WO2015052920A1 - Structured illumination device and structured illumination microscope device - Google Patents

Abstract

An example of this structured illumination device comprises: a spatial light modulator that splits light from a light source into a plurality of light beams; an optical system that forms an interference pattern with all or some of the plurality of light beams, and that irradiates a sample with the interference pattern; and a control unit that controls the spatial light modulator. The control unit outputs a drive signal to the spatial light modulator, and thereby sets a first phase delay amount to a first region of the spatial light modulator and sets a second phase delay amount to a second region such that the intensity ratio between the plurality of light beams contributing to the interference pattern has a predetermined value.

Description

Structured illumination device and structured illumination microscope device

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

A structured illumination microscope that modulates the spatial frequency of the structure of a specimen with illumination light is known as a technique for super-resolution observation of an object (specimen) such as a biological specimen.

This structured illumination microscope illuminates a specimen with a spatially modulated illumination pattern, and contributes high spatial frequency information exceeding the resolution limit included in the specimen structure to the imaging of the microscope optical system. The structured illumination microscope operates on a plurality of modulated image data (hereinafter referred to as “modulated images”) obtained under different illumination patterns to perform demodulation image data (hereinafter referred to as “demodulated image”). Image ”or“ super-resolution image ”).

In particular, in the structured illumination microscope described in Non-Patent Document 1, a two-dimensional spatial light modulator (SLM) is used as a phase diffraction grating for generating a striped illumination pattern. In the SLM, liquid crystal pixels are closely arranged, and the phase delay amount of light in each liquid crystal pixel can be controlled by controlling the voltage applied to each liquid crystal pixel. If the phase delay amount of this SLM is set to a periodic distribution in one direction, a phase diffraction grating appears in the SLM. To switch the orientation and phase of this phase diffraction grating, the voltage distribution applied to the SLM may be switched. Therefore, the structured illumination microscope apparatus described in Non-Patent Document 1 can switch the azimuth and phase of the illumination pattern without mechanical driving.

However, if the SLM that functions as the phase diffraction grating is not properly controlled, the performance of the structured illumination microscope may be deteriorated.

An example of the structured illuminating device of the present invention forms an interference fringe with a spatial light modulator that branches light from a light source into a plurality of light beams, and all or part of the plurality of light beams, and illuminates the sample with the interference fringes. An optical system and a control unit that controls the spatial light modulator are provided. The control unit outputs a drive signal to the spatial light modulator, so that the intensity ratio of the plurality of light beams contributing to the interference fringes becomes a predetermined value, so that the first region of the spatial light modulator is A first phase delay amount is set for the second region, and a second phase delay amount is set for the second region.

An example of the structured illumination microscope apparatus of the present invention includes the example of the structured illumination apparatus of the present invention and an imaging unit that acquires a modulated image that is an image of a sample spatially modulated by interference fringes.

1 is a configuration diagram of a structured illumination microscope apparatus 1. FIG. It is a figure explaining SLM13. It is a figure explaining the relationship between the direction of a phase diffraction grating, and the direction of an interference fringe. It is a figure explaining the 0th-order light shutter 200 and the high-order light cut member 18. FIG. FIG. 6 is a diagram for explaining the function of a half-wave plate 17. It is a figure explaining the phase shift of an interference fringe. It is a figure which shows the relationship between phase difference (PHI) of a phase diffraction grating, and diffraction intensity. 4 is an operation flowchart of the control device 39. It is a modification of the arrangement | positioning attitude | position of SLM13.

[First Embodiment]
Hereinafter, a structured illumination microscope (SIM) 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, a case where the structured illumination microscope apparatus 1 is used in the 3D-SIM mode will be mainly 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, an image sensor 35, a control device 39, an image storage / An arithmetic device 40 and an image display device 45 are provided. Among these, the illumination optical system 10 and the imaging optical system 30 share the objective lens 6 and the dichroic mirror 7.

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 λ ₁ of the first laser light source 101 is longer than the wavelength λ ₂ of the second laser light source 102 (λ ₁ > λ ₂ ). 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 composed of, 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 emission end of the optical fiber 11 in the optical axis AZ direction 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 light beam branching unit 15, a condensing lens 16, a light beam selecting unit 24, a lens 25, and a field of view. A diaphragm 26, a field lens 27, an excitation filter 28, a dichroic mirror 7, and an objective lens 6 are disposed.

The light beam splitting unit 15 includes a two-dimensional liquid crystal spatial light modulator (SLM) 13 that functions as a phase diffraction grating and a liquid crystal drive circuit 15A. Among these, the liquid crystal drive circuit 15 </ b> A is driven and controlled by the control device 39. Here, the SLM refers to a member having a function of giving a predetermined distribution spatially (within the space of the light beam) to the incident light beam.

The light beam selector 24 includes a zero-order light shutter 200, a half-wave plate 17, a high-order light cut member 18, a rotation mechanism 200A, and a wave plate driving circuit 17A. Among these, the rotation mechanism 200A and the wave plate driving circuit 17A are driven and controlled by the control device 39, respectively.

