Structured illumination microscope

Abstract

A structured illumination microscope according to the present embodiment comprises: an excitation light source for generating excitation light; an activation light source that generates activation light to activate a fluorescent substance; an objective lens that focuses the excitation light onto a sample S; a structured illumination optical system that illuminates the sample using the excitation light as a structured illumination pattern; an activation optical system that irradiates the sample with activation 2 through the objective lens; and an imaging optical system that detects fluorescence generated at the sample via the objective lens to capture a fluorescent image of the sample.

Description

Structured illumination microscope

[0001] The present disclosure relates to structured illumination microscopes.

[0002] In the field of fluorescence microscopes, super-resolution microscopes have been developed as a technology that enables observations beyond the spatial resolution of conventional optical systems. Patent Document 1 discloses a structured illumination microscope (SIM) as a super-resolution microscope. Structured illumination microscopes achieve super-resolution by utilizing stripe-pattern illumination. Fluorescent images obtained by stripe illumination contain high-resolution information that cannot be detected by conventional microscopes, and super-resolution images can be generated by image processing. SIM is capable of observing a relatively wide area at low intensity and high speed, making it excellent for observing the structure and dynamics of biological samples.

[0003] WO 2016 / 125281

[0004] However, one of the challenges is that the observation target is limited to thin samples and it is not possible to observe the surface or interior of thick samples such as multi-layered cells or tissues because light emitted from outside the focal plane of the detection (and illumination) objective lens reduces the contrast of the stripe illumination image, making it difficult to generate super-resolution images.

[0005] In recent years, in order to improve the contrast of stripe illumination images and realize SIM observation of the inside of a sample, Patent Document 1 uses activation light (also called activation light) along with structured illumination light (excitation light). Patent Document 1 uses light sheet illumination, which irradiates a sheet of activation light from a direction perpendicular to the irradiation direction of the excitation light (structured illumination light). Light sheet illumination is a sheet of illumination formed on the focal plane of a detection objective lens, perpendicular to its optical axis. By exciting the sample with light sheet illumination that is thinner than the focal depth of the lens, background light can be suppressed.

[0006] Fluorescent substances (fluorescent dyes) are activated by activating light and enter an activated state in which they can emit fluorescence. When the fluorescent substance receives excitation light in the activated state, it emits fluorescence. The states in which the fluorescent substance can emit light and cannot emit light are referred to as ON and OFF states, respectively. Optically switching fluorescent molecules can be switched ON and OFF using activating light. This structured illumination microscope can reduce background light by turning on fluorescent molecules only in the illumination area of ​​the light sheet and performing SIM observations. In this way, by irradiating the fluorescent substance with activating light that turns on the fluorescent substance, it is possible to select the surface capable of emitting fluorescence (hereinafter referred to as SPA; Selective Plane Activation).

[0007] In Patent Document 1, activation light shaped into a sheet by a cylindrical lens is used. Alternatively, activation light shaped into a Bessel beam by an axicon lens or the like is used. Furthermore, the activation light shaped into a Bessel beam is scanned by a deflection mirror. However, when using light sheet illumination with a cylindrical lens or a Bessel beam, the following problems arise.

[0008] With cylindrical lenses, there is a trade-off between the thickness of the light sheet and the field of view. If the light sheet is made thinner to improve background light suppression, the area of ​​the light sheet in the field of view becomes smaller, reducing the effective field of view. Conversely, if the field of view is to be widened, the thickness of the light sheet cannot be made thin enough.

[0009] Bessel beams have high-intensity side lobes, which turn on fluorescent molecules in non-focal planes. As a result, the field of view can be secured, but the effect of suppressing background light is small. Another technique is to form a stripe pattern by scanning the Bessel beam used in light sheet illumination in a discontinuous manner. This method can be realized simply by innovating the illumination method, and has the advantage of not being limited by fluorescent molecules. However, the spatial resolution cannot be improved because the NA of the objective lens for fluorescence excitation cannot be increased.

[0010] The present disclosure has been made in consideration of the above points, and has as its object to provide a structured illumination microscope that can observe a sample with high resolution.

[0011] The structured illumination microscope according to this embodiment includes an excitation light source that generates excitation light for exciting a fluorescent substance contained in a sample; an activation light source that generates activation light for activating the fluorescent substance; a structured illumination optical system that has an objective lens that focuses the excitation light on the sample and illuminates the sample with the excitation light as a structured illumination pattern; an activation optical system that irradiates the sample with the activation light through the objective lens; and an imaging optical system that detects, via the objective lens, fluorescence generated from the sample in which the fluorescent substance is activated, in order to capture a fluorescent image of the sample.

[0012] In the above structured illumination microscope, the activation light source may generate laser light of a wavelength that activates the fluorescent material by two-photon absorption.

[0013] In the above-mentioned structured illumination microscope, the activation optical system may include a spinning disk that forms multiple foci on the sample, and the rotation of the spinning disk may scan the multiple foci, thereby activating the fluorescent material in the focal plane scanned by the multiple foci.

[0014] In the structured illumination microscope described above, the activation light may pass through a portion off the optical axis of the objective lens and be incident on the sample from a direction tilted from the optical axis.

[0015] In the above-mentioned structured illumination microscope, the activation optical system may include a mirror arranged on the side of the sample, and the activation light that passes through the objective lens may be reflected by the mirror and incident on the sample.

[0016] In the above-described structured illumination microscope, the activation optical system may convert the activation light into light sheet illumination and irradiate the sample.

[0017] In the structured illumination microscope, the activation optical system may irradiate the activation light onto the sample as a Bessel beam.

[0018] The above-mentioned structured illumination microscope may further include a first polarizer arranged in the optical path of the activation light and a second polarizer arranged in the optical path of the fluorescence, and the polarization direction of the activation light by the first polarizer and the polarization direction of the fluorescence by the second polarizer may be changed depending on the direction of the structured illumination pattern.

[0019] The above-described structured illumination microscope may further include a filter disposed in the optical path of the fluorescence to partially block or attenuate the fluorescence, the filter being configured to block or attenuate the fluorescence at both ends in a direction parallel to the stripes of the structured illumination pattern.

[0020] The structured illumination microscope according to this embodiment includes an excitation light source that generates excitation light for exciting a fluorescent substance contained in a sample; an activation light source that generates activation light for activating the fluorescent substance by two-photon absorption; a structured illumination optical system that has an objective lens that focuses the excitation light on the sample and irradiates the sample with the excitation light as a structured illumination pattern; an activation optical system that irradiates the sample with the activation light; and an imaging optical system that detects, via the objective lens, fluorescence generated from the sample in a state in which the fluorescent substance is activated, in order to capture a fluorescent image of the sample.

[0021] In the above-mentioned structured illumination microscope, the activation optical system may include a spinning disk that forms multiple foci on the sample, and the rotation of the spinning disk may scan the multiple foci, thereby activating the fluorescent material in the focal plane scanned by the multiple foci.

[0022] The structured illumination microscope according to this embodiment includes: an excitation light source that generates excitation light for exciting a fluorescent substance contained in a sample; an activation light source that generates activation light for activating the fluorescent substance; a structured illumination optical system that is arranged at an angle tilted from the vertical direction and has a first objective lens that focuses the excitation light on the sample and irradiates the sample with the excitation light as a structured illumination pattern; an activation optical system that is arranged at an angle tilted from the vertical direction and has a second objective lens that focuses the activation light and irradiates the sample with the activation light; and an imaging optical system that detects, via the first objective lens, fluorescence generated from the sample in which the fluorescent substance is activated, in order to capture a fluorescent image of the sample.

[0023] In the above structured illumination microscope, the activation light source may generate laser light of a wavelength that activates the fluorescent material by two-photon absorption.

[0024] According to the present disclosure, it is possible to provide a structured illumination microscope that can observe a sample with high resolution by reducing background light.

