JPWO2015033514A1 - Illumination device and structured illumination microscope device - Google Patents

Illumination device and structured illumination microscope device Download PDF

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JPWO2015033514A1
JPWO2015033514A1 JP2014004009A JP2015535297A JPWO2015033514A1 JP WO2015033514 A1 JPWO2015033514 A1 JP WO2015033514A1 JP 2014004009 A JP2014004009 A JP 2014004009A JP 2015535297 A JP2015535297 A JP 2015535297A JP WO2015033514 A1 JPWO2015033514 A1 JP WO2015033514A1
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light beam
light
direction
plurality
illumination
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JP6137324B2 (en
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範夫 三宅
範夫 三宅
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株式会社ニコン
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    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/06Means for illuminating specimens
    • G02B21/08Condensers
    • G02B21/082Condensers for incident illumination only
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/36Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365Control or image processing arrangements for digital or video microscopes
    • G02B21/367Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison

Abstract

In order to improve the acquisition efficiency of a plurality of modulated images necessary for generating a super-resolution image (demodulated image), the structured illumination device of the present invention includes a branching unit that branches light from a light source into a plurality of light beams, A light beam selection member that selects a part of the plurality of light beams, an optical system that illuminates an object with the selected part of the light beam, and a rotation that switches the illumination by rotating the light beam selection member A rotation axis of the light beam selecting member is provided at a position deviating from a branch center of the plurality of light beams, and on each circumference of two or more circles centered on the rotation axis, A plurality of selectors for selecting the light flux are provided at predetermined intervals.

Description

  The present invention relates to an illumination device and a structured illumination microscope device.

  As a technique for super-resolution observation of an observation object such as a biological specimen, there is a technique of modulating the spatial frequency of the structure of the observation object with illumination light (see Patent Document 1).

  In this technique, the object to be observed is illuminated with spatially modulated illumination light, and high spatial frequency information exceeding the resolution limit included in the structure of the object to be observed is contributed to the imaging of the microscope optical system. Further, by switching the spatial illumination pattern and performing operations on a plurality of modulated image data (hereinafter referred to as “modulated images”) obtained under different illumination patterns, demodulated image data (hereinafter referred to as “modulated image data”). (Referred to as “demodulated image” or “super-resolution image”).

US Reissue Patent No. 38307 Specification

  An object of the present invention is to improve the acquisition efficiency of a plurality of modulated images necessary for generating a super-resolution image (demodulated image).

  An example of the illuminating device of the present invention includes a branching unit that branches light from a light source into a plurality of light beams, a light beam selection member that selects a part of the light beams from the plurality of light beams, and the selected part of the light beams. An optical system that illuminates an object, and a rotation mechanism that switches the illumination by rotating the light beam selection member, and a rotation axis of the light beam selection member is deviated from a branching center of the plurality of light beams A plurality of selection units for selecting a light beam are provided at predetermined intervals on each circumference of two or more circles provided at positions and centered on the rotation axis.

  An example of the structured illumination device of the present invention is formed by an example of the illumination device of the present invention, an imaging optical system that forms a modulated image of the object spatially modulated by the interference fringes, and the imaging optical system. And an imaging device that captures the modulated image to generate a modulated image.

1 is a configuration diagram of a structured illumination microscope apparatus 1. FIG. It is a figure explaining the diffraction grating. It is a figure explaining the 0th-order light shutter 200 and the light beam selection member 18. FIG. It is a figure explaining the light beam selection member 18 in the case of θ ′ = 0 °. It is a figure explaining the light beam selection member 18 in the case of θ ′ = 20 °. It is a figure explaining the light beam selection member 18 in (theta) '= 40 degrees. It is a figure explaining the light beam selection member 18 in (theta) '= 60 degrees. It is a figure explaining the light beam selection member 18 in the case of θ ′ = 80 °. It is a figure explaining the light beam selection member 18 in the case of θ ′ = 100 °. It is a figure explaining the function of the polarizing plate 23 and the half-wave plate 17. FIG. It is a figure explaining the function of 15 A of translation mechanisms. It is a timing chart of a prior art example. It is a figure explaining the relationship between the required time t1 and t2.

[First Embodiment]
Hereinafter, a structured illumination microscope apparatus will be described as a first embodiment of the present invention.

  First, the structure of the structured illumination microscope apparatus will be described.

  FIG. 1 is a configuration diagram of the structured illumination microscope apparatus 1. Hereinafter, a case where the structured illumination microscope apparatus 1 is used as a total internal reflection fluorescence microscope (TIRFM) will be described as appropriate. TIRFM is a microscope that observes an extremely thin layer on the surface of a sample (specimen) 5 having fluorescence.

  First, the structure of the structured illumination microscope apparatus 1 will be described.

  As shown in FIG. 1, the structured illumination microscope apparatus 1 includes a laser unit 100, an optical fiber 11, an illumination optical system 10, an imaging optical system 30, a first image sensor 351, and a second image sensor 352. A control device 39, an image storage / arithmetic device 40, and an image display device 45 are provided. The illumination optical system 10 is an epi-illumination type, and the specimen 5 is illuminated using the objective lens 6 and the dichroic mirror 7 of the imaging optical system 30.

  The laser unit 100 includes a first laser light source 101, a second laser light source 102, shutters 1031, 1032, a mirror 105, a dichroic mirror 106, and a lens 107. Each of the first laser light source 101 and the second laser light source 102 is a coherent light source, and the emission wavelengths thereof are different from each other. Here, it is assumed that the wavelength λ1 of the first laser light source 101 is longer than the wavelength λ2 of the second laser light source 102 (λ1> λ2). The first laser light source 101, the second laser light source 102, and the shutters 1031 and 1032 are driven and controlled by the control device 39, respectively.

  The optical fiber 11 is configured by, for example, a polarization-preserving single mode fiber in order to guide the laser light emitted from the laser unit 100. The position of the exit end of the optical fiber 11 in the direction of the optical axis O can be adjusted by the position adjusting mechanism 11A. The position adjusting mechanism 11A is driven and controlled by the control device 39.

  The illumination optical system 10 includes, in order from the emission end side of the optical fiber 11, a collector lens 12, a polarizing plate 23, a light beam branching unit 15, a condensing lens 16, a light beam selecting unit 24, a lens 25, and a field of view. A diaphragm 26, a field lens 27, an excitation filter 28, a dichroic mirror 7, and an objective lens 6 are disposed.

  When a polarization-preserving single mode fiber is used as the optical fiber 11, the polarization plane of the laser light is preserved before and after the optical fiber 11, so that the polarizing plate 23 is not essential, but the laser light This is effective for maintaining the quality of polarized light. On the other hand, when a multimode fiber is used as the optical fiber 11, the polarizing plate 23 is essential.

  The beam splitter 15 includes a diffractive optical element (diffraction grating) 13 and a translation mechanism 15A, and the beam selector 24 includes a zero-order light shutter 200, a half-wave plate 17, and a beam selector. 18, a rotation mechanism 200 </ b> A, a rotation mechanism 17 </ b> A, and a rotation mechanism 18 </ b> A. The translation mechanism 15A, the rotation mechanism 200A, the rotation mechanism 17A, and the rotation mechanism 18A are driven and controlled by the control device 39.

  In the imaging optical system 30, an objective lens 6, a dichroic mirror 7, a barrier filter 31, a second objective lens 32, and a second dichroic mirror 35 are arranged in this order from the sample 5 side.

  The specimen 5 is, for example, fluorescent cells (cells stained with a fluorescent dye) arranged on a parallel flat glass surface, or fluorescent living cells (moving cells stained with a fluorescent dye) present in a petri dish. ) And so on. In this cell, both the first fluorescent region excited by light of wavelength λ1 and the second fluorescent region excited by light of wavelength λ2 are expressed.

