JP6268826B2 - Structured illumination device and structured illumination microscope device - Google Patents

Description

  The present invention relates to a structured illumination apparatus and a structured illumination microscope apparatus.

  In the field of specimen observation and measurement, in order to achieve a resolution that exceeds the performance of the objective lens, the sample is illuminated with spatially modulated illumination light to obtain a modulated image, and the modulation component contained in the modulated image is obtained. A structured illumination microscope (SIM) that generates a super-resolution image of a specimen by removing (demodulating) has been proposed (see, for example, Patent Document 1).

  In particular, in the structured illumination microscope described in Patent Document 1, a light beam emitted from a light source is branched into a plurality of light beams by a diffraction grating, and the light beams are condensed at different positions on the pupil plane of the objective lens, and in the vicinity of the specimen. The interference fringes are used as structured illumination light.

US Reissue Patent No. 38307 Specification

  In the future, when the fluorescent dyes of specimens are diversified, the light source wavelength of the structured illumination microscope will need to be diversified, but the position of the interference fringe where the interference intensity is maximum (observation target surface) in the optical axis direction The position may be shifted depending on the light source wavelength. The present invention has been made in view of such circumstances, and an object of the present invention is to provide a structured illumination device and a structured illumination microscope device that can easily and reliably suppress a shift between wavelengths of an observation target surface. To do.

An example of the structured illuminating device of the present invention includes a branching unit that branches an emitted light beam from a light source into at least two branched light beams, an optical system that forms interference fringes by the two branched light beams on a sample, A phase difference providing member that gives a phase difference to at least two different types of the emitted light beams, and the branching unit is a diffractive optical element that branches the two types of emitted light beams at different angles, and the phase difference applying member, the optical path of the two kinds of the light flux is Ru is disposed at a location spatially separated.

  An example of the structured illumination microscope apparatus of the present invention includes an example of the structured illumination apparatus of the present invention and an imaging optical system that images an observation light beam from the sample modulated by the interference fringes on a photodetector. Prepare.

  ADVANTAGE OF THE INVENTION According to this invention, the structured illumination apparatus and structured illumination microscope apparatus which can suppress the shift | offset | difference between the wavelengths of an observation object surface easily and reliably are implement | achieved.

1 is a configuration diagram of a structured illumination microscope apparatus 1. FIG. It is a figure explaining the diffraction grating. FIG. 6 is a diagram for explaining the function of a half-wave plate 17. It is a figure explaining the function of the light beam selection member. It is a figure explaining the function of the half-wave plate 17 and the light beam selection member 18. FIG. It is a figure explaining the shape of the light beam selection member. It is a figure explaining the function of 15 A of translation mechanisms. It is a figure explaining the relationship between the diffraction angle in the diffraction grating 13, and a wavelength. It is a figure explaining the phase plate 200 of 1st Embodiment. It is a figure explaining the shift | offset | difference of the focal plane of the objective lens 6 with respect to wavelength (lambda) _S , (lambda) _L. Is a diagram showing an extended amount [Delta] d _p required to match the focal plane 5A _S wavelength lambda _S in the focal plane 5A _L of wavelength lambda _L. It is a figure explaining the effect | action of the phase delay part 200B. (A) is the figure which looked at the phase plate 200 of 1st Embodiment from the direction (sample side) along an optical axis, (B) cut | disconnects the phase plate 200 in the plane containing the optical axis O. FIG. (A) is the figure which looked at the modification (rotation type) of the phase plate 200 in 1st Embodiment from the direction along the optical axis O, (B) is a plane containing the optical axis O in the phase plate 200. FIG. It is sectional drawing which can be cut | disconnected. (A) is the figure which looked at the modification (ring type) of the phase plate 200 in 1st Embodiment from the direction along the optical axis O, (B) is a plane containing the optical axis O in the phase plate 200. FIG. It is sectional drawing which can be cut | disconnected. (A) is the figure which looked at the modification (double ring type) of the phase plate 200 in 1st Embodiment from the direction along the optical axis O, (B) is a plane containing the optical axis O in a plane It is sectional drawing formed by cut | disconnecting 200. FIG. It is a figure explaining the shape of the light beam selection member 18 'applied to 3D-SIM. (A) is the figure which looked at the phase plate 200 of 2nd Embodiment from the direction (sample side) along the optical axis O, (B) cut | disconnects the phase plate 200 in the plane containing the optical axis O. FIG. FIG. Height [rho _S is a view different from the delay amount [Delta] d _p will be described different.

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

  FIG. 1 is a configuration diagram of the structured illumination microscope apparatus 1. Hereinafter, the case where the structured illumination microscope apparatus 1 is used as a total internal reflection fluorescence microscope (TIRFM) will also be described. TIRFM can observe a very 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 of the first laser light source 101 is λ _L , the wavelength of the second laser light source 102 is λ _S , and λ _L > λ _S. 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 output 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. For example, a piezo element or the like is used as the position adjustment mechanism 11A.

  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 beam splitter 15, a condenser lens 16, a phase plate 200, a beam selector 24, A lens 25, a field stop 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 half-wave plate 17, a beam selector 18 and a rotation mechanism 24A. And are provided. The translation mechanism 15A and the rotation mechanism 24A 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 λ _L and the second fluorescent region excited by light of wavelength λ _S are expressed. The first fluorescent region, _'to generate a first fluorescence and the second fluorescence region, the central wavelength lambda _S in accordance with the light of wavelength lambda _S' center wavelength lambda _L in accordance with the light of the wavelength lambda _L first of 2. Generate fluorescence.

  When the structured illumination microscope apparatus 1 is used as a TIRFM (total reflection fluorescence microscope), the objective lens 6 is configured as an 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 the frame period (imaging repetition period) of each of the first image sensor 351 and the second image sensor 352 is necessary for imaging time of the image sensor (that is, time required for charge accumulation and charge readout) and for switching the direction of interference fringes. Of time and other required time, it is determined by rate-limiting.

  The control device 39 drives and controls the laser unit 100, the position adjustment mechanism 11A, the translation mechanism 15A, the rotation mechanism 24A, the first image sensor 351, and the second image sensor 352.

  The image storage / arithmetic unit 40 performs a calculation on the image given via the control unit 39, stores the calculated image in an internal memory (not shown), and sends it to the image display unit 45.

