JP2007279287A - Structured illuminating optical system and structured illuminating microscope having the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To capture at high speed information about a super-resolution image formed in a plurality of directions. <P>SOLUTION: The structured illuminating optical system includes: a space modulation element (2), which generates a diffracted luminous flux for forming an interference fringe (F) on a surface (13a) to be observed; and luminous flux rotating means (4 and 42), which is disposed between the space modulation element (2) and the surface (13a) to be observed, and rotates the diffracted luminous flux, running from the space modulation element (2) to the surface (13a), around a normal line of the surface (13a). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a structured illumination optical system that illuminates a surface to be observed with spatially modulated illumination light, and a structured illumination microscope including the structured illumination optical system.

  A high-resolution light beam that exceeds the resolution limit of the imaging optical system by structuring and illuminating the observation object using a spatial modulation element such as a diffraction grating as a technique for super-resolution observation of an observation object such as a biological specimen. (See Patent Documents 1 and 2, etc.). In the structured illumination method, in addition to the method of directly arranging the diffraction grating on the object to be observed, the diffraction grating is inserted into a surface conjugate with the sample surface in the illumination optical system, and the pattern is placed on the object to be observed. A projection method is also effective. In either case, super-resolution observation of the object to be observed is possible by detecting the image (modulated image) of the object that is structured and illuminated and demodulating the modulated image.

However, in order to demodulate the modulation image, data of a plurality of modulation images having different phases is required. For this reason, it is necessary to repeatedly detect the modulation image while shifting the diffraction grating in the direction of the grating pitch. Furthermore, in order to obtain the super-resolution effect in each direction on the object to be observed, it is necessary to rotate the arrangement direction of the diffraction grating and perform such a shift at each rotation position. Therefore, the diffraction grating requires a mechanism that combines a shift actuator and a rotary stage.
JP 11-242189 A US Reissue Patent No. 38307 Specification

  However, when an actuator is combined with a rotary stage, the mechanism and wiring become complicated, so it is difficult to repeat rotation and stop at high speed, and it takes time to obtain necessary data. Incidentally, in order to simplify the mechanism and wiring, it is possible to use a two-dimensional diffraction grating and shift it only in two directions, but the two shift directions and the two grating pitch directions are exactly matched. This is difficult and inadequate in terms of accuracy.

  Therefore, the present invention provides a structured illumination optical system suitable for high-speed acquisition of super-resolution image information in a plurality of directions, and a structured structure capable of high-speed acquisition of super-resolution image information in a plurality of directions. An object is to provide an illumination microscope.

The structured illumination optical system according to the present invention includes a spatial modulation element that forms interference fringes on a surface to be observed, and is disposed between the spatial modulation element and the surface to be observed. And a light beam rotating means for rotating the diffracted light beam traveling toward the normal line of the surface to be observed.
The light beam rotating means includes a reflection optical system that reflects the diffracted light beam an odd number of times and a rotation mechanism that rotates the entire reflection optical system, and the incident optical axis, the emission optical axis, and the rotation of the reflection optical system. The axes are preferably on the same straight line.

The reflection optical system is, for example, an image rotation prism.
A polarizing element that rotates together with the reflection optical system is inserted on the spatial modulation element side of the reflection optical system, and is fixed to the observation surface side on the observation surface side of the reflection optical system. A half-wave plate is inserted, and the direction of the transmission axis of the polarizing element is set so that the polarization direction of the diffracted light beam is P-polarized light or S-polarized light with respect to the reflecting surface of the reflective optical system. The direction of the optical axis of the two-wave plate is set so as to form an equal angle with respect to both the direction perpendicular to the separation direction of the diffracted light beam emitted from the reflection optical system and the direction of the transmission axis of the polarizing element. It is desirable.

Moreover, the structured illumination optical system of the present invention may further include phase changing means for changing the phase of the interference fringes.
The phase changing means is, for example, a shift mechanism that shifts the spatial modulation element in the modulation direction.
The structured illumination microscope of the present invention includes any one of the structured illumination optical systems of the present invention and an imaging optical system that forms an image of light from the observation surface on which the interference fringes are formed. It is characterized by that.

  The structured illumination microscope of the present invention may further include an image sensor that captures an image of the surface to be observed that is imaged by the imaging optical system, and a computer that performs arithmetic processing on the output of the image sensor. .

  According to the present invention, a structured illumination optical system suitable for high-speed acquisition of super-resolution image information in a plurality of directions and a structure capable of acquiring high-resolution image information in a plurality of directions at high speed. Realization of an integrated illumination microscope.