  The first fluorescent region generates the first fluorescence having the center wavelength λ1 ′ in response to the light having the wavelength λ1, and the second fluorescent region generates the second fluorescence having the center wavelength λ2 ′ in response to the light having the wavelength λ2. Let

  When the structured illumination microscope apparatus 1 is used as a TIRFM (total reflection fluorescence microscope), the objective lens 6 is configured as an all immersion type (oil immersion type) objective lens. That is, the gap between the objective lens 6 and the glass of the specimen 5 is filled with the immersion liquid (oil).

  Each of the first image sensor 351 and the second image sensor 352 is a two-dimensional image sensor composed of a CCD, a CMOS, or the like. When each of the first imaging element 351 and the second imaging element 352 is driven by the control device 39, an image formed on each of the imaging surface 361 of the first imaging element 351 and the imaging surface 362 of the second imaging element 352. Is captured and an image is generated. Images generated by the first image sensor 351 and the second image sensor 352 are taken into the image storage / arithmetic device 40 via the control device 39. Note that each of the first imaging element 351 and the second imaging element 352 can repeat image generation (imaging) at a predetermined frame period. The frame period (imaging repetition period) of each of the first image sensor 351 and the second image sensor 352 is the image capture time of the image sensor (that is, the time required for charge accumulation and charge readout), the time required for switching the direction of the interference fringes, Among other required times, it is determined by rate limiting, and is, for example, 30 msec, 60 msec, etc. (details will be described later).

  The control device 39 drives and controls the laser unit 100, the position adjustment mechanism 11A, the translation mechanism 15A, the rotation mechanism 200A, the rotation mechanism 17A, the rotation mechanism 18A, the first image sensor 351, and the second image sensor 352.

  The image storage / arithmetic unit 40 performs demodulation calculation for each wavelength on the image given via the control unit 39, and stores the calculated image (super-resolution image for each wavelength) in an internal memory (not shown). At the same time, it is sent to the image display device 45.

  Next, the behavior of laser light in the structured illumination microscope apparatus 1 will be described.

  When the laser beam (first laser beam) having the wavelength λ1 emitted from the first laser light source 101 enters the mirror 105 via the shutter 1031, it reflects from the mirror 105 and enters the dichroic mirror 106. On the other hand, the laser light having the wavelength λ2 (second laser light) emitted from the second laser light source 102 enters the beam splitter 106 via the shutter 1032 and is integrated with the first laser light. The first laser beam and the second laser beam emitted from the dichroic mirror 106 enter the incident end of the optical fiber 11 through the lens 107.

  The control device 39 controls the shutters 1031 and 1032 of the laser unit 100 to change the wavelength (= light source wavelength) of the laser light incident on the incident end of the optical fiber 11 between the long wavelength λ1 and the short wavelength λ2. The light source wavelength can be set to both the long wavelength λ1 and the short wavelength λ2.

  The laser light incident on the incident end of the optical fiber 11 propagates inside the optical fiber 11 and generates a point light source at the output end of the optical fiber 11. The laser light emitted from the point light source is converted into a parallel light beam by the collector lens 12 and enters the diffraction grating 13 via the polarizing plate 23, and then is diffracted light beam of each order (hereinafter referred to as “diffracted light beam group”). Fork. When the diffracted light beam group is incident on the condenser lens 16, the diffracted light beam group is focused on each position of the pupil conjugate plane 6 </ b> A ′ by receiving the condensing action of the condenser lens 16.

  Here, the pupil conjugate plane 6A ′ is the focal position (rear focal position) of the condenser lens 16, and the pupil 6A of the objective lens 6 described later with respect to the field lens 27 and the lens 25 (± first-order diffracted light is condensed). Position). The lens 16 is arranged so that the focal position (rear focal position) of the lens 16 coincides with the pupil conjugate plane 6A '. It should be noted that the concept of “conjugate position” includes a position determined by a person skilled in the art in consideration of design requirements such as aberration and vignetting of the objective lens 6, the field lens 27, and the lens 25. .

  In addition, since the laser light emitted from the optical fiber 11 is basically linearly polarized, the polarizing plate 23 can be omitted, but it is effective for surely cutting off the excess polarization component. Further, in order to increase the utilization efficiency of the laser light, it is desirable that the axis of the polarizing plate 23 coincides with the polarization direction of the laser light emitted from the optical fiber 11.

  Now, the diffracted light beams of the respective orders directed toward the pupil conjugate plane 6A ′ enter the light beam selection unit 24 disposed in the vicinity of the pupil conjugate plane 6A ′.

  Here, when the structured illumination microscope apparatus 1 is used as a TIRFM (total reflection fluorescence microscope), the light beam selection unit 24 includes only one pair of diffracted light beams (here, ± 1) among the incident diffracted light beams of each order. (Only the next diffracted light beam) is selectively passed.

  The ± first-order diffracted light beam that has passed through the light beam selecting unit 24 forms a conjugate plane with the diffraction grating 13 near the field stop 26 by the lens 25. Thereafter, each of the ± first-order diffracted light beams is converted into convergent light by the field lens 27, further reflected by the dichroic mirror 7 after passing through the excitation filter 28, and condensed at different positions on the pupil plane 6 </ b> A of the objective lens 6. To do.

  Each of the ± first-order diffracted light beams collected on the pupil plane 6A becomes a parallel light beam when emitted from the tip of the objective lens 6 and interferes with each other on the surface of the sample 5 to form interference fringes. This interference fringe is used as structured illumination light.

  Further, when the structured illumination microscope apparatus 1 is used as a TIRFM (total reflection fluorescence microscope), the incident angle when entering the surface of the specimen 5 is the total reflection condition (TIRF condition) which is an evanescent field generation condition. Fulfill.

  In order to satisfy the TIRF condition, the condensing point of the ± 1st-order diffracted light beam on the pupil plane 6A only needs to be located in a predetermined annular zone located on the outermost periphery of the pupil plane 6A. In this case, an evanescent field due to interference fringes is generated near the surface of the specimen 5.

  When the specimen 5 is illuminated with such interference fringes, a moire fringe corresponding to the difference between the periodic structure of the interference fringes and the periodic structure of the fluorescent region on the specimen 5 appears. Since the structure is shifted to a lower frequency side than the original frequency, the fluorescence indicating this structure is directed toward the objective lens 6 at an angle smaller than the original angle. Therefore, when the specimen 5 is illuminated by the interference fringes, even the high-frequency structural information of the fluorescent region is transmitted by the objective lens 6.

  When the fluorescence generated in the sample 5 enters the objective lens 6, it is converted into parallel light by the objective lens 6, then passes through the dichroic mirror 7 and the barrier filter 31, and enters the second dichroic mirror 35. The first fluorescence having the wavelength λ1 ′ incident on the second dichroic mirror 35 is reflected by the second dichroic mirror 35, and the second fluorescence having the wavelength λ2 ′ incident on the second dichroic mirror 35 is transmitted through the second dichroic mirror 35. To do.

  The first fluorescence reflected from the second dichroic mirror 35 forms a modulated image of the first fluorescence region on the imaging surface 361 of the first imaging element 351, and the second fluorescence transmitted through the second dichroic mirror 35 is second A modulated image of the second fluorescent region is formed on the imaging surface 362 of the imaging element 352.

  The modulated image of the first fluorescent region formed on the imaging surface 361 and the modulated image of the second fluorescent region formed on the imaging surface 362 are individually imaged by the first imaging device 351 and the second imaging device 352, and A modulated image of one fluorescent region and a modulated image of the second fluorescent region are generated.

  The modulated image of the first fluorescent region and the modulated image of the second fluorescent region are taken into the image storage / arithmetic device 40 via the control device 39. Further, each of the captured modulated image of the first fluorescent region and the modulated image of the second fluorescent region is subjected to a demodulation operation in the image storage / arithmetic device 40, so that the demodulated image (super solution) of the first fluorescent region is obtained. Image) and a demodulated image (super-resolution image) of the second fluorescent region are generated. These super-resolution images are stored in an internal memory (not shown) of the image storage / arithmetic device 40 and are sent to the image display device 45. As the demodulation operation, for example, a method disclosed in US Pat. No. 8,115,806 is used.

