Super-resolution microscope and method for controlling super-resolution microscope

Abstract

The present invention performs super-resolution imaging with high spatial resolution in a deep part of a sample. This super-resolution microscope (1) comprises: a first emission part (37) that emits excitation light (21); second emission parts (31a-38d) that emit deactivation light (22); an objective lens (4) that collects the deactivation light such that the center of the excitation light and the center of the deactivation light are displaced from each other in a sample (5) including a phosphor; and a zoom lens (2) that is disposed between the first emission part and the second emission parts, and the objective lens, the zoom lens (2) being capable of changing the magnification thereof.

Description

Super-resolution microscope and method for controlling the same

[0001] The present invention relates to a super-resolution microscope and a control method thereof.

[0002] STED microscopy is a type of super-resolution microscope capable of capturing super-resolution images of biological samples. STED microscopy is rapidly gaining popularity because it can easily capture ultra-high-resolution images of the surface layers of biological samples. However, conventional STED microscopes are limited in that they can only observe the surface layers of biological samples, such as planarly cultured cells. This is primarily due to the difficulty of sufficiently focusing the donut-shaped STED light used in STED microscopy deep within biological samples. The donut-shaped STED light has a spiral wavefront with zero intensity at its center. However, inside biological samples with non-uniform refractive index distribution, this spiral wavefront breaks down, resulting in a non-zero intensity at the center of the donut shape.

[0003] Efforts have been made to improve STED microscopes so that they can acquire super-resolution images of deep areas of biological samples. As an example, Non-Patent Document 1 discloses a STED microscope that combines Bessel beams and STED light to enable the acquisition of super-resolution images of areas at a certain depth in biological samples.

[0004] Wentao Yu et. al. :"Super-resolution deep imaging with hollow Bessel beam STED microscopy", Laser & Photonics Review, 2015

[0005] However, it is difficult to obtain super-resolution images of deep areas in biological samples using the technique of Non-Patent Document 1. There is also room for improvement in the spatial resolution of super-resolution images of deep areas in biological samples.

[0006] One aspect of the present invention aims to perform super-resolution imaging with high spatial resolution deep within a sample.

[0007] A super-resolution microscope according to one aspect of the present invention is configured to include a first exit section that emits first excitation light that excites a phosphor, a second exit section that emits first quenching light that quenches the phosphor, an objective lens that focuses the first quenching light from the second exit section so that the center of the first excitation light from the first exit section and the center of the first quenching light from the second exit section are offset from each other in a sample containing the phosphor, and a zoom lens that is arranged between the first exit section, the second exit section, and the objective lens and has a variable magnification.

[0008] A method for controlling a super-resolution microscope according to one aspect of the present invention includes a first emission step of emitting excitation light that excites a phosphor, a second emission step of emitting deactivation light that deactivates the phosphor, a changing step of changing the magnification of a zoom lens onto which the excitation light and the deactivation light are incident, and a focusing step of focusing the deactivation light on a sample containing the phosphor so that the center of the excitation light and the center of the deactivation light are offset from each other.

[0009] According to one aspect of the present disclosure, super-resolution imaging with high spatial resolution can be performed deep within a sample.

[0010] FIG. 1 is a diagram showing a part of the configuration of a super-resolution microscope according to one embodiment of the present invention. FIG. 2 is a diagram showing other parts of the configuration of the super-resolution microscope. FIG. 3 is a diagram showing focused excitation light and multiple deactivation light spots on a sample. FIG. 4 is a diagram showing spot positions when the magnification of the zoom lens is increased compared to FIG. 2 , and the configuration of the super-resolution microscope. FIG. 5 is a diagram showing excitation light spots and deactivation light spots on a sample. FIG. 6 is a diagram showing a part of the configuration of a super-resolution microscope according to one embodiment of the present invention. FIG. 7 is a diagram showing excitation light spots and deactivation light spots on a sample formed by the super-resolution microscope. FIG. 8 is a diagram showing a part of the configuration of a super-resolution microscope according to one embodiment of the present invention. FIG. 9 is a schematic diagram showing a part of the configuration of a super-resolution microscope. FIG. 10 is a plan view showing the configuration of a mask. FIG. 11 is a schematic diagram showing a part of the configuration of a super-resolution microscope. FIG. 12 is a diagram showing the point spread function of deactivation light near a focus. FIG. 13 is a cross-sectional view showing a schematic configuration of a light guide of another modified example.

[0011] [Embodiment 1] (Configuration of Super-Resolution Microscope 1) FIG. 1 is a diagram showing a portion of the configuration of a super-resolution microscope 1 according to this embodiment. FIG. 2 is a diagram showing other portions of the configuration of the super-resolution microscope 1. The super-resolution microscope 1 is a STED (STimulated Emission Depletion) microscope that performs super-resolution imaging of a sample 5 (e.g., a biological sample) to acquire a super-resolution image of the sample 5. The super-resolution microscope 1 includes an excitation light source 11, a deactivation light source 12, a first input unit 31, a second input unit 32, a branching unit 33, a first optical fiber 34, multiple second optical fibers 35, a light guide 36, a zoom lens 2, galvanometer mirrors 3a and 3b, an objective lens 4, a dichroic mirror 6, a band-pass filter 7, and a detector 8. The super-resolution microscope 1 further includes relay lenses 41, 43, and 45, a mirror 44, and the like as an optical system for guiding the excitation light and deactivation light.

[0012] The excitation light source 11 emits excitation light 21 that excites fluorescent molecules (phosphors) contained in the sample 5. The deactivation light source 12 emits deactivation light 22 (STED light) that deactivates the excited fluorescent molecules. The excitation light 21 and the deactivation light 22 have different wavelengths. A pulsed laser light source can be used as the excitation light source 11 and the deactivation light source 12. A CW (continuous wave) laser light source can also be used as the excitation light source 11 and the deactivation light source 12.

[0013] The first input unit 31 is a member (e.g., a connector connecting a light source and an optical fiber) to which the excitation light 21 is input. The excitation light 21 input to the first input unit 31 passes through a first optical fiber 34 and is input to the light guide 36. The first optical fiber 34 is connected to the light guide 36.

[0014] The second input portion 32 is a member (e.g., a connector connecting a light source and an optical fiber) to which the deactivation light 22 is input. The deactivation light 22 input to the second input portion 32 is input to the branching portion 33. The plurality of second optical fibers 35 are connected to a light guide 36.

[0015] The branching unit 33 is, for example, a branching coupler that branches the input light into multiple (four in this example) beams. A plurality of second optical fibers 35 are connected to the output end of the branching unit 33. The multiple deactivation beams 22, which have been divided with equal intensities, are input to the light guide 36 through the multiple second optical fibers 35.

[0016] The light guide 36 guides and emits the excitation light 21 and the plurality of deactivation light beams 22. Here, the light guide 36 is an optical fiber bundle formed by bundling together a first optical fiber 34 and a plurality of second optical fibers 35. The light guide 36 may be a multi-core fiber or a light guide block having a plurality of cores.

[0017] 1 and 2 also show schematic diagrams of the output end surface of the light guide 36. The light guide 36 is formed with a first exit portion 37 and multiple second exit portions 38a-38d. The first exit portion 37 is connected to the first optical fiber 34 and emits the excitation light 21. The multiple second exit portions 38a-38d are connected to multiple second optical fibers 35, respectively, and emit the deactivation light 22. The multiple second exit portions 38a-38d are arranged around the first exit portion 37. The positional relationship between the first exit portion 37 and the multiple second exit portions 38a-38d directly corresponds to the positional relationship between the spot of the excitation light 21 and the spots of the deactivation light 22 focused on the sample 5. The second exit portion 38a and the second exit portion 38c sandwich the first exit portion 37 in the vertical direction. In the horizontal direction, second emission portion 38b and second emission portion 38d sandwich first emission portion 37. Here, four second emission portions 38a to 38d surround first emission portion 37, but this is not limited thereto, and two or more second emission portions may surround or sandwich first emission portion 37. Note that first emission portion 37 and multiple second emission portions may be configured using a beam splitter, a mirror, or the like instead of branch portion 33 and light guide 36.