[First Embodiment]
A first embodiment of the present invention will be described. This embodiment is an embodiment of a microscope apparatus capable of super-resolution observation.
First, the configuration of the microscope apparatus will be described.
FIG. 1 is a schematic configuration diagram of the microscope apparatus. As shown in FIG. 1, this microscope apparatus includes a laser light source 1, an optical fiber 1a, a collector lens 2, a phase-type / transmission-type diffraction grating 3 having a one-dimensional periodic structure, a linearly polarizing plate 31, an incident optical axis, and the like. Dove prism 4, half-wave plate 32, lens 5, zeroth-order light cut stop 6, lens 7, field stop 8, lens 9, excitation filter 10, dichroic mirror 11, objective Lens 12, sample 13 such as a living body labeled with a fluorescent dye, barrier filter 14, second objective lens 15, imaging device 21 such as a CCD camera, control / arithmetic unit 22 such as a circuit or a computer, an image such as a liquid crystal display A display device 23, an actuator 41 such as a piezo element, and a rotary stage 42 are arranged.

  The light beam from the laser light source 1 is guided by the optical fiber 1a and generates a secondary light source on the end face 1A of the optical fiber 1a. The light beam from the secondary light source is converted into parallel light by the collector lens 2 and illuminates the diffraction grating 3. The diffracted light beams of the respective orders generated in the diffraction grating 3 pass through the linear polarizing plate 31 to become linearly polarized light and enter the incident surface 4 b of the Dove prism 4. When the diffracted light beam enters the dove prism 4 from the entrance surface 4 b, it is totally reflected on the bottom surface 4 a of the dove prism 4 and then exits from the exit surface 4 c of the dove prism 4. The diffracted light beam passes through the half-wave plate 32 to change the polarization direction, and is condensed by the lens 5 at each position on the arrangement surface of the 0th-order light cut stop 6 for each order. This arrangement surface is a conjugate surface with respect to the end surface 1A of the optical fiber 1a and the lenses 2 and 5.

Among the diffracted light beams, the 0th-order diffracted light beam and the second-order and higher-order diffracted light beams are shielded by the 0th-order light cut diaphragm 6, and only the ± 1st-order diffracted light beams pass through the 0th-order light cut diaphragm 6. The ± first-order diffracted light beams are condensed on the arrangement surface of the field stop 8 by the lens 7 and then enter the dichroic mirror 11 through the lens 9 and the excitation filter 10.
The ± first-order diffracted light beams incident on the dichroic mirror 11 are reflected by the dichroic mirror 11 and condensed on the rear focal plane of the objective lens 12, and then emitted as parallel light from the front end side of the objective lens 12. It enters the sample surface 13a at a predetermined angle and interferes with each other (two-beam interference). Due to the two-beam interference, a striped interference fringe F is formed on the sample surface 13a. As a result, the sample surface 13a is illuminated (structured illumination) with spatially modulated illumination light.

  On the specimen surface 13a illuminated by the interference fringes F, the fluorescent dye is excited and emits fluorescence. When the sample surface 13a at this time is viewed from the objective lens 12 side, moire fringes are observed. The moire fringes are formed by the fine structure of the specimen 13 and the pattern of the interference fringes F. The fine structure of the specimen 13 is expressed in a spatial frequency band that is lower by the spatial frequency of the pattern of the interference fringes F. Is. Therefore, even the fluorescence having a high spatial frequency structure exceeding the resolution limit of the objective lens 12 is captured by the objective lens 12.

The fluorescence captured by the objective lens 12 passes through the dichroic mirror 11 and the barrier filter 14, and then forms a modulated image 16 on the sample surface 13a by the second objective lens 15. The modulated image 16 is detected as an image by the imaging device 21 and is taken into the control / arithmetic device 22.
In the above microscope apparatus, the diffraction grating 3 that determines the pattern of the interference fringes F can be shifted by the actuator 41 in the direction Db perpendicular to the grating lines. When the diffraction grating 3 is shifted in this direction Db, the interference fringe F changes only the phase while maintaining the spatial frequency of the pattern.

  Further, the Dove prism 4 arranged at the rear stage of the diffraction grating 3 can be rotated around the optical axis by a rotary stage 42. When the Dove prism 4 rotates, the ± first-order diffracted light beams emitted from the Dove prism 4 rotate around the optical axis while maintaining the angular relationship of the respective rays. At this time, the ± first-order diffracted light beams incident on the sample surface 13a also rotate around the optical axis Z while maintaining the angular relationship between the light beams. Therefore, the interference fringe F rotates only in that direction while maintaining the pattern.