  Next, the diffraction grating 13 will be described in detail.

  2A is a view of the diffraction grating 13 as viewed from the direction of the optical axis O, and FIG. 2B shows the positional relationship of the condensing points formed on the pupil conjugate plane by the ± first-order diffracted light beams. FIG. 2A is a schematic diagram, the structure period of the diffraction grating 13 illustrated in FIG. 2A is not necessarily the same as the actual structure period. In addition, the condensing point here is a barycentric position of an area having 80% or more of the maximum intensity. Therefore, the illumination optical system 10 of the present embodiment does not need to collect the light beam until a complete condensing point is formed.

As shown in FIG. 2A, the diffraction grating 13 is a diffraction grating having a periodic structure in a plurality of different directions within a plane perpendicular to the optical axis O of the illumination optical system 10. Here, the diffraction grating 13 is a three-way diffraction grating having a periodic structure in each of the first direction V 1 , the second direction V 2 , and the third direction V 3 that are different by 60 °, and the period of these periodic structures is Are common.

  The material of the diffraction grating 13 is, for example, glass. The periodic structure of the diffraction grating 13 is either a concentration-type periodic structure formed using density (transmittance) or a phase-type periodic structure formed using steps (phase difference). However, the phase difference type periodic structure is preferable in that the diffraction efficiency of the + 1st order diffracted light is higher.

Parallel beam incident on such grating 13, a first diffracted light flux group branched toward the first direction V 1, and the second diffracted light flux group branched toward the second direction V 2, branches toward the third direction V 3 Converted into the third diffracted light beam group.

  The first diffracted light beam group includes a 0th order diffracted light beam and a ± 1st order diffracted light beam. Among these, the ± 1st order diffracted light beam having the same order travels in a symmetric direction with respect to the optical axis O, and 0 The next diffracted light beam travels along the optical axis O.

  Similarly, the second diffracted light beam group includes a 0th-order diffracted light beam and a ± 1st-order diffracted light beam, and of these, the ± 1st-order diffracted light beams having common orders travel in a symmetric direction with respect to the optical axis O. Then, the 0th-order diffracted light beam travels along the optical axis O.

  Similarly, the third diffracted light beam group includes a 0th-order diffracted light beam and a ± 1st-order diffracted light beam, and of these, the ± 1st-order diffracted light beams having common orders travel in a symmetric direction with respect to the optical axis O. Then, the 0th-order diffracted light beam travels along the optical axis O.

  The ± 1st order diffracted light beam of the first diffracted light beam group, the ± 1st order diffracted light beam of the second diffracted light beam group, and the ± 1st order diffracted light beam of the third diffracted light beam group are converted into the pupil conjugate plane 6A by the condenser lens 16 described above. 'Are condensed at different positions.

As shown in FIG. 2B, the condensing points 14d and 14g of the ± first-order diffracted light beams of the first diffracted light beam group are symmetric with respect to the optical axis O, and the arrangement direction of the condensing points 14d and 14g is It corresponds to the first direction V 1.

Further, the focal point 14c of ± 1-order diffracted light flux of the second diffracted light beam group, 14f is symmetrical with respect to the optical axis O, the condensing point 14c, the arrangement direction of 14f, corresponding to the second direction V 2 Yes. In addition, the distance from the condensing points 14c and 14f of the second diffracted light beam group to the optical axis O is the same as the distance from the condensing points 14d and 14g of the first diffracted light beam group to the optical axis O.

Further, the focal point 14b of the ± 1-order diffracted light flux of the third diffracted light flux group, 14e are symmetric with respect to optical axis O, the condensing point 14b, the arrangement direction of the 14e, corresponding to the third direction V 3 Yes. In addition, the distance from the condensing points 14b and 14e of the third light beam group to the optical axis O is the same as the distance from the condensing points 14d and 14g of the first diffracted light beam group to the optical axis O.

  As shown in FIG. 2B, the condensing point 14a of the 0th-order diffracted light beam of each of the first to third diffracted light beam groups is located on the optical axis O.

The diffraction grating 13 described above can be translated by a translation mechanism 15A (see FIG. 1) composed of a piezo motor or the like. The direction of translational movement of the diffraction grating 13 by the translation mechanism 15A is a direction perpendicular to the optical axis O of the illumination optical system 10, and is in the first direction V 1 , the second direction V 2 , and the third direction V 3 described above . The direction is non-perpendicular to each. When the diffraction grating 13 is translated in this direction, the phase of the interference fringes shifts (details will be described later).

  Next, the 0th-order light shutter 200 will be described in detail.

  FIG. 3A is a diagram for explaining the 0th-order optical shutter 200. As shown in FIG. 3A, the 0th-order optical shutter 200 is a spatial filter formed by forming a circular light shielding part 200C on a part of a circular transparent substrate.

  The light-shielding part 200C of the 0th-order light shutter 200 covers the optical path (condensing point 14a) of the 0th-order diffracted light beam common to the first to third diffracted light beam groups, and the non-light-shielding part (transmission part) of the 0th-order light shutter 200 200B) covers the optical paths (condensing points 14b to 14g) of the ± first-order diffracted light beams of the first to third diffracted light beam groups.

  The zero-order light shutter 200 can be rotated around a straight line (axis AR) parallel to and away from the optical axis O of the illumination optical system 10 by a rotation mechanism 200A (see FIG. 1). is there.

  The rotation mechanism 200A includes, for example, a rotation shaft (not shown) that holds the zero-order light shutter 200 and can rotate around the axis AR, and a motor (not shown) that applies a rotation force to the rotation shaft. (Rotary motor). When this motor is driven, the rotation shaft rotates, and the zero-order light shutter 200 rotates about the axis AR.

  When the rotation angle of the 0th-order optical shutter 200 is set to the reference angle (0 °) shown in FIG. 3A, the light-shielding portion 200C is inserted into the optical path of the 0th-order diffracted light beam. When the rotation angle is set to a predetermined angle (for example, 30 °) that deviates from the reference angle, the light shielding portion 200C is deviated from the optical path of the 0th-order diffracted light beam.

  Therefore, if the rotation angle of the 0th-order light shutter 200 is switched between 0 ° and 30 °, the 0th-order diffracted light beam remains on while the ± 1st-order diffracted light beam of each of the first to third diffracted light beam groups remains on. Only can be turned on / off.

  However, regardless of whether the rotation angle of the 0th-order light shutter 200 is the reference angle (0 °) or the predetermined angle (30 °), the light blocking portion 200C of the 0th-order light shutter 200 has the first to third diffraction. It is assumed that the optical path of the ± first-order diffracted light beam of each group of light beam groups is not blocked.

  Although the 0th-order light shutter 200 is a rotatable spatial filter here, the 0th-order light shutter 200 may be configured by a slidable spatial filter or a liquid crystal element that is fixedly arranged. Note that if the orientation of the liquid crystal element is electrically controlled, the refractive index anisotropy of the liquid crystal element can be controlled, so that the liquid crystal element can function as the zero-order light shutter 200.

  Next, the light beam selection member 18 will be described in detail.

  FIG. 3B is a diagram illustrating the light beam selection member 18. As shown in FIG. 3B, the light beam selection member 18 includes a circular opaque substrate (mask substrate), a plurality of circular openings 18a, a plurality of circular openings 18b, a plurality of circular openings 18c, and a plurality of circular openings. It is a spatial filter formed by forming a portion 18d, a plurality of circular openings 18e, a plurality of circular openings 18f, and a plurality of circular openings 18g.

  The light beam selection member 18 can be rotated by a rotation mechanism 18A (see FIG. 1). The rotation axis of the light beam selection member 18 is parallel to the optical axis O of the illumination optical system 10, and its optical axis. Deviated from O. Incidentally, although the rotation axis of the light beam selection member 18 does not need to coincide with the axis AR of the zero-order optical shutter 200, it is assumed here that it coincides with the axis AR for simplicity.