[0018] The excitation light 21 emitted from the first exit portion 37 and the plurality of deactivation light beams 22 emitted from the plurality of second exit portions 38 a to 38 d are incident on the zoom lens 2 .

[0019] The zoom lens 2 is a lens group capable of changing the magnification. The range of magnification that the zoom lens 2 can change may include not only magnifications greater than 1 (enlargement) but also magnifications less than 1 (reduction). The zoom lens 2 only needs to have the function of changing the magnification, whether enlargement or reduction. The zoom lens 2 is disposed on the optical path between the objective lens 4 and the first exit portion 37 and the plurality of second exit portions 38a to 38d. The excitation light 21 and the plurality of quenching light beams 22 emitted from the zoom lens 2 are imaged on an image forming plane. FIG. 2 also shows the positional relationship between the excitation light 21 and the plurality of quenching light beams 22 on the image forming plane. The excitation light 21 and the plurality of quenching light beams 22 emitted from the zoom lens 2 are incident on the galvanometer mirror 3a.

[0020] The galvanometer mirrors 3a and 3b change the angles of their reflecting surfaces to change (adjust) the directions of the reflected excitation light 21 and the plurality of deactivation light beams 22. For example, the galvanometer mirror 3a scans in the X direction, and the galvanometer mirror 3b scans in the Y direction. This allows the excitation light 21 and the plurality of deactivation light beams 22 to scan over the sample 5. The excitation light 21 and the plurality of deactivation light beams 22 emitted from the galvanometer mirrors 3a and 3b are incident on the dichroic mirror 6.

[0021] The dichroic mirror 6 transmits the excitation light 21 and the plurality of quenching light beams 22 and reflects the fluorescence 27 from the fluorescent molecules. The excitation light 21 and the plurality of quenching light beams 22 that have passed through the dichroic mirror 6 are incident on the objective lens 4.

[0022] The objective lens 4 focuses the excitation light 21 and the multiple quenching lights 22 on the sample 5 (inside the sample 5). The objective lens 4 focuses the multiple quenching lights 22 from the multiple second exit portions 38a to 38d at different positions on the sample 5 around the excitation light from the first exit portion 37. In other words, the objective lens 4 focuses the multiple quenching lights 22 from the multiple second exit portions 38a to 38d so that the center of the excitation light from the first exit portion 37 is offset from the center of each quenching light 22 from each of the second exit portions 38a to 38d. FIG. 2 also shows the positional relationship between the spot of the excitation light 21 and the spots of the multiple quenching lights 22 focused on the sample 5. Note that each spot has a certain degree of spread due to the diffraction limit, light scattering, the refractive index distribution of the sample 5, and the like.

[0023] The excitation light 21 and the plurality of quenching lights 22 emitted from the light guide 36 travel along the same path (the path including the zoom lens 2 and the objective lens 4) from the light guide 36 to the sample 5, and are irradiated onto the sample 5. Therefore, the center of the excitation light 21 and the centers of the plurality of quenching lights 22 on the sample 5 are shifted from each other, reflecting the positions of the first emission part 37 and the plurality of second emission parts 38a to 38d on the light guide 36.

[0024] Of the fluorescent molecules excited by the excitation light 21, those irradiated with the deactivation light 22 are deactivated by stimulated emission and do not emit fluorescence. Fluorescent molecules excited by the excitation light 21 but not irradiated with the deactivation light 22 emit fluorescence 27. The fluorescence 27 from the fluorescent molecules is reflected by the dichroic mirror 6 and enters the bandpass filter 7.

[0025] The bandpass filter 7 transmits the fluorescence 27 and blocks light of other wavelengths (including the excitation light 21 and the quenching light 22). The fluorescence 27 that has passed through the bandpass filter 7 is incident on the detector 8.

[0026] The detector 8 detects the incident fluorescence 27. The detector 8 may be, for example, a photomultiplier tube (PMT), a CMOS, or a CCD. The super-resolution microscope 1 detects the fluorescence 27 with the detector 8 while two-dimensionally scanning the positions of the spots of the excitation light 21 and the multiple quenching lights 22 with the galvanometer mirrors 3a and 3b. This allows the super-resolution microscope 1 to obtain a super-resolution image of the sample 5. Note that instead of scanning with the galvanometer mirrors 3a and 3b, scanning may be performed by displacing a stage on which the sample 5 is placed.

[0027] The sample 5 contains fluorescent molecules. The sample 5 may be a biological sample or a non-biological sample. The sample 5 may also be a small, thick artificial tissue (such as an artificial organ) made from human, animal, or plant cells. In this case, the super-resolution microscope 1 can acquire, for example, the results of a drug response to the artificial tissue as a super-resolution image.

[0028] (Example of Irradiation Light) Fig. 3 is a diagram showing the spots of the focused excitation light 21 and the multiple quenching lights 22a to 22d on the sample 5. Fig. 3 shows an example of the excitation light 21 and the multiple quenching lights 22a to 22d irradiated by the super-resolution microscope 1.

[0029] In the example shown in FIG. 3 , the super-resolution microscope 1 irradiates a sample 5 containing fluorescent molecules with excitation light 21 that excites the fluorescent molecules. Furthermore, the super-resolution microscope 1 irradiates the sample 5 with multiple quenching lights 22a-22d, each focused at a different position around the center of the excitation light 21. This causes fluorescent molecules outside the central portion of the irradiation position of the excitation light 21 to be quenched by stimulated emission due to the action of the quenching lights 22a-22d. As a result, fluorescence 27 is emitted only from the central portion of the irradiation position of the excitation light 21. In other words, the emission region 23 of the fluorescence 27 of the super-resolution microscope 1 can be made smaller than the emission region 24 of the fluorescence 27 when only the excitation light 21 is irradiated. By detecting the fluorescence 27 from such a narrow emission region 23, the super-resolution microscope 1 can increase the spatial resolution of imaging based on fluorescence detection and achieve super-resolution imaging.

[0030] The super-resolution microscope 1 irradiates the specimen 5 with multiple deactivation beams 22a-22d, which individually form spots, rather than the conventional doughnut-shaped deactivation beam. The deactivation beams 22a-22d, which individually form spots, do not significantly disrupt the phase of light due to non-uniformity in the refractive index of the specimen 5. Therefore, each deactivation beam 22a-22d can form a sufficiently focused spot even deep within the specimen 5 (e.g., approximately 100 μm to 10 mm). This allows fluorescent molecules located around the center of irradiation by the excitation beam 21 to be appropriately deactivated by stimulated emission. As a result, the emission region 23 of the fluorescence 27 generated deep within the specimen 5 can be made as small as in the surface layer, thereby increasing the spatial resolution of super-resolution imaging deep within the specimen 5 as in the surface layer.

[0031] Here, depending on the sample 5 or the depth, non-uniformity in the refractive index may cause the spots of the excitation light 21 and the quenching light 22a to 22d to become blurred and spread deep inside. If the spots of the multiple quenching light 22a to 22d spread and cover the entire spot of the excitation light 21, the emission region 23 of the fluorescence 27 will disappear.