Incidentally, the Dove prism 4 is an image rotation prism that is generally arranged in a parallel system, and the rotation angle θ of the Dove prism 4 and the rotation angle θ ′ of the image (here, the direction of separating ± first-order diffracted light beams) , The rotation angle of the interference fringe F.) is θ ′ = 2θ.
Here, since the incident surface 4b and the exit surface 4c of the Dove prism 4 are inclined with respect to the optical axis, the optical path lengths of the light beams having different incident angles are different, and aberration is generated. Both pupil conjugates are not often used in general microscopes that correct aberrations. However, since this microscope apparatus uses the laser light source 1 as a light source, the ± first-order diffracted light beam that contributes to the interference fringes F is a light beam having a single wavelength with a sufficiently long coherence length.

Therefore, even if an optical path difference is given from the Dove prism 4 to each ray of the ± first-order diffracted light beam, as long as the optical path difference is shorter than the coherence length, the + 1st-order diffracted light beams interfere with each other on the sample surface 13a. Stripes F can be formed.
On the other hand, on the condensing surface of the ± 1st order diffracted light beam, that is, the pupil conjugate surface (such as the surface on which the 0th order light cut stop 6 is disposed), if a light path difference depending on the angle occurs in the light beam condensed there, it is formed on the sample surface 13a. The contrast of the interference fringes F to be reduced is lowered.

  Therefore, in this microscope apparatus, the insertion location of the Dove prism 4 is between the diffraction grating 3 and the lens 5, which is a parallel system position with respect to the pupil conjugate (see FIG. 1). In this microscope apparatus, the position between the lens 7 and the field stop 8 or between the field stop 8 and the lens 9 is also a parallel system position with respect to pupil conjugation. Since the system is often housed in the housing of the microscope main body and there is no space for placing the rotary stage 42, the position between the diffraction grating 3 and the lens 5 is most suitable as the insertion location of the Dove prism 4. It is thought that there is.

  Further, the linearly polarizing plate 31 and the half-wave plate 32 arranged on the incident side and the exit side of the Dove prism 4 are optically inserted to maintain the contrast of the interference fringes F regardless of the rotation of the Dove prism 4. It is an element. Among these, the linearly polarizing plate 31 can be rotated by the rotary stage 42 together with the Dove prism 4, and the half-wave plate 32 is not rotated by the rotary stage 42. The relationship between the linearly polarizing plate 31 and the half-wave plate 32 and the dove prism 4 will be described in detail later.

Next, the operation of the control / arithmetic unit 22 will be described.
The control / arithmetic unit 22 acquires necessary data by executing the following procedures (1) to (4).
(1) The rotation stage 42 is controlled to set the rotation angle θ of the Dove prism 4 and the linearly polarizing plate 31 to 0 ° (reference position).

(2) The actuator 41 and the imaging device 21 are controlled, the phase of the interference fringe F is changed in three stages, and one image of the modulated image 16 is acquired under each phase.
(3) The rotation stage 42 is controlled to set the rotation angle θ of the Dove prism 4 and the linearly polarizing plate 31 to + 60 °, and the procedure (2) is executed.
(4) The rotation stage 42 is controlled to set the rotation angle θ of the Dove prism 4 and the linearly polarizing plate 31 to −60 °, and the procedure (2) is executed.

When the necessary data is acquired by the above procedure, the control / arithmetic unit 22 performs a known image processing calculation on the data and acquires a super-resolution image of the specimen 13. This super-resolution image is displayed on the image display device 23.
Next, the relationship between the linearly polarizing plate 31 and the half-wave plate 32 and the dove prism 4 will be described.
In order to increase the contrast of the interference fringes F, it is desirable that the ± first-order diffracted light beams incident on the specimen surface 13a be S-polarized light. This is because the interference intensity of two-beam interference is 1 when the two beams are S-polarized light, but the contrast is cos (2φ ₀ ) depending on the incident angle φ ₀ when the two beams are P-polarized light. . When the two light beams are non-polarized light, the contrast is the average of the two. Therefore, the mixing of P-polarized light with respect to finite φ ₀ is not preferable because the contrast of the interference fringes F is lowered and the accuracy of super-resolution observation is lowered. For this reason, the ± first-order diffracted light beam with respect to the specimen surface 13a needs to be set to S-polarized light.

However, in this microscope apparatus, since the ± 1st-order diffracted light beam is rotated by the Dove prism 4, it is necessary to devise a technique for keeping the polarization direction of the ± 1st-order diffracted light beam with respect to the specimen surface 13a as S-polarized light. This is why the linearly polarizing plate 31 that rotates together with the dove prism 4 and the non-rotating half-wave plate 32 are inserted.
FIG. 2 is a perspective view showing a state around the Dove prism 4. Here, each angle around the optical axis is represented by an angle with respect to the grating line direction of the diffraction grating 3. FIG. 2 shows a state where the rotation angle θ of the Dove prism 4 and the linear polarizing plate 31 is 0 °.