[0025] 1 is a diagram showing a simulation result of an observation image obtained with structured illumination. FIG. 2 is a diagram showing a simulation result of an observation image with SPA-SIM that combines structured illumination and light sheet illumination. FIG. 3 is a schematic diagram showing an example of SPA-SIM that combines structured illumination and light sheet illumination. FIG. 4 is a schematic diagram showing an example of SPA-SIM that combines structured illumination and Bessel beam illumination. FIG. 5 is a schematic diagram showing an example of SPA-SIM that combines structured illumination and Bessel beam illumination. FIG. 6 is a schematic diagram showing an example of SPA-SIM that combines structured illumination and Bessel beam illumination. FIG. 7 is a schematic diagram showing the optical system of a structured illumination microscope according to a first embodiment. FIG. 8 is a diagram showing an imaging result with SPA-SIM. FIG. 9 is a schematic diagram showing the optical system of a structured illumination microscope according to a first modification of the first embodiment. FIG. 10 is a diagram showing an imaging result with SPA-SIM by two-photon absorption. FIG. 11 is a diagram showing an imaging result with SPA-SIM by two-photon absorption. FIG. 12 is a schematic diagram showing the optical system of a structured illumination microscope according to a second embodiment. FIG. 13 is a diagram for explaining multifocal scanning using a spinning disk. 1 is a diagram schematically illustrating an optical system of a structured illumination microscope according to a second modification of the second embodiment. FIG. 2 is a diagram schematically illustrating an optical system of a structured illumination microscope according to a third modification of the second embodiment. FIG. 3 is a diagram schematically illustrating an optical system of a structured illumination microscope according to a fourth modification of the second embodiment. FIG. 4 is a diagram schematically illustrating an optical system of a structured illumination microscope according to a fifth modification of the second embodiment. FIG. 5 is a diagram for explaining an illumination pattern of structured illumination light and its polarization state. FIG. 6 is a diagram for explaining a polarization direction and a neutral density filter. FIG. 7 is a diagram illustrating a fluorescent image of mitochondria in a living cell and its spatial resolution. FIG. 8 is a diagram illustrating a fluorescent image inside a cell spheroid and its spatial resolution. FIG. 9 is a diagram illustrating a fluorescent image inside a cell spheroid. FIG. 10 is a diagram for explaining an optical system in which activation light is a stripe pattern. FIG. 11 is a diagram for explaining the stripe pattern of activation light and the stripe pattern of excitation light. FIG. 12 is a diagram for explaining the stripe pattern of activation light and the stripe pattern of excitation light.

[0026] Below, embodiments to which the present invention can be applied are described. The following description is for describing embodiments of the present invention, and the present invention is not limited to the following embodiments. For clarity of explanation, the following description has been omitted and simplified as appropriate. Furthermore, a person skilled in the art would be able to easily modify, add, or convert each element of the following embodiments within the scope of the present invention. Note that elements with the same reference numerals in each drawing indicate similar elements, and descriptions thereof will be omitted as appropriate.

[0027] First, a structured illumination microscope (SIM) will be described with reference to FIG. 1. FIG. 1 is a diagram for explaining the basic principle of a structured illumination microscope. FIG. 1 is a diagram showing the results of a simulation in which structured illumination is performed using an objective lens 132 with NA=0.8. In the following description, the optical axis direction of the objective lens 132 is the z direction. The directions orthogonal to the z direction are the x direction and the y direction, respectively. The x direction and the y direction are orthogonal to each other.

[0028] FIG. 1 shows the observed images and intensity profiles of samples S with different thicknesses. Specifically, the observed images and intensity profiles are shown when structured illumination is applied to a flat disk with a diameter of 10 μm and a sphere with a diameter of 10 μm. Structured illumination light L1 is incident on the sample S via an objective lens, and signal light L3 from the sample S is detected via an objective lens 132. Here, the structured illumination light L1 forms a stripe pattern with a constant width repeated. The longitudinal direction of the stripe pattern is the y direction. In other words, the stripe pattern of the structured illumination light L1 consists of bands of a constant width repeatedly arranged in the x direction. In the observed image of a thin disk, high-contrast stripes appear. On the other hand, in the observed image of a thick sphere, the stripe contrast is reduced.

[0029] In addition, in a structured illumination microscope, the fluorescence emission can be controlled by selective plane activation (SPA) using a light sheet illumination. For example, as shown in Figure 2, the light sheet illumination light is used as activation light L2 that controls the fluorescence emission of a switching fluorescent probe (switching fluorescent protein).

[0030] Figure 2 shows the simulation results when activation light L2 is irradiated onto a sample S from the side (x direction) of the sample. The activation light L2 is irradiated onto the sample S from the x direction. The optical axis direction of the objective lens 132 for the structured illumination light L1 and the signal light L3 is the z direction. Therefore, the structured illumination light L1 is irradiated onto the sample S from the z direction. The structured illumination light L1 forms a stripe pattern on the sample S. The stripe pattern is formed by two-beam interference or three-beam interference at the focal plane of the objective lens. In Figure 2, the activation light L2 is a light sheet parallel to the xy plane.

[0031] The sample S is stained with a switching fluorescent probe (hereinafter also referred to as a fluorescent substance or fluorescent molecule). The fluorescent substance is activated in the plane irradiated with the activation light L2. The fluorescent substance is activated and turned ON in the xy plane irradiated with the activation light L2. The focal plane of the objective lens 132 is aligned with the xy plane of the light sheet of the activation light L2. By doing so, the structured illumination light L1 excites the fluorescent substance that is turned ON in the focal plane of the objective lens. Fluorescence that becomes signal light L3 is generated from this xy plane. Meanwhile, in planes other than the focal plane, the fluorescent substance is not activated and is therefore in the OFF state. Therefore, even when the structured illumination light L1 is irradiated, no fluorescence is generated from planes other than the focal plane. Fluorescence that becomes background light is not generated at positions off the focal plane.

[0032] Specifically, signal light L3 is generated according to the product of the intensity of activation light L2 and the intensity of structured illumination light L1. Furthermore, in an actual sample, signal light L3 is detected which is the product of the intensity of activation light L2 and the intensity of structured illumination light L1 multiplied by the distribution of fluorescent materials in the sample.

[0033] The structured illumination microscope according to this embodiment irradiates activation light L2. In this way, the fluorescent material is activated and enters an ON state in which it can emit light. Fluorescence is emitted only from the surface of the sample that is in the ON state. The surface irradiated with activation light L2 is made to coincide with or intersect with the focal plane of the objective lens. Background light from outside the focal plane can be reduced. High contrast can be obtained even when observing a thick sample S. Thick samples can be observed with high resolution. A configuration in which activation light is applied to a structured illumination microscope is called SPA-SIM.

[0034] Next, the activation light of the SPA-SIM applicable to this embodiment will be described with reference to FIGS. 3 to 6. FIGS. 3 to 6 are schematic diagrams showing the configuration near the sample in the SPA-SIM and its illumination pattern. In FIGS. 3 to 6, the optical axis of the objective lens 132 for the structured illumination light and the signal light (observation light) is set to the z direction. FIGS. 3 to 6 also show a schematic configuration in which the activation light L2 is irradiated onto the sample S from the x direction. In other words, the optical axis of the optical path of the activation light L2 is parallel to the x direction.

[0035] In Figure 3, the activation light L2 is irradiated onto the sample S in a sheet-like form. In Figure 3, the light sheet is parallel to the xy plane. In the yz cross section, the activation light L2 is rectangular, with the y direction as the longitudinal direction and the z direction as the transverse direction. This allows fluorescent substances in a specific xy plane to be turned on. In other words, the position where fluorescence is generated can be limited in the z direction.

[0036] The illumination patterns are shown when the NA of the lens of the activation light L2 is set to 0.1 and 0.67. When NA = 0.1, the field of view can be widened. Since the size of the activation light L2 in the z direction cannot be reduced, it becomes difficult to obtain high resolution. On the other hand, when NA = 0.67, the activation light L2 expands before and after the focal position, limiting the area in which the fluorescent material is in the ON state. Therefore, although high resolution can be obtained, it becomes difficult to widen the field of view in the xy plane.

[0037] In Fig. 4, the activation light L2 is converted into a Bessel beam and irradiated onto the sample S. In this case, side lobes are generated, causing background light to be detected. This may result in a decrease in the contrast of the fluorescence image obtained with the stripe pattern structured illumination light L1.

