described based on the configuration and operation of the microscope MS. FIG. 7 is a flowchart showing the observation method of the first embodiment. For each part of the microscope MS, refer to FIGS. 1 to 6 as appropriate. In step S1, the control unit 7 sets the phase of the first light L1. The controller 7 sets the phase of the first light L1 to the scheduled phase. Here, the scheduled phases are the first phase, the second phase, and the third phase shown in FIG. 6, and the control unit 7 sets the phase of the first light L1 to the first phase.

In step S2, the control unit 7 starts scanning the sample S with the first light L1 and the second light L2. The process of step S2 includes the process of step S3 and the process of step S4. The first irradiation unit 1 irradiates the first area L1 with the first light L1. The second irradiation unit 2 irradiates the second region A2 with the second light L2. The first irradiation unit 1 and the second irradiation unit 2 change the position of the first region A1 and the position of the second region A2 of the sample surface SF by scanning of the scanning unit 3, while performing the process of step S2 and the process of step S3. Perform processing in parallel. At least a part of the process of step S4 may be executed after the process of step S3.

In step S5, the detection unit 4 detects the fluorescence L. In step S6, the control unit 7 determines whether or not the detection of the fluorescence L in the predetermined area is completed. The predetermined region is, for example, a region preset as a region to be observed. The control unit 7 determines whether or not the scanning with the first light L1 and the second light with respect to the predetermined region is completed based on information (setting information or the like) that controls the scanning unit 3. When the control unit 7 determines that the scanning of the predetermined area is not completed, it determines that the detection of the fluorescence L in the predetermined area is not completed (Step S6; No). When the control unit 7 determines that the detection of the fluorescence L in the predetermined area is not completed (step S6; No), the control unit 7 repeatedly executes the detection of the fluorescence L in step S5.

When the control unit 7 determines that the scanning of the predetermined region is completed, it determines that the detection of the fluorescence L in the predetermined region is completed (step S6; Yes). When the control unit 7 determines that the detection of the fluorescence L on the predetermined region is completed (step S6; Yes), the scanning of the sample S by the first light L1 and the second light L2 is stopped in step S7. In step S8, the control unit 7 determines whether to change the phase of the first light L1. When the processing of steps S2 to S7 has not been completed for a part of the planned phases (eg, some phases for a plurality of phases, remaining phases, etc.), the control unit 7 determines the first light L1. It is determined that the phase of is changed (step S8; Yes).

When it is determined that the phase of the first light L1 is changed (step S8; Yes), the control unit 7 sets the first light L1 to the next phase among the plurality of phases set in step S1. Here, the control unit 7 sets the first light L1 to the second phase. Then, the microscope MS executes the processing from step S2 to step S8. When it is determined that the phase of the first light L1 is changed next (step S8; Yes), the control unit 7 sets the first light L1 to the next phase in step S1. Here, the control unit 7 sets the first light L1 to the third phase. Then, the microscope MS executes the processing from step S2 to step S8.

When determining that the phase of the first light L1 is not changed (step S8; No), the control unit 7 causes the image processing unit 5 to perform image processing. In step S9, the image processing unit 5 generates an image (for example, a high-resolution composite image) based on the detection result (plurality of images) of the fluorescence L. The image processing unit 5 generates a second image having a resolution different from that of the first image, for example, based on the first image Im1, the first image Im2, and the first image Im3 described in FIG. 6B. For example, the image processing unit 5 generates, as the second image, a super-resolution image having a higher resolution than the first image.

For example, when the second image is generated using the same number of captured images as the conventional SIM, the microscope MS according to the above-described embodiment can acquire a higher resolution image than the conventional SIM. Further, the microscope MS according to the embodiment can omit the operation or the configuration for changing the direction of the illumination light, as compared with the conventional SIM for changing the direction of the illumination light.

In the present embodiment, the first period during which the first irradiation unit 1 emits the first light L1 overlaps at least part of the second period during which the second irradiation unit 2 emits the second light L2. The second irradiation unit 2 irradiates the second light L2 in parallel with at least a part of the irradiation of the first light L1 by the first irradiation unit 1. The detection unit 4 executes the detection in the period in which the first period and the second period overlap in the detection period. In this case, the microscope MS continuously scans the fluorescent material in the region where the fluorescence L is detected by the irradiation of the first light L1 by continuously scanning the sample S with the first light L1 and the second light L2, for example. Can be deactivated. In this case, the microscope MS simplifies the process of controlling the timing of irradiating the first light L1 and the timing of irradiating the second light L2.

The first irradiation unit 1 may emit continuous light as the first light L1 or may emit pulsed light. The second irradiation unit 2 may emit continuous light as the second light L2 or may emit pulsed light. The scanning unit 3 may continuously scan the first light L1 and the second light L2, or may perform stepwise scanning. Note that, for example, the second irradiation unit 2 irradiates the pair of second regions A2 with the second light L2 whose intensity is changed in the second direction D2. Moreover, the 1st irradiation part 1 irradiates the 1st light L1 which changed intensity|strength between a pair of 2nd area|region A2.

Here, a case where the scanning unit 3 scans in steps will be described. The scanning unit 3 sets the scanning mirror 24 at the first angle so that the area A0 is at the first position (scanning position). Then, with the scanning mirror 24 set to the first angle, the first irradiation section 1 irradiates the first area L1 with the first light L1 and the second irradiation section 2 forms the second area L2 with the second light L2. Irradiate A2. Then, the detection unit 4 detects the fluorescence L while the sample S is irradiated with the first light L1 and the second light L2. Then, the scanning unit 3 sets the scanning mirror 24 at the second angle so that the area A0 becomes the second position different from the first position after the detection by the detecting unit 4 is completed. Then, the detection unit 4 detects the fluorescence L in a state where the scanning unit 3 sets the area A0 at the second position (scanning position). Then, with the scanning mirror 24 set to the second angle, the first irradiation unit 1 irradiates the first region L1 with the first light L1, and the second irradiation unit 2 emits the second light L2 into the second region. Irradiate A2. Then, the detection unit 4 detects the fluorescence L while the sample S is irradiated with the first light L1 and the second light L2. The microscope MS detects the fluorescence L generated in the predetermined region of the sample S by repeating such processing.

The wavelength of the excitation light may be the same as the wavelength of the deactivating light. For example, the first light L1 is activation light that activates the fluorescent substance, and the second light L2 serves as both excitation light that excites the fluorescent substance and inactivation light that inactivates the fluorescent substance. May be. In this case, the light amount of the second light L2 with which the sample S is irradiated as the excitation light may be different from the light amount of the second light with which the sample S is irradiated as the deactivating light. The light amount of the second light L2 can be adjusted by changing one or both of the intensity of the second light L2 and the irradiation time.

The wavelength of the excitation light may be different from both the wavelength of the activation light and the wavelength of the deactivation light. In this case, the microscope MS irradiates the activation light to the region including at least a part of the first region A1 to which the excitation light is radiated as the first light L1. Then, the microscope MS irradiates the excitation light as the first light L1 and the inactivation light as the second light L2, and the detection unit 4 detects the fluorescence L. Moreover, when the wavelength of the excitation light is different from the wavelength of the activation light, the first irradiation unit 1 may irradiate the activation light as the first light L1. For example, the microscope MS emits activation light as the first light L1. Then, the microscope MS irradiates at least a part of the first region A1 irradiated with the first light L1 with the excitation light, irradiates the inactivation light as the second light L2, and the fluorescence L is detected by the detection unit 4. To detect.

The first irradiation unit 1 emits, as the first light L1, excitation light that excites the fluorescent substance contained in the sample S, and the second irradiation unit 2 emits the second light L2 as the first light L1. The fluorescent substance may be irradiated with stimulated emission light that causes stimulated emission. In this case, the excited fluorescent substance is irradiated with the second light L2 to generate stimulated emission light, and is brought into a ground state, so that generation of the fluorescence L is suppressed.

In addition, in the present embodiment, the region A0 in which the fluorescence L is generated is a region longer than the second direction D2 in the first direction D1, but may be a region having the same size as the second direction D2 in the first direction D1. However, the area may be shorter in the first direction D1 than in the second direction D2. The scanning unit 3 scans the first light L1 and the second light L2 in the second direction D2, but also scans the first light L1 and the second light L2 in the first direction D1 and the second direction D2. Good. For example, the microscope MS may be a microscope that uses localized fringe illumination with the first light L1 and the second light L2.