  The rotation mechanism 18A includes, for example, a rotation shaft that holds the light beam selection member 18 and is rotatable around the axis AR, and a motor (rotation motor) (not shown) that supplies rotational force to the rotation shaft. And are provided. When this motor is driven, the rotation shaft rotates and the light beam selection member 18 rotates about the axis AR.

Moreover, if detached thus the axis AR of the luminous flux selecting member 18 from the optical axis O of the illumination optical system 10, the axis of ± 1-order diffracted light beam (convergence point 14d, 14 g) which is branched to the first direction V 1 and distance to the AR, ± 1-order diffracted light beam (convergence point 14c, 14f) which branches in a second direction V 2 and the distance from to the axis AR, ± 1-order diffracted light beam (converging branched in the third direction V 3 The distances from the light spots 14b, 14e) to the axis AR can be shifted from each other.

Thus, ± 1-order diffracted light beam (convergence point 14d, 14 g) which is branched to the first direction V 1 and sweep trajectory drawn in the light beam selecting member 18, ± 1 order diffraction branched in the second direction V 2 light flux (focus point 14c, 14f) and sweep trajectory drawn light beam selecting member 18, the sweep trajectory ± 1-order diffracted light beam (the focal point 14b, 14e) branching the third direction V 3 draws the light beam selecting member 18 Can be separated from each other.

  Here, it is assumed that the distance to the axis AR is shifted between all the diffracted light beams (all the condensing points 14a to 14d). In this case, the trajectories are separated between all the diffracted light beams (all the condensing points 14a to 14d).

  The trajectory Oa shown in FIG. 3B is the trajectory of the focal point 14a, the trajectory Ob is the trajectory of the focal point 14b, the trajectory Oc is the trajectory of the focal point 14c, and the trajectory Od is the focal point. 14d, the trajectory Oe is the trajectory of the condensing point 14e, the trajectory Of is the trajectory of the condensing point 14f, and the trajectory Og is the trajectory of the condensing point 14g.

  On the track Oa of the light beam selection member 18, 18 openings 18a are arranged at equal intervals with an angular period of 20 °. These 18 openings 18a open the condensing point 14a every time the light beam selection member 18 rotates 20 °. The condensing point 14a can be appropriately shielded by the above-described 0th-order light shutter 200 regardless of the rotation angle of the light beam selection member 18, but in FIG. A state in which light is not shielded by 200 is assumed.

  On the track Ob of the light beam selection member 18, six openings 18b are arranged at equal intervals with an angular period of 60 °. These six openings 18b open the condensing point 14b every time the light beam selection member 18 rotates 60 °.

  On the track Oc of the light beam selection member 18, six openings 18c are arranged at equal intervals with an angular period of 60 °. These six openings 18c open the condensing point 14c every time the light beam selection member 18 rotates 60 °.

  On the track Od of the light beam selection member 18, six openings 18d are arranged at equal intervals with an angular period of 60 °. These six openings 18d open the condensing point 14d every time the light beam selection member 18 rotates 60 °.

  On the track Oe of the light beam selection member 18, six openings 18e are arranged at equal intervals with an angular period of 60 °. These six openings 18e open the condensing point 14e every time the light beam selection member 18 rotates 60 °.

  On the orbit Of of the light beam selection member 18, six openings 18f are arranged at equal intervals with an angular period of 60 °. These six openings 18f open the condensing point 14f every time the light beam selection member 18 rotates 60 °.

  On the track Og of the light beam selecting member 18, six openings 18g are arranged at equal intervals with an angular period of 60 °. These six openings 18g open the condensing point 14g every time the light beam selection member 18 rotates 60 °.

  Moreover, the size of each opening part 18a-18g is set to the size equivalent to the spot of each condensing point (code | symbol 14a-14g of FIG. 3).

  However, at least the sizes of the openings (openings 18b to 18g) related to the ± 1st-order diffracted light beams are large enough to cope with switching of the light source wavelength or increasing the number of light sources. And This is because the diffraction angle of the ± 1st-order diffracted light beam in the diffraction grating 13 depends on the light source wavelength, so the height from the optical axis O of the illumination optical system 10 to the condensing points 14b, 14c, 14d, 14e, 14f, and 14g is Depends on the light source wavelength. Incidentally, if the light source wavelength is set to λ, the structural period of the diffraction grating 13 is set to P, and the focal length of the lens 16 is set to fc, the condensing points 14b, 14c, 14d, and 14e from the optical axis O of the illumination optical system 10 are set. , 14f, and 14g are represented by D∝2fcλ / P.

In addition, the total formation position of the 18 openings 18a around the axis AR, the overall formation position of the 6 openings 18b around the axis AR, and the six openings 18c around the axis AR The total formation position, the total formation position of the six openings 18d around the rotation axis AR, the total formation position of the six openings 18e around the axis AR, and the six around the axis AR The relationship between the overall formation position of the aperture 18f and the overall formation position of the six openings 18g around the axis AR is that the condensing point simultaneously opened by the light beam selection member 18 is the first direction V. 1 , the second direction V 2 , and the third direction V 3 are set so as to be limited only to the condensing points arranged in one direction. It is shown in FIG. 3 (B), focusing point 14d which is arranged in the first direction V 1, 14a, a state in which only 14g is opened.

  Hereinafter, the rotation angle of the light beam selection member 18 around the axis AR is set as θ ′, and the rotation angle θ ′ as shown in FIG. 4 is set as θ ′ = 0 °. Further, the arrangement direction of the condensing points opened by the light beam selection member 18 is expressed by an angle around the optical axis O, which is referred to as “light beam selection angle θ”, and the light beam selection angle θ as shown in FIG. = 0.

First, as shown in FIG. 4, when the rotation angle θ ′ of the light beam selection member 18 is 0 °, the light beam selection angle θ is 0 ° (the arrangement direction of the condensing points is the first direction V 1 ).

Next, as shown in FIG. 5, when the rotation angle θ ′ of the light beam selection member 18 is 20 °, the light beam selection angle θ is 60 ° (the arrangement direction of the condensing points is the second direction V 2 ).

Next, as shown in FIG. 6, when the rotation angle θ ′ of the light beam selection member 18 is 40 °, the light beam selection angle θ is 120 ° (the arrangement direction of the condensing points is the third direction V 3 ).

Next, as shown in FIG. 7, when the rotation angle θ ′ of the light beam selection member 18 is 60 °, the light beam selection angle θ is 180 ° (the arrangement direction of the condensing points is the first direction V 1 ).

Next, as shown in FIG. 8, when the rotation angle θ ′ of the light beam selection member 18 is 80 °, the light beam selection angle θ is 240 ° (the arrangement direction of the condensing points is the second direction V 2 ).

Next, as shown in FIG. 9, when the rotation angle θ ′ of the light beam selection member 18 is 100 °, the light beam selection angle θ is 300 ° (the arrangement direction of the condensing points is the third direction V 3 ).

  Accordingly, in this embodiment, the branch direction of the selected diffracted light beam is rotated at an angular period of 60 ° only by rotating the light beam selection member 18 around the axis AR at an angular period of 20 ° (that is, at an angular period of 60 °). The direction of the interference fringes can be rotated at an angular period of 60 °.

  Here, a conventional example will be described for comparison. In the conventional example, a unidirectional diffraction grating having a periodic structure extending in a single direction is used as the diffraction grating, and the diffraction grating is rotated at an angular period of 60 ° in order to switch the direction of the interference fringes at an angular period of 60 °. I was letting. As shown in FIG. 12, the required time t1 for rotating the direction of the interference fringes by 60 ° is as long as about 80 ms, whereas the charge accumulation time of the image sensor is about 5 ms, for example. For example, it is as short as about 10 to 35 ms. For this reason, in the conventional example, when determining the frame period of the image sensor, the required time t1 for rotating the direction of the interference fringes by 60 ° is rate-limiting, and it is difficult to shorten the frame period.