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

Laser light having a wavelength lambda _L emitted from the first laser light source 101 (first laser beam) is incident to the mirror 105 through the shutter 1031, and reflecting mirrors 105 and enters the dichroic mirror 106. On the other hand, the laser light (second laser light) having the wavelength λ _S 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. When the control device 39 controls the laser unit 100, the wavelength of the laser light (= light source wavelength) incident on the incident end of the optical fiber 11 is switched between the long wavelength λ _L and the short wavelength λ _S , The wavelength can be set to both the long wavelength λ _L and the short wavelength λ _S.

  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 is incident on the diffraction grating 13 via the polarizing plate 23, and is branched into diffracted light beams of respective orders. When these incident diffracted light beams (hereinafter referred to as “diffracted light beam groups”) enter the condenser lens 16, they are condensed by the condenser lens 16 at different positions on the pupil conjugate plane 6 </ b> A ′.

  Here, the pupil conjugate plane 6 </ b> A ′ is a position conjugate with the pupil 6 </ b> A (position where ± 1st-order diffracted light is collected) of the objective lens 6 with respect to the field lens 27 and the lens 25. The condenser lens 16 is arranged so that the focal position (rear focal position) of the condenser lens 16 coincides with the pupil conjugate plane 6A ′. However, the concept of “conjugate position” here includes a position determined by a person skilled in the art in consideration of design necessary matters such as aberration and vignetting of the objective lens 6, the field lens 27, and the lens 25. Shall.

  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 reliably 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.

  The diffracted light beams of respective orders toward the pupil conjugate plane 6A ′ are passed through the phase plate 200 disposed in the vicinity of the pupil conjugate plane 6A ′ to the light beam selection unit 24 that is also disposed in the vicinity of the pupil conjugate plane 6A ′. Incident.

  Here, when the structured illumination microscope apparatus 1 of the present embodiment is used as a TIRFM (total reflection fluorescent microscope), the light beam selection unit 24 selects only one pair of diffracted light beams among the incident diffracted light beams of each order ( Here, only ± first-order diffracted light flux) 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. The excitation filter 28 has a function of removing autofluorescence generated in the optical path so far.

  The ± first-order diffracted light beams collected on the pupil plane 6A become parallel light beams when emitted from the tip of the objective lens 6 and overlap 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 of the present embodiment is used as a TIRFM (total reflection fluorescence microscope), the incident angle when entering the surface of the specimen 5 depends on the evanescent field generation condition (total reflection condition). Should be met. Hereinafter, the total reflection condition is referred to as “TIRF condition”.

  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 is incident on 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 λ _L ′ incident on the second dichroic mirror 35 reflects the second dichroic mirror 35, and the second fluorescence having the wavelength λ _S ′ incident on the second dichroic mirror 35 is reflected by the second dichroic mirror 35. Transparent.

  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 output.

  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 known demodulation operation in the image storage / arithmetic device 40, and the demodulated image of the first fluorescent region ( A super-resolution 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 a known 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 diagram of the diffraction grating 13 viewed from the direction of the optical axis O, and FIG. 2B is a diagram illustrating the positional relationship between the condensing points formed on the pupil conjugate plane by the ± first-order diffracted light beams. It is. 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.

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. The material of the diffraction grating 13 is, for example, glass. 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 120 °, and the period of the periodic structure Is assumed to be common between these three directions.

  The periodic structure of the diffraction grating 13 is either a concentration-type periodic structure formed using concentration (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, and of these, the ± 1st-order diffracted light beams having common orders travel in a symmetric direction with respect to 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. To do.

  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. To do.

  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 brought into the pupil conjugate plane 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.

  Here, assuming that the wavelength of the laser light emitted from the optical fiber 11 is λ, the structural period of the diffraction grating 13 is P, and the focal length of the lens 16 is fc, the condensing points 14b, 14c, 14d, and 14e from the optical axis O. , 14f, and 14g are represented by the following formula.

D∝2fcλ / P
Therefore, when the wavelength of the laser beam is changed, a shift occurs in the distance from the optical axis O to the condensing points 14b, 14c, 14d, 14e, 14f, and 14g.
In addition, a condensing point here means the gravity center position of the area | region which has an intensity | strength 80% or more of the maximum intensity | strength. 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.

The diffraction grating 13 described above can be translated by a translation mechanism 15A (see FIG. 1) composed of a piezoelectric 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 fringe shifts (details will be described later).

  Next, functions of the half-wave plate 17 and the light beam selection member 18 will be described in detail.

  FIG. 3 is a diagram for explaining the function of the half-wave plate 17, and FIG. 4 is a diagram for explaining the function of the light beam selection member 18.

  As shown in FIG. 3, the half-wave plate 17 is a wave plate that sets the polarization direction of the incident diffracted light beam of each order. As shown in FIG. 4, the light beam selection member 18 includes first to third light beams. It is a mask that selectively allows only one group of ± first-order diffracted light beams to pass through.

  The half-wave plate 17 and the light beam selection member 18 can be rotated around the optical axis O by a rotation mechanism 24A (see FIG. 1).

  The rotation mechanism 24A includes, for example, a first gear formed around the light beam selection member 18, a second gear (not shown) that meshes with the first gear, and a not shown connected to the second gear. Motor (rotary motor). When this motor is driven, the second gear rotates, the rotational force is transmitted to the first gear, and the light beam selection member 18 rotates around the optical axis O.

  The rotation mechanism 24A rotates the light beam selecting member 18 to switch the selected ± first-order diffracted light beam between the first to third diffracted light beam groups, and in conjunction with the light beam selecting member 18, 1 / By rotating the two-wave plate 17, the polarization direction when the selected ± first-order diffracted light beams enter the sample 5 is maintained as S-polarized light. That is, the half-wave plate 17 and the light beam selection member 18 switch the direction of interference fringes while maintaining the state of interference fringes. Hereinafter, the conditions for maintaining the stripe state will be specifically described.

First, the direction of the fast axis (fast axis) of the half-wave plate 17 is with respect to the branch direction (any _{one of} the first direction V ₁ to the third direction V ₃ ) of the selected ± first-order diffracted light beam. It is necessary to set the polarization direction of the ± first-order diffracted light beams to be vertical. Here, 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. It is.

  The opening pattern of the light beam selection member 18 includes a first opening portion 19 and a second opening portion 20 that individually allow one and the other of the ± first-order diffracted light beams belonging to the same diffracted light beam group to pass therethrough. The length of each of the first opening 19 and the second opening 20 around the optical axis O is set such that a diffracted light beam linearly polarized in the above-described direction can pass therethrough. Therefore, each shape of the 1st opening part 19 and the 2nd opening part 20 is a shape close | similar to partial ring zone shape (fan shape).