The microscope MS may not include one or both of the first light source 11 and the second light source 31. For example, the microscope MS may be provided without the first light source 11. The first light source 11 may be attached to the microscope MS when the microscope MS is used. The first light source 11 may be replaceable (eg, attachable or detachable) with respect to the first irradiation unit 1. Further, the microscope MS may be provided without the second light source 31. The second light source 31 may be attached to the microscope MS when the microscope MS is used. The second light source 31 may be replaceable (eg, attachable or detachable) with respect to the second irradiation unit 2.

[Second Embodiment]
Next, a second embodiment will be described. In the present embodiment, configurations similar to those of the above-described embodiments are designated by the same reference numerals, and description thereof will be omitted or simplified. FIG. 8 is a diagram showing a microscope according to the second embodiment. In the present embodiment, at least a part of the optical system (illumination optical system) is shared by the first irradiation unit 1 and the second irradiation unit 2. The first irradiation unit 1 includes a first light source 11 and an optical system 51. The optical system 51 corresponds to the first optical system 12 of FIG. 1 and irradiates the first region A1 of the sample surface SF with the first light L1 emitted from the first light source 11. The second irradiation unit 2 includes a second light source 31 and an optical system 51. The optical system 51 corresponds to the second optical system 32 of FIG. 1 and irradiates the second region A2 of the sample surface SF with the second light L2 emitted from the second light source 31. The optical system 51 is shared by the first irradiation unit 1 and the second irradiation unit 2. The first irradiation unit 1 includes a part (eg, the optical system 51) of the second irradiation unit 2. The second irradiation unit 2 also includes a part (eg, the optical system 51) of the first irradiation unit 1.

The optical system 51 differs from the first optical system 12 in FIG. 1 in the configuration between the first light source 11 and the lens 22. The optical system 51 differs from the second optical system 32 of FIG. 1 in the configuration between the second light source 31 and the lens 22. The optical system 51 includes a dichroic mirror 52, a lens 53, a phase adjusting unit 54, an optical member 55, a branching unit 15, a lens 56, a mask 57, a lens 58, a lens 59, and a mirror 60. Prepare

The dichroic mirror 52 of FIG. 8 is arranged at a position where the first light L1 emitted from the first light source 11 is incident and the second light L2 emitted from the second light source 31 is incident. The dichroic mirror 52 is similar to the dichroic mirror 21 shown in FIG. The first light L1 emitted from the first light source 11 is wavelength-selectively transmitted through the dichroic mirror 52 and is incident on the lens 53. The second light L2 emitted from the second light source 31 is wavelength-selectively reflected by the dichroic mirror 52 and enters the lens 53.

The lens 53 is, for example, a collimator. The lens 53 converts the first light L1 emitted from the first light source 11 into parallel light. The lens 53 also converts the second light L2 emitted from the second light source 31 into parallel light. The first light L1 and the second light L2 that have passed through the lens 53 enter the phase adjustment unit 54, respectively. The configuration from the phase adjusting unit 54 to the lens 58 will be described below with reference to FIGS. 9 to 12.

First, the optical path of the first light L1 will be described. 9A and 9B are views showing a first irradiation unit and a second irradiation unit of the second embodiment. FIG. 9A is a view of the optical path of the first light L1 from the first light source 11 to the lens 58 as seen from the X direction. FIG. 9B is a diagram of the optical path of the first light L1 from the first light source 11 to the lens 58 as viewed from the Y direction. The phase adjusting unit 54 is arranged at or near the position of the pupil conjugate plane P5. The phase adjustment unit 54 is an optical member that adjusts the phase based on the wavelength of incident light.

10(A) and 10(B) are diagrams showing the phase adjusting unit of the second embodiment. FIG. 10A shows the first light L1 before and after entering the phase adjustment unit 54. Further, FIG. 10B shows the second light L2 before and after entering the phase adjustment unit 54. The phase adjusting unit 54 has the same shape as the phase adjusting unit 34 of FIG. 4, and each unit of the phase adjusting unit 54 is represented by the same symbol as that of FIG.

First, the first light L1 will be described. In the following description, the first light L1 incident on the region 34a is represented by reference symbol L1a, and the first light L1 incident on the region 34b is represented by reference symbol L1b as appropriate. The phase adjustment unit 54 adjusts the phase difference between the first light L1a and the first light L1b. When the phase difference between the first light L1a and the first light L1b is close to an even multiple of π or an even multiple of π, the intensity distribution of the first light L1 on the sample surface SF is the same as that without the phase modulator 54. An intensity distribution is obtained. The phase adjustment unit 54 adjusts the phase difference between the first light L1a and the first light L1b to, for example, an even multiple of π. The first portion 37 and the second portion 38 have such a refractive index and dimensions that the phase difference between the first light L1a emitted from the region 34a and the first light L1a emitted from the region 34b is an even multiple of π. The optical characteristics are set.

Here, the dimension of the second portion 38 in the optical axis direction of the optical system 51 is represented by d, the refractive index of the first portion 37 with respect to the wavelength λ1 of the first light L1 is represented by n11, and the refractive index of the second portion 38. Is represented by n12. Also, the phase difference between the first light L1a emitted from the region 34a and the first light L1b emitted from the region 34b is represented by W1, and is an integer (-3, -2, -1, 0, 1, 2, 3,...・・) is represented by N. The phase difference W1 is set to W1=2N×π, and λ1, n11, n12, and d are set so as to satisfy the following expression (1).
W1=2N×π=2π(n11-n12)d/λ1 (1)

Next, the second light L2 will be described. The first portion 37 and the second portion 38 have such a refractive index and dimensions that the phase difference between the second light L2a emitted from the region 34a and the second light L2b emitted from the region 34b is an odd multiple of π. The optical characteristics are set. Here, the refractive index of the first portion 37 with respect to the wavelength λ2 of the second light L2 is represented by n21, and the refractive index of the second portion 38 with respect to the wavelength λ2 of the second light L2 is represented by n22. Further, the phase difference between the second light L2a emitted from the region 34a and the second light L2b emitted from the region 34b is represented by W2, and is an integer (-3, -2, -1, 0, 1, 2, 3,...・・) is represented by M. The phase difference W2 is set to W2=(2M−1)×π, and λ2, n21, n22, and d are set so as to satisfy the following expression (2).
W2=(2M−1)×π=2π(n21−n22)d/λ2 (2)

The phase adjusting unit 54 according to the present embodiment, based on λ1 which is the wavelength of the first light L1 and λ2 which is the wavelength of the second light L2, the refractive index and size of the first portion 37, and the second portion 38. The refractive index and dimensions of are set. For example, the phase adjusting unit 54 may satisfy the above equations (1) and (2) or minimize the residual error for the above equation (1) and the residual error for the above equation (2). Is configured. For example, the residual relating to the equation (1) is represented by ΔW1. ΔW1 is a value obtained by substituting the values of N, λ1, n11, n12, and d into the following equation (3).
ΔW1=2Nπ−2π(n11−n12)d/λ1 (3)
In addition, the residual relating to the equation (2) is represented by ΔW2. ΔW2 is a value obtained by substituting the values of M, λ2, n21, n22, and d into the following equation (4).
ΔW2=2(M−1)×π−2π(n21−n22)d/λ2 (4)
Further, the evaluation function regarding the residual is defined by the following expression (5).
ΔW=|ΔW1|×K1+|ΔW2|×K2 (5)
K1 and K2 in the equation (5) are weighting coefficients. The phase adjustment unit 54 is configured such that the value of the evaluation function ΔW is equal to or less than the threshold value.

Returning to the description of FIGS. 9A and 9B, the first light L1 emitted from the phase adjusting unit 54 enters the optical member 55. In the optical member 55, the power corresponding to the second direction D2 is stronger than the power corresponding to the first direction D1. The power corresponding to the second direction D2 is a refracting power that deflects a light beam in the X direction corresponding to the second direction D2. The power corresponding to the first direction D1 is a refracting power that deflects a light beam in the Y direction corresponding to the first direction D1. The optical member 55 is an astigmatism member that acts on the first light L1 to generate astigmatism and acts on the second light L2 to generate astigmatism. The optical member 55 is, for example, a cylindrical lens. The optical system 51 includes, instead of the optical member 55, an astigmatism optical system that acts on the first light L1 to generate astigmatism and acts on the second light L2 to generate astigmatism. Good.