In the imaging optical system 30, an objective lens 6, a dichroic mirror 7, a barrier filter 31, and a second objective lens 32 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 the light of wavelength λ ₁ and the second fluorescent region excited by the light of wavelength λ ₂ are expressed. First fluorescent region, _'to generate a first fluorescence and the second fluorescence region, the central wavelength lambda ₂ in accordance with the wavelength lambda ₂ of the _light' center wavelength lambda ₁ in accordance with the wavelength lambda ₁ of the optical second fluorescence Is generated.

The image sensor 35 is a two-dimensional image sensor composed of a CCD, a CMOS, or the like. When the image pickup device 35 is driven by the control device 39, the image pickup device 35 picks up an image formed on the image pickup surface 36 of the image pickup device 35 and generates an image. The image generated by the image sensor 35 is taken into the image storage / arithmetic device 40 via the control device 39. The imaging element 35 can repeat image generation (imaging) at a predetermined frame period. The frame period (imaging repetition period) of the image sensor 35 is set to 30 msec, 60 msec, or the like, for example. The frame period (imaging repetition period) of the image sensor 35 is determined by the rate-determining among the imaging time of the image sensor (that is, the time required for charge accumulation and charge readout), the time required for switching the direction of interference fringes, and other required times. It is done.

The control device 39 drives and controls the first laser light source 101, the second laser light source 102, the shutters 1031, 1032, the position adjustment mechanism 11A, the liquid crystal drive circuit 15A, the rotation mechanism 200A, the wave plate drive circuit 17A, and the image sensor 35. To do.

When the image storage / arithmetic unit 40 performs a demodulation operation on the image generated by the image sensor 35 to generate a super-resolution image, the image storage / arithmetic unit 40 stores the super-resolution image in an internal memory (not shown) and an image display unit. 45.

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

When the laser beam (first laser beam) having the wavelength λ ₁ emitted from the first laser light source 101 enters the mirror 105 through the shutter 1031, the laser beam is reflected by the mirror 105 and enters the dichroic mirror 106. On the other hand, laser light (second laser light) having a wavelength λ ₂ emitted from the second laser light source 102 enters the beam splitter 106 via the shutter 1032 and is integrated with the first laser light. The first laser beam and the second laser beam emitted from the dichroic mirror 106 enter the incident end of the optical fiber 11 through the lens 107.

The control device 39 controls the first laser light source 101, the second laser light source 102, and the shutters 1031 and 1032 to increase the emission wavelength of the laser unit 100, that is, the light source wavelength λ of the structured illumination microscope device 1. It is possible to switch between wavelength λ ₁ and short wavelength λ ₂ .

The laser light incident on the incident end of the optical fiber 11 propagates inside the optical fiber 11 to generate 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 SLM 13 via the polarizing plate 23, and branches into a diffracted light beam of each order (hereinafter referred to as “diffracted light beam group”). (Details will be described later). When the diffracted light beams of the respective orders included in the diffracted light beam group enter the condenser lens 16, the diffracted light beams are condensed at each position of the pupil conjugate plane 6 </ b> A ′ by receiving the condensing action of the condenser lens 16.

Here, the pupil conjugate plane 6A ′ is a position conjugate with the pupil plane 6A of the objective lens 6 with respect to the field lens 27 and the lens 25 (a position where the diffracted light beams of respective orders are individually collected). 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.

When a polarization plane preserving single mode fiber is used as the optical fiber 11, the polarizing plate 23 can be omitted, but it is effective for reliably cutting off an 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. Incidentally, when a multimode fiber is used as the optical fiber 11, the polarizing plate 23 is essential. When the SLM 13 is used as a diffraction grating, it is necessary to set the polarization direction of the light beam incident on the SLM 13 to an appropriate direction.

Now, the diffracted light beam group directed toward the pupil conjugate surface 6A 'enters the light beam selector 24 disposed in the vicinity of the pupil conjugate surface 6A'.

When the structured illumination microscope apparatus 1 is used in the 3D-SIM mode, the light beam selection unit 24 selects only three diffracted light beams (only the 0th-order diffracted light beam and the ± 1st-order diffracted light beams) from the incident diffracted light beam group. Let it pass. The 0th-order light shutter 200 of the light beam selection unit 24 has a function of turning on / off the 0th-order diffracted light beam as necessary. The high-order light cut member 18 of the light beam selection unit 24 has a second or higher order higher next time. There is a function of always blocking the folded light beam (details will be described later).

The 0th-order diffracted light beam and the ± 1st-order diffracted light beam that have passed through the light beam selection unit 24 form a conjugate plane with the SLM 13 near the field stop 26 by the lens 25. Thereafter, each of the 0th-order diffracted light beam and the ± 1st-order diffracted light beam is converted into convergent light by the field lens 27, further passes through the excitation filter 28, is reflected by the dichroic mirror 7, and is mutually reflected on the pupil plane 6 </ b> A of the objective lens 6. Concentrate at different positions.