[0038] In Fig. 5, the activation light L2, which is a Bessel beam, is scanned in the y direction. In this case, a strong side lobe is generated at the z position where the Bessel beam is irradiated. In Figs. 4 and 5, the activation light L2 is a laser beam with a wavelength of 405 nm.

[0039] 6 shows an example in which the activation light L2, which is a Bessel beam, has a two-photon absorption wavelength. Specifically, the activation light L2 is a laser beam with a wavelength of 780 nm. The fluorescent material is turned on by two-photon absorption of the laser beam with a wavelength of 780 nm. Furthermore, the activation light L2 is scanned in the y direction as in FIG. 6.

[0040] Two-photon absorption is a nonlinear optical effect proportional to the square of the laser light intensity. The absorption cross section of two-photon absorption is very small, making it a very unlikely phenomenon. Two-photon absorption becomes prominent when the laser light intensity is very high. Essentially, two-photon absorption occurs only at the focal position of the activation light L2 focused by a lens (not shown in FIG. 6 ). This allows the fluorescent material to be turned on in a spatially small area. Then, by scanning the focal position of the activation light L2 in the y- or xy-direction, the fluorescent material in the desired xy-plane can be turned on. This reduces background light from areas other than the desired xy-plane. Therefore, the contrast of the fluorescent image obtained with the stripe-pattern structured illumination light L1 can be increased, resulting in high resolution. Furthermore, scanning the focal position in the xy-direction allows a wide field of view to be obtained.

[0041] First Embodiment A structured illumination microscope 1 according to a first embodiment will be described with reference to Fig. 7. Fig. 7 is a schematic diagram showing the optical system of the structured illumination microscope 1. The structured illumination microscope 1 mainly includes a structured illumination optical system 100, an excitation light source 101, an activation optical system 200, an activation light source 201, and an imaging optical system 300.

[0042] First, a description will be given of the activation optical system 200 for guiding the activation light L2 to the sample S. The activation optical system 200 irradiates the activation light L2 onto the sample S from obliquely above.

[0043] The activation optical system 2300 includes an AO modulator 202 , a mirror 203 , a mirror 204 , a lens 205 , a mirror 206 , a cylindrical lens 231 , a lens 232 , a mirror 233 , a slit 234 , a lens 235 , a lens 236 , and an objective lens 237 .

[0044] The activation light source 201 generates activation light L2. The activation light source 201 generates, for example, a CW (Continuous Wave) laser beam having a wavelength of 405 nm. The activation light L2 from the activation light source 201 is incident on the AO modulator 202. The AO modulator 202 is an acousto-optic element that modulates the activation light L2. For example, the AO modulator 202 deflects the activation light L2 to switch between irradiating the activation light L2 on the sample S and irradiating the activation light L2 on the sample S.

[0045] A mirror 204 is removably disposed in the optical path of the activation light L2 from the AO modulator 202. The mirror 204 is provided to switch the optical path of the activation light L2. For example, when it is desired to observe the sample S with a wide field of view, the mirror 204 is inserted into the optical path. When wide-field observation is performed, the activation light L2 may be used as illumination light (excitation light) without structured illumination. When it is desired to observe the sample S with SPA-SIM, the mirror 204 is removed from the optical path.

[0046] When mirror 204 is inserted into the optical path, activation light L2 is reflected by mirror 203 and enters lens 205. Activation light L2 refracted by lens 205 is reflected by mirror 206 and enters slit 234. Note that mirror 233 is disposed in the optical path so as to be insertable and detachable. When mirror 204 is inserted into the optical path, mirror 233 is removed from the optical path. Therefore, activation light L2 from mirror 206 enters slit 234.

[0047] When the mirror 204 is removed from the optical path, the activation light L2 from the AO modulator 202 is incident on the cylindrical lens 231. The activation light L2 from the cylindrical lens 231 is incident on the mirror 233 via the lens 232. Note that when the mirror 204 is removed from the optical path, the mirror 233 is inserted into the optical path. The mirror 233 reflects the activation light L2 from the lens 232 toward the slit 234.

[0048] The activation light L2 passing through the slit 234 is incident on the sample S via the lens 235, the lens 236, and the objective lens 237. The slit 234 is positioned conjugate with the pupil of the objective lens 237, allowing the effective NA of the activation light to be controlled. The objective lens 237 is, for example, an immersion objective lens with a magnification of 28.6x and an NA of 0.7. A medium W with a refractive index higher than that of air is provided between the objective lens 237 and the sample S. Specifically, the medium W is a liquid such as water or immersion oil. In the activation optical system 200, the activation light L2 is shaped into a light sheet by the cylindrical lens 231, the slit 234, etc. The sheet-shaped activation light L2 can be irradiated onto the sample S. The activation light L2 serves as light sheet illumination. The light sheet is a plane tilted from the optical axis of the objective lens 132 of the structured illumination optical system 100.

[0049] When the activation light L2 is irradiated, the fluorescent material in the sample S is turned on. Therefore, when the sample S is irradiated with structured illumination light L1 (described later), fluorescence is generated from the fluorescent material in the region that is turned on.

[0050] A structured illumination optical system 100 for guiding structured illumination light L1 to a sample S will be described. The structured illumination optical system 100 is an optical system for irradiating the sample S with structured illumination light L1 from obliquely above. Note that the structured illumination light L1 is incident on the sample S from a different direction than the activation light L2. Furthermore, the structured illumination optical system 100 shapes the structured illumination light L1 into a stripe pattern on the sample S in order to perform structured illumination.

[0051] The structured illumination optical system 100 includes an AO modulator 120, a mirror 107, a λ / 2 plate 108, a polarizing beam splitter 110, a spatial light modulator 111, a λ / 2 plate 121, a lens 112, a polarization control element 122, a mask 113, a lens 114, a dichroic mirror 116, a lens 131, and an objective lens 132.

[0052] The excitation light source 101 is, for example, a laser light source that generates a CW laser light with a wavelength of 488 nm. The laser light generated by the excitation light source 101 is referred to as structured illumination light L1. The structured illumination light L1 serves as excitation light that excites the fluorescent material in the sample S. The structured illumination light L1 from the excitation light source 101 is incident on the AO modulator 120.

[0053] The AO modulator 120 is an acousto-optical element that switches between the presence and absence of structured illumination light L1. The structured illumination light L1 from the AO modulator 120 is reflected by a mirror 107 and enters a λ / 2 plate 108. The λ / 2 plate 108 polarizes the structured illumination light L1 in a desired polarization direction. Therefore, the structured illumination light L1 that passes through the λ / 2 plate 108 is reflected by a polarizing beam splitter 110 and enters a spatial light modulator 111.

[0054] The spatial light modulator 111 spatially modulates the structured illumination light L1. The spatial light modulator 111 is, for example, an LCOS-SLM (Liquid Crystal On Silicon-Spatial Light Modulator) device. The spatial light modulator 111 has a plurality of pixels, and a voltage is applied to each pixel. The spatial light modulator 111 can control the wavefront of the structured illumination light L1. As a result, a desired structured illumination pattern can be formed on the sample S. Furthermore, when the structured illumination light L1 is reflected by the spatial light modulator 111, the polarization direction changes. Therefore, the structured illumination light L1 from the spatial light modulator 111 passes through the polarizing beam splitter 110.

[0055] The structured illumination light L1 that passes through the polarizing beam splitter 110 passes through a λ / 2 plate 121 and is polarized in a desired direction. The structured illumination light L1 from the λ / 2 plate 121 is incident on a polarization control element 122 via a lens 112. The polarization control element 122 is, for example, a retarder (phase plate) that converts linearly polarized light to radially polarized light. The structured illumination light L1 that passes through the polarization control element 122 is incident on a mask 113. The mask 113 has an aperture for partially transmitting light. For example, the mask 113 has two circular apertures that are symmetrically arranged with respect to the optical axis. A fringe pattern is formed on the sample S by interference between the +1st-order diffracted light from one aperture and the −1st-order diffracted light from the other aperture.