[0032] FIG. 4 shows the spot positions and the configuration of the super-resolution microscope 1 when the magnification of the zoom lens 2 is increased compared to FIG. 2 . Here, the zoom lens 2 is positioned so that the imaging plane of the zoom lens 2 faces the sample 5. That is, the lens of the zoom lens 2 that is normally located on the subject side faces the light guide 36. In this case, increasing the magnification of the zoom lens 2 widens the distance (the distance between the centers) between the excitation light 21 and the deactivation light 22 on the imaging plane. Similarly, the distance between the centers of the spots of the excitation light 21 and the deactivation light 22 focused on the sample 5 also widens. Therefore, even if the spot of the deactivation light 22 becomes blurred and widens, the spot of the deactivation light 22 does not overlap the center of the spot of the excitation light 21. For example, by repeating the steps of detecting fluorescence 27 using the super-resolution microscope 1 and changing the magnification of the zoom lens 2, it is possible to identify the limit (lower limit) of the magnification at which fluorescence 27 can be detected and the light-emitting region 23 is minimized. Therefore, the super-resolution microscope 1 can perform super-resolution imaging with an appropriately high spatial resolution by adjusting (changing) the magnification of the zoom lens 2 according to the sample 5 or the depth of the observation position in the sample 5. The super-resolution microscope 1 can appropriately observe a wide range of samples 5 and a wide range of depths without replacing or changing the position of optical elements.

[0033] (Example of irradiation pattern of deactivating light) The light guide 36 may be configured to include two second exit portions 38 a, 38 c positioned on either side of the first exit portion 37. This can improve spatial resolution in the direction in which the spots of the two deactivating lights 22 are aligned.

[0034] The light guide 36 may be configured to include three or more second exit portions positioned to surround the first exit portion 37 so that the multiple spots of the deactivation light 22 completely surround the spot of the excitation light 21. For example, three second exit portions may be arranged at the vertices of an equilateral triangle. For example, six second exit portions may be arranged at the vertices of a regular hexagon surrounding the first exit portion 37. By adjusting the magnification of the zoom lens 2, the spots of the deactivation light 22 can be arranged to completely surround the periphery of the central portion of the spot of the excitation light 21, leaving only the central portion.

[0035] The light guide 36 may be configured to include a first exit portion 37 and a single second exit portion 38a. Even in this case, the spot of the quenching light 22 can be partially overlapped with the spot of the excitation light 21 by adjusting the magnification of the zoom lens 2. Even in this case, the light-emitting region 23 of the fluorescence 27 can be made smaller.

[0036] (Timing of irradiation with quenching light) Figure 5 is a diagram showing spots of excitation light 21 and quenching light 22 on sample 5. The excitation light 21 and the plurality of quenching light rays 22 may be irradiated onto sample 5 simultaneously. However, it is preferable to end irradiation of sample 5 with the plurality of quenching light rays 22 after irradiation of sample 5 with excitation light 21 has ended. Detector 8 only needs to detect fluorescence 27 irradiated during the period when sample 5 is irradiated with the plurality of quenching light rays 22, or during the period from the time when the plurality of quenching light rays 22 are irradiated onto sample 5 until the fluorescence emission ends.

[0037] When multiple quenching lights 22 surround the excitation light 21, there is an overlapping region where the spots of the multiple quenching lights 22 overlap. If interference occurs between the quenching lights 22, the intensity of the quenching lights 22 may become zero or small in the overlapping region. This may result in insufficient quenching of fluorescent molecules in the overlapping region. As a result, the emission region 23 of the fluorescence 27 may expand, potentially reducing spatial resolution.

[0038] To prevent interference between the deactivation lights 22, during a period when one of the multiple second emission sections 38a to 38d is not irradiating the sample 5 with the deactivation light 22, at least one of the other second emission sections irradiates the sample 5 with the deactivation light 22. Specifically, the super-resolution microscope 1 irradiates the sample 5 with multiple deactivation lights 22 whose spots overlap each other at different times. For example, the super-resolution microscope 1 sets a different period for irradiating the multiple deactivation lights 22a and 22c and a different period for irradiating the multiple deactivation lights 22b and 22d. The multiple deactivation lights 22a and 22c whose spots do not overlap each other may be irradiated onto the sample 5 simultaneously. Alternatively, the irradiation periods of all of the deactivation lights 22a to 22d may be set different from each other.

[0039] For example, if the deactivating light 22a-22d is a pulsed laser with a pulse width of 500 ps, ​​the super-resolution microscope 1 shifts the irradiation periods by approximately 500 ps. This can be easily achieved by varying the lengths of the second optical fibers 35 connecting the branching portion 33 and the light guide 36. By increasing the lengths of the second optical fibers 35 connected to the second exit portions 38a-38d by, for example, approximately 10 cm each, the irradiation periods of the deactivating light 22 can be shifted by approximately 500 ps each.

[0040] (Other Application Examples) The spatial resolution can be further improved by applying various known microscopy methods to the super-resolution microscope. Examples of such microscopy methods include gated-STED microscopy and saturated excitation (SAX) microscopy. In a super-resolution microscope employing gated-STED microscopy, the detector 8 detects long-lived portions of the fluorescence 27 generated by irradiation with a pulsed laser. For example, the fluorescence lifetime of the central portion of the excited region is longer than that of the peripheral portion. Therefore, by detecting only the fluorescence 27 at the end of the fluorescence emission period, the detector 8 can detect only the fluorescence 27 emitted from the central portion of the light-emitting region 23. This allows for even higher spatial resolution.

[0041] The super-resolution microscope 1 can observe not only the deep part of the sample 5 but also the surface layer of the sample 5 .

[0042] The super-resolution microscope 1 may be configured to omit the zoom lens 2 and stagger the irradiation timings of the multiple deactivation rays 22. With this configuration, even if the spot of the deactivation rays 22 spreads deep within the sample 5, interference between the deactivation rays 22 can be prevented, and super-resolution imaging with high spatial resolution can be performed deep within the sample.

[0043] In the super-resolution microscope 1, a microlens array may be disposed between the objective lens 4 and the zoom lens 2. This allows a configuration in which a set of excitation light 21 and a plurality of quenching lights 22 are focused at a plurality of positions on the sample 5. In this case, a camera having a plurality of light-receiving pixels is required as the detector 8. This allows information on a plurality of positions on the sample 5 to be obtained simultaneously.

[0044] (Variation) Two-photon excitation may be used to excite fluorescent molecules. In this case, the excitation light source 11 is a pulsed laser light source. In two-photon excitation, the wavelength of the deactivation light 22 is shorter than that of the excitation light 21, and the wavelength of the fluorescence 27 is shorter than that of the deactivation light 22. When the excitation light 21 excites fluorescent molecules by two-photon excitation, the excitation light 21 and the deactivation light 22 may have the same wavelength.

[0045] The zoom lens 2 may be positioned so that the image plane of the zoom lens 2 is located on the light guide 36 side. In this case, the lens of the zoom lens 2 that is normally located on the subject side is located on the sample 5 side. In this case, when the magnification of the zoom lens 2 is reduced, the distance between the centers of the spots of the excitation light 21 and the quenching light 22 focused on the sample 5 increases.

[0046] [Embodiment 2] Another embodiment of the present invention will be described below. For convenience of explanation, the same reference numerals will be used to designate components having the same functions as those described in the above embodiment, and the description thereof will not be repeated.

[0047] 6 is a diagram showing a portion of the configuration of a super-resolution microscope 1a according to this embodiment. The super-resolution microscope 1a includes an excitation light source 11, a deactivation light source 12, a first input section 31, a second input section 32, a first branch section 33a, a second branch section 33b, a plurality of first optical fibers 34a, 34b, a plurality of second optical fibers 35a, 35b, a first light guide 36a, and a second light guide 36b. The super-resolution microscope 1a also includes the zoom lens 2, galvanometer mirrors 3a, 3b, an objective lens 4, a dichroic mirror 6, a band-pass filter 7, and a detector 8, which are configured as shown in FIG. 2. In the super-resolution microscope 1a, the configuration from the zoom lens 2 to the detector 8 is the same as that of the first embodiment.

[0048] The first branching unit 33a is, for example, a branching coupler that branches the input light into multiple beams (two beams in FIG. 6 ). A plurality of first optical fibers 34a, 34b are connected to the output end of the first branching unit 33a. The multiple pumping light beams 21, which have been split with equal intensities, are input to a first light guide 36a and a second light guide 36b through the multiple first optical fibers 34a, 34b. The first optical fiber 34a is connected to the first light guide 36a. The first optical fiber 34b is connected to the second light guide 36b.