  Here, as shown in FIG. 3A, the rotation angle of the half-wave plate 17 when the direction of the fast axis of the half-wave plate 17 is parallel to the direction of the axis of the polarizing plate 23, The rotation angle of the half-wave plate 17 is used as a reference (hereinafter referred to as “first reference position”).

  The rotation angle of the light beam selection member 18 when the light beam selection direction of the light beam selection member 18 (= the branch direction of the selected ± first-order diffracted light beam) is perpendicular to the axis direction of the polarizing plate 23 The rotation angle of the member 18 is used as a reference (hereinafter referred to as “second reference position”).

  At this time, as shown in FIG. 3B, the rotation angle of the half-wave plate 17 from the first reference position is controlled to one half of the rotation angle of the light beam selection member 18 from the second reference position. It should be.

  That is, when the rotation angle of the half-wave plate 17 from the first reference position is θ / 2, the rotation angle of the light beam selection member 18 from the second reference position is set to θ.

Therefore, the rotation mechanism 24A (see FIG. 1) selects the ± first-order diffracted light beam (the branch direction is the first direction V ₁ ) of the first diffracted light beam group, as shown in FIG. When the light beam selection direction of the light beam selection member 18 is rotated rightward from the second reference position by the rotation angle θ ₁ , the fast axis direction of the half-wave plate 17 is changed to the right from the first reference position. Is rotated by a rotation angle θ _1/2 .

At this time, the polarization directions of the diffracted light beams of the respective orders before passing through the half-wave plate 17 are parallel to the direction of the axis of the polarizing plate 23 as shown by the broken line double arrow in FIG. On the other hand, the polarization directions of the diffracted light beams of the respective orders after passing through the half-wave plate 17 rotate to the right by the rotation angle θ _1, so the polarization directions of the selected ± 1st order diffracted light beams are 4A, it is perpendicular to the branching direction (first direction V ₁ ) of the ± 1st-order diffracted light beams, as indicated by the solid line double arrow in FIG.

Further, as shown in FIG. 4B, the rotation mechanism 24A (see FIG. 1) selects the ± first-order diffracted light beam (the branch direction is the second direction V ₂ ) of the second diffracted light beam group, If a light beam selection direction of the light beam selecting member 18 is rotated by the rotation angle theta ₂ from the second reference position to the right, the direction of the fast axis of the 1/2-wavelength plate 17, right from the first reference position only the rotation angle theta _2/2 is rotated.

At this time, the polarization direction of the diffracted light beam of each order before passing through the half-wave plate 17 is parallel to the direction of the axis of the polarizing plate 23 as shown by the broken line in FIG. 4B. On the other hand, the polarization direction of each order of the diffracted light beam after passing through the half-wave plate 17 is rotated to the right by the rotation angle θ _2, so the polarization direction of the selected ± 1st order diffracted light beam Is perpendicular to the branching direction (second direction V ₂ ) of the ± first-order diffracted light beams, as indicated by the solid double arrows in FIG.

Further, as shown in FIG. 4C, the rotation mechanism 24A (see FIG. 1) selects the ± first-order diffracted light beam (the branch direction is the third direction V ₃ ) of the third diffracted light beam group, When the light beam selection direction of the light beam selection member 18 is rotated from the second reference position to the left (seen from the sample side; the same applies hereinafter) by the rotation angle θ ₃ , the direction of the fast axis of the half-wave plate 17 and it is rotated by the rotation angle theta _3/2 to the left from the first reference position.

At this time, the polarization directions of the diffracted light beams of the respective orders before passing through the half-wave plate 17 are parallel to the direction of the axis of the polarizing plate 23, as indicated by the broken line in FIG. 4C. On the other hand, the polarization direction of each order diffracted light beam after passing through the half-wave plate 17 rotates to the left by the rotation angle θ _3, so the polarization direction of the selected ± 1st order diffracted light beam Is perpendicular to the branching direction (third direction V ₃ ) of the ± first-order diffracted light beams, as indicated by the actual double arrows in FIG.

  Therefore, the rotation mechanism 24A only needs to interlock the half-wave plate 17 and the light beam selection member 18 with a gear ratio of 2: 1.

  In the above description, the rotatable half-wave plate 17 is used to keep the ± first-order diffracted light beam incident on the specimen 5 as S-polarized light. Instead, a fixed 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, so that the fast axis as a half-wave plate can be rotated around the optical axis. Incidentally, there are other methods for keeping the ± first-order diffracted light beam incident on the specimen 5 to be S-polarized light.

  FIG. 5 is a diagram illustrating the functions of the half-wave plate 17 and the light beam selection member 18 described above. In FIG. 5, a double arrow surrounded by a circular frame indicates the polarization direction of the light beam, and a double arrow surrounded by a square frame indicates the axial direction of the optical element.

  Also, as shown in FIG. 6, a plurality of (six in the example shown in FIG. 6) notches 21 are formed on the outer peripheral portion of the light beam selecting member 18, and the rotation mechanism 24A includes these notches 21. A timing sensor 22 for detecting the notch 21 is provided. Accordingly, the rotation mechanism 24A can detect the rotation angle of the light beam selection member 18, and thus the rotation angle of the half-wave plate 17.

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

  FIG. 7 is a diagram illustrating the function of the translation mechanism 15A.

  First, in order to enable the above-described demodulation operation, for example, a plurality of modulated images with different interference fringe phases, which are modulated images related to the same sample 5, are 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. 7A, 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 needed to the phase of the interference fringe is shifted by a desired shift amount φ is the case the light beam selected direction of the light beam selecting unit 24 is the first direction V _1, the second direction When it is V ₂ and when it is the third direction V ₃ , it is not necessarily the same.

As shown in FIG. 7B, the structural period of each of the first direction V ₁ , second direction V ₂ , and 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} Shi DOO 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 wavelength 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 interference fringes can be shifted by one period simply by shifting the diffraction grating 13 by half a period (because the fringe period of the interference fringes made of ± 1st order diffracted light is twice the structural period of the diffraction grating 13. Equivalent to.).

  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 (in this embodiment, M = 3 and a = 2).

  Next, the laser unit 100, the objective lens 6, the diffraction grating 13, and the phase plate 200 of this embodiment will be described in detail.