[0056] The structured illumination light L1 from the mask 113 is refracted by the lens 114 and enters the dichroic mirror 116. The dichroic mirror 116 is a beam splitter that splits the structured illumination light L1 and the signal light L3 according to wavelength. The dichroic mirror 116 reflects light of the excitation light wavelength and transmits light of the fluorescence wavelength. The structured illumination light L1 reflected by the dichroic mirror 116 enters the sample S via the lens 131 and the objective lens 132. The objective lens 132 is, for example, an immersion objective lens with a magnification of 25x and an NA of 11. The optical axis of the objective lens 132 is tilted from the vertical direction. The objective lens 132 is, for example, tilted 30° or 60° from an upright orientation. The objective lens 132 focuses the structured illumination light L1 on the sample S, forming a structured illumination stripe pattern on the sample S.

[0057] The imaging optical system 300 will now be described. The imaging optical system 300 includes an objective lens 132, a lens 131, a dichroic mirror 116, filters 311 and 312, and a camera 305. The objective lens 132, the lens 131, and the dichroic mirror 116 are common to the structured illumination optical system 100.

[0058] Fluorescence generated in the sample S is incident on the objective lens 132 as signal light L3. The signal light L3 is incident on the dichroic mirror 116 via the objective lens 132 and the lens 131. The signal light L3 passes through the dichroic mirror 116 and is incident on the filters 311 and 312. The filter 311 is a long-pass filter and has wavelength characteristics that transmit light of the fluorescent wavelength. The filter 312 is a filter that cuts the activation light L2. Therefore, the signal light L3 passes through the filters 311 and 312 and is incident on the camera 305. In this way, the imaging optical system 300 detects the fluorescence, which is the signal light L3, via the objective lens 132.

[0059] The lens 131 forms an image of the sample S on the camera 305. The camera 305 is a two-dimensional photodetector such as a charge coupled device (CCD) sensor or a complementary metal oxide semiconductor (CMOS) image sensor. The camera 305 has a plurality of pixels arranged along the X and Y directions, for example. The camera 305 captures an image of the sample S illuminated by the structured illumination light L1. The camera 305 outputs data of the captured image (also referred to as a fluorescent image) to the processing device 330.

[0060] The processing device 330 constructs a super-resolution image based on the structured illumination fluorescence image of the sample S. For example, the structured illumination optical system 100 changes the direction of the structured illumination stripe pattern. The camera 305 captures fluorescence images in the direction of each stripe pattern. The processing device 330 can emphasize high-frequency components by demodulating the multiple fluorescence images. The processing device 330 then calculates a super-resolution image of the sample S based on the harmonic components. Since known techniques can be used for image formation using structured illumination and its image processing, detailed explanations will be omitted.

[0061] Figure 8 shows the results of imaging with a structured illumination microscope using activation light (SPA-SIM). As comparative examples, Figure 8 also shows the results of imaging with structured illumination without activation light L2 (SIM) and with normal illumination (widefield). The fluorescent image shows mitochondria from fixed HeLa cells. The fluorescent protein used is Skylan-NS.

[0062] In fluorescence images taken with SPA-SIM, background light can be suppressed more effectively than in fluorescence images taken with standard SIM. Even with thick samples, background light from planes other than the focal plane can be suppressed. Therefore, even with thick samples S such as multi-layered cells or tissues, the interior of the sample S can be observed with high resolution. Furthermore, a wide field of view can be imaged with high resolution.

[0063] Modification 1 A structured illumination microscope 1 according to Modification 1 of the first embodiment will be described with reference to FIG. 9 . The structured illumination microscope 1 of Modification 1 includes an activation light source 261 that generates activation light L4 at a two-photon absorption wavelength. Furthermore, an activation optical system 200 shapes the activation light L2, L4 into a Bessel beam. Note that the configuration other than the activation optical system 200 is the same as that shown in FIG. 7 , and therefore description thereof will be omitted where appropriate. For example, the structured illumination optical system 100 and the imaging optical system 300 have the same configuration as that shown in FIG. 7 .

[0064] The activation optical system 200 includes an activation light source 261, a mirror 263, an axicon lens 264, a lens 265, and an optical scanner 266. The activation light source 261 generates activation light L4, which is a pulsed laser beam. The activation light L4 is, for example, a femto-pulse laser beam with a wavelength of 780 nm. In the sample S, two-photon absorption of the activation light L4 turns the fluorescent substance into an ON state.

[0065] The mirrors 263, 204, and 233 are removably inserted into the optical path. This allows switching between activation by two-photon absorption and activation by one-photon absorption. Specifically, when activation by two-photon absorption or one-photon absorption is performed, the mirror 233 is removed from the optical path. When the mirror 263 is removed from the optical path of the activation light L4, activation by two-photon absorption is performed. When the mirrors 263 and 204 are inserted into the optical path of the activation light L2, activation by one-photon absorption is performed. When the mirror 233 is inserted into the optical path and the mirror 204 is removed from the optical path of the activation light L2, wide-field observation is performed.

[0066] The following describes the case where a fluorescent substance is activated by two-photon absorption of activation light L4. In this case, mirrors 263 and 233 are removed from the optical path. The activation light L4 is incident on an AO modulator 262. The AO modulator 262 modulates the activation light L4. The activation light L4 modulated by the AO modulator 262 is incident on an optical scanner 266 via an axicon lens 264 and a lens 265. The axicon lens 264 is provided to convert the activation light L2 and L4 into a Bessel beam.

[0067] The optical scanner 266 is, for example, a galvanometer mirror, and scans the activation light L4. This allows the focal position of the activation light L4 on the sample S to be changed. Therefore, the area where the fluorescent material is in the ON state can be changed. The activation light L4 is then incident on the sample S via the lens 235, the lens 236, and the objective lens 237. The objective lens 237 focuses the activation light L4 on the sample S. The fluorescent material is in the ON state at the position where the activation light L4 is focused by the objective lens 237. When the activation light L4 turns the fluorescent material into the ON state, the structured illumination optical system 100 illuminates the sample S with structured illumination light L1. This allows a fluorescent image of the sample S to be captured with high resolution.

[0068] Furthermore, when the fluorescent material is turned ON by one-photon absorption of the activation light L2, mirrors 204 and 263 are inserted into the optical path, and mirror 233 is removed from the optical path. Therefore, the activation light L2 from the AO modulator 202 is reflected by mirrors 204 and 263 and enters the axicon lens 264. The activation light L2 from the axicon lens 264 propagates along the same optical path as the activation light L4 and is focused on the sample S. The optical scanner 266 scans the activation light L2. This allows the focal position of the activation light L2 on the sample S to be changed.

[0069] Figure 10 shows the results of imaging using SPA-SIM with two-photon absorption. Sample S is a HeLa cell histone H2B. The fluorescent protein used to label sample S is rsGamillus-S. Figure 10 shows a fluorescent image captured using SPA-SIM with two-photon absorption, a fluorescent image captured using SIM, and a fluorescent image captured with a wide field of view. By turning on the fluorescent protein through two-photon absorption, only the internal structure of sample S can be made to emit light. This suppresses the reduction in contrast due to background light, allowing for the acquisition of high-resolution super-resolution images.

[0070] Figure 11 shows the results of imaging using SPA-SIM with two-photon absorption. Sample S is histone H2B inside a cell spheroid (diameter: approximately 100 μm, depth: 43 μm). The fluorescent protein used to label sample S is rsGamillus-S. Figure 11 shows a fluorescent image captured using SPA-SIM with two-photon absorption, a fluorescent image captured using SIM, and a fluorescent image captured with a wide field of view.

[0071] With activation by one-photon absorption, it is difficult to detect fringes, making it impossible to observe the inside of the sample. On the other hand, with activation by two-photon absorption, only the internal structure of the sample S can be made to emit light. This makes it possible to suppress the reduction in contrast caused by background light, thereby enabling the acquisition of high-resolution super-resolution images.