[0049] The second branching unit 33b is, for example, a branching coupler that branches the input light into multiple beams (eight beams in FIG. 6 ). A plurality of second optical fibers 35a, 35b are connected to the output end of the second branching unit 33b. The multiple deactivation beams 22, which have been divided with equal intensities, are input to the first light guide 36a and the second light guide 36b through the multiple second optical fibers 35a, 35b. The multiple second optical fibers 35a are connected to the first light guide 36a. The multiple second optical fibers 35b are connected to the second light guide 36b.

[0050] The first light guide 36a and the second light guide 36b guide and emit the excitation light 21 and the plurality of deactivation light beams 22, respectively. Here, the first light guide 36a is an optical fiber bundle formed by bundling a first optical fiber 34a and a plurality of second optical fibers 35a into one unit. The second light guide 36b is an optical fiber bundle formed by bundling a first optical fiber 34b and a plurality of second optical fibers 35b into one unit. Note that the first light guide 36a and the second light guide 36b may be multicore fibers or light guide blocks having multiple cores. Alternatively, the first light guide 36a and the second light guide 36b may be an integrated light guide.

[0051] 6 also shows a schematic diagram of the output end surfaces of the first light guide 36a and the second light guide 36b. The first light guide 36a has a first exit portion 37a and multiple second exit portions 38aa-38ad formed therein. The first exit portion 37a is connected to the first optical fiber 34a and emits the excitation light 21. The multiple second exit portions 38aa-38ad are connected to multiple second optical fibers 35a, respectively, and emit the deactivation light 22. The positional relationship between the multiple second exit portions 38aa-38ad and the first exit portion 37a is the same as the positional relationship between the multiple second exit portions 38aa-38d and the first exit portion 37 in the first embodiment.

[0052] The second light guide 36b is formed with a third exit portion 37b and a plurality of fourth exit portions 38ba-38bd. The third exit portion 37b is connected to the first optical fiber 34b and emits the excitation light 21. The plurality of fourth exit portions 38ba-38bd are connected to the plurality of second optical fibers 35b, respectively, and emit the deactivation light 22. The positional relationship between the plurality of fourth exit portions 38ba-38bd and the third exit portion 37b is the same as the positional relationship between the plurality of second exit portions 38aa-38ad and the first exit portion 37a.

[0053] The excitation light 21 emitted from the first exit 37a and the third exit 37b and the plurality of deactivation light rays 22 emitted from the plurality of second exits 38aa to 38ad and the plurality of fourth exits 38ba to 38bd are incident on the zoom lens 2.

[0054] For simplicity, the super-resolution microscope 1a will be described using a configuration including two light guides (a first light guide 36a and a second light guide 36b), but this is not limiting. The super-resolution microscope 1a may also be configured to include three or more light guides of similar configurations aligned in a predetermined direction. The super-resolution microscope 1a may also be configured to include four or more light guides of similar configurations arranged two-dimensionally.

[0055] 7 is a diagram showing the spots of excitation light 21 and deactivation light 22 formed on the sample 5 by the super-resolution microscope 1a. The objective lens 4 focuses the spots of deactivation light 22aa-22ad emitted from the second exit portions 38aa-38ad at different positions around the spot of excitation light 21a emitted from the first exit portion 37a. Similarly, the objective lens 4 focuses the spots of deactivation light 22ba-22bd emitted from the fourth exit portions 38ba-38bd at different positions around the spot of excitation light 21b emitted from the third exit portion 37b. The centers of the excitation light 21b and the deactivation light 22ba-22bd on the sample 5 are offset from each other, reflecting the positions of the third exit portion 37b and the fourth exit portions 38ba-38bd on the second light guide 36b. Furthermore, reflecting the positions of the first exit portion 37a in the first light guide 36a and the third exit portion 37b in the second light guide 36b, the centers of the excitation light 21a and the excitation light 21b in the sample 5 are shifted from each other.

[0056] (Timing of Irradiation) The super-resolution microscope 1a irradiates the sample 5 with the excitation light 21a from the first emission unit 37a and the quenching light 22aa-22ad from the plurality of second emission units 38aa-38ad. Thereafter, the super-resolution microscope 1a irradiates the sample 5 with the excitation light 21b from the third emission unit 37b and the quenching light 22ba-22bd from the plurality of fourth emission units 38ba-38bd. The super-resolution microscope 1a irradiates the sample 5 with the excitation light 21b from the third emission unit 37b during a period when the excitation light 21a from the first emission unit 37a is not irradiating the sample 5. This allows the detector 8 to detect fluorescence 27 from a certain region generated by the excitation light 21a and fluorescence 27 from a different region generated by the excitation light 21b at mutually different timings.

[0057] More preferably, the super-resolution microscope 1a starts irradiating the sample 5 with excitation light 21b from the third emission section 37b after a period of time equal to or greater than the fluorescence lifetime of the fluorescent molecules contained in the sample 5 has elapsed since the irradiation of the sample 5 with excitation light 21a from the first emission section 37a has ended.

[0058] For example, if the fluorescence lifetime of the fluorescent molecules (the period during which fluorescence emission continues after irradiation with excitation light has ended) is 20 ns, irradiation of the sample 5 with excitation light 21b from the third exit portion 37b begins 20 ns or more after the excitation light 21a has finished being emitted from the first exit portion 37a. This can be easily achieved by making the length of the first optical fiber 34a connecting the first branch portion 33a and the first light guide 36a different from the length of the first optical fiber 34b connecting the first branch portion 33a and the second light guide 36b. Furthermore, when multiple light guides are provided, the irradiation periods of the excitation light can be shifted sequentially.

[0059] The super-resolution microscope 1a detects fluorescence 27 from multiple regions using multiple excitation light beams 21a, 21b, and then scans the position of each spot in the X and Y directions using galvanometer mirrors 3a, 3b, etc., to obtain a two-dimensional super-resolution image. Therefore, the super-resolution microscope 1a can obtain a super-resolution image in a shorter time than when a super-resolution image is obtained by simply scanning a single excitation light spot two-dimensionally using galvanometer mirrors 3a, 3b. Because the super-resolution microscope 1a can stagger the timing at which fluorescence 27 is generated in multiple light-emitting regions, the detector 8 does not require multiple light-receiving pixels. The X and Y directions are perpendicular to each other in the observation plane.

[0060] In the second embodiment, the zoom lens 2 can be omitted.

[0061] [Embodiment 3] Another embodiment of the present invention will be described below. For convenience of explanation, the same reference numerals will be used to designate components having the same functions as those described in the above embodiment, and the description thereof will not be repeated.

[0062] 8 is a diagram showing a portion of the configuration of a super-resolution microscope 1b according to this embodiment. The super-resolution microscope 1b includes a modulator 9, a first excitation light source 11a, a second excitation light source 11b, a deactivation light source 12, a third input section 31a, a fourth input section 31b, a second input section 32, a second branch section 33b, a plurality of first optical fibers 34a, 34b, a plurality of second optical fibers 35a, 35b, a first light guide 36a, and a second light guide 36b. The super-resolution microscope 1b also includes the zoom lens 2, galvanometer mirrors 3a, 3b, an objective lens 4, a dichroic mirror 6, a bandpass filter 7, and a detector 8, which are configured as shown in FIG. 2. In the super-resolution microscope 1b, the configuration from the zoom lens 2 to the detector 8 is the same as that of the first embodiment.