Each of the 0th-order diffracted light beam and the ± 1st-order diffracted light beam collected on the pupil plane 6A becomes a parallel light beam when exiting from the tip of the objective lens 6, interferes with each other on the surface of the sample 5, and forms interference fringes. Form. This interference fringe is used as structured illumination light.

When the specimen 5 is illuminated with such an interference fringe, the specimen 5 is spatially modulated, and a moire fringe corresponding to the difference between the periodic structure of the interference fringe and the periodic structure of the fluorescent region in the specimen 5 appears. Since the high frequency structure in the fluorescent region is shifted to the lower frequency side than the original frequency, the fluorescence indicating this structure is directed to 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 passes through the second objective lens 32 and passes through the second imaging lens 35. A modulated image of the fluorescent region is formed on the imaging surface 36. The modulated image is imaged by the image sensor 35 to generate a modulated image. The modulated image is taken into the image storage / arithmetic device 40 via the control device 39. Further, the modulation image is demodulated by the image storage / calculation device 40, and a demodulated image (super-resolution image) of the fluorescent region is generated. The super-resolution image is stored in an internal memory (not shown) of the image storage / arithmetic device 40 and is sent to the image display device 45. As the demodulation operation, for example, a method disclosed in US Pat. No. 8,115,806 is used.

Next, the SLM 13 will be described in detail.

FIG. 2A is a schematic diagram in which a part of the SLM 13 is enlarged. As shown in FIG. 2A, the SLM 13 is a reflection-type spatial light modulator, and includes a circuit layer 13c such as CMOS in which pixel circuits are two-dimensionally arranged, a liquid crystal layer 13a made of nematic liquid crystal, and the like, wavelengths λ ₁ and λ _2. And a protective layer 13b transparent to the light. That is, the SLM 13 includes a two-dimensional liquid crystal member in which pixels made of liquid crystal elements are densely arranged in each of two directions orthogonal to each other. The SLM 13 is directed toward the protective layer 13b with respect to the incident light beam, and the normal of the surface (incident surface) of the protective layer 13b forms an angle of, for example, 45 ° with respect to the principal ray of the incident light beam.

Note that the configuration of the SLM may be a configuration used for regular reflection. In that case, it is desirable to spatially separate the incident light on the SLM and the reflected light from the SLM by a combination of a half mirror, a polarizing beam splitter, and a wave plate.

When a voltage is applied to each pixel circuit in the circuit layer 13c, the orientation of each pixel (Pixel) in the liquid crystal layer 13a changes, and the refractive index of each pixel changes. When the refractive index of each pixel of the liquid crystal layer 13a changes, the phase delay amount of light reflected by each pixel of the liquid crystal layer 13a changes.

Therefore, if the voltage value applied to each pixel circuit of the circuit layer 13c is controlled, the phase distribution of the incident light beam with respect to the SLM 13 can be controlled. Note that the amplitude distribution and polarization direction distribution of the incident light beam with respect to the SLM 13 do not change at all.

As such a reflective spatial light modulator, for example, X10468 of Hamamatsu Photonics, LC-R720 of HOLOEYE, or the like can be applied. In addition, although a reflective spatial light modulator is used as the SLM 13 here, a transmissive spatial light modulator may be used. Further, although the liquid crystal method is used here as the light modulation method, another method in which the refractive index of the optical path is variable may be used.

Now, the liquid crystal drive circuit 15A (see FIG. 1) described above sets the refractive index distribution of the liquid crystal phase 13a to a periodic distribution in one direction under the control of the control device 39 (see FIG. 1). Thereby, for example, a one-way phase diffraction grating as shown in FIG. 2B is displayed on the SLM 13. It is the function of this phase diffraction grating that splits the incident light beam into the diffracted light beam group.

Note that the grating pattern of the phase diffraction grating displayed on the SLM 13 cannot actually be visually observed. However, for the sake of explanation, the grating pattern of the phase diffraction grating is visualized below. In addition, the fact that the phase delay amount distribution of at least an effective light incident region of the SLM 13 is set to be a periodic distribution is expressed as “a phase diffraction grating is displayed on the SLM 13”.

FIG. 2B is a schematic view of the phase diffraction grating displayed on the SLM 13 as viewed from the sample side. In FIG. 2B, a pixel region 13A having a relatively low refractive index (a pixel region having a relatively small phase delay amount) is represented in white, and a pixel region 13B having a relatively high refractive index (a relative phase delay amount). A large pixel area) is shown in gray. In FIG. 2B, the grating period (structure period) P of the phase diffraction grating is drawn larger than the actual one.

Here, the phase delay amount imparted to the pixel region 13A having a relatively low refractive index is a constant value within the region, and the phase delay amount imparted to the pixel region 13B having a relatively high refractive index is a constant value within the region. It is. In this case, the SLM is the same as a rectangular uneven diffraction grating.

Note that the pattern of the SLM phase delay amount distribution may be a pattern in which anti-aliasing is applied only to the boundary region of each pixel region of the SLM. Again, the SLM is included in the rectangular concept.

However, the present invention is not limited to this, and the distribution of the phase delay amount in the SLM may be a sinusoidal distribution.