As shown in FIG. 2, in this microscope apparatus, the transmission axis a31 of the linear polarizing plate 31 and the bottom surface 4a of the Dove prism 4 are parallel, and the direction of the optical axis a32 of the half-wave plate 32 is 0 ° (that is, a diffraction grating). 3 (parallel to the grid line 3) (condition 1) is satisfied.
First, the behavior of light rays when θ = 0 ° will be described.
The separation direction D1 of the ± first-order diffracted light beams L + 1 and L−1 emitted from the diffraction grating 3 is 90 ° because it is orthogonal to the grating lines. When θ = 0 °, the direction of the transmission axis a31 of the linearly polarizing plate 31 is also 0 °. Therefore, the polarization direction P1 of the ± first-order diffracted light beams L + 1 and L−1 transmitted through the linearly polarizing plate 31 is also 0 °. These ± 1st-order diffracted light beams L + 1 and L−1 are reflected by the bottom surface 4a of the Dove prism 4 in the S-polarized state, and are emitted from the Dove prism 4 while being S-polarized. Since θ = 0 °, the separation direction D1 ′ of the ± 1st-order diffracted light beams L + 1 ′ and L−1 ′ emitted from the Dove prism 4 is 90 ° as in the case of entering the Dove prism 4.

When these ± first-order diffracted light beams L + 1 ′ and L−1 ′ pass through the half-wave plate 32, their polarization directions P1 are reversed with the optical axis a32 as the symmetry axis, and changed to P1 ′. . The direction of the optical axis a32 is 0 °, and when θ = 0 °, the polarization direction P1 is 0 °. Therefore, the polarization direction P1 ′ after inversion is 0 ° similarly to the polarization direction P1 before inversion.
Therefore, when θ = 0 °, the separation direction D1 ′ of ± first-order diffracted light beams L + 1 ′ and L−1 ′ emitted from the half-wave plate 32 is 90 °, and their polarization direction P1 ′ is 0 °. And they are orthogonal. As a result, the ± first-order diffracted light beams L + 1 ′ and L−1 ′ incident on the sample surface 13a become S-polarized light with respect to the sample surface 13a.

2, the exit side optical path A of the diffraction grating 3, the exit side optical path B of the linearly polarizing plate 31, the exit side optical path C of the Dove prism 4, and the exit side optical path D of the half-wave plate 32 are viewed from the sample side. 3 (A), (B), (C), and (D), respectively.
Hereinafter, the change in the separation direction D of the ± first-order diffracted light beams L + 1 and L-1 and the change in the polarization direction P0 will be individually described with reference to FIG.

  First, the diffraction grating 3 and the dove prism 4 are related to the separation direction D. Immediately after passing through the diffraction grating 3 (FIG. 3A), the separation direction D is set to D1, and immediately after passing through the Dove prism 4 (FIG. 3C), the separation direction D is D1 '. Of these, the diffraction grating 3 has a function of setting the separation direction D to 90 ° regardless of θ, and the Dove prism 4 has a function of rotating the separation direction D by 2θ according to θ. However, when θ = 0 °, the amount of rotation by the Dove prism 4 is 0 °, so the total amount of rotation in the separation direction D is 90 °.

  On the other hand, the linearly polarizing plate 31 and the half-wave plate 32 are related to the polarization direction P0. Immediately after passing through the linear polarizing plate 31 (FIG. 3B), the polarization direction P0 is set to P1, and immediately after passing through the half-wave plate 32 (FIG. 3D), the polarization direction P0 is set to P1 ′. The Among these, the linearly polarizing plate 31 has a function of setting the polarization direction P0 to θ, and the half-wave plate 32 has a function of inverting the polarization direction P0 with respect to the optical axis a32 regardless of θ. However, when θ = 0 °, the rotation amount of the polarization direction P0 by the linearly polarizing plate 31 is 0 °, and at this time, the polarization direction P1 and the direction of the optical axis a32 of the half-wave plate 32 coincide. The rotation amount of the polarization direction P0 by the half-wave plate 32 is also 0 °. Therefore, when θ = 0 °, the total rotation amount in the polarization direction P0 is 0 °.

In general, during total reflection at the bottom surface 4a of the Dove prism 4, a phase difference occurs between the P-polarized component and the S-polarized component, and the polarization state changes. However, in condition 1 of the present embodiment, the axis a31 of the polarizing plate. And the bottom surface 4a of the Dove prism 4 are made parallel, and the incident light to the Dove prism 4 is set to only S-polarized light, so that the polarization is maintained before and after total reflection and is not related to the polarization direction P0.
As a result of the above, a difference of 90 ° occurs between the total rotation amount in the separation direction D and the total rotation amount in the polarization direction P0, and it is clear that the separation direction D and the polarization direction P0 are finally orthogonal.