  However, in this embodiment, the rotation angle period of the member for switching the direction of the interference fringes (here, the light beam selection member 181) is suppressed to 20 °, so that the direction of the interference fringes is rotated by 60 °. The required time t2 is significantly shorter than the required time t1 of the conventional example. The area of the triangle OAt1 shown in FIG. 13 corresponds to the rotation amount (60 °) of the diffraction grating in the conventional example, and the area of the triangle OA't2 is the rotation amount (20 °) of the light beam selection member 18 in this embodiment. It corresponds to. The required time t2 of the present embodiment is a time such that the area of the triangle OA't2 becomes 1/3 of the area of the triangle OAt1 (however, the moment of inertia around the optical axis of the diffraction grating of the conventional example and the present embodiment) It is assumed that the moment of inertia around the axis AR of the luminous flux selection member 18 is equal.) Therefore, although the required time t2 of this embodiment is longer than 1/3 of the required time t1 of the conventional example, it is much shorter than the required time t1 of the conventional example (for example, t2 = 5 msec). Therefore, in the present embodiment, the frame period of the image sensor can be shortened by the amount of time required.

  Next, functions of the polarizing plate 23 and the half-wave plate 17 will be described in detail.

  FIG. 10 is a diagram for explaining the functions of the polarizing plate 23 and the half-wave plate 17.

  The polarizing plate 23 and the half-wave plate 17 are used for controlling the polarization state of the diffracted light beam incident on the specimen 5 to S-polarized light. This is because the contrast of the interference fringes can be kept high by controlling the diffracted light beam incident on the specimen 5 to be S-polarized light.

First, as shown in FIG. 10 (A), when the branch direction of the diffracted light flux group selected by the light beam selecting member 18 is the first direction V 1 was, the polarization direction of the diffracted light flux group in FIG. 10 (A) The direction V 1 ′ indicated by the dotted arrow should be taken. The direction V 1 ′ is a direction obtained by rotating the direction V 1 about the optical axis O by 90 °.

Next, as shown in FIG. 10 (B), when the branch direction of the diffracted light flux group selected by the light beam selecting member 18 is the second direction V 2, the polarization direction of the diffracted light beam group, and FIG. 10 (B) The direction V 2 ′ shown by the dotted arrow in FIG. The direction V 2 ′ is a direction obtained by rotating the direction V 2 by 90 ° around the optical axis O.

Next, as shown in FIG. 10 (C), when the branch direction of the diffracted light flux group selected by the light beam selecting member 18 is the third direction V 3, the polarization direction of the diffracted light flux group, FIG. 10 (C) The direction V 3 ′ indicated by the dotted arrow should be taken. This direction V 3 ′ is a direction obtained by rotating the direction V 3 around the optical axis O by 90 °.

  Therefore, in this embodiment, the axial direction of the polarizing plate 23 is fixed in a predetermined direction, and the half-wave plate 17 is rotated around the optical axis O by the rotation mechanism 17A (see FIG. 1). .

  The rotation mechanism 17A includes, for example, a holding member (not shown) that holds the half-wave plate 17 and that can rotate around the optical axis O, and that is formed around the holding member (not shown). A first gear, a second gear (not shown) that meshes with the first gear, and a motor (rotary motor) (not shown) connected to the second gear are provided. When this motor is driven, the second gear rotates, the rotational force is transmitted to the first gear, and the half-wave plate 17 rotates around the optical axis O.

Here, the axial direction of the polarizing plate 23 is fixed to be the same as the direction V 2 ′ as indicated by the white arrow. In this case, the polarization direction of the diffracted light beam group at the time of incidence on the half-wave plate 17 is the same as the direction V 2 ′.

Therefore, as shown in FIG. 10 (A), when the branch direction of the diffracted light flux group chosen a first direction V 1 was, the direction of the fast axis of the 1/2-wavelength plate 17, FIG. 10 (A) As indicated by the solid line arrow, the direction V 2 ′ and the direction V 1 ′ may be set in a bisecting direction. The fast axis of the half-wave plate 17 is a direction in which the amount of phase delay when light polarized in the direction of the axis passes through the half-wave plate 17 is minimized.

Further, as shown in FIG. 10 (B), when the branch direction of the diffracted light flux group selected a second direction V 2, the direction of the fast axis of the 1/2-wavelength plate 17, in FIG. 10 (B) as shown by a solid line arrow, it suffices same as set to the direction V 2 '.

Further, as shown in FIG. 10 (C), when the branch direction of the diffracted light flux group selected a third direction V 3, the direction of the fast axis of the 1/2-wavelength plate 17, in FIG. 10 (C) As indicated by the solid line arrow, the direction V 1 ′ and the direction V 3 ′ may be set in a bisecting direction.

Therefore, when the rotation angle of the half-wave plate 17 around the optical axis O is θ ″, and the rotation angle θ ″ when the branching direction is the first direction V 1 is θ ″ = − 30 °. When the branch direction is the second direction first direction V 2 (FIG. 10B), the rotation angle θ ″ is set to 0 °, and when the branch direction is the third direction V 3 (FIG. 10). In (C)), the rotation angle θ ″ may be set to + 30 °.

  That is, in the present embodiment, the rotation mechanism 18A rotates when the rotation angle θ ′ of the light beam selection member 18 is switched from “0 °” to “20 °” to “40 °” with an angle period of 20 °. The mechanism 17A may switch the rotation angle θ ″ of the half-wave plate 17 from “−30 °” → “0 °” → “+ 30 °” with an angular period of 30 °.

  In the present embodiment, it is assumed that the time required to switch the rotation angle θ ″ of the half-wave plate 17 by one angular period (here, 30 °) is less than 5 msec. This is approximately the same as the time required to switch 18 by one angular period (20 ° in this case).

  As described above, in the present embodiment, the time required for switching the direction of the interference fringes (here, the time required for switching the light beam selection member 18 by one angular period) is suppressed, so It is desirable that the time required for switching the rotation angle of the phase axis by one angular period is also suppressed to the same or less.

  For this purpose, for example, instead of the rotatable half-wave plate 17, a fixedly arranged liquid crystal element may be used, and the liquid crystal element may function as the half-wave plate 17. If the orientation of the liquid crystal element is electrically controlled, the refractive index anisotropy of the liquid crystal element can be controlled at high speed, so that the fast axis as a half-wave plate can be rotated at high speed. Incidentally, there are other methods for keeping the diffracted light beam incident on the specimen 5 in S-polarized light (described later).

  Next, the function of the translation mechanism 15A (see FIG. 1) will be described in detail.

  FIG. 11 is a diagram illustrating the function of the translation mechanism 15A.

  First, in the above-described demodulation operation, for example, two or more modulated images having the same specimen 5 and the interference fringes in the same direction and having different phases of the interference fringes may be used. This is because the modulated image generated by the structured illumination microscope apparatus 1 includes a 0th-order modulation component, a + 1st-order modulation component, − This is because a primary modulation component is included, and these three unknown parameters need to be made known by demodulation calculation.

Therefore, the translation mechanism 15A shifts the phase of the interference fringes, as shown in FIG. 11A, in the direction perpendicular to the optical axis O of the illumination optical system 10 and the first direction V 1 described above. The diffraction grating 13 is shifted in a direction (x direction) that is non-perpendicular to all of the second direction V 2 and the third direction V 3 .

However, the shift amount L of the diffraction grating 13 necessary for shifting the phase of the interference fringes by a desired shift amount φ is the first in the light beam selection direction (= the branch direction of the selected diffraction light beam group) by the light beam selection unit 24. and when 1 is the direction V 1, and when the second is the direction V 2, at the time the third is the direction V 3, not necessarily the same as.