Specifically, the phase delay amount of the pixel region 13A having a relatively low refractive index is a value distributed in a sine wave shape and may be set to include a minimum value, and has a relatively high refractive index. The phase delay amount of the pixel region 13B is a value distributed in a sine wave shape, and may be set to include the maximum value.

In addition, when connecting the values of a plurality of pixels in order to form the phase delay amount distribution of the image regions 13A and 13B, when viewed locally, the sinusoidal distribution is discontinuous, but as a whole SLM, What is necessary is just to consider that it is distributed in the shape of a sine wave.

Here, when the phase delay amount of the pixel region 13A and the phase delay amount of the pixel region 13B are constant values and are different from each other (hereinafter, rectangular wave mode), the phase delay amount of the pixel region 13A is sine. The difference from the case where the value is distributed in a wave shape and includes the maximum value and the phase delay amount of the pixel region 13B is distributed in a sine wave shape and includes the minimum value (hereinafter referred to as sine wave mode) is as follows. It is as follows.

The first phase delay amount and the second phase delay amount so that the ratio of the intensity of the 0th order diffracted light beam, the intensity of the + 1st order diffracted light beam, and the intensity of the −1st order diffracted light beam is, for example, 0.7: 1: 1. Is set, the difference between the first phase delay amount and the second phase delay amount due to the distribution shape of the phase delay amount is smaller in the rectangular wave mode than in the sine wave mode.

Further, the phase delay amount of the pixel region 13A having a relatively low refractive index is a value distributed in an inverted trapezoidal shape (the upper side is long and the lower side is short), and the lower side is set to a minimum value. In particular, the phase delay amount of the pixel region 13B having a high refractive index is a value distributed in a trapezoidal shape (the upper side is short and the lower side is long), and the upper side may be set to a maximum value. Furthermore, in the case of a trapezoid (inverted trapezoid), the line connecting the upper side and the lower side is usually a straight line.

The light beam incident on the SLM 13 is converted into a diffracted light beam group branched in the direction V of the periodic structure of the phase diffraction grating. This 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 beam having the same order travels in a symmetric direction with respect to the optical axis AZ. The folded light beam travels along the optical axis AZ. These 0th-order diffracted light beam and ± 1st-order diffracted light beam are condensed at different positions on the pupil conjugate plane 6A ′. As shown in FIG. 2C, the condensing point 14a of the 0th-order diffracted light beam is located on the optical axis AZ, and the condensing points 14b and 14c of the ± 1st-order diffracted light beams are symmetric with respect to the optical axis AZ. The arrangement direction of the condensing points 14c, 14a, 14b is the same as the branching direction V of the diffracted light beam group.

Note that the “focusing point” mentioned here is the position of the center of gravity of an area having 80% or more of the maximum intensity. Therefore, the illumination optical system 10 of the present embodiment does not need to collect the light beam until a complete condensing point is formed.

Further, the structural period P of the phase diffraction grating is set to an appropriate value P ₀ such that the respective condensing points 14b and 14c of the ± first-order diffracted light beams are located on the outermost circumference of the pupil conjugate plane 6A ′. According to the appropriate value P ₀ , the fringe period of the interference fringes (FIG. 2D) is appropriately reduced (in FIG. 2D, the structural period P is drawn larger than the actual period).

Further, in the present embodiment, the refractive index difference Δn of the phase diffraction grating, that is, the difference between the refractive index of the pixel region 13A and the refractive index of the pixel region 13B is calculated from the phase diffraction grating based on the equation (1) described later. The ratio of the intensity of the emitted 0th-order diffracted light beam, the intensity of the + 1st-order diffracted light beam, and the intensity of the −1st-order diffracted light beam is set to an appropriate value Δn ₀ such as 0.7: 1: 1. According to this appropriate value Δn ₀ , the contrast of the demodulated image (super-resolution image) becomes optimal (details will be described later).

The reason for this is as follows.

That is, when the intensity ratio of the 0th-order diffracted light and the 1st-order diffracted light is 1: 1, the magnitude of the Fourier spectrum of the second-order OTF in the 3D-SIM mode using structured illumination by three-beam interference is two beams. This is because it is lower than the Fourier spectrum of the second-order OTF in the 2D-SIM mode using structured illumination due to interference. The reason is that, in the 3D-SIM mode, the first-order OTF is generated due to the interference fringes of the 0th-order diffracted light and the + 1st-order diffracted light, and the interference fringes of the 0th-order diffracted light and the + 1st-order diffracted light. The size is reduced. Here, the second-order OTF means OTF caused by interference fringes of + 1st order diffracted light and −1st order diffracted light.

Therefore, in order to improve the contrast of the high frequency component of the demodulated image (super-resolution image), it is necessary to make the intensity of the 0th-order diffracted light beam lower than the intensity of the ± 1st-order diffracted light beam.

Further, the liquid crystal driving circuit 15A described above changes the display direction of the phase diffraction grating, for example, from FIG. 3A1 to FIG. 3 by switching the refractive index distribution of the liquid crystal layer 13a under the control of the control device 39 (see FIG. 1). 3 (A2) → As shown in FIG. 3 (A3), switching is performed in three ways with an angular period of 60 °.