Next, the behavior of light rays when θ = 60 ° will be described.
FIG. 4 is a perspective view showing a state around the Dove prism 4 (when θ = 60 °).
The separation direction D1 of the ± first-order diffracted light beams L + 1 and L-1 emitted from the diffraction grating 3 is 90 ° regardless of θ. However, when θ = 60 °, the direction of the transmission axis a31 of the linearly polarizing plate 31 is 60 °. Therefore, the polarization direction P1 of the ± first-order diffracted light beams L + 1 and L−1 transmitted through the linearly polarizing plate 31 is 60 °. Since the linearly polarizing plate 31 and the Dove prism 4 have the same posture, these ± 1st-order diffracted beams L + 1 and L-1 are reflected in the S-polarized state on the bottom surface 4a of the Dove prism 4, and remain as the S-polarized dove prism. Eject from 4. Since θ = 60 °, the separation direction of the ± first-order diffracted light beams L + 1 ′ and L−1 ′ emitted from the Dove prism 4 is rotated by 2 × 60 ° = 120 °, and the first separation direction D1 (90 ° ), The separation direction D1 ′ is 90 ° + 120 ° = 210 °.

When these ± first-order diffracted light beams L + 1 ′ and L−1 ′ pass through the half-wave plate 32, their polarization directions P1 are reversed with the optical axis a32 as the symmetry axis, and changed to P1 ′. . While the direction of the optical axis a32 is 0 ° regardless of θ, the polarization direction P1 is 60 ° when θ = 60 °, so the polarization direction P1 ′ after inversion is 120 °. .
Therefore, when θ = 60 °, the separation direction D1 ′ of the ± first-order diffracted light beams L + 1 ′ and L−1 ′ emitted from the half-wave plate 32 is 210 °, and their polarization direction P1 ′ is 120 °. And they are orthogonal. As a result, the ± first-order diffracted light beams L + 1 ′ and L−1 ′ incident on the sample surface 13a become S-polarized light with respect to the sample surface 13a.

In FIG. 4, the exit side optical path A of the diffraction grating 3, the exit side optical path B of the linear polarizing plate 31, the exit side optical path C of the Dove prism 4, and the exit side optical path D of the half-wave plate 32 are viewed from the sample side. 5A, 5B, 5C, and 5D, respectively.
Hereinafter, the change in the separation direction D of the ± first-order diffracted light beams L + 1 and L-1 and the change in the polarization direction P0 will be individually described with reference to FIG.

  First, the diffraction grating 3 and the dove prism 4 are related to the separation direction D. Immediately after passing through the diffraction grating 3 (FIG. 5A), the separation direction D is set to D1, and immediately after passing through the Dove prism 4 (FIG. 5C), the separation direction D is D1 '. Of these, the diffraction grating 3 has a function of setting the separation direction D to 90 ° regardless of θ, and the Dove prism 4 has a function of rotating the separation direction D by 2θ according to θ. In particular, when θ = 60 °, the amount of rotation by the Dove prism 4 is 2θ = 120 °. Therefore, when θ = 60 °, the total rotation amount in the separation direction D is 90 ° + 120 ° = 210 °.

  On the other hand, the linearly polarizing plate 31 and the half-wave plate 32 are related to the polarization direction P0. After passing through the linear polarizing plate 31 (FIG. 5B), the polarization direction P0 is set to P1, and immediately after passing through the half-wave plate 32 (FIG. 5D), the polarization direction P0 becomes P1 '. Among these, the linearly polarizing plate 31 has a function of setting the polarization direction P0 to θ, and the half-wave plate 32 has a function of inverting the polarization direction P0 with respect to the optical axis a32 regardless of θ. In particular, when θ = 60 °, the rotation amount of the polarization direction P0 by the linearly polarizing plate 31 is 60 °, and at this time, the angle between the polarization direction P1 and the direction of the optical axis a32 of the half-wave plate 32 is 60 °. Therefore, the rotation amount of the polarization direction P0 by the half-wave plate 32 is 60 °. Therefore, when θ = 60 °, the total rotation amount in the polarization direction P0 is 120 °.

As a result, there is a difference of 90 ° between the total rotation amount in the separation direction D and the total rotation amount in the polarization direction P0, and it is clear that the separation direction D and the polarization direction P0 are finally orthogonal. .
Although not shown, the state of the periphery of the dove prism 4 when θ = −60 ° is obtained by inverting FIGS. 4 and 5 with the separation direction D1 as the axis of symmetry. Therefore, even when θ = −60 °, the ± first-order diffracted light beams L + 1 ′ and L−1 ′ incident on the sample surface 13a become S-polarized light with respect to the sample surface 13a.