First, the wavelength λ _L of the first laser light source 101 mounted on the laser unit 100 is 561 nm belonging to the visible light region, whereas the wavelength λ _S of the second laser light source 102 is 405 nm belonging to the ultraviolet light region. Assume that

  On the other hand, it is assumed that the correction wavelength range of axial chromatic aberration of the objective lens 6 is in the range of 435 nm to 656 nm and is corrected with reference to the d-line (= 588 nm).

That is, the correction wavelength range of axial chromatic aberration of the objective lens 6 covers the wavelength λ _L (= 561 nm) of the first laser light source 101, but covers the wavelength λ _S (= 405 nm) of the second laser light source 102. Assume that you have not.

In this case, the amount of axial chromatic aberration of the objective lens 6 with respect to the wavelength λ _S (= 405 nm) is, for example, about several μm on the sample side.

For this reason, the position (observation target surface) where the interference intensity of the interference fringes becomes maximum in the optical axis O direction is shifted between the wavelength λ _L (= 561 nm) and the wavelength λ _S (= 405 nm).

In order to suppress this deviation, for example, every time the operating wavelength is switched between the wavelength λ _L (= 561 nm) and the wavelength λ _S (= 405 nm), the output end of the optical fiber 11 or the optical axis of the diffraction grating 13 is used. A method of adjusting the position in the O direction is also conceivable.

However, this method not only takes time for position adjustment, but also has a drawback that observation using the wavelength λ _L (= 561 nm) and observation using the wavelength λ _S (= 405 nm) cannot be performed simultaneously.

Therefore, in this embodiment, as shown in FIGS. 8A and 8B, the diffraction angle in the diffraction grating 13 is a ± first-order diffracted light beam having a wavelength λ _L (= 561 nm) and a wavelength λ _S (= 405 nm). Therefore, the condensing point of the ± 1st order diffracted light beam having the wavelength λ _L (= 561 nm) and the condensing point of the ± 1st order diffracted light beam having the wavelength λ _S (= 405 nm) are different. But take advantage of slipping.

Specifically, by placing the structural period of the diffraction grating 13 (pitch) and p, wavelength lambda _L ± 1 diffraction angle theta _L-order diffracted light _beam, the diffraction angle theta _S of ± 1-order diffracted light flux of wavelength lambda _S is It is expressed as follows.

sin θ _L = λ _L / p,
sin θ _S = λ _S / p
When placing the focal length of the lens 16 and f _16, the wavelength lambda _L ± 1 height h _L from the optical axis O of the converging point P _L to form the diffracted light beam pupil conjugate plane 6A 'of _the wavelength lambda the height h _S from the optical axis O of the converging point P _S of ± 1-order diffracted light beam _S is formed on the pupil conjugate plane 6A 'is expressed as follows.

h _L = f ₁₆ × sin θ _L ,
h _S = f ₁₆ × sin θ _S
Using this, in the present embodiment, by arranging the phase plate 200 on the pupil conjugate plane 6A ′, the phase of the ± first-order diffracted light beam of wavelength λ _S and the phase of the ± first-order diffracted light beam of wavelength λ _L Is given a phase difference. That is, in the present embodiment, the phase delay amount of the phase plate 200 in the vicinity of the converging point P _S of wavelength lambda _S, larger than the phase delay of the phase plate 200 in the vicinity of the converging point P _L having a wavelength lambda _L Set. In other words, in the present embodiment, the optical path length of ± 1-order diffracted light flux of wavelength lambda _S, in the optical path length of ± 1-order diffracted light flux of the wavelength lambda _L, for imparting an optical path length difference.

  As the phase plate 200 having such a function, for example, a phase plate 200 configured as shown in FIG.

The phase plate 200, the wavelength lambda _L, lambda on a substrate 200A made of a transparent parallel flat plate with respect to _S, the wavelength lambda _L, a transparent phase delay unit 200B relative lambda _S, which was partially formed is there. The material of the substrate 200A is, for example, glass, and the phase delay unit 200B is configured by a thin film such as SiO ₂ .

  Note that, for example, techniques such as etching and vapor deposition are preferably applied to the formation of the phase delay portion 200B on the substrate 200A. This is because, according to techniques such as etching and vapor deposition, the shape and thickness of the phase delay unit 200B can be set with sufficient accuracy.

The formation destination of the phase delay unit 200B in the substrate 200A, the wavelength lambda covers converging point P _S of ± 1-order diffracted light flux of the _S, and the wavelength lambda _L ± 1 converging point P _L-order diffracted light beam It is off. Further, the formation destination of the phase delay unit 200B in the substrate 200A is deviated from the condensing point P ₀ of the 0th-order diffracted light beam (common between the wavelength λ _S and the wavelength λ _L ).

Note that the reason why the formation destination of the phase delay unit 200B is removed from the condensing point P0 of the _0th-order diffracted light beam is because the structured illumination microscope apparatus 1 is assumed to be used as a 3D-SIM (described later). . In 3D-SIM, it is necessary to guide not only the ± 1st-order diffracted light beam but also the 0th-order diffracted light beam to the specimen 5.

The thickness d _c of the optical axis O direction of the phase delay unit 200B in advance to a value which cancels the wavelength lambda _S, lambda deviation of the focal plane of the illumination optical system 10 for _L (shift of the focal plane of the sample side) Is set.

Here, the wavelength lambda _S, lambda and the deviation of the focal plane of the illumination optical system 10 for _L, a unique axial chromatic aberration in the illumination optical system 10 (= [Delta] d _c objective from the condenser lens 16 when it is zero lens 6 This is a deviation caused by axial chromatic aberration generated in the optical system up to.

  However, the axial chromatic aberration inherent in the illumination optical system 10 is substantially determined by the axial chromatic aberration inherent in the objective lens 6.

  Therefore, for the sake of simplicity, the axial chromatic aberration caused by the optical system other than the objective lens 6 (lenses 16, 25, 27) is regarded as zero, and only the axial chromatic aberration specific to the objective lens 6 is considered.

Now, compensation wavelength range of specific longitudinal chromatic aberration in the objective lens 6, as described above, whereas the covers wavelength lambda _L, not covering the wavelength lambda _S, an objective lens for the wavelength lambda _S The axial chromatic aberration of 6 is about several μm on the specimen side.

Therefore, the thickness d _c of the phase delay unit 200B in this embodiment, may be set to a value that can correct the longitudinal chromatic aberration of the order of the number [mu] m.