When the direction of the periodic structure of the phase diffraction grating is V ₁ as shown in FIG. 3 (A1), the arrangement direction of the condensing points 14c, 14a, and 14b on the pupil conjugate plane 6A ′ as shown in FIG. is the same as the direction _{V 1.}

Incidentally, assuming that the wavelength of the laser light emitted from the optical fiber 11 is λ, the grating period of the SLM 13 is P, and the focal length of the lens 16 is fc, the distance D from the optical axis AZ to the condensing points 14b and 14c is expressed by the following equation: It is represented by

D∝2fcλ / P
When the direction of the periodic structure of the phase diffraction grating is V ₂ as shown in FIG. 3 (A2), the arrangement direction of the condensing points 14c, 14a, and 14b on the pupil conjugate plane 6A ′ as shown in FIG. is the same as the direction _{V 2.} The distance of the focal point 14c, from 14b to the optical axis AZ is the same as the distance when the direction of the periodic structure is _{V 1} (see FIG. 3 (B1)).

When the direction of the periodic structure of the phase diffraction grating is V ₃ as shown in FIG. 3 (A3), the arrangement direction of the condensing points 14c, 14a, and 14b on the pupil conjugate plane 6A ′ is as shown in FIG. is the same as the direction _{V 3.} The distance of the focal point 14c, from 14b to the optical axis AZ is the same as the distance when the direction of the periodic structure is _{V 1} (see FIG. 3 (B1)).

Therefore, the liquid crystal driving circuit 15A of the present embodiment changes the direction of the interference fringes formed on the specimen 5 to an angular period of 60 ° as shown in FIG. 3 (C1) → FIG. 3 (C2) → FIG. 3 (C3). You can switch between three ways. Moreover, in this embodiment, since the SLM 13 is used as the phase diffraction grating, the direction of the interference fringes is switched at high speed.

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

FIG. 4A is a diagram for explaining the zero-order light shutter 200. As shown in FIG. 4A, the 0th-order optical shutter 200 is a spatial filter formed by forming a circular light shielding part 200C on a part of a circular transparent substrate.

The light-shielding part 200C of the 0th-order optical shutter 200 covers the optical path (condensing point 14a) of the 0th-order diffracted light beam, and the non-light-shielding part (transmission part 200B) of the 0th-order light shutter 200 is the optical path of ± first-order diffracted light flux. The entire region that can be formed (that is, the region where the condensing points 14b and 14c can be formed) is covered.

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

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

When the rotation angle of the 0th-order light shutter 200 is set to the reference angle (0 °) shown in FIG. 4, the light-shielding portion 200C is inserted into the optical path (condensing point 14a) of the 0th-order diffracted light beam, and the 0th-order light When the rotation angle of the shutter 200 is set to a predetermined angle (for example, 30 °) that deviates from the reference angle, the light shielding part 200C is deviated from the optical path (condensing point 14a) of the 0th-order diffracted light beam.

Therefore, if the rotation angle of the 0th-order light shutter 200 is switched between the reference angle (0 °) and the predetermined angle (30 °), the 0th-order diffracted light beam is turned on / off while the ± 1st-order diffracted light beam remains on. can do. 4A shows a state in which the 0th-order diffracted light beam is turned off, and FIG. 1 shows a state in which the 0th-order diffracted light beam is turned on. Incidentally, when the 0th-order diffracted light beam is turned off, the structured illumination microscope apparatus 1 is set to the 2D-SIM mode, and when the 0th-order diffracted light beam is turned on, the structured illumination microscope apparatus 1 is set to the 3D-SIM mode. .

It should be noted that the light shielding portion 200C of the 0th-order light shutter 200 has a ± 1st-order diffracted light beam regardless of whether the rotation angle of the 0th-order light shutter 200 is a reference angle (0 °) or a predetermined angle (30 °). It is assumed that a region that can be an optical path (that is, a region where the condensing points 14b and 14c can be formed) is not blocked.

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

Next, the high-order light cut member 18 will be described in detail.

FIG. 4B is a diagram illustrating the high-order light cut member 18. As shown in FIG. 4B, the high-order light cut member 18 is a spatial filter formed by forming a circular opening 18a and a ring-shaped opening 18b on a circular opaque substrate (mask substrate).

In the high-order light cut member 18, the circular opening 18 a covers the optical path of the 0th-order diffracted light beam (condensing point 14 a), and the ring-shaped opening 18 b is an area that can be an optical path of the ± 1st-order diffracted light beam (that is, The region where the condensing points 14b and 14c can be formed). In the high-order light cut member 18, a region that can be an optical path of a second-order or higher-order diffracted light beam is a light shielding portion (non-opening portion).

It should be noted that the high-order light cut member 18 may be omitted when the intensity of the second-order and higher-order diffracted light beams generated by the SLM 13 is sufficiently weak.

Next, the function of the half-wave plate 17 (see FIG. 1) will be described in detail.