[0072] In the first embodiment and its first modification, the activation light L2 and L4 are incident on the sample S from a different direction from the structured illumination light L1. Specifically, the objective lens 132 of the structured illumination optical system 100 is tilted from an upright position. The objective lens 237 of the activation optical system 200 is also tilted from an upright position. The objective lenses 132 and 237 are each tilted. This allows the plane that turns the fluorescent material ON to be tilted from the focal plane of the objective lens 132. This reduces background light from other than the focal plane of the objective lens 132. Since the reduction in contrast of the observed image can be suppressed, high-resolution observation is possible.

[0073] As described above, in this embodiment, the structured illumination microscope 1 includes a structured illumination optical system 100 , an excitation light source 101 , an activation optical system 200 , an activation light source 201 , and an imaging optical system 300 .

[0074] The excitation light source 101 generates excitation light for exciting a fluorescent substance contained in the sample S. The activation light source 201 generates activation light for activating the fluorescent substance. The structured illumination optical system 100 has an objective lens 132, and irradiates the sample S with the excitation light as a structured illumination pattern. The objective lens 132 is disposed at an angle inclined from the vertical direction, and focuses the excitation light on the sample S.

[0075] The activation light source 201 has an objective lens 237 and irradiates the sample S with activation light L2. The objective lens 237 is positioned at an angle tilted from the vertical direction to focus the activation light. The objective lens 237 is also positioned at a different angle from the objective lens 132. The imaging optical system 300 detects, via the objective lens 132, fluorescence emitted from the sample in which the fluorescent substance is activated. This allows a fluorescent image of the sample to be captured. A processing device 330 constructs a super-resolution image from multiple fluorescent images captured by changing the direction of the structured illumination pattern.

[0076] This reduces background light from outside the focal plane, preventing a decrease in the contrast of the fluorescent image, and thus enabling high-resolution observation of even thick samples.

[0077] Second Embodiment In a structured illumination microscope according to a second embodiment, a common objective lens 132 is used by the structured illumination optical system 100 and the activation optical system 200. That is, activation light L2 and structured illumination light L1 are collected by the common objective lens 132 and irradiated onto the sample S. The structured illumination microscope according to the second embodiment will be described with reference to Fig. 12 . Fig. 12 is a schematic diagram showing the optical system of the structured illumination microscope 1.

[0078] The structured illumination optical system 100 includes a lens 102, a pinhole plate 103, a lens 104, a mirror 107, a polarizing beam splitter 110, a spatial light modulator 111, a lens 112, a mask 113, a lens 114, a lens 115, a dichroic mirror 116, and an objective lens 132. Note that descriptions of the same components as those in the first embodiment and its modifications will be omitted where appropriate.

[0079] The excitation light source 101 is a CW laser light source that generates structured illumination light L1. The structured illumination light L1 is condensed by a lens 102 and enters a pinhole plate 103. A pinhole is formed on the optical axis of the pinhole plate 103. Therefore, the structured illumination light L1 condensed by the lens 102 passes through the pinhole plate 103.

[0080] The structured illumination light L1 from the pinhole plate 103 is incident on a mirror 107 via a lens 104. The structured illumination light L1 reflected by the mirror 107 is reflected by a polarizing beam splitter 110 and incident on a spatial light modulator 111. The spatial light modulator 111 is an LCOS-SLM device or the like, and controls the wavefront of the structured illumination light L1.

[0081] The structured illumination light L1 modulated by the spatial light modulator 111 passes through the polarizing beam splitter 110 and enters the lens 112. The structured illumination light L1 from the lens 112 passes through a mask 113, lenses 114, and 115 and enters the dichroic mirror 116. The dichroic mirror 116 reflects the structured illumination light L1 toward the objective lens 132. The objective lens 132 focuses the structured illumination light L1 on the sample S. A structured illumination stripe pattern is formed on the sample S by the spatial light modulator 111, the mask 113, and the like. The objective lens 132 is disposed directly below the sample S. The z direction, which is the optical axis direction of the objective lens 132, is parallel to the vertical direction.

[0082] 12 shows the aperture pattern of the mask 113 in the structured illumination optical system 100. In the case of two-dimensional SIM, the mask 113 has six circular apertures. The six circular apertures are positioned at equal intervals of 60° in the circumferential direction. In the case of three-dimensional SIM, a circular aperture is added on the optical axis, so the mask has seven circular apertures.

[0083] The activation optical system 200 will now be described. The activation optical system 200 includes a lens 212, a pinhole plate 213, a lens 214, a spinning disk 215, a lens 216, and a dichroic mirror 217.

[0084] The activation light source 201 is a pulsed laser light source that generates activation light L4. Here, the activation light L4 has a wavelength that turns on the fluorescent material by two-photon absorption. For example, the wavelength of the activation light L4 is 560 nm.

[0085] Activation light L4 from activation light source 201 is incident on spinning disk 215 via lens 212, pinhole plate 213, and lens 214. A pinhole is formed on the optical axis of pinhole plate 213. Therefore, activation light L4 condensed by lens 212 passes through pinhole plate 213.

[0086] A microlens array 215a for forming multiple focal points is formed on the spinning disk 215. The spinning disk 215 is a rotating disk that scans the multiple focal points. The configuration of the spinning disk 215 will be described later. The activation light L4 from the spinning disk 215 is incident on a dichroic mirror 217 via a lens 216.

[0087] The dichroic mirror 217 is a beam splitter that splits light according to wavelength. The dichroic mirror 217 reflects light with the wavelength of the activation light L4 and transmits light with the fluorescence wavelength. Therefore, the dichroic mirror 217 reflects the activation light L4 toward the objective lens 132. The dichroic mirror 116 also reflects light with the excitation light wavelength and transmits light with the fluorescence wavelength and the wavelength of the activation light L4. Therefore, the activation light L4 passes through the dichroic mirror 116 and enters the objective lens 132. The objective lens 132 focuses the activation light L4 on the sample S.

[0088] Here, a microlens array 215a for forming multiple focal points is arranged on the spinning disk 215. The configuration of the spinning disk 215 will be described with reference to FIG. 13. FIG. 13 is a diagram for explaining activation light L4 passing through the spinning disk 215. Furthermore, FIG. 13 shows the xz-plane distribution of light intensity obtained by multifocal scanning. Specifically, the distribution at a single focal point, the sheet distribution obtained by multifocal scanning, and the distribution of the stripe pattern of structured illumination light are shown.

[0089] The spinning disk 215 has a microlens array 215a. In the microlens array 215a, a plurality of microlenses 215b are arranged in a spiral. The activation light L4 is split into a plurality of sub-beams L4s by the plurality of microlenses 215b. A single focus by one microlens 215b results in a minute spot distribution.

[0090] The microlens array 215a forms multifocal points MS on the sample S. The rotation axis of the spinning disk 215 is parallel to the optical axis. By rotating the spinning disk 215, the multifocal points MS of the activation light L4 are spin-scanned. When the spinning disk 215 rotates, the xy positions of the multifocal points MS on the sample S change. By the multifocal scanning, the activation light L4 becomes a sheet distribution on the sample S that is parallel to the xy plane.

[0091] As described above, two-photon absorption occurs only at the focal position of the activation light L4. Therefore, the fluorescent material is in the ON state at the focal plane of the objective lens 132. In other words, the fluorescent material is in the OFF state at planes other than the focal plane of the objective lens 132. By multi-focal scanning, the fluorescent material can be turned ON in a sheet-like region.

[0092] The fluorescent material is turned on by two-photon absorption, and then irradiated with structured illumination light L1. The structured illumination light L1 forms a stripe pattern on the focal plane. Activating the fluorescent material through two-photon absorption allows fluorescence to be generated from the focal plane of the objective lens 132. Meanwhile, the fluorescent material is in the off state in the xy plane other than the focal plane of the objective lens 132. This prevents fluorescence from being generated in the xy plane other than the focal plane, thereby suppressing background light.

[0093] The imaging optical system 300 will now be described. The imaging optical system 300 includes an objective lens 132, a dichroic mirror 116, a dichroic mirror 217, a filter 311, a filter 312, a lens 303, and a camera 305.