Will now be described in detail the relationship between the thickness d _c of the axial chromatic aberration and a phase delay portion 200B of the objective lens 6.

FIG. 10 is a diagram for explaining the shift of the focal plane of the objective lens 6 with respect to the wavelengths λ _S and λ _L.

Here, for simplicity, it is assumed that the magnification from the pupil conjugate plane 6A ′ to the pupil plane 6A is 1. In this case, the size of the pupil plane 6A is equal to the size of the pupil conjugate plane 6A ′, the size of the image (not shown) of the phase plate 200 projected onto the pupil conjugate plane 6A ′ is also equal to the size of the phase plate 200, the height of ± 1 converging point P _L-order diffracted light with a wavelength lambda _L in the pupil plane 6A is h _L becomes, ± 1 height of the focal point P _S-order diffracted light of wavelength lambda _S in the pupil plane. 6A h _S It becomes.

  As described above, the condensing point does not need to be a complete condensing point, and usually has a predetermined size (area). Therefore, the size (area) of the phase delay unit 200B is a predetermined size. It is necessary to ensure that it can cover the condensing point.

Further, in FIG. 10, in order to clarify that the focal plane on the sample side of the objective lens 6 is shifted between the wavelengths λ _L and λ _S , the optical path of the + 1st order diffracted light beam (with the wavelengths λ _L and λ _S ). The optical path of the 0th-order diffracted light beam (common between the wavelength λ _S and the wavelength λ _L ) toward the sample 5 is also drawn.

First, as shown in FIG. 10, the focal plane of the objective lens 6 by the light of wavelength λ _L is set to 5A _L , and the focal plane of the objective lens 6 by the light of wavelength λ _S is set to 5A _S.

Further, as shown in FIG. 10, relative to the focal plane 5A _L of wavelength lambda _L, the deviation of the focal plane 5A _S wavelength lambda _S, is denoted by [Delta] d _o.

In the present embodiment, in order to make this shift Δd _o zero, the phase delay unit 200B is disposed only in the optical path of the ± first-order diffracted light beam of the wavelength λ _S and the optical axis of the condensing point P _S viewed from the sample side. Extend the virtual distance in the O direction.

Figure 11 is a diagram showing an extended amount [Delta] d _p required to match the focal plane 5A _S wavelength lambda _S focal plane 5A _L of wavelength lambda _L. This extension amount Δd _p is expressed as follows by the “defocus aberration equation”.

The unit of the delay amount Δd _{p is} the unit of the phase of the wavelength λ _S. Further, [rho _S is a representation of the height h _S of the converging point P _S in units of numerical aperture, n _o is the refractive index of the specimen side of the medium of the objective lens 6 (where immersion liquid) is there.

On the other hand, the thickness of the phase delay unit 200B _{d c} Distant, placing the refractive index of the phase delay unit 200B and _{n c,} the phase delay amount Δd of the phase delay unit 200B is provided to the light of the wavelength lambda _S, as follows Represented.

Thus, in this embodiment, may be set thickness d _c of the phase delay unit 200B as [Delta] d is equal to [Delta] d _p. In other words, the thickness d _c of the phase delay unit 200B may be set so as to satisfy the following equation (3).

According to this setting, as shown in FIG. 12, the virtual distance of the focal point P _S viewed from the specimen side is moderately extended, the focal plane 5A _S wavelength lambda _S coincides with the focal plane 5A _L wavelength lambda _L . In FIG. 12, what is indicated by a symbol P _s ′ is a virtual image of the condensing point P _S as viewed from the sample side.

Thus, for example, wavelength lambda _S, lambda focal plane _5A S of _L, deviation [Delta] d _o of 5A _L is 0.2 mm, the refractive index _{n o} of the immersion liquid is 1.515, the refractive index of the phase delay unit 200B n _c is 1.5168, if the height [rho _S of the focal point _{P S} was 1.4, setting equation (3), the thickness _{d c} of the phase delay unit 200B to 0.36mm I understand that it should be done. The thickness of the phase delay unit 200B having such a thickness can be sufficiently manufactured by a conventional technique such as etching or vapor deposition.

Incidentally, by setting the thickness d _c of the phase delay portion 200B so as to satisfy the equation (3), theoretically, it is possible to exactly match the focal plane of the different wavelengths, the focus between multiple wavelengths The deviation of the surface can be allowed up to about 1/4 of the resolution in the optical axis direction. Specifically, it can be accepted up to about 60 nm in the specimen 5, and the focal planes coincide with each other when the deviation is this level. Can be considered.

By the way, in this embodiment, the direction of the interference fringes is switched between the above-described three directions (the first direction V ₁ , the second direction V ₂ , and the third direction V ₃ ). In order to cope with this, it is assumed that the phase delay unit 200B having the above-described configuration is formed on each of the three directions on the phase plate 200 (see FIG. 13).

  FIG. 13A is a view of the phase plate 200 as viewed from the direction along the optical axis O (sample side), and FIG. 13B shows the phase plate 200 cut along a plane including the optical axis O. FIG.

In this example, the phase delay unit 200B is formed in each of three directions (first direction V ₁ , second direction V ₂ , and third direction V ₃ ), and one of the phase delay units 200B is branched. direction covers converging point P _S pair, which is the first direction V _1, the other phase delay portion 200B covers the converging point P _S pair branch direction is the second direction V ₂ the remainder of the phase delay unit 200B, the branch direction covers converging point P _S pair is a third direction V _3.

[Modification of First Embodiment]
In the first embodiment, the phase plate 200 is disposed in the vicinity of the pupil conjugate plane 6A ′ as shown in FIG. 1, but needless to say, it may be in the vicinity of the pupil plane 6A. Locations of at least the phase plate 200 may be any place where the optical path of ± 1-order diffracted light flux of wavelength lambda _S, and the optical path of ± 1-order diffracted light flux of wavelength lambda _L are spatially separated.

  In the first embodiment, the phase plate 200 formed by forming the phase delay unit 200B on the substrate 200A is disposed in the optical path in order to give a phase difference between the plurality of light beams. However, only from the phase delay unit 200B. A phase plate (that is, a phase plate having no substrate) may be arranged in the optical path.