The first light L1 emitted from the optical member 55 enters the branch portion 15. The branching portion 15 is arranged at or near the position of the sample conjugate plane SF6. The first light L1 is diffracted by the branching unit 15 and branched in the Y direction. The first light L1 is split into a plurality of diffracted lights by the splitting unit 15, and enters the lens 56. The lens 56 is arranged such that the light source side focus thereof is at the position of the sample conjugate plane SF6 or in the vicinity thereof. The first light L1 emitted from the lens 56 enters the mask 57. The mask 57 is arranged at the position of the pupil conjugate plane P6 or in the vicinity thereof. The mask 57 is arranged at the position of the focus of the lens 56 on the sample side or in the vicinity thereof.

11A to 11C are views showing the mask of the second embodiment. The mask 57 is a light blocking member having wavelength selectivity. In the mask 57, the transmittance of the first light L1 and the transmittance of the second light L2 are different in the area on the mask 57. The mask 57 includes an opening 57a, an opening 57b, and a light shield 57c. The opening 57a is arranged at a position where the +1st-order diffracted light of the first light L1 branched by the branching unit 15 is incident and a position where the −1st-order diffracted light is incident. The size of the opening 57a in the Y direction corresponding to the first direction D1 of the sample surface SF shown in FIG. 9 is smaller than the size in the X direction corresponding to the second direction D2.

The opening 57b is arranged at a position where the 0th-order diffracted light of the first light L1 branched by the branching portion 15 is incident. The size of the opening 57b in the Y direction corresponding to the first direction D1 of the sample surface SF shown in FIG. 9 is smaller than the size in the X direction corresponding to the second direction D2.

FIG. 11B is a diagram showing the transmittance with respect to the wavelength in the opening 57a. The opening 57a transmits the first light L1 and blocks the second light L2. The transmittance of the opening 57a for the wavelength λ1 of the first light L1 is higher than the transmittance of the second light L2 for the wavelength λ2. FIG. 11C is a diagram showing the transmittance with respect to the wavelength of light in the opening 57b. The opening 57b blocks the first light L1 and transmits the second light L2. The opening 57b has a lower transmittance for the wavelength λ1 of the first light L1 than that for the wavelength λ2 of the second light L2. The light shielding portion 57c in FIG. 11A is a portion around the opening 57a and a portion around the opening 57b. The light blocking portion 57c has a lower transmittance for both the wavelength of the first light L1 and the wavelength of the second light L2 than the openings 57a and 57b.

Returning to the description of FIGS. 9A and 9B, the first light L1 transmitted through the mask 57 enters the lens 58. The lens 58 is arranged such that the position of the focus on the light source side is the position of the mask 57 or the position in the vicinity thereof. The lens 58 is arranged such that the position of the focal point on the sample side is the position of the sample conjugate plane SF7 or a position in the vicinity thereof. The first light L1 emitted from the lens 58 is condensed on the sample conjugate plane SF7.

Next, the optical path of the second light L2 will be described. 12A and 12B are views showing the optical path of the second irradiation section of the second embodiment. FIG. 12A is a diagram of the optical path of the second light L2 from the second light source 31 to the lens 58 as seen from the X direction. FIG. 12B is a diagram of the optical path of the second light L2 from the second light source 31 to the lens 58 as viewed from the Y direction. The second light L2 emitted from the second light source 31 is reflected by the dichroic mirror 52 and enters the lens 53. The second light L2 emitted from the lens 53 enters the phase adjustment unit 54, and the phase difference between the second light L2a and the second light L2b is adjusted as described with reference to FIG.

The second light L2 emitted from the phase adjustment unit 54 enters the optical member 55 (eg, a cylindrical lens). As shown in FIG. 12A, the second light L2 is converted into parallel light on the YZ plane by the optical member 55 and is incident on the branch portion 15. As shown in FIG. 12B, the second light L2 is condensed on the XZ plane by the optical member 55 and is incident on the branch portion 15. The second light L2 is diffracted by the branching portion 15 and branched in the Y direction.

The second light L2 emitted from the branch portion 15 is condensed by the lens 56 and is incident on the mask 57. The 0th-order diffracted light of the second light L2 branched by the branching unit 15 enters the opening 57b shown in FIG. As shown in FIG. 11C, the opening 57b has a relatively high transmittance with respect to the wavelength λ2 of the second light L2. The second light L2 entering the opening 57b passes through the opening 57b and enters the lens 58. The +1st-order diffracted light and the -1st-order diffracted light of the second light L2 split by the splitting unit 15 enter the opening 57a shown in FIG. As shown in FIG. 11B, the opening 57a has a relatively low transmittance for the wavelength λ2 of the second light L2. The second light L2 incident on the opening 57a is blocked by the opening 57a.

If the region of the mask 57 on which the first-order diffracted light of the first light L1 is incident is different from the region of the mask 57 on which the first-order diffracted light of the second light L2 is incident (eg, when they are separated), the mask 57 In, the region where the first-order diffracted light of the first light L1 is incident may be used as the opening portion, and the region where the first-order diffracted light of the second light L2 is incident may be used as the light shielding portion. This opening need not be wavelength selective.

The second light L2 that has entered the lens 58 is converted into parallel light on the YZ plane and enters the sample conjugate plane SF7, as shown in FIG. Further, the second light L2 incident on the lens 58 is condensed on the sample conjugate plane SF7 on the XZ plane, as shown in FIG. 12(B).

Returning to the description of FIG. 8, the first light L1 emitted from the lens 58 enters the lens 59. The first light L1 emitted from the lens 59 is reflected by the mirror 60 and enters the lens 22. Similar to the first embodiment, the first light L1 emitted from the lens 22 is reflected by the dichroic mirror 23 and passes through the scanning mirror 24, the lens 25, the lens 26, and the lens 27, and the first region of the sample surface SF. A1 is irradiated.

Further, the second light L2 emitted from the lens 58 enters the lens 59. The second light L2 emitted from the lens 59 is reflected by the mirror 60 and enters the lens 22. Similarly to the first embodiment, the second light L2 emitted from the lens 22 is reflected by the dichroic mirror 23 and passes through the scanning mirror 24, the lens 25, the lens 26, and the lens 27, and the second region of the sample surface SF. A2 is irradiated.

The microscope MS according to the present embodiment has a large number of configurations shared by the first optical system 12 and the second optical system 32 as compared with the first embodiment, and the number of parts of the optical system can be reduced. The microscope MS according to the present embodiment can reduce the cost required for alignment of, for example, a lens by reducing the number of optical system components.

[Third Embodiment]
Next, a third embodiment will be described. In the present embodiment, configurations similar to those of the above-described embodiments are designated by the same reference numerals, and description thereof will be omitted or simplified. FIGS. 13A and 13B are views showing the first irradiation unit of the third embodiment. In this embodiment, the configuration of the branching unit 15 is different from that of the above embodiment.

In this embodiment, the 1st irradiation part 1 can change the fringe period of the 1st light L1. The first irradiation unit 1 includes a lens 13, a polarizing element 75, an optical member 14, a branch unit 15, a lens 16, and a mask 17 on the light emission side of the first light source 11. The lens 13, the optical member 14 (for example, a cylindrical lens), the lens 16, and the mask 17 are the same as those in the first embodiment, and the description thereof will be omitted or simplified.

The branch unit 15 includes a polarizing element 76 and an SLM 77 (spatial light modulator). FIG. 13A shows the optical path of the first light L1 from the first light source 11 to the SLM 77 in the first irradiation section 1. Further, FIG. 13B shows the optical path of the first light L1 from the SLM 77 to the mask 17 in the first irradiation section 1.

The polarization element 76 includes a polarization separation film 78. The polarization separation film 78 is tilted with respect to the optical axis 12A of the first optical system 12. The angle formed by the polarization separation film 78 and the optical axis 12A is, for example, 45°. The polarization separation film 78 has an optical characteristic that P-polarized light for the polarization separation film 78 is transmitted and S-polarized light for the polarization separation film 78 is reflected. In the present embodiment, "P-polarized light with respect to the polarization separation film 78" is appropriately referred to as "P-polarized light", and "S-polarized light with respect to the polarization separation film 78" is appropriately represented as "S-polarized light".

The first light source 11 is, for example, a laser diode, and emits the P-polarized first light L1. The polarizing element 75 transmits P-polarized light and blocks S-polarized light. The polarizing element 75 is, for example, a polarizing plate that absorbs S polarized light, but may be a polarizing element that reflects S polarized light. The polarizing element 75 is arranged in the optical path between the first light source 11 and the polarizing element 76. In FIG. 13A, the polarization element 75 is arranged in the optical path between the lens 13 and the optical member 14.