[0094] Fluorescence emitted from the focal plane is incident on the objective lens 132 as signal light L3. The signal light L3 from the objective lens 132 passes through the dichroic mirrors 116 and 217 and is incident on the filter 311. The filter 311 is a long-pass filter that transmits the fluorescent wavelength. The filter 312 is a filter for cutting the activation light. The signal light L3 from the filter 312 is imaged on the camera 305 by the lens 303.

[0095] The camera 305 detects the signal light L3 that has passed through the filters 311 and 312. This makes it possible to capture a fluorescent image of the sample S illuminated by the structured illumination light L1. The structured illumination microscope 1 captures the fluorescent images by changing the direction of the stripe pattern. A processing device (see FIG. 7) demodulates the multiple fluorescent images to obtain a super-resolution image.

[0096] In this way, the objective lens 132 is common to the structured illumination optical system 100 and the activation optical system 200. This simplifies the device configuration, making it easy to adjust the optical axis, etc.

[0097] In this embodiment, by using multiple microlenses 215b, multiple focal points can be formed simultaneously on the sample S. Two-photon excitation localizes the distribution in three dimensions. In other words, the fluorescent material is not activated in the xy plane other than the focal plane. Therefore, the fluorescent material is in the ON state on the focal plane, and the fluorescent material can remain in the OFF state outside the focal plane. This reduces background light from outside the focal plane, preventing a decrease in contrast. Therefore, the sample S can be observed with high resolution.

[0098] Modification 2 A structured illumination microscope 1 according to Modification 2 will be described with reference to FIG. 14 . FIG. 14 is a schematic diagram showing the optical system of the structured illumination microscope 1. In Modification 2, the wavelength of the activation light L is a one-photon absorption wavelength. The activation light L2 may be a pulsed laser beam or a CW laser beam. The activation light L2 is incident on the sample S via the objective lens 132. The activation light L2 is a light sheet.

[0099] In the structured illumination microscope 1 of Modification 2, the activation optical system 200 has a different configuration from that of Embodiment 2. Specifically, the configuration within the dashed rectangular frame in Fig. 14 is different from the configuration in Fig. 12. The basic configuration other than the activation optical system 200 is the same as that in Fig. 12, and therefore a description thereof will be omitted where appropriate.

[0100] The activation optical system 200 is provided with a cylindrical lens 241 and a lens 242 instead of the spinning disk 215 and the lens 216. The cylindrical lens 241 focuses the activation light L2 into a line shape in the xy plane. The activation light L2 from the cylindrical lens 241 is incident on the dichroic mirror 217 via the lens 242. The activation light L2 reflected by the dichroic mirror 217 is then irradiated onto the sample S via the dichroic mirror 116 and the objective lens 132.

[0101] The activation light L2 incident on the sample S will be described in detail below. Fig. 14 shows a cross-sectional configuration of the sample surface and the vicinity of the objective lens 132. Specifically, xz cross section and yz cross section around the objective lens 132 are shown. The sample S is placed on the focal plane FP of the objective lens 132.

[0102] The focal plane FP is a plane parallel to the xy plane. The cylindrical lens focuses the activation light L2. Therefore, the activation light L2 becomes a sheet-like illumination extending in the y direction. The light sheet is a plane tilted from the optical axis of the objective lens 132. The light sheet is a plane including the y direction. The light sheet is also a plane including an inclined direction tilted from the x direction and the z direction. In the area irradiated by the light sheet, the fluorescent material is turned ON.

[0103] Furthermore, the activation light L2 is incident on only a portion of the pupil of the objective lens 132 at a position outside the optical axis OX. Specifically, the activation light L2 is incident on the pupil of the objective lens 132 at a position shifted in the -x direction from the optical axis OX of the objective lens 132. The objective lens 132 refracts the activation light L2 in the +x direction. Therefore, the activation light L2 emitted from the objective lens 132 is incident on the sample S from a direction tilted from the optical axis OX. The activation light L2 intersects with the optical axis OX at the focal plane FP.

[0104] This arrangement suppresses the generation of background light. That is, it is possible to prevent the fluorescent material in the field of view from being turned on in the xy plane other than the focal plane FP of the structured illumination light L1. This prevents a decrease in the contrast of the fluorescent image, allowing the sample S to be imaged with high resolution.

[0105] Modification 3 A structured illumination microscope 1 according to Modification 3 will be described with reference to FIG. 15 . FIG. 15 is a schematic diagram showing the configuration of the optical system of the structured illumination microscope 1. In Modification 3, the wavelength of the activation light L is a one-photon absorption wavelength. The activation light L2 may be a pulsed laser beam or a CW laser beam. The activation light L2 is incident on the sample S via the objective lens 132. The activation light L2 is not a light sheet.

[0106] In the structured illumination microscope 1 of Modification 3, the activation optical system 200 has a configuration different from that of Modification 2. Specifically, the configuration within the dashed rectangular frame in Fig. 15 is different from the configuration in Fig. 14. The basic configuration other than the activation optical system 200 is the same as in Figs. 12 and 14, so a description thereof will be omitted where appropriate. The activation optical system 200 includes a mirror 243, an optical scanner 244, a lens 245, and a lens 246.

[0107] The activation light L2 from the lens 214 is reflected by a mirror 243 and enters an optical scanner 244. The optical scanner 244 is a galvanometer mirror that scans the activation light. The activation light L2 scanned by the optical scanner 244 enters a dichroic mirror 217 via lenses 245 and 246. Note that the activation light L2 may be a Bessel beam by providing an axicon lens in the activation optical system 200.

[0108] The optical scanner 244 scans the activation light L2 in the Y direction on the focal plane FP. This makes it possible to change the area of ​​the sample S where the fluorescent material is turned on. This makes it possible to suppress a decrease in the contrast of the fluorescent image, as in the third modification, and thus to image the sample S with high resolution.

[0109] Modification 4 A structured illumination microscope 1 according to Modification 4 will be described with reference to Fig. 16 . Fig. 16 is a schematic diagram showing the configuration of the optical system of the structured illumination microscope 1. In the structured illumination microscope 1 of Modification 4, the activation optical system 200 differs from that of Modification 2. Specifically, the configuration within the dashed rectangular frame in Fig. 16 differs from that of Fig. 14 . The basic configuration other than the activation optical system 200 is the same as in Figs. 12 and 14 , and therefore description thereof will be omitted where appropriate.

[0110] In the fourth modification, the wavelength of the activation light L2 is a one-photon absorption wavelength. Therefore, the activation light L2 may be a pulsed laser beam or a CW laser beam. The activation light L2 is incident on the sample S via the objective lens 132.

[0111] In Modification 4, an optical scanner 251, an optical scanner 252, a lens 253, and a tunable lens 254 are added to the activation optical system 200 compared to the configuration of Modification 2. Furthermore, a mirror 256 is disposed on the exit side of the objective lens 132. The configuration other than these is the same as that of Modification 2, and therefore description thereof will be omitted as appropriate.

[0112] The activation light L2 from the lens 242 is reflected by the optical scanners 251 and 252 and enters the lens 253. The optical scanners 251 and 252 are galvanometer mirrors that scan the activation light L2. The activation light L2 from the lens 253 enters the dichroic mirror 217 via the tunable lens 254.

[0113] A mirror 256 is provided near the objective lens 132. The mirror 256 is a micromirror and is arranged to the side of the sample S. In the z direction, the mirror 256 and the sample S are arranged at the same position, and the mirror 256 is arranged at an angle of 45° with respect to the z direction. Therefore, the activation light L2 reflected by the mirror 256 travels in the x direction and is incident on the sample S.

[0114] Optical scanners 251 and 252 adjust the irradiation position of the activation light L2 on the sample S in the z and y directions. In addition, a tunable lens 254 can adjust the position in the x direction.

[0115] In this way, it is possible to change the region where the fluorescent material is turned on, and therefore, as in the second modification, it is possible to suppress a decrease in the contrast of the fluorescent image, and it is possible to image the sample S with high resolution.