In the first embodiment, in order to give a difference in the phase delay amount of ± 1 ± 1-order diffracted light flux of the phase delay and the wavelength lambda _S-order diffracted light flux of the wavelength lambda _L, ± 1-order wavelength lambda _L While inserting the phase delay portion 200B only one of the optical path of the ± 1-order diffracted light flux of the light path and the wavelength lambda _S of diffracted light beams, ± 1-order diffracted light path and the wavelength lambda _S of ± 1-order diffracted light flux of wavelength lambda _L The phase delay unit 200 is provided in both the optical path of the luminous flux.
B may be inserted.

  In that case, a thickness difference may be provided between the phase delay unit 200B inserted in one and the phase delay unit 200B inserted in the other. Alternatively, instead of providing a difference in thickness, a difference in refractive index may be provided. Or you may provide the difference of the combination of thickness and refractive index.

  Further, the phase plate 200 of the first embodiment is provided with three sets of the phase delay units 200B in order to cope with the switching of the direction of the interference fringes (see FIG. 13), but only one set of the phase delay units 200B. While providing, you may further provide the mechanism which rotates the whole phase plate 200 around the optical axis O (refer FIG. 14). In that case, the control device 39 described above may link the rotation angle of the phase plate 200 with the rotation angle of the light beam selector 18.

  Further, the phase plate 200 of the first embodiment is provided with three sets of the phase delay units 200B (see FIG. 13) in order to cope with the switching of the direction of the interference fringes, but one of these phase delay units 200B. You may comprise a part or all by a common member. For example, as illustrated in FIG. 15, all the phase delay units 200 </ b> B may be configured with a common member (for example, a ring-shaped member). When such a phase delay unit 200B is employed, adjustment of the rotation angle around the optical axis O of the phase plate 200 becomes easy (basically, adjustment of the rotation angle is not necessary).

In the first embodiment, since the number of light source wavelengths is “2” and the number of image pickup devices is “2”, observation by one of the two wavelengths λ _L and λ _S and observation by the other are simultaneously performed. It can be carried out. However, in the first embodiment, the number of image sensors may be 1, and observation with one of the two wavelengths λ _L and λ _S and observation with the other may be performed sequentially.

  In the first embodiment, the number of light source wavelengths is “2”, but it goes without saying that the number may be extended to 3 or more.

  Further, when two or more light source wavelengths out of three or more light source wavelengths are out of the above-described correction wavelength region (here, 435 nm to 656 nm), a phase delay unit suitable for each of the two or more light source wavelengths. 200B may be formed on the phase plate 200.

For example, three types of wavelengths λ _L , λ _S1 , and λ _S2 are used as the light source wavelengths. If two of these wavelengths λ _S1 and λ _S2 are out of the correction wavelength range, for example, as shown in FIG. Both the phase delay unit 200B-1 suitable for the wavelength λ _S1 and the phase delay unit 200B- ₂ suitable for the wavelength λ _S2 are formed on the phase plate 200.

Among these, the height from the optical axis O of the formation position of the phase delay unit 200B-1 is set to be the same as the height from the optical axis O of the condensing point of the wavelength λ _S1 , and the phase delay unit 200B-2 is formed. The height of the position from the optical axis O is set to be the same as the height from the optical axis O of the condensing point of wavelength λ _S2 .

Further, the thickness of the phase delay unit 200B-1 in the optical axis O direction is set to a thickness for matching the focal plane of the wavelength λ _{S1 with} the focal plane of the wavelength λ _L , and the light of the phase delay unit 200B-2 The thickness in the direction of the axis O is set to a thickness for making the focal plane of the wavelength λ _S2 coincide with the focal plane of the wavelength λ _L.

  In FIG. 16, the two phase delay units 200 </ b> B- 1 and 200 </ b> B- 2 are configured as separate members, but needless to say, they may be configured as the same member.

  The phase plate shown in FIG. 16 is a modification of the phase plate (ring type) shown in FIG. 15, but the phase plate shown in FIG. 13 (6-block type) and the phase plate shown in FIG. Needless to say, the rotary type may be similarly modified.

  In the first embodiment, the phase plate 200 can be inserted into and removed from the optical path. When at least one of the light source wavelengths is out of the correction wavelength range (435 nm to 656 nm in this case), the phase plate 200 is inserted into the optical path. When all of the light source wavelengths are within the correction wavelength range (435 nm to 656 nm in this case), the phase plate 200 may be separated from the optical path. However, since the phase plate 200 of this embodiment can make the focal plane coincide between all wavelengths, there is no problem even if it is always inserted.

  In the first embodiment, the diffraction grating 13 (see FIG. 2A) that simultaneously generates a plurality of diffracted light flux groups having different branching directions is used as means for branching the light flux emitted from the light source. However, a diffraction grating (one-direction diffraction grating) that generates only one group of common diffracted light beams may be used. In this case, however, a mechanism for rotating the one-way diffraction grating around the optical axis O is provided to switch the direction of the interference fringes.

  In that case, a non-rotating 0th-order light cut mask may be used instead of the rotatable light beam selector 18. The 0th-order light cut mask has a mask portion disposed in an area that can be an optical path of a second-order or higher-order diffracted light beam, and an opening is disposed in an area that can be an optical path of a ± 1st-order diffracted light beam. This is a mask in which a mask portion is arranged in a region that becomes an optical path of a folded light beam.

  Further, in the first embodiment, in order to keep the ± first-order diffracted light beam incident on the specimen 5 to be S-polarized light, the half-wave plate 17 that can be rotated around the optical axis O is used. A quarter wavelength plate and a quarter wavelength plate capable of rotating around the optical axis O may be used. However, in that case, the rotation angle of the quarter-wave plate with respect to the first reference position is set to be the same as the rotation angle of the light beam selection member 18 with reference to the second reference position.

  Moreover, although 1st Embodiment demonstrated the case where the structured illumination microscope apparatus 1 was utilized as a total reflection fluorescence microscope (TIRFM), the structured illumination microscope apparatus 1 is replaced with a three-dimensional structured illumination microscope apparatus (3D-SIM). : 3D-Structured Illumination Microscopy).

  However, when the structured illumination microscope apparatus 1 is used as a 3D-SIM, the 0th-order diffracted light beam generated by the diffraction grating 13 is incident on the sample 5 together with the ± 1st-order diffracted light without being cut at the pupil conjugate plane 6A ′. . For this purpose, for example, a light beam selection member 18 ′ shown in FIG. 17 may be used instead of the light beam selection member 18 shown in FIG. 6. This light beam selection member 18 'is the same as the light beam selection member 18 shown in FIG. 6, but is provided with an opening 29 through which the 0th-order diffracted light beam passes. The opening 29 is formed in the vicinity of the optical axis O, and the shape of the opening 29 is, for example, a circle. According to such a light beam selection member 18 ', not only the ± 1st order diffracted light beam but also the 0th order diffracted light beam can contribute to the interference fringes.