As shown in FIG. 11B, the structural period of each of the first direction V 1 , the second direction V 2 , and the third direction V 3 of the diffraction grating 13 is P, and the shift direction (x direction) of the diffraction grating 13 is set. And the first direction V 1 is θ 1 , the shift direction of the diffraction grating 13 (x direction) and the second direction V 2 is θ 2 , and the shift direction of the diffraction grating 13 (x putting an angle of theta 3 direction) and the third direction V 3, x-direction shift amount L 1 of the diffraction grating 13 requires the light beam selected direction is the first direction V 1 was, L 1 = φ × P / (a × 4π × | cosθ 1 |) is represented by, x-direction shift amount L 2 of the diffraction grating 13 requires the light beam selected direction is the second direction V 2 is, L 2 = phi × P / (a × 4π × | cosθ 2 |) is expressed in, the light beam selected direction is x direction of the diffraction grating 13 requires the third is the direction V 3 Shift amount L 3 is, L 3 = φ × P / represented by (a × 4π × | | cosθ 3).

That is, the shift amount L in the x direction of the diffraction grating 13 necessary for setting the phase shift amount of the interference fringes to a desired value φ is the light beam selection direction (first direction V 1 , second direction V 2 , third direction). Any one of V 3 ) and the angle θ formed by the x direction is expressed as in Expression (1).

L = φ × P / (a × 4π × | cos θ |) (1)
Incidentally, the shift amount L in the x direction of the diffraction grating 13 necessary for setting the phase shift amount φ of the interference fringes to 2π is P / (a × 2 × | cos θ |). This is an amount corresponding to a half period of the diffraction grating 13. In other words, the phase of the structured illumination light can be shifted by one period only by shifting the diffraction grating 13 by a half period (because the fringe period of the structured illumination light composed of ± first-order diffracted light is the structural period of the diffraction grating 13. Is equivalent to twice.).

  However, a = 1 (when M = 1, 2) and a = 2 (when M = 3). M is the number of directions of the periodic structure of the diffraction grating 13.

  Next, the operation procedure of the control device 39 will be described.

  The control device 39 acquires data necessary for generating a super-resolution image by executing the following procedures (1) to (5).

  (1) The control device 39 sets the emission wavelength (light source wavelength) of the laser unit 100 to both of the two wavelengths λ1 and λ2. Further, the 0th-order diffracted light beam is turned off by driving the rotation mechanism 200A.

  (2) The control device 39 drives the rotation mechanisms 18A and 17A to set the rotation angle θ ′ of the light beam selection member 18 to 0 ° and the rotation angle θ ″ of the half-wave plate 17 Is set to -30 °.

  (3) The control device 39 drives the translation mechanism 15A to shift the phase of the interference fringes in a plurality of stages, and under each of these phases, the laser unit 100, the first image sensor 351, and the second image sensor. By driving the element 352, a plurality of images are acquired for each wavelength.

  (4) The control device 39 drives the rotation mechanisms 18A and 17A to change the rotation angle θ ′ of the light beam selection member 18 by + 20 ° and at the same time the rotation angle θ ″ of the half-wave plate 17 Is changed by + 30 °, and the control device 39 executes step (3) under the setting.

  (5) By driving the rotation mechanisms 18A and 17A, the rotation angle θ ′ of the light beam selection member 18 is changed by + 20 °, and the rotation angle θ ″ of the half-wave plate 17 is changed by + 30 °. Then, the control device 39 executes the procedure (3) under the setting (the procedure (5)).

  As described above, in this embodiment, the direction of interference fringes can be switched at high speed, so that the procedures (4) and (5) can be speeded up. Therefore, in this embodiment, the acquisition speed of data necessary for super-resolution can be increased.

  Note that the control device 39 of the present embodiment repeats a series of processes consisting of the procedures (1) to (5), and the image storage / arithmetic apparatus 40 of the present embodiment supervises every time the series of processes is completed. An image image may be generated (updated). As described above, in the present embodiment, the series of processing is speeded up, so that the super-resolution image update frequency is also increased.

  However, when a series of processing is repeated, in the second and subsequent processing, the control device 39 may execute the following procedure (1 ') instead of the above-described procedures (1) and (2).

  (1 ′) The controller 39 drives the rotation mechanisms 18A and 17A to change the rotation angle θ ′ of the light beam selection member 18 by + 20 °, and at the same time, the rotation angle θ of the half-wave plate 17 "Is set to -30 °.

[Supplement to the first embodiment]
In the embodiment described above, the diffracted light beam branching pattern (diffraction grating structure) is set so that a plurality of branching optical paths (a plurality of condensing points) are formed around the optical axis O at an angular period of 60 °. The direction of branching of the diffracted light beam incident on the specimen 5 was switched by rotating the light beam selection member 18 with an angular period of 20 °.

  However, the combination of the formation angle period of the branched optical path around the optical axis O (the formation angle period of the condensing point) and the rotation angle period of the light beam selection member 18 around the rotation axis is the latter than the former angle period. As long as the rotation angle period is smaller, other combinations may be set. For example, the formation angle period of the branch optical path around the optical axis O may be 60 °, the rotation angle period of the light beam selection member 18 may be 30 °, the formation angle period of the branch optical path around the optical axis O is 90 °, and the light beam selection. The rotation angle cycle of the member 18 may be 45 °.

  In the above-described embodiment, a transmissive mask member (that is, an opening made of a transmission part and a non-opening part made of a light shielding part) is used as a member (light beam selection member) for selecting a part of the light from a plurality of light fluxes. However, a reflective mask member (that is, a mask member having an opening made of a reflection portion and a non-opening portion made of a light shielding portion) may be used.

  Further, in the transmission type mask member, it is desirable that the transmittance of the opening and the transmittance of the non-opening are 100% and 0%, respectively, but the transmittance of the opening is higher than that of the non-opening. Higher than 100% and 0%, respectively.

  In the reflective mask member, the reflectance of the opening and the reflectance of the non-opening are preferably 100% and 0%, respectively. However, the reflectance of the opening is higher than that of the non-opening. Higher than 100% and 0%, respectively.

  In the above-described embodiment, in order to switch the height from the optical axis O to the condensing point between a height suitable for TIRFM and a height suitable for SIM, the diffraction grating 13 inserted in the optical path is changed. The TIRF-SIM mode diffraction grating and the SIM mode diffraction grating may be switched. The structural period of the TIRF-SIM mode diffraction grating is different from that of the SIM mode diffraction grating.

  Further, in the above-described embodiment, the interference fringes that turn off the 0th-order light diffracted light beam and project it onto the specimen 5 are the two light beam interference fringes (that is, an example in which the structured illumination microscope apparatus 1 is used in the 2D-SIM mode). However, if the 0th-order diffracted light beam is turned on, the interference fringes projected onto the specimen 5 are changed to the three-beam interference fringes (that is, the structured illumination microscope apparatus 1 is used in the 3D-SIM mode). Is possible.

  Thus, the interference fringes generated by the interference of the three diffracted light beams (three-beam interference) are spatially modulated not only in the surface direction of the sample 2 but also in the depth direction of the sample 5. Therefore, according to this interference fringe, a super-resolution effect can be obtained also in the depth direction of the specimen 5.

  However, when the structured illumination microscope apparatus 1 is used in the 3D-SIM mode, in order to adjust the intensity balance between the 0th-order diffracted light beam and the ± 1st-order diffracted light beam, for example, a single optical path of the 0th-order diffracted light beam It is desirable to arrange a neutral density filter (ND filter). In order to realize this, for example, a light reducing film may be provided in each of the 18 openings 18 a in the light beam selection member 18.

  Further, the contents of the demodulation operation to be executed by the image storage / arithmetic apparatus 40 are different between the 2D-SIM mode and the 3D-SIM mode. This is because the modulated image generated in the 2D-SIM mode is superimposed with three components of the fluorescence zero-order modulation component, the fluorescence plus first-order modulation component, and the fluorescence minus first-order modulation component. -The modulated image generated in the SIM mode includes 5th order of the 0th order modulation component of fluorescence, the 1st order modulation component of fluorescence, the 1st order modulation component of fluorescence, the + 2nd order modulation component of fluorescence, and the 2nd order modulation component of fluorescence. This is because the components are superimposed.