The polarizing element 76 is, for example, a polarizing beam splitter (appropriately referred to as PBS). The polarization element 76 of FIG. 13A is a PBS prism and includes a polarization separation film 78. The P-polarized first light L1 transmitted through the polarization element 75 is transmitted through the polarization separation film 78 and is incident on the SLM 77. The SLM 77 has a plurality of regions 79 on which the first light L1 is incident. The SLM 77 is, for example, an SLM using liquid crystal, and the region 79 is one or two or more pixels. The SLM 77 has a variable refractive index or reflectance for each region 79. The SLM 77 can adjust the polarization state of the incident first light L1 for each region 79. The SLM 77 converts P-polarized light incident on a pixel into S-polarized light while a predetermined electric field is applied to the pixel.

FIG. 13B is a diagram showing an optical path of S-polarized light in the first light L1 emitted from the SLM 77. The S-polarized first light L1 emitted from the SLM 77 enters the polarization separation film 78 and is reflected by the polarization separation film 78. The S-polarized first light L1 reflected by the polarization separation film 78 enters the mask 17 via the lens 16. The first light L1 transmitted through the mask 17 is applied to the sample surface SF, as in the first embodiment. On the other hand, the P-polarized light in the first light L1 emitted from the SLM 77 passes through the polarization separation film 78 and is deviated from the optical path toward the sample surface SF.

14(A) to 14(C) are diagrams showing a branching portion of the third embodiment. The SLM 77 is controlled by the controller 7 to adjust the polarization state of the first light L1. For example, the control unit 7 supplies the SLM 77 with data (for example, bitmap data) representing the voltage applied to each of the plurality of regions 79. The SLM 77 sets the voltage pattern in the plurality of regions 79 based on the data supplied from the control unit 7. The fringe period of the first light L1 shown in FIG. 1 depends on the period of the voltage pattern in the plurality of regions 79. The control unit 7 controls the SLM 77 to control the fringe period of the first light L1 shown in FIG.

14B and 14C, the region 79 to which the positive voltage is applied is represented by reference numeral 79a, and the region 79 to which the negative voltage is applied is represented by reference numeral 79b. In FIG. 14B, the symbol ΔPa represents the cycle of the region 79a in the Y direction corresponding to the first direction D1 shown in FIG. The period ΔPa of the area 79a corresponds to the distance between the centers of the areas 79a. Reference numeral PL1a is the stripe pattern of the first light L1 formed on the sample surface SF in FIG. 1, and reference numeral ΔD1a is the stripe period of the first light L1. The fringe period ΔD1a of the first light L1 is determined by the period ΔPa of the area 79a, the magnification of the optical system, and the like.

In FIG. 14C, the symbol ΔPb is the cycle of the region 79a in the Y direction corresponding to the first direction D1 shown in FIG. The period ΔPb of the region 79a corresponds to the distance between the centers of the regions 79a. Reference numeral PL1b is a stripe pattern of the first light L1 formed on the sample surface SF in FIG. 1, and reference numeral ΔD1b is a stripe cycle of the first light L1. The fringe period ΔD1a of the first light L1 is determined by the period ΔPa of the area 79a, the magnification of the optical system, and the like. The period ΔPb of the region 79a in FIG. 14C is longer than the period ΔPb of the region 79a in FIG. 14B, and the stripe period ΔD1b of the first light L1 in FIG. 14C is the same as that in FIG. 14B. It is longer than the fringe period ΔD1a of the first light L1.

In the second image (eg, super-resolution image) generated by the image processing unit 5 illustrated in FIG. 1, the resolution of the second image in the first direction D1 depends on the fringe period of the first light L1. The microscope MS according to the present embodiment can change the resolution corresponding to the first direction D1 in the second image generated by the image processing unit 5 by controlling the fringe period of the first light L1 by the control unit 7.

15A to 15C are diagrams showing the processing of the image processing unit of the third embodiment. In FIGS. 15A to 15C, the reference character ky is the first axis direction in the frequency space corresponding to the first direction D1. The symbol kx is the second axis direction in the frequency space corresponding to the second direction D2. Reference numeral F1 is a region corresponding to the OTF of the optical system of the microscope MS.

Here, the state in which the cycle of the region 79a in the SLM 77 shown in FIG. 14B and the like is set to a predetermined value is called the first state. FIG. 15A is a diagram showing the spatial frequency components obtained in the first state. The image processing unit 5 separates the spatial frequency component using the plurality of first images obtained from the detection result of the detection unit 4 in the first state. The image processing unit 5 arranges the separated spatial frequency components. Reference numeral F3 is a region in which the spatial frequency component is obtained by the image processing unit 5 executing processing based on the detection result of the detection unit 4 in the first state. Reference sign F3y is the range (eg, width) of the region 79 in the first axis direction ky. The spatial frequency at the end of the range F3y is the cutoff frequency corresponding to the first direction D1. The symbol F3x is the range (eg, width) of the region F3 in the second axis direction kx. The spatial frequency at the end of the range F3x is the cutoff frequency corresponding to the second direction D2.

Further, a state in which the period of the region 79a in the SLM 77 is shorter than that in the first state is referred to as a second state. FIG. 15B is a diagram showing the spatial frequency components obtained in the second state. Reference numeral F4 is a region in which the spatial frequency component is obtained by the image processing unit 5 executing processing based on the detection result of the detection unit 4 in the second state. Reference sign F4y is a range (eg, width) of the region F4 in the first axis direction ky. The spatial frequency at the end of the range F4y is the cutoff frequency corresponding to the first direction D1. The symbol F4x is the range (eg, width) of the region F4 in the second axis direction kx. The spatial frequency at the end of the range F4x is the cutoff frequency corresponding to the second direction D2.

The range F4y in the first axis direction ky of the area F4 is wider than the range F3y in the first axis direction ky of the area F3 in FIG. The region F4 has a higher cutoff frequency corresponding to the first direction D1 than the region F3 of FIG. The image processing unit 5 uses the spatial frequency component in the area F4, so that the resolution in the first direction D1 is higher than that in the case where the detection result of the detection unit 4 corresponding to the first state is used (see FIG. 15A). It is possible to generate a second image having a high value.

Further, a state in which the period of the region 79a in the SLM 77 is longer than the first state is referred to as a third state. FIG. 15C is a diagram showing the spatial frequency components obtained in the third state. Reference numeral F5 is a region in which the spatial frequency component is obtained by the image processing unit 5 executing processing based on the detection result of the detection unit 4 in the third state. Reference sign F5y is a range (eg, width) of the region F5 in the first axis direction ky. The spatial frequency at the end of the range F5y is the cutoff frequency corresponding to the first direction D1. The symbol F5x is the range (eg, width) of the region F5 in the second axis direction kx. The spatial frequency at the end of the range F5x is the cutoff frequency corresponding to the second direction D2.

The range F5y in the first axis direction ky of the area F5 is narrower than the range F3y in the first axis direction ky of the area F3 in FIG. The region F5 has a lower cutoff frequency corresponding to the first direction D1 than the region F3 of FIG. The image processing unit 5 uses the spatial frequency component in the region F5 to compare the detection result of the detection unit 4 corresponding to the first state (see FIG. 15A)) with respect to the first direction D1. A second image with low resolution can be generated.

As described above, the microscope MS according to the present embodiment can adjust the resolution corresponding to the first direction D1 in the second image generated by the image processing unit 5. The control unit 7 sets the resolution of the second image in the first direction D1 based on the resolution of the second image in the second direction D2, for example. For example, the control unit 7 controls the SLM 77 so that the resolution of the second image in the first direction D1 and the resolution of the second image in the second direction D2 are uniform (or close to each other).

Here, the control of the control unit 7 when the resolution in the first direction D1 is different from the resolution in the second direction in the second image acquired by the microscope MS will be described. First, the control of the control unit 7 in the case where the resolution of the second image in the first direction D1 is higher than the resolution in the second direction (hereinafter referred to as the first case) will be described.

The first case corresponds to, for example, FIG. Here, it is assumed that the fluorescence detection condition by the microscope MS in FIG. 15B is a condition in which the fringe cycle of the first light L1 is the first cycle. Under this condition, in the area F4 in which the spatial frequency component exists, the range F4y in the first axis direction ky is wider than the range F4x in the second axis direction kx. The image processing unit 5 generates the second image based on the spatial frequency components included in the entire area F4, for example. In this second image, the resolution in the first direction D1 is higher than the resolution in the second direction D2.