  As described above, 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 5 but also in the depth direction of the sample 5. Therefore, according to this interference fringe, a three-dimensional super-resolution image of the sample 5 can be generated.

  In the first embodiment, when the structured illumination microscope apparatus 1 is used as a TIRF-SIM, a combination of a + 1st order diffracted light beam and a −1st order diffracted light beam is used as a diffracted light beam that contributes to interference fringes. Of course, other combinations may be used.

  Moreover, in 1st Embodiment, when using the structured illumination microscope apparatus 1 as 3D-SIM, as a diffracted light beam which contributes to an interference fringe, + 1st order diffracted light beam, −1st order diffracted light beam, and 0th order diffracted light beam Although combinations are used, it goes without saying that other combinations may be used.

[Effects of First Embodiment]
As described above, the structured illuminating device of the first embodiment is branched with the branch portion (diffraction grating 13) that branches the emitted light beam from the light source (laser unit 100) into at least two branched light beams (± first-order diffracted light beams). An optical system (illumination optical system 10) that forms interference fringes due to the two branched light beams on the sample (5), and a phase difference between at least two types of the emitted light beams (λ _L , λ _S ) having different wavelengths. A phase difference providing member (phase plate 200).

Therefore, even if the axial chromatic aberration inherent in the optical system (illumination optical system 10) for the two types of emitted light beams (λ _L , λ _S ) is not zero, the two types of emitted light beams (λ _L , It is possible to match the observation target surface between one and the other of λ _S ).

  In addition, the branch portion (diffraction grating 13) is a diffractive optical element that branches the two types of emitted light beams at different angles, and the phase difference providing member (phase plate 200) is configured to emit the two types of emitted light beams. The optical paths are arranged at spatially separated locations.

In such a place, it is easy to adjust the phase relationship between the two kinds of emitted light beams (λ _L , λ _S ).

  The optical system (illumination optical system 10) includes an objective lens (6), and the phase difference imparting member (phase plate 200) is a pupil plane (6A) or a pupil conjugate plane (of the objective lens (6)). 6A ').

In the pupil plane (6A) or the pupil conjugate plane (6A ′), the spatial separation amount of the two kinds of emitted light beams (λ _L , λ _S ) is maximized, so that the phase relationship can be adjusted most easily. Become.

In addition, the phase difference providing member (phase plate 200) includes a first region (passage region of wavelength λ _S ) that imparts a predetermined phase to one of the two types of emitted light beams, and the two types of emitted light beams. On the other hand, it has a second region (pass region of wavelength λ _L ) that gives a phase different from the predetermined phase.

  The phase difference imparted to the two types of emitted light beams by the phase difference providing member (phase plate 200) is set to a value corresponding to the axial chromatic aberration of the two types of emitted light beams.

  Therefore, the phase difference providing member (phase plate 200) can reliably suppress the deviation of the observation target surface due to the axial chromatic aberration.

  Alternatively, the phase difference imparted to the two types of emitted light beams by the phase difference providing member may be set to a value that matches the focal plane of the sample between the two types of emitted light beams.

  In addition, when the structured illumination microscope apparatus (1) is used as a 3D-SIM, the interference fringes formed by the optical system (illumination optical system 10) are the two branched light beams (± first-order diffracted light beams). This is a three-beam interference fringe formed by three branched beams with another branched beam (0th-order diffracted beam).

  Thus, when the structured illumination microscope apparatus (1) is used as a 3D-SIM, the displacement of the observation target surface becomes a big problem, and therefore the arrangement of the phase difference providing member (phase plate 200) is particularly effective. It is.

  The light source (100) emits the two types of emitted light beams simultaneously or sequentially.

Therefore, the structured illumination device of the first embodiment can perform sequential illumination or simultaneous illumination with the two types of emitted light beams (λ _L , λ _S ).

  Further, the structured illumination device of the first embodiment further includes a switching unit (light beam selection member 18) for switching the direction of the interference fringes formed on the specimen (5) between a plurality of directions, and the phase difference The applying member (phase plate 200) is prepared for each of the plurality of directions.

  Alternatively, the structured illumination device of the first embodiment switches the direction of the phase difference providing member and the first switching unit (light beam selection member 18) that switches the direction of the interference fringes formed on the specimen (5). And a second switching unit (see FIG. 14).

  Moreover, the structured illumination device of the first embodiment further includes a phase shift unit (translation mechanism 15A) that shifts the phase of the interference fringes.

  Therefore, the structured illumination device of the first embodiment can switch the direction and phase of the interference fringes.

  Further, the structured illumination microscope apparatus (1) of the first embodiment and the structured illumination apparatus of the first embodiment and a light detector (imaging) for an observation light beam from the sample (5) modulated by the interference fringes. An imaging optical system (30) that forms an image on the elements (351, 352), and an arithmetic means (image) that calculates a demodulated image of the sample (5) based on an image generated by the photodetector (imaging elements 351, 352). Storage / arithmetic unit 40).

Therefore, according to the structured illumination microscope apparatus (1) of the first embodiment, it is possible to perform super-resolution observation of the same observation target surface in the sample (5) with two types of wavelengths (λ _L , λ _S ). .

In addition, the structured illumination microscope apparatus (1) of the first embodiment has an optical element between the super-resolution observation by one of two kinds of wavelengths (λ _L , λ _S ) and the super-resolution observation by the other. It is not necessary to perform position adjustment (for example, position adjustment in the optical axis O direction of the emission end of the optical fiber 11 or the diffraction grating 13).

Therefore, the structured illumination microscope apparatus (1) of the first embodiment simultaneously performs super-resolution observation using one of two wavelengths (λ _L , λ _S ) and super-resolution observation using the other (simultaneous excitation). (Simultaneous exposure) is also possible.

[Second Embodiment]
Hereinafter, modifications of the first embodiment will be described as a second embodiment of the present invention. Here, only differences from the first embodiment will be described. The difference is in the shape of the phase delay unit 200B formed in the phase plate 200.

  FIG. 18A is a view of the phase plate 200 of the present embodiment as viewed from the direction along the optical axis O (sample side), and FIG. 18B is a plane including the optical axis O. It is sectional drawing which can be cut | disconnected.