[0063] The modulator 9 generates modulation signals and outputs the modulation signals to the first excitation light source 11a and the second excitation light source 11b in order to cause the first excitation light source 11a and the second excitation light source 11b to emit intensity-modulated excitation light 21a and 21b. The modulator 9 outputs a sine wave modulation signal of a first frequency to the first excitation light source 11a. The modulator 9 outputs a sine wave modulation signal of a second frequency, different from the first frequency, to the second excitation light source 11b. The first frequency and the second frequency may be, for example, on the order of MHz. Note that the modulation signals are not limited to sine waves and may be any modulation signals that change periodically.

[0064] The first excitation light source 11a is a CW laser light source that emits excitation light 21a whose intensity is modulated in accordance with a modulation signal. The first excitation light source 11a outputs excitation light 21a whose intensity is modulated at a first frequency. The excitation light 21a whose intensity is modulated at the first frequency is input to the third input port 31a.

[0065] The second excitation light source 11b is a CW laser light source that emits excitation light 21b intensity-modulated in response to a modulation signal. The second excitation light source 11b outputs excitation light 21b intensity-modulated at a second frequency. The excitation light 21b intensity-modulated at the second frequency is input to the fourth input port 31b. The excitation light 21a and the excitation light 21b have the same wavelength.

[0066] The third input portion 31a is a member into which the excitation light 21a is input. The excitation light 21a input to the third input portion 31a passes through the first optical fiber 34a and is input to the first light guide 36a. The first optical fiber 34a connects the third input portion 31a and the first light guide 36a.

[0067] The fourth input portion 31b is a member into which the excitation light 21b is input. The excitation light 21b input to the fourth input portion 31b passes through the first optical fiber 34b and is input to the second light guide 36b. The first optical fiber 34b connects the fourth input portion 31b and the second light guide 36b.

[0068] The configurations of the first light guiding body 36a and the second light guiding body 36b and the configuration relating to the path of the deactivation light 22 are similar to those of the second embodiment shown in Fig. 6. In this embodiment, the deactivation light source 12 is also a CW laser light source.

[0069] In the super-resolution microscope 1b, the excitation light 21a emitted from the first emission part 37a, the excitation light 21b emitted from the third emission part 37b, and the plurality of quenching lights 22aa to 22ad and 22ba to 22bd are simultaneously irradiated onto the sample 5. The detector 8 simultaneously detects both the fluorescence 27 generated by the excitation light 21a and the fluorescence 27 generated by the excitation light 21b.

[0070] The period of intensity modulation is sufficiently longer than the fluorescence lifetime. The fluorescence 27 generated by the excitation light 21a intensity-modulated at the first frequency is intensity-modulated at the first frequency. The fluorescence 27 generated by the excitation light 21b intensity-modulated at the second frequency is intensity-modulated at the second frequency. Therefore, the super-resolution microscope 1b can separate the two by Fourier transforming the intensity signal of the fluorescence 27 detected by the detector 8. Therefore, the super-resolution microscope 1b can simultaneously obtain information on the fluorescence 27 from multiple regions. After detecting the fluorescence 27 from multiple regions using multiple excitation light beams 21a and 21b, the super-resolution microscope 1b scans the position of each spot in the X and Y directions using galvanometer mirrors 3a and 3b, etc., to obtain a two-dimensional super-resolution image. Because the super-resolution microscope 1b separates the fluorescence 27 from multiple light-emitting regions using Fourier transform, the detector 8 does not require multiple light-receiving pixels.

[0071] (Variation) The modulation device 9 may output a first modulated signal of a first frequency to the first excitation light source 11a, and output a second modulated signal of the first frequency, which has a phase different from that of the first modulated signal, to the second excitation light source 11b. The first excitation light source 11a outputs excitation light 21a intensity-modulated at the first frequency. The second excitation light source 11b outputs excitation light 21b intensity-modulated at the first frequency and having a phase of intensity modulation different from that of the excitation light 21a.

[0072] The excitation light 21a and the excitation light 21b irradiated onto the sample 5 are intensity-modulated so that their phases are different from each other. Therefore, the fluorescence 27 generated by the excitation light 21a and the fluorescence 27 generated by the excitation light 21b are intensity-modulated with different phases from each other. The detector 8 detects the fluorescence 27 in synchronization with the respective phases, thereby being able to separate the fluorescence 27 generated by the excitation light 21a from the fluorescence 27 generated by the excitation light 21b.

[0073] Instead of the modulation device 9, the pumping light 21a and 21b emitted from the first pumping light source 11a and the second pumping light source 11b may be modulated using an LN modulator or the like.

[0074] In addition, intensity modulation and / or phase modulation may be performed using a pulsed laser light source.

[0075] [Embodiment 4] Another embodiment of the present invention will be described below. For convenience of explanation, the same reference numerals will be used to designate components having the same functions as those described in the above embodiment, and the description thereof will not be repeated.

[0076] FIG. 9 is a schematic diagram showing part of the configuration of the super-resolution microscope 1. The configuration shown in FIG. 9 is a diagram in which part of the optical system in the super-resolution microscope 1 shown in FIG. 2 is omitted. For example, the relay optical elements (3a to 45), the sample 5, and the elements (6 to 8) for fluorescence detection are not shown in FIG. 9. The direction of the optical axis OA of the excitation light 21 is the Z axis. The X, Y, and Z axes are perpendicular to one another. Reference numeral 100 denotes the point spread function 21p (PSF) of the excitation light 21 and the point spread functions 22pa and 22pc of the two quenching lights 22 in the YZ plane.

[0077] The point spread function 21p of the excitation light 21 is formed to pass through the focal plane FP and extend parallel to the optical axis OA direction in front of and behind the focal plane FP. The center of the point spread function 21p of the excitation light 21 is the focal position.

[0078] The point spread functions 22pa and 22pc of the two deactivation light beams 22 are formed to extend substantially parallel to the optical axis OA direction, similar to the excitation light beam 21, except for their positions on the focal plane FP. The point spread functions 22pa and 22pc of the two deactivation light beams 22 are formed to sandwich the point spread function 21p of the excitation light beam 21 and to partially overlap with the point spread function 21p of the excitation light beam 21. The same is true in the XZ plane. Therefore, the light-emitting region in the X and Y directions is reduced, improving the spatial resolution in the X and Y directions. Meanwhile, the point spread function 21p of the excitation light beam 21 extending in the optical axis OA direction also excites and emits light from phosphors located in front of or behind the focal plane FP as viewed from the objective lens 4. Therefore, the spatial resolution in the Z direction (depth direction) is almost the same as when the deactivation light beam 22 is not present.

[0079] Therefore, in the super-resolution microscope 1c, the point spread functions 22pa and 22pc of the multiple deactivation rays 22 are tilted with respect to the optical axis OA. Specifically, the super-resolution microscope 1c includes a mask disposed at a position where the multiple deactivation rays 22 do not overlap with one another. The multiple deactivation rays 22 do not overlap with one another near the image plane. For example, the mask is disposed at a position 51 near the second exit portion (the end face of the optical fiber) of the light guide 36, or at a position 52 near the image plane formed by the zoom lens 2. Alternatively, the mask may be disposed between the relay lens 43 and the mirror 44 shown in FIG. 2.

[0080] FIG. 10 is a plan view showing the configuration of the mask 50. The mask 50 has a first mask portion 50a, a second mask portion 50b, a third mask portion 50c, and a fourth mask portion 50d. With the excitation light 21 at the center, the first mask portion 50a blocks a portion of the outer side of the deactivation light 22a. The third mask portion 50c blocks a portion of the outer side of the deactivation light 22c. The second mask portion 50b blocks a portion of the inner side of the deactivation light 22b. The fourth mask portion 50d blocks a portion of the inner side of the deactivation light 22d. That is, the mask 50 blocks a portion of the outer side of the deactivation light 22a and 22c arranged in the Y direction on either side of the excitation light 21, and blocks a portion of the inner side of the deactivation light 22b and 22d arranged in the X direction on either side of the excitation light 21. Note that the mask 50 may overlap the excitation light 21 as long as it is a material that transmits the excitation light 21.