In the first case, the control unit 7 changes the fringe period of the first light L1 to the second period which is longer than the first period. The condition that the fringe cycle of the first light L1 is the second cycle corresponds to, for example, the fluorescence detection condition by the microscope MS in FIG. A range F3y in the first axis direction ky of the area F3 in FIG. 15A is narrower than a range F4y in the first axis direction ky of the area F4 in FIG. 15B corresponding to the first cycle.

The image processing unit 5 generates the second image, for example, based on the spatial frequency components included in the entire region F3 in FIG. 15A corresponding to the second period. The resolution of the second image in the first direction D1 is lower than the resolution of the second image in the first direction corresponding to the first period. Regarding the resolution of the second image corresponding to the second cycle in the second direction, the resolution in the first direction D1 is closer to the resolution in the second direction D2 than the resolution of the second image corresponding to the first cycle.

For example, in the area F3 of FIG. 15A corresponding to the second cycle, the range F3y in the first axis direction ky is the same as the range F3x in the second axis direction kx. The image processing unit 5 can generate a second image having the same resolution in the first direction D1 and the second direction D2, for example, based on the spatial frequency components included in the entire region F3.

In this way, the control unit 7 makes the resolution in the first direction D1 of the second image corresponding to the second period closer to the resolution in the second direction D2 than in the second image corresponding to the first period. Thus, the fringe period of the first light L1 is changed to the second period which is longer than the first period. As a result, the microscope MS can acquire, for example, the second image whose resolution isotropic is improved.

Next, the control of the control unit 7 in the case where the resolution of the second image in the first direction D1 is lower than the resolution in the second direction (hereinafter referred to as the second case) will be described. The second case corresponds to, for example, FIG. Here, it is assumed that the fluorescence detection condition by the microscope MS in FIG. 15C is a condition in which the fringe cycle of the first light L1 is the third cycle. Further, under this condition, in the area F5 in which the spatial frequency component exists, the range F5y in the first axis direction ky is narrower than the range F5x in the second axis direction kx. The image processing unit 5 generates, for example, a second image whose resolution in the first direction D1 is lower than that in the second direction D2, based on the spatial frequency components included in the entire area F5.

In the second case, the control unit 7 changes the fringe period of the first light L1 to the fourth period which is shorter than the third period. The condition that the fringe cycle of the first light L1 is the fourth cycle corresponds to, for example, the fluorescence detection condition by the microscope MS in FIG. The range F3y in the first axis direction ky of the area F3 in FIG. 15A is wider than the range F5y in the first axis direction ky of the area F5 in FIG. 15C corresponding to the third cycle.

The image processing unit 5 generates the second image, for example, based on the spatial frequency components included in the entire region F3 in FIG. 15A corresponding to the second period. The resolution of the second image in the first direction D1 is higher than the resolution of the second image in the first direction corresponding to the third period. Regarding the resolution in the second direction of the second image corresponding to the fourth cycle, the resolution in the first direction D1 is closer to the resolution in the second direction D2 than the second image corresponding to the third cycle.

For example, in the region F3 of FIG. 15A corresponding to the fourth period, the range F3y in the first axis direction ky is the same as the range F3x in the second axis direction kx. The image processing unit 5 can generate a second image having the same resolution in the first direction D1 and the second direction D2, for example, based on the spatial frequency components included in the entire region F3.

As described above, the control unit 7 makes the resolution in the first direction D1 of the second image corresponding to the fourth cycle closer to the resolution in the second direction D2, as compared with the second image corresponding to the third cycle. Thus, the fringe period of the first light L1 is changed to the fourth period which is shorter than the third period. As a result, the microscope MS can acquire, for example, the second image whose resolution isotropic is improved.

As described above, the fringe period of the first light L1 may be set based on the resolution in the first direction D1 and the resolution in the second direction D2 of the sample S obtained from the detection result of the detection unit 4. For example, the microscope MS may detect the fluorescence L under the first detection condition and change the fringe period of the first light L1 based on the detection result of the detection unit 4 corresponding to the first detection condition. In this case, the image processing unit 5 may perform frequency analysis based on the detection result of the detection unit 4 corresponding to the first detection condition. For example, the image processing unit 5 includes a first cutoff frequency corresponding to the first direction D1 of the spatial frequency component obtained by performing a Fourier transform on the detection result of the detection unit 4 and a second cutoff frequency corresponding to the second direction D2. And the cutoff frequency.

The control unit 7 sets the condition for detecting the fluorescence L to the second detection condition in which the fringe period of the first light L1 is different from the first detection condition, based on the result of the frequency analysis by the image processing unit 5. May be. The control unit 7 determines the first frequency in the first direction D1 based on the cutoff frequency corresponding to the first direction D1 and the cutoff frequency corresponding to the second direction D2 of the spatial frequency component obtained from the detection result of the detection unit 4. You may control the period in which the intensity|strength of 1 light L1 changes.

The detection unit 4 may detect the fluorescence L under the second detection condition, and the image processing unit 5 may generate the second image based on the detection result of the detection unit 4 corresponding to the second detection condition. .. The image processing unit 5 may or may not generate the second image corresponding to the first detection condition.

The second detection condition is based on at least one of the wavelength of the first light L1, the optical characteristic of the first optical system 12, the wavelength of the second light L2, and the optical characteristic of the second optical system 32. It may be previously derived experimentally or experimentally. In the first embodiment, the detection condition for detecting the fluorescence L by the microscope MS may be set to the second detection condition derived in advance. In this case, the 1st irradiation part 1 does not need to change the fringe period of the 1st light L1.

In the microscope MS, one or both of the wavelength of the first light L1 and the wavelength of the second light L2 may be changed based on the type of the fluorescent substance, for example. For example, one or both of the first light source 11 and the second light source 31 may be replaced based on the type of fluorescent material. In such a case, the microscope MS according to the present embodiment may change the fringe period of the first light L1 based on one or both of the wavelength of the first light L1 and the wavelength of the second light L2. The microscope MS according to the present embodiment may change the fringe period of the first light L1 based on a parameter (eg, wavelength) different from the resolution of the second image.

[Fourth Embodiment]
Next, a fourth embodiment will be described. In the present embodiment, configurations similar to those of the above-described embodiments are designated by the same reference numerals, and description thereof will be omitted or simplified. 16(A) to 16(C) are diagrams showing a second irradiation unit of the fourth embodiment. In the present embodiment, the second irradiation unit 2 sets the pattern of the second light L2 shown in FIG. 1 and the like using the mask 81, and projects this pattern on the sample surface SF (eg, relaying). In the present embodiment, the mask 81 includes an SLM, and the second irradiation unit 2 can change the interval between the pair of second regions A2a and A2b irradiated with the second light L2. In the following description, the mask 81 will be appropriately referred to as SLM81.

FIG. 16A shows the configuration from the second light source 31 to the lens 22 in the second irradiation section 2. The second irradiation unit 2 includes a lens 33, an SLM 81, a lens 36, and a lens 22 on the light emission side of the second light source 31. The configurations of the second light source 31 and the lens 33 and the configurations from the lens 22 to the sample surface SF (see FIG. 1) are the same as those in the first embodiment, and the description thereof will be simplified or omitted.

The second light L2 emitted from the second light source 31 enters the SLM 81 via the lens 33. The SLM 81 is arranged at or near the position of the sample conjugate plane SF4. The SLM 81 has a plurality of regions 82 on which the second light L2 is incident. The SLM 81 is, for example, an SLM using liquid crystal, and the region 82 is one pixel or two or more pixels. The SLM 81 has a variable transmittance for each region 82. The SLM 81 can switch each area 82 between a light blocking state in which the second light L2 is blocked and a transparent state in which the second light L2 is transmitted. The SLM 81 is controlled by the controller 7 to adjust the transmittance. For example, the control unit 7 supplies the SLM 81 with data (for example, bitmap data) representing the voltage applied to each of the plurality of regions 82. The SLM 81 sets the voltage pattern in the plurality of regions 82 based on the data supplied from the control unit 7.

FIGS. 16B and 16C are diagrams showing the transmittance distribution in the SLM 81 and the second region A2 on the sample surface SF, respectively. In FIGS. 14B and 14C, a region 82 having a relatively high transmittance is denoted by reference numeral 82a, and a region having a relatively low transmittance is denoted by reference numeral 82b. For example, the area 82a is the transparent area 82, and the area 82b is the light-blocking area 82. The area 82a has a shape corresponding to the second area A2. The region 82a has a strip shape in which the dimension in the Y direction corresponding to the first direction D1 is larger than the dimension in the X direction corresponding to the second direction D2.