The half-wave plate 17 (see FIG. 1) is used to maintain the polarization state of the diffracted light beam group contributing to the interference fringes as S-polarized light. This is because the contrast of the interference fringes becomes maximum when the polarization state of the diffracted light beam group is S-polarized light.

When the branching direction of the diffracted light beam group is V ₁ as shown in FIG. 5A, the polarization direction of the diffracted light beam group should be the direction V ₁ ′ shown by the dotted arrow in FIG. . This direction V ₁ ′ is a direction obtained by rotating the direction V ₁ by 90 ° around the optical axis AZ.

When the branching direction of the diffracted light beam group is V ₂ as shown in FIG. 5B, the polarization direction of the diffracted light beam group should be the direction V ₂ ′ shown by the dotted arrow in FIG. 5B. . This direction V ₂ ′ is a direction obtained by rotating the direction V ₂ by 90 ° around the optical axis AZ.

As shown in FIG. 5C, when the branching direction of the diffracted light beam group is V ₃ , the polarization direction of the diffracted light beam group should be the direction V ₃ ′ indicated by the dotted arrow in FIG. . This direction V ₃ ′ is a direction obtained by rotating the direction V ₃ by 90 ° around the optical axis AZ.

Therefore, in the present embodiment, the axial direction of the polarizing plate 23 disposed on the upstream side of the SLM 13 is matched with the direction V ₂ ′ in advance, and the progression of the half-wave plate 17 disposed on the downstream side of the SLM 13 is achieved. The direction of the phase axis is appropriately rotated around the optical axis AZ by the wave plate driving circuit 17A (see FIG. 1). The fast axis of the half-wave plate 17 is a direction in which the amount of phase delay when light polarized in the direction of the axis passes through the half-wave plate 17 is minimized.

Further, in this embodiment, since the switching of the branch direction of the diffracted light beam group is accelerated by the SLM 13, it is assumed that the half-wave plate 17 is also composed of a liquid crystal element so that the effect of the acceleration is not impaired. . If the orientation of the liquid crystal element is electrically controlled by the wave plate driving circuit 17A (see FIG. 1), the direction of the fast axis of the half-wave plate 17 is switched at high speed.

When the branching direction of the diffracted light beam group is V ₁ as shown in FIG. 5 (A), the direction of the fast axis of the half-wave plate 17 is set to the direction indicated by the solid line double arrow in FIG. 5 (A). Is done. This direction is 1/2 the polarization direction V _{2 'of} the diffracted light flux group incident has the wavelength plate 17, 1/2 polarization direction V ₁ to the diffracted light flux group emitted from the wavelength plate 17 has It is the direction that bisects'.

5 when the branch direction of the diffracted light beam group as shown in (B) is V _2, the direction of the fast axis of the 1/2-wavelength plate 17, set in the direction indicated by the solid line double arrow in FIG. 5 (B) Is done. This direction is 1/2 the polarization direction V _{2 'of} the diffracted light flux group incident has the wavelength plate 17, 1/2 polarization direction V ₂ should diffracted light flux group emitted from the wavelength plate 17 has Is a direction (= V ₂ ′).

When the branching direction of the diffracted light beam group is V ₃ as shown in FIG. 5C, the direction of the fast axis of the half-wave plate 17 is set to the direction indicated by the solid double-pointed arrow in FIG. Is done. This direction includes the polarization direction V ₂ ′ that the diffracted light beam group incident on the half-wave plate 17 has, and the polarization direction V ₃ that the diffracted light beam group emitted from the half-wave plate 17 should have. It is the direction that bisects'.

Here, the half-wave plate 17 made of a liquid crystal element is used to keep the diffracted light beam group contributing to the interference fringe as S-polarized light. However, in order to keep the diffracted light beam group contributing to the interference fringe as S-polarized light. There are other methods (described later).

Next, the operation of the liquid crystal drive circuit 15A (see FIG. 1) regarding the phase shift of interference fringes will be described in detail.

The above-described demodulation operation requires, for example, a plurality of modulation images (for example, five modulation images) that are the same sample 5 and the modulation images related to the interference fringes in the same direction and have different phases of the interference fringes. 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 the primary modulation component, the + secondary modulation component, and the -2nd modulation component are included, and it is necessary to make these five unknown parameters known by the demodulation operation.

Therefore, the liquid crystal drive circuit 15A shifts the display destination of the phase diffraction grating in the SLM 13 in order to shift the phase of the interference fringes. The shift direction is, for example, a direction (x direction) non-perpendicular to the direction V of the periodic structure of the phase diffraction grating (here, any _one of V ₁ , V ₂ , V ₃ ) as shown in FIG.

Incidentally, the shift amount L in the x direction of the phase diffraction grating necessary for shifting the phase of the interference fringes by φ is determined by the structural period P of the phase diffraction grating and the angle θ shown in FIG. This angle θ is an angle formed by the shift direction (x direction) of the phase diffraction grating and the direction V of the periodic structure (in this case, any _one of V ₁ , V ₂ , and V ₃ ). Further, when the number of phases of interference fringes necessary for the demodulation operation is N, the phase shift period φ is set to 2π / N, for example.