[0081] The distance between the first mask portion 50a and the third mask portion 50c is variable. Similarly, the distance between the second mask portion 50b and the fourth mask portion 50d is variable. This allows the positions of the first mask portion 50a to the fourth mask portion 50d to be adjusted so as to block part of the deactivation rays 22a to 22d, even if the distances between the deactivation rays 22a to 22d are changed by the zoom lens 2.

[0082] 11 is a schematic diagram showing part of the configuration of the super-resolution microscope 1c. Reference numeral 201 denotes one deactivation ray 22a in the YZ plane. A portion (outer side) of the deactivation ray 22a emitted from the zoom lens 2 is blocked by a mask 50, and the remainder (inner side) is focused onto the focal plane FP by the objective lens 4. The deactivation ray 22a is incident on the objective lens 4, biased to one side from the center. Therefore, the deactivation ray 22a is incident on the focal plane FP at a large angle of incidence with respect to the optical axis OA of the excitation light 21.

[0083] Reference numeral 101 denotes the point spread function 22pa of the deactivation light 22a in the YZ plane. The deactivation light 22a is incident on the focal plane FP, biased from one side (the -Y direction). Therefore, the point spread function 22pa of the deactivation light 22a is tilted in the Y direction (first direction) with respect to the optical axis OA of the excitation light 21. Furthermore, on the focal plane FP, the point spread function 22pa of the deactivation light 22a is spaced away from the focus of the excitation light 21 (the position of the optical axis OA) in the -Y direction. The point spread function 22pa of the deactivation light 22a intersects with the optical axis OA of the excitation light 21 behind the focus of the excitation light 21. Note that, because a portion of the deactivation light 22a is blocked, low spatial frequency components are reduced on the focal plane FP. Therefore, the width of the point spread function 22pa in the Y direction is wider than that of reference numeral 100. Although not shown, the same applies to the deactivation light 22c (which is symmetrical to the deactivation light 22a with respect to the optical axis OA of the excitation light 21).

[0084] The angle of incidence of the deactivation light 22a with respect to the optical axis OA of the excitation light 21 is changed by increasing or decreasing the area blocked by the mask 50. This makes it possible to change the slope of the point spread function 22pa of the deactivation light 22a with respect to the optical axis OA of the excitation light 21. The same applies to the other deactivation lights. For example, by increasing the angle of incidence of the deactivation light 22a, the spatial resolution in the direction of the optical axis OA can be improved.

[0085] Reference numeral 202 denotes one deactivation ray 22b in the XZ plane. A portion (inner portion) of the deactivation ray 22b emitted from the zoom lens 2 is blocked by the mask 50, and the remainder (outer portion) is collected on the focal plane FP by the objective lens 4. The deactivation ray 22b is incident on the objective lens 4, biased to one side from the center. Therefore, the deactivation ray 22b is incident on the focal plane FP at a large angle of incidence with respect to the optical axis OA of the excitation light 21.

[0086] Reference numeral 102 denotes the point spread function 22pb of the deactivation light 22b in the XZ plane. The deactivation light 22b is incident on the focal plane FP, biased from one side (the X direction side). Therefore, the point spread function 22pb of the deactivation light 22b is tilted in the −X direction (a second direction different from the first direction) with respect to the optical axis OA of the excitation light 21. Furthermore, on the focal plane FP, the point spread function 22pb of the deactivation light 22b is spaced away from the focus of the excitation light 21 (the position of the optical axis OA) in the −X direction. The point spread function 22pb of the deactivation light 22b intersects with the optical axis OA of the excitation light 21 just before the focus of the excitation light 21. Although not shown in the figure, the same is true for the deactivation light 22d (symmetrical to the deactivation light 22b with respect to the optical axis OA of the excitation light 21).

[0087] 12 is a diagram showing point spread functions 22pa to 22pd of the deactivation light 22a to 22d near the focal points. Reference numeral 103 denotes the point spread function 22pa of the deactivation light 22a and the point spread function 22pc of the deactivation light 22c in the YZ plane. On the focal plane FP, the point spread function 22pa of the deactivation light 22a is spaced in the -Y direction from the focal point of the excitation light 21 (the position of the optical axis OA). Contrary to the deactivation light 22a, the point spread function 22pc of the deactivation light 22c is spaced in the Y direction from the focal point of the excitation light 21 (the position of the optical axis OA) on the focal plane FP. The point spread functions 22pa and 22pc of the two deactivation light rays 22a and 22c intersect (overlap) with each other behind (in the Z direction) the focal point of the excitation light 21 (focal plane FP).

[0088] Reference numeral 104 denotes the point spread function 22pb of the deactivation light 22b and the point spread function 22pd of the deactivation light 22d in the XZ plane. On the focal plane FP, the point spread function 22pb of the deactivation light 22b is spaced in the −X direction from the focus of the excitation light 21 (the position of the optical axis OA). Contrary to the deactivation light 22b, on the focal plane FP, the point spread function 22pd of the deactivation light 22d is spaced in the X direction from the focus of the excitation light 21 (the position of the optical axis OA). The point spread functions 22pb and 22pd of the two deactivation lights 22b and 22d intersect (overlap) with each other in front of (on the −Z direction side of) the focus of the excitation light 21 (focal plane FP).

[0089] In the super-resolution microscope 1c, the point spread functions 22pa to 22pd of the four deactivation light beams 22a to 22d are superimposed three-dimensionally. Therefore, the point spread functions 22pa and 22pc of the two deactivation light beams 22a and 22c sandwich the point spread function 21p of the excitation light beam 21 in the Y direction (partially overlap), and also overlap with the point spread function 21p of the excitation light beam 21 on the Z direction side of the focal point of the excitation light beam 21. The point spread functions 22pb and 22pd of the two deactivation light beams 22b and 22d sandwich the point spread function 21p of the excitation light beam 21 in the X direction (partially overlap), and also overlap with the point spread function 21p of the excitation light beam 21 on the −Z direction side of the focal point of the excitation light beam 21. The point spread functions 22pa to 22pd of the four quenching light beams 22a to 22d also sandwich the point spread function 21p of the excitation light beam 21 in the Z direction. Therefore, the point spread functions 22pa to 22pd of the four quenching light beams 22a to 22d can limit the fluorescence emission region not only in the XY directions but also in the Z direction. Therefore, the super-resolution microscope 1c can also increase the spatial resolution in the Z direction (depth direction).

[0090] The positions of the point spread functions 22pa to 22pd of the deactivation rays 22a to 22d can be changed by changing the magnification of the zoom lens 2. This makes it possible to adjust the size of the light emitting area not only in the XY directions but also in the Z direction.

[0091] Note that additional multiple magnification / reduction lenses may be introduced to increase the distance between the deactivation lights 22a to 22d in order to position the mask 50. For example, a magnification lens and a reduction lens may be positioned between the light guide 36 and the zoom lens 2, and the mask 50 may be positioned between the magnification lens and the reduction lens. The mask 50 can be positioned at a location where the deactivation lights 22a to 22d are separated from each other by the magnification lens.

[0092] (Modification) The shape of the mask is not limited to the above. For example, an iris whose axis is shifted from the center of each deactivation light may be provided as a mask for each deactivation light.

[0093] Instead of a mechanically displaceable shielding member, a liquid crystal element may be used as the mask 50. A pixel array of the liquid crystal element may form an area that transmits the deactivation rays 22a to 22d and an area that blocks the deactivation rays 22a to 22d.