Reference sign ΔQa in FIG. 16B is the interval (for example, the distance between the centers) of the regions 82a in the Y direction corresponding to the second direction D2 of the sample surface SF. Further, the reference sign ΔD2a is the distance (eg, the distance between the centers) between the pair of second regions A2a and A2b in the second direction D2 of the sample surface SF. In the following description, the interval between the pair of second regions A2a and A2b in the second direction D2 of the sample surface SF is appropriately referred to as the interval between the second regions A2. The distance ΔD2a between the second areas A2 depends on the distance ΔQa between the areas 82a in the SLM 81 and the magnification of the optical system.
--- Determined.

Reference sign ΔQb in FIG. 16C is the interval between the regions 82a in the X direction corresponding to the second direction D2 of the sample surface SF. The symbol ΔD2b is the interval between the second regions A2 on the sample surface SF. The distance ΔD2b between the second areas A2 is determined by the distance ΔQb between the areas 82a in the SLM 81 and the magnification of the optical system. The interval ΔQb between the regions 82a in FIG. 16C is narrower than the interval ΔQa between the regions 82a in FIG. 16B, and the interval ΔD2b between the second regions A2 in FIG. It is narrower than the interval ΔD2a between the two areas A2.

The control unit 7 controls the SLM 81 to control the interval between the pair of second regions A2 in the second direction D2. The control unit 7 can set one or both of the position and size of the second region A2 on the sample surface SF by setting the pattern of the region 82a on the SLM 81.

In the second image (eg, super-resolution image) generated by the image processing unit 5 illustrated in FIG. 1, the resolution corresponding to the second direction D2 depends on the interval between the second regions A2. The interval between the pair of second regions A2 in the second direction D2 is set based on the resolution in the first direction D1 and the resolution in the second direction D2 of the sample S obtained from the detection result of the detection unit 4. The microscope MS according to the present embodiment can change the resolution corresponding to the second direction D2 in the second image generated by the image processing unit 5 by controlling the interval between the second areas A2 by the control unit 7.

17A to 17C are diagrams showing the processing of the image processing unit of the fourth embodiment. In FIGS. 17A to 17C, reference character ky is the first axis direction in the frequency space corresponding to the first direction D1. The symbol kx is the second axis direction in the frequency space corresponding to the second direction D2. Reference numeral F1 is a region corresponding to the OTF of the optical system of the microscope MS.

Here, a state in which the interval between the regions 82a in the SLM 81 is set to a predetermined value is referred to as a fourth state. FIG. 17A is a diagram showing the spatial frequency components obtained in the fourth state. Since FIG. 17A is the same as FIG. 15A, the description thereof is omitted.

Further, a state in which the interval between the regions 82a in the SLM 81 is wider than that in the fourth state is referred to as a fifth state. FIG. 17B is a diagram showing the spatial frequency components obtained in the fifth state. Reference numeral F6 is a region in which the spatial frequency component is obtained by the image processing unit 5 executing processing based on the detection result of the detection unit 4 in the fifth state. Reference sign F6y is a range (eg, width) of the region F6 in the first axis direction ky. The spatial frequency at the end of the range F6 is a cutoff frequency corresponding to the first direction D1. The symbol F6x is the range (eg, width) of the region F6 in the second axis direction kx. The spatial frequency at the end of the range F6 is a cutoff frequency corresponding to the second direction D2.

The range F6x of the region F6 in the second axis direction kx is narrower than the range F3x of the region F3 in the second axis direction kx of FIG. 17A. The region F6 of FIG. 17B has a lower cutoff frequency corresponding to the second direction D2 than the region F3 of FIG. 17A. The image processing unit 5 uses the spatial frequency component in the area F6, so that the resolution in the second direction D2 is higher than that in the case where the detection result of the detection unit 4 corresponding to the fourth state is used (see FIG. 17A). It is possible to generate a second image having a low value.

Further, a state in which the interval between the regions 82a in the SLM 81 is narrower than that in the fourth state is referred to as a sixth state. FIG. 17C is a diagram showing the spatial frequency components obtained in the sixth state. Reference numeral F7 is a region in which the spatial frequency component is obtained by the image processing unit 5 executing processing based on the detection result of the detection unit 4 in the sixth state. Reference sign F7y is the range (eg, width) of the region F7 in the first axis direction ky. The spatial frequency at the end of the range F7y is a cutoff frequency corresponding to the first direction D1. The symbol F7x is the range (eg, width) of the region F7 in the second axis direction kx. The spatial frequency at the end of the range F7x is the cutoff frequency corresponding to the second direction D2.

The range F7x in the second axial direction kx of the region F7 corresponding to the sixth state is wider than the range F3x in the second axial direction kx of the region F3 corresponding to the fourth state shown in FIG. 17(A). The area F7 in FIG. 17C has a higher cutoff frequency corresponding to the second direction D2 than the area F3 in FIG. 17A. The image processing unit 5 uses the spatial frequency component in the region F7, so that the resolution in the second direction D2 is higher than that in the case where the detection result of the detection unit 4 corresponding to the fourth state is used (see FIG. 17A). It is possible to generate a second image having a high value.

As described above, the microscope MS according to the present embodiment can adjust the resolution corresponding to the second direction D2 in the second image generated by the image processing unit 5. The control unit 7 sets the resolution of the second image in the second direction D2 based on, for example, the resolution of the second image in the first direction D1. For example, the control unit 7 controls the SLM 81 so that the resolution of the second image in the first direction D1 and the resolution of the second image in the second direction D2 are the same.

Here, the control of the control unit 7 when the resolution in the first direction D1 is different from the resolution in the second direction in the second image acquired by the microscope MS will be described. First, the control of the control unit 7 in the case where the resolution of the second image in the first direction D1 is higher than the resolution in the second direction (hereinafter referred to as the third case) will be described.

The third case corresponds to FIG. 17B, for example. Here, it is assumed that the fluorescence detection condition by the microscope MS in FIG. 17B is a condition in which the interval between the second regions A2 is the first interval. Further, under this condition, in the region F6, the range F6y in the first axis direction ky is wider than the range F6x in the second axis direction kx. The image processing unit 5 generates, for example, a second image in which the resolution in the first direction D1 is higher than the resolution in the second direction D2, based on the spatial frequency components included in the entire area F6 corresponding to the first interval. ..

In the third case, the control unit 7 changes the interval of the second area A2 to the second interval that is narrower than the first interval. The condition that the interval between the second regions A2 is the second interval corresponds to the condition for detecting fluorescence by the microscope MS in FIG. 17A, for example. The range F3x in the second axis direction kx of the area F3 in FIG. 17A is wider than the range F6x in the second axis direction kx of the area F6 in FIG. 17B corresponding to the first interval.

The image processing unit 5 generates the second image, for example, based on the spatial frequency components included in the entire area F3 corresponding to the second interval. The resolution of the second image in the first direction D1 (see FIG. 17A) is higher than the resolution of the second image in the first direction (see FIG. 17B) corresponding to the first interval. As for the resolution in the second direction of the second image corresponding to FIG. 17A, the resolution in the second direction D2 is closer to the resolution in the first direction D1 than in the second image corresponding to FIG. 17B. ..

For example, in the area F3 of FIG. 17A corresponding to the second interval, the range F3x in the second axis direction kx is the same as the range F3y in the first axis direction ky. The image processing unit 5 can generate a second image having the same resolution in the first direction D1 and the second direction D2, for example, based on the spatial frequency components included in the entire region F3.

In this way, the control unit 7 makes the resolution in the second direction D2 of the second image corresponding to the second interval closer to the resolution in the first direction D1 compared to the second image corresponding to the first interval. Thus, the interval of the second region is changed to the second interval that is narrower than the first interval. As a result, the microscope MS can acquire, for example, the second image whose resolution isotropic is improved.

Next, the control of the control unit 7 in the case where the resolution of the second image in the second direction is higher than the resolution in the first direction D1 (hereinafter referred to as the fourth case) will be described. The fourth case corresponds to FIG. 17C, for example. Here, it is assumed that the fluorescence detection condition by the microscope MS in FIG. 17C is a condition in which the interval between the second regions A2 is the third interval. Further, under this condition, the range F7 is such that the range F7x in the second axis direction kx is wider than the range F7y in the first axis direction ky. The image processing unit 5 generates, for example, a second image in which the resolution in the second direction D2 is higher than the resolution in the first direction D1 based on the spatial frequency components included in the entire area F7 corresponding to the third interval. ..