[0116] Modification 5 A structured illumination microscope 1 according to Modification 5 will be described with reference to Fig. 17. Fig. 17 is a schematic diagram showing the configuration of the optical system of the structured illumination microscope 1. In the structured illumination microscope 1 according to Modification 5, a configuration for changing the polarization direction is added. The basic configuration is the same as that of Modification 2, etc., and therefore description thereof will be omitted.

[0117] The structured illumination optical system 100 additionally includes a λ / 2 plate 108, a λ / 2 plate 121, and a polarization control element 122. The λ / 2 plate 108, the λ / 2 plate 121, and the polarization control element 122 are the same as those used in the structured illumination microscope 1 of the first embodiment, that is, the structured illumination microscope 1 in FIG.

[0118] The illumination pattern incident on the polarization control element 122 and its polarization state will be described with reference to Fig. 18. Fig. 18 is an xy plan view showing the illumination pattern incident on the polarization control element 122 and its polarization state. Fig. 18 also shows the polarization state before and after incidence on the polarization control element 122. Fig. 18 also shows the fast axis (principal axis) of the polarization control element 122.

[0119] The polarization control element 122 is a radial polarization conversion element that converts linearly polarized light into radially polarized light. Specifically, the polarization control element 122 has six-division wave plates 122a. Each of the division wave plates 122a is a λ / 2 plate and has a 60° sector shape. The directions of the fast axes (major axes) of the division wave plates 122a are different. The two division wave plates 122d that are symmetrical with respect to the optical axis have their fast axes orthogonal to each other.

[0120] The illumination pattern incident on the polarization control element 122 corresponds to the six-division segmented wave plate 122a. Specifically, the illumination pattern incident on the polarization control element 122 has six circular patterns P. The six circular patterns P are arranged at equal intervals of 60° in the circumferential direction. Therefore, each circular pattern P is incident on a different segmented wave plate 122a.

[0121] 18 , the structured illumination light L1 is assumed to be linearly polarized in the x-direction before entering the polarization control element 122. The structured illumination light L1 after exiting the polarization control element 122 is radially polarized. The structured illumination light L1 from the segmented wave plates 122a symmetrical with respect to the optical axis has parallel polarization directions. Furthermore, the structured illumination light L1 from two adjacent segmented wave plates 122a has polarization directions tilted by 60°.

[0122] The mask 113 has openings of six circular patterns P. Therefore, the light passes through two circular patterns P that are symmetrical with respect to the optical axis of the mask 113. By switching the circular patterns P that pass through the mask 113, the direction of the stripe pattern of the structured illumination light L1 can be changed.

[0123] As shown in Figure 17, a rotating polarizer 249 is added to the activation optical system 200. The rotating polarizer 249 is a polarizer that transmits linearly polarized light components with a predetermined polarization direction. Furthermore, the rotating polarizer 249 is arranged so that it can rotate around the optical axis. This allows the polarization direction of the activation light L2 incident on the sample S to be changed.

[0124] The imaging optical system 300 additionally includes lenses 341 and 342, an ND filter 343, and a rotary polarizer 344. The signal light L3 from the objective lens 132 is incident on the lenses 341 and 342 via the dichroic mirrors 116 and 217. The signal light L3 that has passed through the lenses 341 and 342 is incident on the ND filter 343. The ND filter 343 is provided so as to be rotatable around the optical axis.

[0125] The lenses 341 and 342 form an image on the pupil plane of the objective lens 132 onto the ND filter 343. The ND filter 343 is disposed in the optical path of the signal light L3 and partially blocks or attenuates the fluorescence. Specifically, the ND filter 343 is a neutral density filter having transmittance according to the xy position. In other words, the ND filter 343 has a spatial distribution of transmittance in the xy plane. The ND filter 343 partially blocks or attenuates the signal light L3. Furthermore, by rotating the ND filter 343, the position through which the signal light L3 passes can be changed.

[0126] The signal light L3 that has passed through the ND filter 343 is incident on the rotating polarizer 344. The rotating polarizer 344 is a polarizer that transmits linearly polarized light components in a predetermined polarization direction. Furthermore, the rotating polarizer 344 is arranged so that it can rotate around the optical axis. This allows the polarization direction of the signal light L3 detected by the camera 305 to be changed.

[0127] In the fifth modification, the rotation angles of the mask 113, the rotating polarizer 344, and the rotating polarizer 240 are controlled. In this way, the polarization directions of the activation light L2 and the signal light L3 can be adjusted according to the orientation of the stripe pattern of the structured illumination light L1.

[0128] The orientation direction and polarization direction of fluorescent molecules will be described with reference to FIG. 19 . Fluorescent molecules have a distribution of the direction in which they emit fluorescence depending on their orientation direction. For example, there will be more components perpendicular to the orientation direction of the fluorescent molecules than components parallel to the orientation direction. Here, an example will be described in which fluorescent molecules are oriented in the x direction. When fluorescent molecules are oriented in the x direction, the component of fluorescence emitted from the fluorescent molecules that is emitted in the y direction will be higher. Furthermore, when the direction of the stripe pattern and the orientation direction of the fluorescent molecules coincide, it is possible to include more information for achieving high resolution.

[0129] The polarization directions of the activation light L2 and the signal light L3 are changed depending on the orientation of the stripe pattern of the structured illumination light L1. For example, the orientation of the stripe pattern is made to coincide with the polarization directions of the activation light L2 and the signal light L3. In this case, the mask 113, the rotating polarizer 344, and the rotating polarizer 249 can be rotated in unison. For example, when the orientation of the stripe pattern is set to 0°, 60°, and 120° and three fluorescent images are captured, the angles of the rotating polarizer 344 and the rotating polarizer 249 are also set to 0°, 60°, and 120°. This can improve resolution.

[0130] In the structured illumination microscope 1, the orientation of the stripe pattern is changed to capture multiple fluorescent images. The imaging optical system 300 includes a rotating polarizer 344 arranged in the optical path of the signal light L3. The activation optical system 200 includes a rotating polarizer 249 arranged in the optical path of the activation light L2. The polarization direction of the signal light L3 by the rotating polarizer 344 and the polarization direction of the activation light L2 by the rotating polarizer 249 are changed according to the orientation of the structured illumination pattern. In Variation 5, the orientation of the stripe pattern is aligned with the polarization directions of the activation light L2 and the signal light L3. This can improve resolution.

[0131] When the stripe pattern is formed along the x direction, the fluorescent image contains information that increases the resolution in the y direction. On the other hand, when the stripe pattern is formed along the x direction, the fluorescent image does not contain information that increases the resolution in the x direction. Therefore, light from both ends in the direction parallel to the orientation direction of the fluorescent molecules may become noise that reduces the contrast.

[0132] Therefore, the ND filter 343 blocks light at both ends in the x direction. The ND filter 343 has light-shielding regions 343a at both ends in the x direction and high-transmittance regions 343b at both ends in the y direction. The signal light L3 passing through both ends in the x direction contains almost no information required for achieving high resolution. By making both ends in the x direction light-shielding regions 343a, the ND filter 343 partially blocks the signal light L3. This makes it possible to suppress a decrease in the contrast of the fluorescent image.

[0133] The ND filter 343 is rotated according to the direction of the stripe pattern. That is, the rotation angle of the ND filter 343 is set according to the orientation of the stripe pattern. For example, the ND filter 343 is rotated so that the transmittance of the signal light L3 is low at both ends in the direction parallel to the stripe pattern. In this way, a decrease in contrast can be prevented, and the sample S can be observed with high resolution.

[0134] The results obtained using the structured illumination microscope are shown below. Figure 20 shows a fluorescent image of mitochondria in a living cell. The fluorescent protein used to label sample S is Skylan-NS. Figure 20 also shows a graph evaluating the in-plane (xy) resolution and the optical axis (z) resolution.

[0135] Figure 21 shows a fluorescent image of actin inside a cell spheroid (diameter 160 μm, depth 34 μm). Furthermore, Figure 21 shows a graph evaluating the in-plane (xy) resolution and the optical axis (z) resolution.