  Further, since the number of modulation components to be superimposed on the modulated image is different between the 2D-SIM mode and the 3D-SIM mode, the number of frames of the modulated image to be acquired by the control device 43 is also different.

  In the embodiment described above, the diffraction grating 13 is shifted in order to shift the phase of the interference fringes. However, instead of shifting the diffraction grating 13, the optical path length difference of the ± first-order diffracted light beams may be changed. . In that case, for example, the phase plate may be inserted into and removed from at least one of the optical path of the + 1st order diffracted light beam and the optical path of the −1st order diffracted light beam.

  However, since the relationship between the thickness of the phase plate and the amount of phase shift differs depending on the wavelength used, a plurality of phase plates with different thicknesses are attached to the turret, and these phase plates are selectively transferred to the optical path according to the light source wavelength. It may be inserted.

  In the above-described embodiment, the number of light source wavelengths is 2, but it may be 1 or may be extended to 2 or more.

  In the above-described embodiment, the half-wave plate 17 that can rotate around the optical axis O is used in order to keep the ± first-order diffracted light beam incident on the sample 5 as S-polarized light. Alternatively, a combination of a quarter wave plate and a quarter wave plate that can rotate around the optical axis O may be used.

  In that case, the direction of the fast axis of the fixedly arranged quarter wave plate is set so as to form an angle of 45 ° with respect to the polarization direction of the diffracted light beam incident on the quarter wave plate. The direction of the fast axis of the possible quarter-wave plate is set so as to form an angle of 45 ° with respect to the polarization direction that the diffracted light beam emitted from the quarter-wave plate should have.

  However, in the case of using a rotatable quarter wavelength plate, it is necessary to rotate the quarter wavelength plate at an angular period of 60 °, so in order to further increase the direction switching speed of interference fringes, Use a half-wave plate that can be rotated rather than a quarter-wave plate that can be rotated (rotation angle period is 30 °), or a liquid crystal element that is fixedly arranged instead of the wave plate (phase advance) It is desirable to use an electric control for the direction of the shaft.

  In the above-described embodiment, a combination of ± first-order diffracted light and zero-order diffracted light is used as diffracted light for forming interference fringes (two-beam interference fringes in 2D-SIM mode or three-beam interference fringes in 3D-SIM mode). However, other combinations may be used. In order to form a three-beam interference fringe, three-beam interference is generated by three diffracted lights having equal intervals of diffraction orders. For example, a combination of zero-order diffracted light, first-order diffracted light, and second-order diffracted light , A combination of ± 2nd order diffracted light and 0th order diffracted light, a combination of ± 3rd order diffracted light and 0th order diffracted light, and the like can be used.

  The illumination optical system 10 of any of the above-described examples is configured by an epi-illumination optical system using the objective lens 6, but is not limited thereto, and is a transmission / reflection illumination optical system using a condenser lens instead of the objective lens 6. It may be configured. In this case, the focal point is formed on the pupil plane of the condenser lens.

[Summary of Embodiment]
As described above, the structured illumination device (illumination optical system 10) of the present embodiment allows a plurality of branched light beams (collected light beams) having different branch directions around a predetermined axis (optical axis O) to be emitted from a light source (laser unit 100). A predetermined part for selecting only a predetermined number of branched light beams (condensing points 14d, 14g, etc.) in a predetermined branch direction among the branched light beams (diffraction grating 13) and a plurality of branched light beams. A light beam selection member (18) having at least one set of a plurality of selection parts (openings), and an optical system (condensing lens) for forming interference fringes by a predetermined number of selected branched light beams on the sample (5) 25, the field lens 27, the objective lens 6) and the light beam selecting member (18) are rotated around the axis (AR) deviated from the optical axis of the optical system, and the combination of a predetermined number of selected branched light beams is switched. The direction of the interference fringes A branching light beam that includes a rotating mechanism (18A) for switching and moves on the light beam selection member (18) as the light beam selection member (18) rotates, and that contributes to the formation of interference fringes ( One or more selection portions (openings) are formed on each locus (sweep orbits Oa to Og and the like) of the condensing points 14a to 14g and the like.

  Therefore, the structured illumination device (illumination optical system 10) of the present embodiment can keep the rotation angle period of the light beam selection member (18) necessary for switching the direction of interference fringes small.

  Further, in the structured illumination device (illumination optical system 10) of the present embodiment, a branched light beam that moves on the light beam selection member (18) as the light beam selection member (18) rotates, and forms interference fringes. Two or more selection portions (openings) are formed on each trajectory (sweep trajectories Oa to Og, etc.) of the branched light flux that contributes to.

  Therefore, the structured illumination device (illumination optical system 10) of the present embodiment can further reduce the rotation angle period of the light beam selection member (18) necessary for switching the direction of the interference fringes.

  Further, in the structured illumination device (illumination optical system 10) of the present embodiment, at least two (± first-order diffracted light beams) out of a predetermined number of branched light beams selected before switching and a predetermined number selected after switching. The trajectories are separated from each other between at least two of the branched light beams (± first-order diffracted light beams).

  Further, in the structured illumination device (illumination optical system 10) of the present embodiment, the trajectories are set to be separated from each other among the plurality of branched light beams (condensing points 14b to 14g).

  Therefore, the degree of freedom of arrangement of the selection portion (opening) to be formed in the light beam selection member (18) is increased, and it becomes easy to keep the rotation angle period of the light beam selection member (18) small.

  Further, in the structured illumination device (illumination optical system 10) of the present embodiment, the plurality of branched light beams (condensing points 14a to 14g) are branched at a first angular period around the optical axis (O). A light beam (condensing points 14b to 14g) is included, and the angular period of rotation is a second angular period smaller than the first angular period.

  Therefore, the rotation angle cycle of the light beam selection member (18) is smaller than the branch angle cycle of the plurality of branched light beams (condensing points 14b to 14g).

  For example, in the structured illumination device (illumination optical system 10) of the present embodiment, if the first angular period is 60 ° and the second angular period is 20 °, the light beam selection member (18) is rotated. The moving angle period is 1/3 of the branching angle period of the plurality of branched light beams (condensing points 14b to 14g).

  Further, the structured illumination device (illumination optical system 10) of the present embodiment further includes a phase shift unit (translation mechanism 15A) that shifts the phase of the interference fringes.

  Further, the structured illumination microscope apparatus (1) of the present embodiment includes an illumination device (illumination optical system 10) of the present embodiment and an imaging element that captures a modulated image that is an image of a sample spatially modulated by interference fringes. 351, 352).

  Further, the structured illumination microscope apparatus (1) of the present embodiment further includes a calculation hand (image storage / calculation apparatus 40) that generates a demodulated image of the sample based on the modulated image.

  Therefore, the structured illumination microscope apparatus (1) of the present embodiment can efficiently generate a demodulated image.

[Second Summary]
Hereinafter, this embodiment will be described from another viewpoint.

  The illumination device (illumination optical system 10) of the present embodiment includes a branching unit (such as the diffraction grating 13) that branches light from the light source (laser unit 100) into a plurality of light beams (such as the condensing points 14a to 14g), and a plurality of units. A light beam selecting member (18) for selecting a part of the light flux (condensing points 14d, 14g, etc.) from the light flux, and an optical system (condensing lens 25) for illuminating the object (sample 5) with the selected part of the light flux. , Field lens 27, objective lens 6 and the like) and a rotation mechanism (18A) for switching illumination by rotating the light beam selection member, and the light beam selection member has a plurality of rotation axes (axis AR). A selector for selecting a light beam is provided on each circumference of two or more circles (sweep orbits Ob, Oc, etc.) that are provided at positions deviating from the branching center (optical axis O) of the light beam and centering on the rotation axis. A plurality of openings) are provided at predetermined intervals.

  Further, in the illumination device (illumination optical system 10) of the present embodiment, a selection unit (opening portion) that selects a light beam and a light beam are not selected on each circumference of two or more circles (sweep orbits Ob, Oc, etc.). Non-selection parts (light-shielding parts) are provided alternately.