In the third case, the control unit 7 changes the interval of the second area A2 to the fourth interval that is wider than the third interval. The condition that the interval between the second areas A2 is the fourth interval corresponds to, for example, the condition for detecting fluorescence by the microscope MS in FIG. The range F3x in the second axis direction kx of the region F3 in FIG. 17A is narrower than the range F7x in the second axis direction kx of the region F7 in FIG. 17C corresponding to the fourth interval.

The image processing unit 5 generates the second image, for example, based on the spatial frequency component included in the entire area F3 corresponding to the fourth interval. The resolution of the second image in the first direction D1 (see FIG. 17A) is lower than the resolution of the second image in the first direction (see FIG. 17C) corresponding to the fourth interval. As for the resolution in the second direction of the second image corresponding to FIG. 17A, the resolution in the second direction D2 is closer to the resolution in the first direction D1 than in the second image corresponding to FIG. 17C. ..

For example, in the region F3 of FIG. 17A corresponding to the fourth interval, the range F3x in the second axis direction kx is the same as the range F3y in the first axis direction ky. The image processing unit 5 can generate a second image having the same resolution in the first direction D1 and the second direction D2, for example, based on the spatial frequency components included in the entire region F3.

In this way, the control unit 7 makes the resolution in the second direction D2 of the second image corresponding to the fourth interval closer to the resolution in the first direction D1 compared to the second image corresponding to the third interval. As described above, the interval of the second region is changed to the fourth interval that is wider than the third interval. As a result, the microscope MS can acquire, for example, the second image whose resolution isotropic is improved.

As described above, the interval between the second areas A2 may be set based on the resolution in the first direction D1 and the resolution in the second direction D2 of the sample S obtained from the detection result of the detection unit 4. For example, the microscope MS may detect the fluorescence L under the third detection condition, and change the interval between the second regions A2 based on the detection result of the detection unit 4 corresponding to the third detection condition. In this case, the image processing unit 5 may perform frequency analysis based on the detection result of the detection unit 4 corresponding to the third detection condition. For example, the image processing unit 5 includes a first cutoff frequency corresponding to the first direction D1 of the spatial frequency component obtained by performing a Fourier transform on the detection result of the detection unit 4 and a second cutoff frequency corresponding to the second direction D2. And the cutoff frequency.

The control unit 7 sets the condition for detecting the fluorescence L to the fourth detection condition in which the interval between the second regions A2 is different from the third detection condition, based on the result of the frequency analysis by the image processing unit 5. Good. The control unit 7 determines the first frequency in the first direction D1 based on the cutoff frequency corresponding to the first direction D1 and the cutoff frequency corresponding to the second direction D2 of the spatial frequency component obtained from the detection result of the detection unit 4. You may control the space|interval of 2 area|region A2. The detection unit 4 may detect the fluorescence L under the fourth detection condition, and the image processing unit 5 may generate the second image based on the detection result of the detection unit 4 corresponding to the fourth detection condition. .. The image processing unit 5 may or may not generate the second image corresponding to the third detection condition.

The fourth detection condition is theoretical based on at least one of the wavelength of the first light L1, the optical characteristic of the first optical system 12, the wavelength of the second light L2, and the optical characteristic of the second optical system 32. It may be previously derived experimentally or experimentally. In the first embodiment, the detection condition for detecting the fluorescence L by the microscope MS may be set to the previously derived fourth detection condition. In this case, the 1st irradiation part 1 does not need to change the space|interval of 2nd area|region A2. In this case, the mask 81 does not have to be the SLM.

In the microscope MS, one or both of the wavelength of the first light L1 and the wavelength of the second light L2 may be changed based on the type of the fluorescent substance, for example. For example, one or both of the first light source 11 and the second light source 31 may be replaced based on the type of fluorescent material. In such a case, the microscope MS according to the present embodiment may change the interval between the second regions A2 based on one or both of the wavelength of the first light L1 and the wavelength of the second light L2. The microscope MS according to the present embodiment may change the interval between the second regions A2 based on a parameter (eg, wavelength) different from the resolution of the second image.

[Fifth Embodiment]
Next, a fifth embodiment will be described. In the present embodiment, configurations similar to those of the above-described embodiments are designated by the same reference numerals, and description thereof will be omitted or simplified. The microscope MS according to the present embodiment can change the fringe period of the first light L1 as described in the third embodiment, and can change the interval between the second regions A2 as described in the fourth embodiment. .. The microscope MS adjusts the illumination condition based on the resolution obtained from the detection result of the detection unit 4. The illumination condition includes one or both of the fringe period of the first light L1 and the interval of the second area A2. The detection unit 4 detects the fluorescence L emitted from the sample S in a state where the illumination condition is adjusted. The image processing unit 5 generates an image based on the detection result of the detection unit 4 when the illumination condition is adjusted.

FIG. 18 is a flowchart showing the operation of the microscope of the fifth embodiment. For each part of the microscope MS, refer to FIG. 1, FIG. 14, FIG. Further, the processing of steps S1 to S8 of FIG. 18 is the same as that of FIG. 7, and the description thereof will be omitted or simplified.

As described with reference to FIGS. 6A and 6B, the microscope MS detects the fluorescence L by the processes of steps S1 to S8 under a plurality of conditions in which the phases of the first light L1 are different. In step S11, the image processing unit 5 Fourier transforms the detection result of the fluorescence by the detection unit 4. In step S12, the image processing unit 5 calculates the first cutoff frequency corresponding to the first direction D1 and the second cutoff frequency corresponding to the second direction D2 of the spatial frequency component obtained by performing the Fourier transform. ..

In step S13, the control unit 7 determines whether to adjust the resolution of the generated second image. For example, the control unit 7 determines to adjust the resolution when the first cutoff frequency and the second cutoff frequency calculated by the image processing unit 5 satisfy a predetermined condition (step S13; Yes). The above-mentioned predetermined condition is, for example, a condition in which the ratio between the first cutoff frequency and the second cutoff frequency is outside the predetermined range, but may be other conditions.

When determining to adjust the resolution (step S13; Yes), the control unit 7 adjusts one or both of the stripe period of the first light L1 and the interval of the second area A2 in step S14. For example, the control unit 7 executes the process of step S14 based on the first cutoff frequency and the second cutoff frequency calculated by the image processing unit 5. For example, the control unit 7 sets the lower cutoff frequency of the first cutoff frequency and the second cutoff frequency closer to the higher cutoff frequency and the fringe period of the first light L1 and the second region. Adjust one or both of the intervals of A2.

Here, it is assumed that the first cutoff frequency is higher than the second cutoff frequency. In this case, the control unit 7 controls the SLM 81 shown in FIGS. 16A to 16C so that the second cutoff frequency approaches the first cutoff frequency to narrow the interval of the second region A2. .. The control unit 7 controls the SLM 77 shown in FIGS. 14A to 14C so that the first cutoff frequency approaches the second cutoff frequency to lengthen the fringe period of the first light L1. May be. For example, when the interval between the transmissive regions 82a is the lower limit value (eg, 1 pixel) and the first cutoff frequency and the second cutoff frequency do not satisfy the predetermined condition, for example, the control unit 7 The fringe period of one light L1 may be lengthened.

Here, it is assumed that the first cutoff frequency is lower than the second cutoff frequency. In this case, the control unit 7 controls the SLM 77 shown in FIGS. 14A to 14C so that the first cutoff frequency approaches the second cutoff frequency to shorten the fringe period of the first light L1. To do. The control unit 7 controls the SLM 81 shown in FIGS. 16A to 16C so that the second cutoff frequency approaches the first cutoff frequency to widen the interval of the second region A2. Good. For example, the control unit 7 sets the fringe period of the first light L1 to the lower limit value (for example, the limit at which the shifted spatial frequency component can be detected via the optical system), and the first cutoff frequency and the second cutoff frequency. When the cutoff frequency does not satisfy the predetermined condition, the interval between the second areas A2 may be widened.

The control unit 7 repeats the processing of steps S1 to S13 after the processing of step S14. When the control unit 7 determines that the resolution is not adjusted (step S11; No), the image processing unit 5 determines in step S9 the image based on the detection result of the detection unit 4 obtained in the processes of step S1 to step S8. To generate. The process of step S9 is the same as that of FIG.

The control unit 7 may determine in step S13 whether or not to adjust the resolution of the second image to be generated based on the designation by the user. For example, the microscope MS executes the processing from step S1 to step S9 as in FIG. 7, displays the image (eg, super-resolution image) generated by the image processing unit 5 on the display device, and adjusts the resolution. The user's designation of whether or not to accept may be accepted. The control unit 7 may determine not to adjust the resolution when the user specifies not to adjust the resolution or when the user does not specify to adjust the resolution. Further, the control unit 7 may determine to adjust the resolution when the user specifies that the resolution should be adjusted, or when the user does not specify that the resolution is not adjusted. In this case, the microscope MS may execute the processes of step S11, step S12, and step S14 after step S13 when the control unit 7 determines to adjust the resolution.