As a result, in this microscope apparatus, when the rotation angle θ is switched to 0 °, + 60 °, and −60 ° in the steps (1) to (4) described above, the contrast of the interference fringes F is reliably kept high. Can do. Therefore, necessary data can be acquired with high accuracy.
Next, the 0th-order light cut stop 6 will be described.
FIG. 6 is a view of the zero-order light cut stop 6 as seen from the optical axis direction. 6A shows a state when θ = 0 °, FIG. 6B shows a state when θ = 60 °, and FIG. 6C shows a state when θ = −60 °. . As shown in FIG. 6, in this microscope apparatus, the separation directions of the ± first-order diffracted light beams L + 1 ′ and L-1 ′ are set in three ways: 0 °, + 120 °, and −120 °. There are also three ways of converging the light beams L + 1 ′ and L−1 ′ (6 places in total).

  For this reason, the opening 6C of the 0th-order light cut stop 6 needs to cover all of these six condensing locations. In FIG. 6, reference numeral Z denotes an optical axis, reference numeral 6A denotes a ring-shaped light-shielding plate that shields second-order and higher-order diffracted light, and reference numeral 6B denotes light-shielding zero-order diffracted light. A circular light shielding plate is denoted by reference numeral 6D, and an arm for fixing the light shielding plate 6A and the light shielding plate 6B to each other (this arm 6D may be composed of the same member as the light shielding plates 6A and 6B). .) The arm 6D is provided to configure the zero-order light cut stop 6 as a single component in which the light shielding plate 6A and the light shielding plate 6B are arranged on the same plane. As shown in 6 (A), (B), and (C), it is necessary to be disposed at a position that does not reach any of the six condensing portions of ± first-order diffracted light beams L + 1 ′ and L−1 ′.

If a 0th-order light cut stop is formed by patterning a light-shielding part having the same shape as the light-shielding plates 6A and 6B on the glass substrate, the arm 6D can be omitted, but excess reflected light is generated on the surface of the glass substrate. Since it may occur and give noise to the interference fringes F, it is most desirable that the 0th-order light cut stop 6 is composed of a combination of a light shielding plate and an arm.
As described above, in this microscope apparatus, in order to rotate the direction of the interference fringe F, the Dove prism 4 is inserted in the subsequent stage of the diffraction grating 3 and is rotated by the rotary stage 42. Therefore, it is sufficient that the diffraction grating 3 is shifted by the actuator 41 and does not need to be placed on the rotary stage. Thus, if the rotation target of the rotary stage 42 and the shift target of the actuator 41 are different, the mechanism and wiring of both can be simplified, so that the rotation and stop of the rotary stage 42 can be repeated at high speed. It is. Therefore, the control / arithmetic unit 22 of the microscope apparatus can perform the above-described procedures (1) to (4) at high speed and can obtain a super-resolution image at high speed.

In addition, since the microscope apparatus includes the linearly polarizing plate 31 that rotates together with the Dove prism 4 and the appropriately arranged half-wave plate 32, the contrast of the interference fringes F on the specimen surface 13a is kept high. Therefore, the acquisition accuracy of the super-resolution image is also increased.
(Modification)
In this microscope apparatus, condition 1: “the transmission axis a31 of the linear polarizing plate 31 and the bottom surface 4a of the Dove prism 4 are parallel, and the direction of the optical axis a32 of the half-wave plate 32 is 0 ° (that is, the diffraction grating). However, even if the condition is changed to any one of the condition 2, condition 3, and condition 4 described below, the same effect can be obtained.

<Condition 2>
FIGS. 7A, 7B, 7C, and 7D are views showing condition 2, and show optical paths A, B, C, and D when condition 2 is satisfied as viewed from the sample side. (However, the illustration shows only θ = 0 °). As shown in FIG. 7, the condition 2 is that “the transmission axis a31 of the linearly polarizing plate 31 and the bottom surface 4a of the Dove prism 4 are parallel, and the direction of the optical axis a32 of the half-wave plate 32 is 90 ° (diffraction grating 3 Is perpendicular to the grid line.

  Thus, even when the optical axis a32 of the half-wave plate 32 is set to 90 °, the half-wave plate 32 is provided if the polarization direction P1 of the light incident on the half-wave plate 32 is the same. The polarization direction P1 ′ of the light emitted from (= inverted P1 with the optical axis a32 as the symmetry axis) is the same as when the optical axis a32 is set to 0 °. Therefore, the same effect as condition 1 can be obtained by this condition 2.