[0094] FIG. 13 is a cross-sectional view showing a schematic configuration of a light guide 36c according to another modification. In FIG. 13, only the deactivation light 22a is depicted as the laser beam. A light guide 36c may be used instead of a mask 50, so that the deactivation light 22a-22d enter the focal plane FP at an angle relative to the optical axis OA of the excitation light 21. The light guide 36c is formed with a first exit portion 37c and multiple second exit portions 38ca, 38cc. The first exit portion 37c emits the excitation light 21. The core end faces of the multiple second exit portions 38ca, 38cc are formed at an angle relative to the core end face of the first exit portion 37c. This causes the direction of the deactivation light 22a emitted from the second exit portion 38ca to be tilted outward relative to the optical axis OA of the excitation light 21. This can achieve the same effect as, for example, placing a mask 50 at the position indicated by the dotted line in the figure. Similarly, the direction of the deactivation light emitted from the second emission portion 38cc is also tilted outward with respect to the optical axis OA of the excitation light 21.

[0095] The end faces of the cores of the two second exit portions (not shown) are tilted inward (toward the optical axis OA of the excitation light 21). The direction of the deactivation light emitted from the two second exit portions is tilted inward with respect to the optical axis OA of the excitation light 21. By tilting the direction of the deactivation light in this way, the point spread function of the excitation light can be three-dimensionally surrounded by the point spread functions of multiple deactivation lights, as shown in FIG. 12. Unlike a configuration in which the deactivation light 22 is blocked by a mask 50, the configuration using the light guide 36c does not involve a loss of intensity of the deactivation light 22. Therefore, the intensity of the deactivation light 22 can be increased.

[0096] The light guide 36c and the mask 50 may be used together. The direction of the deactivation light 22 emitted by the light guide 36c may be tilted, and the tilt of the deactivation light 22 may be further adjusted by the mask 50. The area blocked by the mask 50 can be reduced by the amount of tilt caused by the light guide 36c. Therefore, the intensity of the deactivation light 22 can be made stronger than when only the mask 50 is used.

[0097] Alternatively, instead of tilting the direction of the deactivation light using the light guide 36c, the direction of the deactivation light may be tilted by an optical element. For example, a diffractive beam splitter (multi-beam generator) may be used to generate multiple deactivation light beams traveling in different directions from a single deactivation light beam. For example, after multiple deactivation light beams traveling in different directions are generated by the diffractive beam splitter, they may be merged with the excitation light using a dichroic mirror or the like in the optical system. Alternatively, instead of a diffractive beam splitter, an SLM (spatial light modulator) may be used to generate multiple deactivation light beams traveling in different directions.

[0098] Alternatively, a hologram may be used to position the point spread function of the deactivation light so as to surround the point spread function of the excitation light. For example, a hologram may be placed in the optical path of the deactivation light to form a real image of the deactivation light surrounding the focal point of the excitation light.

[0099] A wavefront control device may be disposed in the optical system for adaptive optics. A deformable mirror, an SLM, or the like may be used as the wavefront control device. Furthermore, the wavefront of a single deactivation beam may be controlled by the wavefront control device so that the deactivation beam is focused at multiple positions. This allows the formation of point spread functions of multiple deactivation beams surrounding the focal point of the excitation beam. Of course, the wavefront control device may also perform optical compensation for the excitation beam and multiple deactivation beams. This allows the respective spots of the excitation beam and multiple deactivation beams to be reduced deep within the sample 5.

[0100] However, a configuration using a hologram or a wavefront control device may be affected by the refractive index distribution of the sample 5. In contrast, a configuration using the mask 50 or the light guide 36c is relatively less affected by the refractive index distribution of the sample 5.

[0101] The above-mentioned method may be combined with fluorescence lifetime measurement to apply a technique called Gated STED, which measures only the long-lived component. This allows obtaining only the fluorescence from the central portion, further improving spatial resolution.

[0102] The above-mentioned method may also be applied to a technique called RESOLFT. For example, the on / off state of fluorescent molecules is switched using the above-mentioned excitation light and quenching light. As a result, only fluorescent molecules in an area smaller than the diffraction limit that are not irradiated with quenching light are turned on. Fluorescence may also be measured by irradiating the on-state fluorescent molecules with laser light for measuring a different fluorescence. This method allows measurements to be performed using weaker STED light, thereby reducing damage to cells.

[0103] The above-described super-resolution microscopes 1, 1a, 1b, and 1c can also be applied to rigid endoscopes that perform fluorescence measurement by laser scanning.

[0104] [Summary] The super-resolution microscope according to aspect 1 of the present invention comprises a first exit section that emits first excitation light that excites a phosphor, a second exit section that emits first quenching light that deactivates the phosphor, an objective lens that focuses the first quenching light from the second exit section so that the center of the first excitation light from the first exit section and the center of the first quenching light from the second exit section are offset from each other in a sample containing the phosphor, and a zoom lens that is arranged between the first exit section, the second exit section, and the objective lens and is capable of changing the magnification.

[0105] A super-resolution microscope according to Aspect 2 of the present invention may be configured in the above-described Aspect 1 such that the first exit portion and the second exit portion are formed in a single light guide.

[0106] A super-resolution microscope according to a third aspect of the present invention may be configured in the second aspect described above, wherein the light guide is an optical fiber bundle.

[0107] A super-resolution microscope according to aspect 4 of the present invention may be configured in any one of aspects 1 to 3 above, to include a plurality of second exit sections, and the objective lens may be configured to focus a plurality of the first quenching lights from the plurality of second exit sections at different positions around the first excitation light from the first exit section in a sample containing the phosphor.

[0108] A super-resolution microscope according to aspect 5 of the present invention may be configured in the above-mentioned aspect 4 such that, during a period in which one of the plurality of second exit sections is not irradiating the first deactivation light onto the sample, at least one of the other second exit sections irradiates the first deactivation light onto the sample.

[0109] A super-resolution microscope according to a sixth aspect of the present invention is any of the first to fifth aspects described above, and further comprises: a third exit section that emits second excitation light that excites the phosphor; and a fourth exit section that emits second quenching light that deactivates the phosphor; wherein the objective lens may be configured to focus the first excitation light emitted from the first exit section and the second excitation light emitted from the third exit section at different positions on a sample containing the phosphor; and to focus the second quenching light from the fourth exit section so that the center of the second excitation light from the third exit section and the center of the second quenching light from the fourth exit section are shifted from each other on the sample containing the phosphor; and wherein the third exit section irradiates the sample with the second excitation light during a period when the first exit section is not irradiating the sample with the first excitation light.

[0110] A super-resolution microscope according to aspect 7 of the present invention may be configured in the above-described aspect 6 such that the third exit unit starts irradiating the sample after a period equal to or greater than the fluorescence lifetime of the phosphor has elapsed since the first exit unit finished irradiating the sample.

[0111] A super-resolution microscope according to aspect 8 of the present invention is any of aspects 1 to 4 described above, and further comprises a third exit section that emits second excitation light that excites the phosphor, and a fourth exit section that emits second quenching light that deactivates the phosphor, wherein the objective lens may be configured to focus the first excitation light emitted from the first exit section and the second excitation light emitted from the third exit section at different positions in a sample containing the phosphor, and to focus the second quenching light from the fourth exit section so that the centers of the second excitation light from the third exit section and the second quenching light from the fourth exit section are shifted from each other in the sample containing the phosphor, and the first excitation light emitted from the first exit section and the second excitation light emitted from the third exit section may be intensity-modulated at different frequencies.

[0112] A super-resolution microscope according to aspect 9 of the present invention is, in any of aspects 1 to 4 above, provided with a third exit section that emits second excitation light that excites the phosphor, and a fourth exit section that emits second quenching light that deactivates the phosphor, wherein the objective lens may be configured to focus the first excitation light emitted from the first exit section and the second excitation light emitted from the third exit section at different positions in a sample containing the phosphor, and to focus the second quenching light from the fourth exit section so that the centers of the second excitation light from the third exit section and the second quenching light from the fourth exit section are shifted from each other in the sample containing the phosphor, and the first excitation light emitted from the first exit section and the second excitation light emitted from the third exit section may be intensity-modulated so that they have different phases from each other.