In the above-described embodiment, the control device 6 includes, for example, a computer system. The control device 6 reads the observation program stored in the storage unit 8 and executes various processes according to the observation program. In this observation program, for example, the first light of which the intensity is cyclically changed in the first direction of the sample surface on which the sample where the first light is irradiated and fluorescence is generated is arranged With respect to the control of irradiating the area and the second area set to the first side and the second side opposite to the first side in the second direction intersecting the first direction with respect to at least a part of the first area, , Control of irradiating the sample with the second light that suppresses the generation of fluorescence, control of scanning the sample surface in the second direction with the first light and the second light, and scanning of the first light and the second light. And a control for detecting the fluorescence generated based on the above. The above observation program causes the computer to control the cycle of changing the intensity of the first light in the first direction based on the resolution in the first direction and the resolution in the second direction of the sample obtained from the detection result of the detection. One or both of the control of the gap in the second direction between the second region on the first side and the second region on the second side may be executed. The observation program may be provided by being recorded in a computer-readable storage medium (eg, non-transitory tangible media).

Note that the technical scope of the present invention is not limited to the modes described in the above embodiments and the like. One or more of the requirements described in the above embodiments and the like may be omitted. In addition, the requirements described in the above-described embodiments and the like can be appropriately combined. Further, as far as legally permitted, the disclosure of all documents cited in the above-described embodiments and the like is incorporated as a part of the description of the text.

1... 1st irradiation part, 2... 2nd irradiation part, 3... scanning part, 4... detection part, 5... image processing part, 11... 1st light source, 12... ..First optical system, 14... Optical member, 15... Branching portion (diffraction grating), 32... Second optical system, 34... Phase adjusting unit, 54... Phase adjusting unit, 55... Optical member, A1... 1st area|region, A2... 2nd area|region, D1... 1st direction, D2... 2nd direction, L... Fluorescence, L1... 1 light, L2... second light, MS... microscope, S... sample

Claims (25)

A first irradiator that irradiates the first region of the sample surface with the first light whose intensity changes periodically in the first direction of the sample surface on which the sample irradiated with the first light and generating fluorescence is arranged. When,
A second irradiation unit that irradiates the second region of the sample surface with the second light that changes in intensity in the second direction intersecting the first direction and suppresses the generation of the fluorescence;
A scanning unit that scans the sample surface in the second direction with the first light and the second light;
A microscope comprising: a detection unit configured to detect the fluorescence generated based on the scanned first light and the second light. The first light with which at least a part of the first region is irradiated is a light flux that is longer in the first direction than in the second direction,
The microscope according to claim 1. The second irradiating section is configured with respect to at least a part of the first region with respect to the second region set on a first side in the second direction and a second side opposite to the first side. Irradiating the second light,
The microscope according to claim 1 or 2. The second irradiating unit irradiates the pair of second regions set at positions sandwiching at least a part of the first region in the second direction with the second light.
The microscope according to any one of claims 1 to 3. At least a part of the first region is a region including a position where the intensity of the second light becomes minimum in the second direction,
The microscope according to any one of claims 1 to 4. A position where the intensity of the second light becomes maximum in the second direction is set outside at least a part of the first region,
The microscope according to any one of claims 1 to 5. The first irradiation unit,
A first optical member in which the power corresponding to the second direction is stronger than the power corresponding to the first direction;
A branching unit for branching the first light into a plurality of light beams,
A mask arranged at a position where the plurality of light beams branched by the branch portion are incident,
A first optical system for irradiating the sample surface with interference fringes generated by interference of at least a part of the plurality of light fluxes passing through the mask,
The microscope according to any one of claims 1 to 6. The second irradiation unit,
A second optical member in which the power corresponding to the second direction is stronger than the power corresponding to the first direction;
A phase adjustment unit that includes a first region and a second region on which the second light is incident, and that imparts a phase difference between the second light incident on the first region and the second light incident on the second region. When,
A second optical system for irradiating the sample surface with interference fringes due to the interference of the second light to which the phase difference has been imparted by the phase adjuster.
The microscope according to any one of claims 1 to 7. At least a part of the optical system is common to the first irradiation unit and the second irradiation unit,
The optical system is
An optical member in which the power corresponding to the second direction is stronger than the power corresponding to the first direction,
The second light and the sixth region, which include a fifth region and a sixth region on which the first light and the second light are incident, and which are incident on the fifth region of the first light and the second light, respectively. A phase adjuster for selectively imparting a phase difference to the second light incident on
A mask including a region that transmits the first light and blocks the second light, and a region that blocks the first light and transmits the second light.
The microscope according to any one of claims 1 to 8. A cycle in which the intensity of the first light changes in the first direction is set based on the resolution of the sample in the first direction and the resolution of the second direction obtained from the detection result of the detection unit. The microscope according to any one of claims 1 to 9. The microscope according to any one of claims 1 to 10, further comprising a control unit that controls a cycle in which the intensity of the first light changes in the first direction. The control unit has a resolution in the second direction of the sample obtained from the detection result of the detection unit under the condition that the cycle of the intensity of the first light in the first direction is the first cycle. Changing the cycle of the intensity of the first light in the first direction to a second cycle longer than the first cycle when the resolution is lower than the resolution;
The microscope according to claim 11. An image processing unit that generates an image based on the detection result of the detection unit acquired under the condition that the cycle of the intensity of the first light in the first direction is the second cycle,
The microscope according to claim 12. The second region on the first side and the second region on the second side based on the resolution in the first direction and the resolution in the second direction of the sample obtained from the detection result of the detection unit. The microscope according to any one of claims 1 to 13, wherein an interval in the second direction is set. The microscope according to any one of claims 1 to 14, further comprising a control unit configured to control a gap in the second direction between the second region on the first side and the second region on the second side. .. When the resolution in the second direction of the sample obtained from the detection result of the detection unit is lower than the resolution in the first direction under the condition that the interval is the first interval, the controller sets the interval to the first interval. Change to a second interval that is shorter than one interval,
The microscope according to claim 15. The microscope according to claim 16, further comprising an image processing unit that generates an image based on a detection result of the detection unit acquired under the condition that the interval is the second interval. The intensity of the first light in the first direction based on the cutoff frequency corresponding to the first direction and the cutoff frequency corresponding to the second direction of the spatial frequency component obtained from the detection result of the detection unit. The control unit that controls one or both of a cycle in which the second changes on the first side and an interval in the second direction between the second area on the first side and the second area on the second side. Item 18. The microscope according to any one of items 17. Calculating a first cutoff frequency corresponding to the first direction and a second cutoff frequency corresponding to the second direction of the spatial frequency component obtained by performing a Fourier transform on the detection result of the detection unit,
A cycle in which the intensity of the first light changes based on the first cutoff frequency and the second cutoff frequency, the second region on the first side and the second side on the second direction. The image processing unit that generates an image based on a detection result of the detection unit in a state in which one or both of the interval between the second region and the second region is set is included in any one of claims 1 to 18. The microscope described. The microscope according to claim 1, further comprising an image processing unit that generates an image based on a detection result of the detection unit. The first irradiation unit irradiates, as the first light, excitation light that excites a fluorescent substance contained in the sample,
The second irradiating unit irradiates, as the second light, inactivating light that inactivates the fluorescent material excited by the first light,
The microscope according to any one of claims 1 to 20. The excitation light has the same wavelength as the activation light that activates the fluorescent substance,
The microscope according to claim 21. The wavelength of the first light is different from the wavelength of the second light,
The microscope according to claim 21 or 22. The first irradiation unit irradiates, as the first light, excitation light that excites a fluorescent substance contained in the sample,
The second irradiation unit irradiates, as the second light, stimulated emission light that causes the fluorescent material excited by the first light to cause stimulated emission,
The microscope according to any one of claims 1 to 20. Irradiating the first region of the sample surface with the first light whose intensity changes periodically in the first direction of the sample surface on which the sample that is irradiated with the first light and emits fluorescence is arranged;
Irradiating the second region of the sample surface with second light whose intensity changes in a second direction intersecting the first direction and which suppresses the generation of the fluorescence;
Scanning the sample surface in the second direction with the first light and the second light;
Detecting the fluorescence generated based on the scanned first light and the second light.