<Condition 3>
FIGS. 8A, 8B, 8C, and 8D are views showing condition 3, and show optical paths A, B, C, and D when condition 3 is satisfied as seen from the sample side. (However, the illustration shows only θ = 0 °). As shown in FIG. 8, the condition 3 is “the transmission axis a31 of the linearly polarizing plate 31 and the bottom surface 4a of the dove prism 4 are perpendicular and the direction of the optical axis a32 of the half-wave plate 32 is 45 °”.

  As described above, when the transmission axis a31 of the linearly polarizing plate 31 is perpendicular to the bottom surface 4a of the dove prism 4, the light reflected by the bottom surface 4a becomes P-polarized light. However, under this condition 3, the direction of the optical axis a32 of the half-wave plate 32 is set to 45 °, so that the polarization direction P1 ′ of the light that has passed through the half-wave plate 32 is set to the separation direction D1 ′. Can be vertical. Therefore, the same effect as condition 1 can be obtained by this condition 3.

<Condition 4>
FIGS. 9A, 9B, 9C, and 9D are views showing condition 4, and the optical paths A, B, C, and D when the condition 4 is satisfied are viewed from the sample side. (However, the illustration shows only θ = 0 °). As shown in FIG. 9, the condition 4 is “the transmission axis a31 of the linearly polarizing plate 31 and the bottom surface 4a of the dove prism 4 are perpendicular, and the direction of the optical axis a32 of the half-wave plate 32 is −45 °”. . That is, in condition 3, it is equal to the direction of the optical axis a32 of the half-wave plate 32 rotated by 90 °. Therefore, even under this condition 4, the same effect as the condition 3, that is, the same effect as the condition 1 is obtained.

  Furthermore, when the above conditions 1, 2, 3, and 4 are summarized, at least the present microscope apparatus has the following condition, that is, “the direction of the transmission axis a31 of the linearly polarizing plate 31 is ± first-order diffracted light beams L + 1, L−. The polarization direction of 1 is P-polarized light or S-polarized light with respect to the bottom surface 4 a of the Dove prism 4, and the direction of the optical axis a 32 of the half-wave plate 32 is ± first-order diffraction that has passed through the Dove prism 4. It is only necessary to satisfy “the same angle is formed with respect to both the non-separating direction of the light beams L + 1 ′ and L−1 ′ and the direction of the transmission axis a31 of the linearly polarizing plate 31”.

However, the “non-separation direction” here is a direction perpendicular to the separation direction D1 ′ and corresponding to the fringe direction of the interference fringes F.
In this microscope apparatus, the 0th-order light cut stop 6 is used. However, when the intensity of the 0th-order diffracted light beam is sufficiently small and does not affect the interference fringes F, it may be omitted.
[Second Embodiment]
A second embodiment of the present invention will be described. This embodiment is also an embodiment of a microscope apparatus. Here, only differences from the first embodiment will be described. The difference lies in the types of light source and diffraction grating, the type of image rotation prism, and the insertion position.

FIG. 10 is a schematic configuration diagram of the microscope apparatus. As shown in FIG. 10, in this microscope apparatus, a discharge light source 101 such as a mercury lamp is arranged instead of the laser light source 1, a density type diffraction grating 103 is arranged instead of the phase type diffraction grating 3, and a dove prism. Instead of 4, an Abbe prism 104 is arranged.
Unlike the Dove prism 4, the Abbe prism 104 has an entrance surface 104c and an exit surface 104e perpendicular to the optical axis. The light beam that has entered the Abbe prism 104 from the incident surface 104c is sequentially reflected by the three surfaces 104b, 104a, and 104d of the Abbe prism 104, and then exits from the surface 104e while maintaining the angular relationship between the light beams.

Such an Abbe prism 104 has an advantage that the degree of freedom of the insertion portion is high because no aberration occurs even if the insertion portion is not a parallel light beam. Incidentally, in FIG. 10, the insertion place of the Abbe prism 104 is between the lens 5 and the 0th-order light cut stop 6. However, the insertion location is not limited to this position.
The Abbe prism 104 can also be rotated around its optical axis by the rotary stage 42, similarly to the Dove prism 4 of the first embodiment. The relationship between the Abbe prism 104, the linearly polarizing plate 31, and the half-wave plate 32 is set similarly to the relationship between the Dove prism 4, the linearly polarizing plate 31, and the half-wave plate 32 in the first embodiment. .

Since this Abbe prism 104 does not give a phase difference between incident light beams having different angles, this microscope apparatus is suitable for super-resolution observation even though the discharge light source 101 having a short coherence length is used as the light source. Interference fringes F can be generated.
Further, in the present microscope apparatus, since the discharge light source 101 having low monochromaticity is used, the density type diffraction grating 103 is used as the diffraction grating in accordance therewith. This is because the density type diffraction grating 103 is different from the phase type diffraction grating in that the diffraction intensity does not depend on the wavelength. However, since the density type diffraction grating 103 generates a 0th-order diffracted light beam having a relatively strong intensity, the 0th-order light cut stop 6 is essential in this microscope apparatus.