[0113] A super-resolution microscope according to aspect 10 of the present invention may be configured in the above-mentioned aspect 4 such that the point spread functions of two of the first quenching lights from the plurality of second exit portions intersect with each other on the optical axis of the first excitation light and just before the focus of the first excitation light, and the point spread functions of the other two of the first quenching lights from the plurality of second exit portions intersect with each other on the optical axis of the first excitation light and just behind the focus of the first excitation light.

[0114] A super-resolution microscope according to aspect 11 of the present invention may be configured in the above-mentioned aspect 10 such that the point spread functions of the two first quenching lights are inclined in a first direction perpendicular to the optical axis of the first excitation light, and the point spread functions of the other two first quenching lights are inclined in a second direction perpendicular to the optical axis, different from the first direction, with respect to the optical axis of the first excitation light.

[0115] A super-resolution microscope according to aspect 12 of the present invention may be configured in the above-mentioned aspects 10 or 11, further comprising a mask that blocks a portion of the first quenching light in order to tilt the point spread function of the first quenching light with respect to the optical axis of the first excitation light.

[0116] A super-resolution microscope according to Aspect 13 of the present invention may be configured in the above-described Aspect 12, wherein the mask is positioned so that the first quenching lights do not overlap each other.

[0117] A super-resolution microscope according to aspect 14 of the present invention may be configured in any one of aspects 10 to 13 above, wherein the first exit portion and the plurality of second exit portions are formed in a single light guide, and an end face of a core constituting the second exit portion is inclined with respect to an end face of a core constituting the first exit portion in order to tilt the point spread function of the first quenching light with respect to the optical axis of the first excitation light.

[0118] A method for controlling a super-resolution microscope according to aspect 15 of the present invention includes a first emission step of emitting excitation light that excites a phosphor, a second emission step of emitting deactivation light that deactivates the phosphor, a changing step of changing the magnification of a zoom lens onto which the excitation light and the deactivation light are incident, and a focusing step of focusing the deactivation light on a sample containing the phosphor so that the center of the excitation light and the center of the deactivation light are shifted from each other.

[0119] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0120] REFERENCE SIGNS LIST 1, 1a, 1b, 1c super-resolution microscope 2 zoom lens 3a, 3b galvanometer mirror 4 objective lens 5 sample 6 dichroic mirror 7 band-pass filter 8 detector 9 modulator 11 excitation light source 11a first excitation light source 11b second excitation light source 12 deactivation light source 21, 21a, 21b excitation light 21p, 22pa, 22pb, 22pc, 22pd point spread function 22, 22a, 22aa, 22b, 22ba, 22c, 22d deactivation light 31 first input section 31a third input section 31b fourth input section 32 second input section 33 branch section 33a first branch section 33b second branch section 34, 34a, 34b first optical fiber 35, 35a, 35b Second optical fiber 36, 36c Light guide 36a First light guide 36b Second light guide 37, 37a, 37c First emission section 37b Third emission section 38a to 38d, 38aa to 38ad, 38ca, 38cc Second emission section 38ba to 38bd Fourth emission section 50 Mask

Claims

1. A super-resolution microscope comprising: a first emission section that emits first excitation light that excites a phosphor; a second emission section that emits first quenching light that quenches the phosphor; an objective lens that focuses the first quenching light from the second emission section so that the center of the first excitation light from the first emission section and the center of the first quenching light from the second emission section are offset from each other in a sample containing the phosphor; and a zoom lens that is positioned between the first emission section, the second emission section, and the objective lens and has an adjustable magnification.

2. The super-resolution microscope according to claim 1, wherein the first exit section and the second exit section are formed in a single light guide.

3. The super-resolution microscope according to claim 2, wherein the light guide is an optical fiber bundle.

4. A super-resolution microscope as described in claim 1, comprising a plurality of second exit sections, and wherein the objective lens focuses the plurality of first quenching lights from the plurality of second exit sections at mutually different positions around the first excitation light from the first exit section in a sample containing the fluorescent material.

5. A super-resolution microscope as described in claim 4, wherein, during a period when one of the plurality of second emission sections is not irradiating the first deactivation light onto the sample, at least one of the other second emission sections irradiates the first deactivation light onto the sample.

6. A super-resolution microscope according to any one of claims 1 to 5, comprising: a third emission section that emits second excitation light that excites the phosphor; and a fourth emission section that emits second deactivation light that deactivates the phosphor, wherein the objective lens: focuses the first excitation light emitted from the first emission section and the second excitation light emitted from the third emission section at different positions in a sample containing the phosphor; and focuses the second deactivation light from the fourth emission section so that the center of the second excitation light from the third emission section and the center of the second deactivation light from the fourth emission section are shifted from each other in the sample containing the phosphor; and the third emission section irradiates the sample with the second excitation light during a period when the first emission section is not irradiating the sample with the first excitation light.

7. A super-resolution microscope as described in claim 6, wherein the third emission section starts irradiating the sample after a period of time equal to or greater than the fluorescence lifetime of the phosphor has elapsed since the first emission section finished irradiating the sample.

8. A super-resolution microscope according to any one of claims 1 to 4, comprising: a third emission section that emits second excitation light that excites the phosphor; and a fourth emission section that emits second deactivation light that deactivates the phosphor, wherein the objective lens: focuses the first excitation light emitted from the first emission section and the second excitation light emitted from the third emission section at different positions in a sample containing the phosphor; and focuses the second deactivation light from the fourth emission section so that the center of the second excitation light from the third emission section and the center of the second deactivation light from the fourth emission section are shifted from each other in the sample containing the phosphor; and the first excitation light emitted from the first emission section and the second excitation light emitted from the third emission section are intensity-modulated at different frequencies.

9. A super-resolution microscope according to any one of claims 1 to 4, comprising: a third emission section that emits second excitation light that excites the phosphor; and a fourth emission section that emits second deactivation light that deactivates the phosphor, wherein the objective lens: focuses the first excitation light emitted from the first emission section and the second excitation light emitted from the third emission section at different positions in a sample containing the phosphor; and focuses the second deactivation light from the fourth emission section so that the centers of the second excitation light from the third emission section and the second deactivation light from the fourth emission section are shifted from each other in the sample containing the phosphor; and the first excitation light emitted from the first emission section and the second excitation light emitted from the third emission section are intensity-modulated so that they have different phases from each other.

10. A super-resolution microscope as described in claim 4, wherein the point spread functions of two of the first quenching lights from the plurality of second exit portions intersect with each other on the optical axis of the first excitation light and before the focus of the first excitation light, and the point spread functions of the other two of the first quenching lights from the plurality of second exit portions intersect with each other on the optical axis of the first excitation light and behind the focus of the first excitation light.

11. A super-resolution microscope as described in claim 10, wherein the point spread functions of the two first quenching lights are inclined in a first direction perpendicular to the optical axis of the first excitation light, and the point spread functions of the other two first quenching lights are inclined in a second direction perpendicular to the optical axis, different from the first direction, with respect to the optical axis of the first excitation light.

12. A super-resolution microscope as described in claim 10 or 11, comprising a mask that blocks a portion of the first quenching light to tilt the point spread function of the first quenching light with respect to the optical axis of the first excitation light.

13. The super-resolution microscope according to claim 12, wherein the mask is positioned so that the first quenching lights do not overlap each other.

14. A super-resolution microscope as described in claim 10 or 11, wherein the first exit section and the plurality of second exit sections are formed in a single light guide, and the end face of the core constituting the second exit section is inclined with respect to the end face of the core constituting the first exit section in order to tilt the point spread function of the first quenching light with respect to the optical axis of the first excitation light.

15. A method for controlling a super-resolution microscope, comprising: a first emission step of emitting excitation light that excites a phosphor; a second emission step of emitting deactivation light that deactivates the phosphor; a change step of changing the magnification of a zoom lens onto which the excitation light and the deactivation light are incident; and a focusing step of focusing the deactivation light on a sample containing the phosphor so that the center of the excitation light and the center of the deactivation light are offset from each other.

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