Next, the operation of the liquid crystal drive circuit 15A (see FIG. 1) regarding the switching of the light source wavelength λ will be described in detail.

The intensity ratio between the 0th-order diffracted light beam and the ± 1st-order diffracted light beam that contributes to the interference fringes basically depends on the phase difference Φ of the phase diffraction grating displayed on the SLM 13. The phase difference Φ of the phase diffraction grating is a difference between the phase delay amount given by the pixel region 13A and the phase delay amount given by the pixel region 13B with respect to the incident light beam of the SLM 13.

Here, the relationship between the phase difference Φ of the phase diffraction grating and the diffraction intensity of each order generated in the phase diffraction grating is as shown in FIG. The “diffraction intensity” here refers to the intensity of the diffracted light beam generated when the intensity of the incident light beam with respect to the phase diffraction grating is “1”.

7 that only the 0th-order diffracted light beam is generated and no other-order diffracted light beam is generated at the phase difference Φ = 0.

Further, according to FIG. 7, at the phase difference Φ = 2 [rad] (where the curves intersect), the intensity ratio of the 0th-order diffracted light beam, the + 1st-order diffracted light beam, and the −1st-order diffracted light beam is 1: 1: 1. It turns out that it becomes.

Further, according to FIG. 7, it can be seen that in the phase difference Φ = π≈3 [rad], the 0th-order diffracted light beam is not generated, but the ± 1st-order diffracted light beam and the higher-order diffracted light beam are generated.

Therefore, according to FIG. 7, the phase difference Φ such that the ratio of the intensity of the 0th-order diffracted light beam, the intensity of the + 1st-order diffracted light beam, and the intensity of the −1st-order diffracted light beam is 0.7: 1: 1 is It can be seen that ≈2.2.

The curve in FIG. 7 shows a case where the phase distribution is a sine wave mode. When the phase distribution is a rectangular wave mode, the phase difference Φ for setting the ratio of the intensity of the 0th-order diffracted light beam, the intensity of the + 1st-order diffracted light beam, and the intensity of the −1st-order diffracted light beam to 0.7: 1: 1, for example. Since the phase distribution is smaller than that in the sine wave mode, it is necessary to change the phase value set in the SLM.

The phase difference Φ of the phase diffraction grating is determined by the refractive index difference Δn of the phase diffraction grating. The refractive index difference Δn of the phase diffraction grating is a difference between the refractive index of the pixel region 13A and the refractive index of the pixel region 13B in the SLM 13.

However, the phase difference Φ of the phase diffraction grating depends not only on the refractive index difference Δn of the phase diffraction grating but also on the wavelength λ of the incident light beam. This is expressed by the following formula (1).

Φ = 2π · Δn · d / λ (1)
In the formula (1), d is the thickness (constant) of the liquid crystal layer 13a of the SLM 13 in the optical axis AZ direction.

Therefore, the appropriate value Δn ₀ of the refractive index difference Δn of the phase diffraction grating is slightly different when the light source wavelength λ is the long wavelength λ ₁ and when it is the short wavelength λ ₂ .

Therefore, in the present embodiment, a function indicating the relationship between the ratio of the intensity of the 0th-order diffracted light beam, the intensity of the + 1st-order diffracted light beam and the intensity of the −1st-order diffracted light beam (intensity ratio) and the refractive index difference Δn for each wavelength λ. It is assumed that the table is stored in advance in a storage unit (not shown) in the control device 39.

In this case, the control device 39 can obtain the appropriate value Δn ₀ of the refractive index difference Δn by referring to this function table according to the required intensity ratio and the used wavelength λ. As a result, the appropriate value of the drive signal (voltage value) to be given to the SLM 13 by the liquid crystal drive circuit 15A is determined.

Here, in order to determine the appropriate value Δn ₀ of the refractive index difference Δn according to the required intensity ratio and the wavelength used λ (that is, to determine the appropriate value of the drive signal (voltage value) to be applied to the SLM 13. ), “Function table for obtaining refractive index difference Δn from intensity ratio and wavelength λ used” is used, but other tables may be used. For example, two functions, “a function table for obtaining the phase difference Φ from the intensity ratio” and “a function table for obtaining the refractive index difference Δn from the used wavelength λ and the phase difference Φ”, may be used.

Further, here, the information to be stored in the storage unit is a function table, but part or all of the information to be stored in the storage unit may be the function itself. For example, equation (1) may be used instead of “a function table for obtaining the refractive index difference Δn from the used wavelength λ and the phase difference Φ”.

However, it is preferable to use a function table because an appropriate value Δn ₀ of the refractive index difference Δn (and thus an appropriate value of the voltage value to be applied to the SLM 13) can be derived at high speed.

The liquid crystal drive circuit 15A (see FIG. 1) of the present embodiment combines the voltage value applied to the pixel region 13A and the voltage value applied to the pixel region 13B of the SLM 13 under the control of the control device 39 (see FIG. 1). By controlling the above, the refractive index difference Δn of the phase diffraction grating is appropriately adjusted.