[0136] Figure 22 shows a fluorescent image of Lifeact inside a cell spheroid (diameter: 200 μm, depth: 69 μm). The fluorescent protein used to label sample S is rsGamillus-S. Figure 22 shows fluorescent images captured with a wide field of view, SIM, and SPA-3DSIM (3D SIM).

[0137] FIG. 23 is a diagram showing a structured illumination microscope according to a sixth modification. In the sixth modification, an optical system for forming the activation light into a desired illumination pattern is added. Therefore, the activation optical system 200 includes a polarizing beam splitter 274, a spatial light modulator 275, a mask 278, and the like. The basic configuration of the structured illumination microscope is similar to that described above, and therefore a description thereof will be omitted. For example, the structured illumination optical system 100 has the same configuration as that shown in FIG. 12 , and therefore a description thereof will be omitted. Furthermore, in the activation optical system 200, the configuration from the activation light source 201 to the spinning disk 215 is similar to that shown in FIG. 12 , and therefore a description thereof will be omitted.

[0138] 23, a mirror 271, a lens 272, a lens 273, a polarizing beam splitter 274, a spatial light modulator 275, a lens 276, a mask 278, and a lens 279 are added to the configuration of FIG.

[0139] The activation light L4 from the spinning disk 215 is reflected by a mirror 271. The activation light L4 reflected by the mirror 271 passes through a lens 272 and a lens 273 and enters a polarizing beam splitter 274. The activation light L4 reflected by the polarizing beam splitter 110 enters a spatial light modulator 275. The spatial light modulator 275 spatially modulates the activation light L4. The spatial light modulator 275 may be the same as the spatial light modulator 111.

[0140] The spatial light modulator 275 can control the wavefront of the activation light L4. Therefore, the activation light L4 can form a desired structured illumination pattern on the sample S. Furthermore, when the activation light L4 is reflected by the spatial light modulator 275, the polarization direction of the activation light L4 changes. Therefore, the activation light L4 from the spatial light modulator 275 passes through the polarizing beam splitter 274.

[0141] The activation light L4 from the polarizing beam splitter 274 is incident on a mask 278 via a lens 276. The mask 278 has a pattern similar to that of the mask 113. The activation light L4 forms a stripe pattern on the sample S. The mask 278 is a rotating disk, similar to the mask 113.

[0142] The activation light L4 that has passed through the mask 278 is incident on the dichroic mirror 217 via lenses 279 and 280. Therefore, similar to FIG. 12 , the activation light L4 is incident on the sample S via the objective lens. Furthermore, the activation optical system 200 is provided with a spatial light modulator 275 and a mask 278. Therefore, at the sample S, the activation light L4 forms an illumination pattern that corresponds to the illumination pattern of the structured illumination light L1.

[0143] 24, the activation light L4 forms a stripe pattern. On the sample S, the spatial frequency of the activation light L4 is the same as the spatial frequency of the structured illumination light L1. Furthermore, the phase of the stripe pattern of the activation light L4 matches the phase of the stripe pattern of the structured illumination light L1.

[0144] The activation light L4 and the structured illumination light L1 illuminate the sample S with the same stripe pattern. The effective excitation light distribution is the product of the spatial distribution of the structured illumination light L1 and the spatial distribution of the activation light L1. In this way, stripes that enable detection of information with higher resolution can be formed. Therefore, the spatial resolution can be further improved.

[0145] 25, the phase of the stripe pattern of the activation light L4 and the phase of the stripe pattern of the structured illumination light L1 are shifted by 90°. Excitation and activation are performed using the phase-shifted stripe pattern. By illuminating the sample S in this manner, luminescence can be suppressed. Since illumination can be achieved with a stripe pattern having a substantially higher frequency, spatial resolution can be further improved.

[0146] The invention made by the inventor has been specifically described above based on an embodiment, but it goes without saying that the present invention is not limited to the above embodiment and can be modified in various ways without departing from the gist of the invention.

[0147] This application claims priority based on Japanese Patent Application No. 2023-087075, filed on May 26, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0148] 1 Structured illumination microscope 100 Structured illumination optical system 101 Excitation light source 102 Lens 103 Pinhole plate 104 Lens 107 Mirror 108 λ / 2 plate 110 Polarizing beam splitter 111 Spatial light modulator 112 Lens 120 AO modulator 121 λ / 2 plate 122 Polarization control element 113 Mask 114 Lens 116 Dichroic mirror 131 Lens 132 Objective lens 200 Activation optical system 201 Activation light source 202 AO modulator 203 Mirror 204 Mirror 212 Lens 213 Pinhole plate 214 Lens 215 Spinning disk 215a Microlens array 215b Microlens 216 Lens 222 Mirror 221 Lens 232 Lens 231 Cylindrical lens 232 Lens 233 Mirror 234 Slit 235 Lens 236 Lens 237 Objective lens 271 Mirror 272 Lens 273 Lens 274 Polarizing beam splitter 275 Spatial light modulator 276 Lens 300 Imaging optical system 303 Lens 305 Camera 311 Filter 312 Filter S Sample W Liquid (medium) L1 Structured illumination light L2 Activation light L3 Signal light L4 Activation light

Claims

1. an excitation light source that generates excitation light for exciting a fluorescent substance contained in the sample; an activation light source that generates activation light for activating the fluorescent material; a structured illumination optical system having an objective lens that focuses the excitation light on the sample and illuminates the sample with the excitation light as a structured illumination pattern; an activation optical system that irradiates the activation light onto the sample through the objective lens; an imaging optical system that detects, via the objective lens, fluorescence generated from the sample in a state in which the fluorescent substance is activated, in order to capture a fluorescent image of the sample; the activation optics comprises a spinning disk that forms multiple foci on the sample; A structured illumination microscope, wherein the multi-focal point is scanned by the rotation of the spinning disk, thereby activating the fluorescent material in the focal plane scanned by the multi-focal point.

2. 2. The structured illumination microscope of claim 1, wherein the activation light source generates laser light at a wavelength that activates the fluorescent material by two-photon absorption.

3. 2. The structured illumination microscope according to claim 1, wherein the activation light passes through a portion off the optical axis of the objective lens and is incident on the sample from a direction tilted from the optical axis.

4. the activation optical system includes a mirror disposed laterally of the sample; 2. The structured illumination microscope according to claim 1, wherein the activation light that has passed through the objective lens is reflected by the mirror and is incident on the sample.

5. The structured illumination microscope according to claim 3 or 4, wherein the activation optical system converts the activation light into light sheet illumination and irradiates the sample.

6. 4. The structured illumination microscope according to claim 3, wherein the activation optical system irradiates the sample with the activation light as a Bessel beam.

7. a first polarizer disposed in the optical path of the activation light; a second polarizer disposed in the optical path of the fluorescent light; 2. The structured illumination microscope of claim 1, wherein the polarization direction of the activation light by the first polarizer and the polarization direction of the fluorescence by the second polarizer are changed depending on the direction of the structured illumination pattern.

8. a filter disposed in an optical path of the fluorescent light to partially block or attenuate the fluorescent light; 7. The structured illumination microscope according to claim 6, wherein the filter blocks or attenuates the fluorescence at both ends in a direction parallel to the stripes of the structured illumination pattern.

9. The structured illumination microscope according to claim 1 , wherein the activation optical system irradiates the sample with the activation light in an illumination pattern corresponding to the structured illumination pattern of the excitation light.

10. an excitation light source that generates excitation light for exciting a fluorescent substance contained in the sample; an activation light source that generates activation light for activating the fluorescent material; a structured illumination optical system that is disposed at an angle inclined from a vertical direction and has a first objective lens that focuses the excitation light onto the sample, and that irradiates the sample with the excitation light as a structured illumination pattern; an activation optical system that is disposed at an angle inclined from the vertical direction and has a second objective lens that focuses the activation light and irradiates the activation light onto the sample; and an imaging optical system that detects, via the first objective lens, fluorescence generated from the sample in a state in which the fluorescent substance is activated, in order to capture a fluorescent image of the sample.

11. 11. The structured illumination microscope of claim 10, wherein the activation light source generates laser light at a wavelength that activates the fluorescent material by two-photon absorption.