  As shown in FIGS. 18A and 18B, the thickness of the phase delay unit 200B of this embodiment in the direction of the optical axis O depends on the distance from the optical axis O, specifically, the thickness. The length is set to increase as the distance from the optical axis O increases.

Because, firstly, the condensing point P _S (the condensing point of the ± 1st-order diffracted light beam with the wavelength λ _S ) to be covered by the phase delay unit 200B has a certain spread. It depends on the size of the fiber end of the optical fiber 1. Therefore, the height [rho _S from the optical axis O to the focal point P _S, having a slight extent.

Second, if the height [rho _S are different, since the delay amount [Delta] d _p require different (see curved dotted line in FIG. 19.), Varies the thickness d _c required phase delay unit 200B. Specifically, as when the height [rho _S is large, the thickness d _c required increases.

Thus, for example, a NA of 1.49 of the objective lens 6, and σ illumination is 0.05, the height [rho _S of the focal point _{P S} is in the range of 1.325 to 1.4 assumptions Then, the thickness _{d c} necessary for light is high [rho _S 1.4 is 0.36 mm, the thickness _{d c} necessary for height [rho _S is 1.325 rays, 0.30 mm (See Equation (3)).

Therefore, for example, a gradient in the range of d _c = 0.30 mm to 0.36 mm is given to the phase delay unit 200B of the present embodiment in the range of ρ _S = 1.325 to 1.4.

As described above, the structured illumination device (100, 10) according to the second embodiment is the same as the structured illumination device according to the first embodiment, except that the first region (passage band of wavelength λ _S ) and the second region (wavelength). The phase of at least one of the λ _L passbands) changes as the distance from the optical axis O increases.

Specifically, at least one of the phases of the (passband wavelength lambda _S) a first region and said second region (passband wavelength lambda _L) is set larger as the distance from the optical axis O The

Accordingly, the phase difference imparting member in the second embodiment (phase member 200B) can address at least one of the spread of the light flux of the light flux and the wavelength lambda _L of wavelength lambda _S.

  Note that the second embodiment can be modified in the same manner as the first embodiment. For example, the phase plate 200 in the second embodiment may be configured as a rotary type as shown in FIG. 14A, or may be configured as a ring type as shown in FIG. A double ring type as shown in FIG. For example, instead of arranging the phase plate 200 formed by forming the phase delay unit 200B on the substrate 200A in the optical path, a phase plate consisting only of the phase delay unit 200B (that is, a phase plate having no substrate) is arranged in the optical path. May be.

  In addition, the illumination optical system 10 of the first embodiment or the second embodiment is configured by the epi-illumination optical system using the objective lens 6, but is not limited to this, and transmission / reflection illumination using a condenser lens instead of the objective lens 6. You may comprise with an optical system. In this case, the focal point is formed on the pupil plane of the condenser lens.

  In the first embodiment or the second 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, three-beam interference fringes). 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. , 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.

[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, 13 ... Diffraction grating, 16 ... Condensing lens, 200 ... Phase plate, 18 ... Light beam selection member, 25 ... Lens, 26 ... Field stop, 27 ... Field lens, 28 ... Excitation filter, 7 ... Dichroic mirror, 6 ... Objective lens, 5 ... Sample, 6A ... Pupil plane, 6A '... Pupil conjugate plane

Claims (14)

A branching portion for branching the emitted light beam from the light source into at least two branched light beams;
An optical system for forming an interference fringe on the sample by the two branched light beams branched;
A phase difference imparting member that imparts a phase difference to at least two types of the emitted light beams having different wavelengths ;
The branch portion is a diffractive optical element that branches the two kinds of emitted light beams at different angles,
The phase difference imparting member, the optical path of the two kinds of the light flux is Ru is disposed at a location spatially separated
Structured lighting device. The structured lighting device according to claim 1 ,
The optical system includes an objective lens,
The phase difference providing member is disposed in the vicinity of the pupil plane or the pupil conjugate plane of the objective lens.
Structured lighting device. The structured lighting device according to claim 1 or 2 ,
The phase difference imparting member provides a first region that imparts a predetermined phase to one of the two types of emitted light beams, and a second region that imparts a phase different from the predetermined phase to the other of the two types of emitted light beams. And having an area of
Structured lighting device. The structured lighting device according to claim 3 .
The phase of at least one of the first region and the second region changes with distance from the optical axis.
Structured lighting device. The structured lighting device according to claim 4 .
The phase of at least one of the first region and the second region is set to increase with distance from the optical axis.
Structured lighting device. In the structured lighting device according to any one of claims 1 to 5 ,
The phase difference imparted to the two types of emitted light beams by the phase difference providing member is set to a value corresponding to the axial chromatic aberration of the two types of emitted light beams.
Structured lighting device. In the structured lighting device according to any one of claims 1 to 5 ,
The phase difference imparted to the two types of emitted light beams by the phase difference providing member is set to a value that matches the focal plane of the sample between the two types of emitted light beams.
Structured lighting device. In the structured lighting device according to any one of claims 1 to 7 ,
The interference fringes formed by the optical system are:
It is a three-beam interference fringe formed by three branch beams of the two branch beams and another branch beam.
Structured lighting device. In the structured lighting device according to any one of claims 1 to 8 ,
The light source is
The two kinds of emitted light beams are emitted simultaneously or sequentially.
Structured lighting device. In the structured lighting device according to any one of claims 1 to 9 ,
A switching unit that switches the direction of the interference fringes formed on the specimen between a plurality of directions;
The phase difference providing member is prepared for each of the plurality of directions.
Structured lighting device. In the structured lighting device according to any one of claims 1 to 9 ,
A first switching unit that switches a direction of the interference fringes formed on the specimen;
A second switching unit that switches the direction of the phase difference imparting member;
Further structured illumination device equipped with. In the structured lighting device according to any one of claims 1 to 11 ,
A phase shift unit for shifting the phase of the interference fringes;
Structured lighting device. The structured lighting device according to any one of claims 1 to 12 ,
An imaging optical system that forms an image on the light beam of the observation light beam from the sample modulated by the interference fringes;
Structured illumination microscope equipped with. The structured illumination microscope apparatus according to claim 13 ,
The apparatus further includes an arithmetic unit that calculates a demodulated image of the sample based on the image generated by the photodetector.
Structured illumination microscope.

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