  In the illumination device (illumination optical system 10) of the present embodiment, circles (sweep trajectories Oa to Og) are provided corresponding to the plurality of light beams (condensing points 14a to 14g), respectively.

Further, the plurality of light beams include a plurality of light beam groups branched in mutually different directions (directions V 1 , V 2 , V 3, etc.). There is only one direction of light beam group, and the object is illuminated with interference fringes from the one direction light beam group.

Further, circles (sweep trajectories) are provided corresponding to at least two directions (directions V 1 , V 2, etc.) of the plurality of light beam groups.

A circle (sweep trajectory) is provided corresponding to each direction (direction V 1 , V 2 , V 3 ) of the plurality of light beam groups.

  In addition, the direction angle period of the plurality of light beam groups around the optical axis of the optical system is the first angle period, and the formation angle period of the plurality of selection units around the rotation axis is the first angle period. The second angular period is smaller than the period.

The first angular period is 60 °, and the second angular period is 20 °. The branch portion is a diffractive optical element.

  The illumination device (illumination optical system 10) of the present embodiment further includes a phase shift unit (translation mechanism 15A) that shifts the phase of the interference fringes.

  Further, the structured illumination microscope apparatus (1) of the present embodiment, together with the illumination apparatus (illumination optical system 10) of the present embodiment, an imaging optical system (30) that forms a modulated image of an object spatially modulated by interference fringes. ) And an image sensor (such as an image sensor 351) that captures a modulated image formed by the imaging optical system and generates a modulated image.

  The structured illumination microscope apparatus (1) of the present embodiment further includes a calculation unit (image storage / calculation apparatus 40) that generates a demodulated image of an object based on the modulated image generated by the image sensor.

[Others]
Note that the requirements of the above-described embodiments can be combined as appropriate. Some components may not be used. In addition, as long as it is permitted by law, the disclosure of all publications and US patents relating to the devices cited in the above embodiments and modifications are incorporated herein by reference.

  DESCRIPTION OF SYMBOLS 1 ... Structured illumination microscope apparatus, 100 ... Laser unit, 11 ... Optical fiber, 10 ... Illumination optical system, 30 ... Imaging optical system, 351 ... 1st image sensor, 352 ... 2nd image sensor, 39 ... Control apparatus, DESCRIPTION OF SYMBOLS 40 ... Image memory | storage / arithmetic apparatus, 45 ... Image display apparatus, 12 ... Collector lens, 23 ... Polarizing plate, 15 ... Light beam branching part, 16 ... Condensing lens, 24 ... Light beam selection part, 25 ... Lens, 26 ... Field stop 27 ... Field lens, 28 ... Excitation filter, 7 ... Dichroic mirror, 6 ... Objective lens, 5 ... Sample, 17 ... Half-wave plate, 18 ... Light beam selection member, 17A ... Rotating mechanism, 18A ... Rotating mechanism , 200 ... 0th order light shutter, 200A ... rotation mechanism, 15A ... translation mechanism

An example of a lighting device of the present invention is used, the number in the light of the respective plurality of directions from the light source and a branch unit for branching the plurality of light beams, and a light flux selection element for selecting a plurality of light beams in a predetermined direction from said plurality of light beams and, a lighting device comprising an illumination optical system for illuminating an object in the interference fringes generated by the plurality of light beams selected, and a rotation mechanism which Ru is rotated the beam selecting member, wherein the light beam selecting member The rotation axis is provided at a position deviated from the optical axis of the illumination optical system, and on each circumference of two or more circles centering on the rotation axis, a selector for selecting a light beam has a predetermined interval. The angular period in the direction in which the light is branched by the branching part is a first angular period, and the angular period of the selection part provided on the circumference of at least one of the circles is 2 and the second angular period is the first angular period. Smaller than the angle period.
Another example of the illumination device of the present invention includes a branching unit that branches light from a light source into a plurality of light beams in a plurality of directions, and a light beam selection member that selects a plurality of light beams in a predetermined direction from the plurality of light beams. An illumination optical system that illuminates an object with interference fringes generated by the selected plurality of light beams, and a rotation mechanism that rotates the light beam selection member. A rotation axis of the member is provided at a position deviated from the optical axis of the illumination optical system, and a selection unit for selecting a light beam is provided on each circumference of two or more circles centered on the rotation axis. A plurality of angular intervals in the direction in which the light is branched by the branching portion is a first angular cycle, and an angular cycle in which the light beam selection member rotates is a second angular cycle, The second angular period is smaller than the first angular period.

An example of the structured illumination device of the present invention includes an example of the illumination device of the present invention or another example , an imaging optical system that forms a modulated image of the object spatially modulated by the interference fringes, and the imaging optics An imaging device that captures the modulated image formed by the system and generates a modulated image.

Claims (12)

  1. A branching section for splitting light from the light source into a plurality of light fluxes;
    A light beam selection member that selects a part of the light fluxes from the plurality of light fluxes;
    An optical system for illuminating an object with the selected partial luminous flux;
    A rotation mechanism that switches the illumination by rotating the light beam selection member,
    The rotation axis of the light beam selection member is provided at a position deviated from the branch center of the plurality of light beams,
    A lighting device, wherein a plurality of selection units for selecting a light beam are provided at predetermined intervals on each circumference of two or more circles centered on the rotation axis.
  2. The lighting device according to claim 1.
    The circle is provided corresponding to each of the plurality of light beams.
  3. The lighting device according to claim 2,
    On each circumference of the two or more circles, a selection unit that selects a light beam and a non-selection unit that does not select a light beam are alternately provided.
  4. The lighting device according to claim 1.
    The plurality of light beams include a plurality of light beam groups branched in different directions,
    Of the plurality of light flux groups, only one light flux group is selected as the partial light flux,
    The illumination device is characterized in that the object is illuminated with interference fringes by the light flux group in one direction.
  5. The lighting device according to claim 4.
    The illuminating device, wherein the circle is provided corresponding to at least two directions of the plurality of light flux groups.
  6. The lighting device according to claim 4.
    The circle is provided corresponding to each direction of the plurality of light flux groups.
  7. In the illuminating device as described in any one of Claims 4-6,
    The direction angle period of the plurality of light beam groups around the optical axis of the optical system is a first angle period,
    The illumination device characterized in that a formation angle period of the plurality of selection units around the rotation axis is a second angle period smaller than the first angle period.
  8. The lighting device according to claim 7.
    The first angular period is 60 °;
    The lighting device, wherein the second angular period is 20 °.
  9. In the illuminating device as described in any one of Claims 4-8,
    The branch portion is
    An illumination device, which is a diffractive optical element.
  10. In the illuminating device as described in any one of Claims 4-9,
    The illumination apparatus further comprising a phase shift unit that shifts a phase of the interference fringes.
  11. The lighting device according to any one of claims 4 to 10,
    An imaging optical system for forming a modulated image of the object spatially modulated by the interference fringes;
    An image sensor that captures the modulated image formed by the imaging optical system to generate a modulated image;
    A structured illumination microscope apparatus comprising:
  12. The structured illumination microscope apparatus according to claim 11,
    A structured illumination microscope apparatus, further comprising: an arithmetic unit that generates a demodulated image of the object based on the modulated image generated by the imaging device.
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JP2001272603A (en) * 2000-03-24 2001-10-05 Olympus Optical Co Ltd Optical device
JP2010266406A (en) * 2009-05-18 2010-11-25 Hitachi Kokusai Electric Inc Measuring device
WO2013108626A1 (en) * 2012-01-18 2013-07-25 株式会社ニコン Structured illumination device, structured illumination microscope device, and structured illumination method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001272603A (en) * 2000-03-24 2001-10-05 Olympus Optical Co Ltd Optical device
JP2010266406A (en) * 2009-05-18 2010-11-25 Hitachi Kokusai Electric Inc Measuring device
WO2013108626A1 (en) * 2012-01-18 2013-07-25 株式会社ニコン Structured illumination device, structured illumination microscope device, and structured illumination method

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