[Others]
In each of the above-described embodiments, an image rotating prism such as a Dove prism or an Abbe prism is used to rotate the ± first-order diffracted light beam. However, a reflective optical system having the same function is configured by combining an odd number of mirrors. However, it may be used in place of the image rotation prism.
In each of the above-described embodiments, a one-dimensional and transmission diffraction grating is used as the spatial modulation element. However, a reflection diffraction grating, a two-dimensional diffraction grating, and other spatial modulation elements may be used.

  In each of the above-described embodiments, the ± first-order diffracted light beam is rotated in order to rotate the direction of the interference fringe F. Instead of rotating the light beam, the sample 13 is placed on a rotary stage and is reflected by light. You may rotate around an axis. However, rotating the light beam is more preferable than rotating the specimen 13 in terms of high positioning accuracy before and after the rotation.

It is a schematic block diagram of the microscope apparatus of 1st Embodiment. It is a perspective view which shows the mode of the periphery of the dove prism 4 (when (theta) = 0 degree). It is the figure which looked at the optical path A, B, C, D of FIG. 2 from the sample side. It is a perspective view which shows the mode of the periphery of the dove prism 4 (when (theta) = 60 degrees). It is the figure which looked at optical path A, B, C, D of Drawing 4 from the sample side. It is the figure which looked at the 0th-order light cut stop 6 from the optical axis direction. It is the figure which looked at optical path A, B, C, D when the condition 2 is satisfied from the sample side (when θ = 0 °). It is the figure which looked at optical path A, B, C, D when the condition 3 is satisfied from the sample side (when θ = 0 °). The optical paths A, B, C, and D when the condition 4 is satisfied are viewed from the sample side (when θ = 0 °). It is a schematic block diagram of the microscope apparatus of 2nd Embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Laser light source, 1a ... Optical fiber, 2 ... Collector lens, 3 ... Diffraction grating, 31 ... Linear polarizing plate, 4 ... Dove prism, 32 ... 1/2 wavelength plate, 6 ... 0th-order light cut stop, 7 ... Lens , 8 ... Field stop, 9 ... Lens, 10 ... Excitation filter, 11 ... Dichroic mirror, 12 ... Objective lens, 13 ... Sample, 14 ... Barrier filter, 15 ... Second objective lens, 21 ... Imaging device, 41 ... Actuator, 42 ... Rotation stage

Claims (8)

A spatial modulation element that generates a diffracted light beam for forming interference fringes on the surface to be observed;
A light beam rotating means disposed between the spatial modulation element and the surface to be observed, and rotating the diffracted light beam from the spatial modulation element toward the surface to be observed around a normal line of the surface to be observed;
A structured illumination optical system comprising: The light beam rotating means includes
A reflection optical system that reflects the diffracted light beam an odd number of times, and a rotation mechanism that rotates the entire reflection optical system,
The structured illumination optical system according to claim 1, wherein an incident optical axis, an outgoing optical axis, and a rotation axis of the reflection optical system exist on the same straight line. The reflective optical system is
The reflection optical system according to claim 2, wherein the reflection optical system is an image rotation prism. On the spatial modulation element side of the reflective optical system,
A polarizing element that rotates together with the reflection optical system is inserted,
On the observed surface side of the reflective optical system,
A half-wave plate fixed to the observed surface side is inserted,
The direction of the transmission axis of the polarizing element is:
The polarization direction of the diffracted light beam is set to be P-polarized light or S-polarized light with respect to the reflective surface of the reflective optical system,
The direction of the optical axis of the half-wave plate is
3. The apparatus according to claim 2, wherein the angle is set to be equal to both a direction perpendicular to a separation direction of the diffracted light beam emitted from the reflection optical system and a direction of a transmission axis of the polarizing element. The structured illumination optical system according to claim 3. The structured illumination optical system according to any one of claims 1 to 4, further comprising phase changing means for changing a phase of the interference fringes. The phase changing means includes
The structured illumination optical system according to claim 5, wherein the structured illumination optical system is a shift mechanism that shifts the spatial modulation element in the modulation direction. The structured illumination optical system according to any one of claims 1 to 6,
A structured illumination microscope comprising: an imaging optical system that forms an image of light from the observation surface on which the interference fringes are formed. An image sensor that captures an image of the surface to be observed formed by the imaging optical system;
The structured illumination microscope according to claim 7, further comprising: a computer that performs arithmetic processing on an output of the image sensor.

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