Specifically, the liquid crystal drive circuit 15A, a phase diffraction grating when when the light source wavelength lambda is longer wavelengths lambda ₁ is a refractive index difference [Delta] n of the phase grating in [Delta] n _01, the light source wavelength lambda is short wavelength lambda ₂ Is set to Δn ₀₂ .

However, [Delta] n _01, when the zero-order diffracted light flux intensity and + 1st-order diffracted light flux intensity and -1 the ratio of the intensity of the diffracted light flux for example 0.7 light source wavelength lambda is λ _{1: 1: 1} and It is an appropriate value. According to this appropriate value Δn ₀₁ , the contrast of the demodulated image (super-resolution image) having the wavelength λ ₁ is optimal.

On the other hand, [Delta] n _02, when the zero-order diffracted light flux intensity and + 1st-order diffracted light flux intensity and -1 the ratio of the intensity of the diffracted light flux for example 0.7 light source wavelength lambda is λ _2: 1: 1 and It is an appropriate value. According to this appropriate value Δn ₀₂ , the contrast of the demodulated image (super-resolution image) having the wavelength λ ₂ is optimal.

Therefore, in this embodiment, the contrast of the demodulated image (super-resolution image) is maintained high regardless of the light source wavelength λ.

Incidentally, the relationship between Δn ₀₁ and Δn ₀₂ is expressed by the following equation (2).

Δn ₀₁ / λ ₁ = Δn ₀₂ / λ ₂ (2)
Because it is as described above λ _1> λ _2, a Δn _01> Δn _02.

Next, the procedure for acquiring data necessary for two types of super-resolution images with different wavelengths will be described.

FIG. 8 is an operation flowchart of the control device 39. Hereafter, each step of FIG. 8 is demonstrated in order.

Step S1: The control device 39 sets the structured illumination microscope apparatus 1 to the 3D-SIM mode by setting the rotation angle of the zero-order light shutter 200 to the reference angle via the rotation mechanism 200A. Further, the control device 39 sets the light source wavelength λ of the structured illumination microscope apparatus 1 to the long wavelength λ ₁ via the laser unit 100.

Step S2: The control device 39 displays the phase diffraction grating on the SLM 13 by driving the SLM 13 via the liquid crystal drive circuit 15A. The refractive index difference Δn of this phase diffraction grating is Δn ₀₁ , the direction V of the periodic structure of the phase diffraction grating is V ₁ , and the structural period P of the phase diffraction grating is P ₀ . Further, the control device 39 drives the half-wave plate 17 via the wave plate drive circuit 17A, so that the fast axis of the half-wave plate 17 is in the direction of the solid double arrow in FIG. Set.

Step S3: The control device 39 shifts the display destination of the phase diffraction grating in the SLM 13 in N ways with a predetermined shift cycle via the liquid crystal drive circuit 15A, so that the phase of the interference fringes is N with the shift cycle φ = 2π / N. And N modulated images are acquired by driving the laser unit 100 and the image sensor 35 under these N phases.

Step S4: The control device 39 drives the SLM 13 via the liquid crystal drive circuit 15A, thereby switching the direction V of the periodic structure of the phase diffraction grating to V ₂ and a half wavelength via the wave plate drive circuit 17A. When the fast axis of the half-wave plate 17 is switched in the direction of the solid double arrow in FIG. 5B by driving the plate 17, step S3 is executed.

Note that the refractive index difference Δn and the structural period P are common between the phase diffraction grating displayed on the SLM 13 in step S4 and the phase diffraction grating displayed on the SLM 13 in step S2.

Step S5: the control device 39, by driving the SLM13 through the liquid crystal drive circuit 15A, switches the direction V of the periodic structure of the phase grating to _{V 3,} 1/2 wavelength through a wavelength plate drive circuit 17A When the fast axis of the half-wave plate 17 is switched in the direction of the solid double arrow in FIG. 5C by driving the plate 17, step S3 is executed.

Note that the refractive index difference Δn and the structural period P are common between the phase diffraction grating displayed on the SLM 13 in step S5 and the phase diffraction grating displayed on the SLM 13 in step S2.

Step S6: the control device 39 switches the light source wavelength lambda of the structured illumination microscope apparatus 1 from a long wavelength lambda ₁ through the laser unit 100 to the shorter wavelength lambda _2.

Step S7: The control device 39 displays the phase diffraction grating on the SLM 13 by driving the SLM 13 via the liquid crystal drive circuit 15A. The refractive index difference Δn of this phase diffraction grating is Δn ₀₂ , the direction V of the periodic structure of the phase diffraction grating is V ₁ , and the structural period P of the phase diffraction grating is P ₀ . Further, the control device 39 drives the half-wave plate 17 via the wave plate drive circuit 17A, so that the fast axis of the half-wave plate 17 is in the direction of the solid double arrow in FIG. Switch.

Step S8: The control device 39 shifts the display destination of the phase diffraction grating in the SLM 13 in N ways with a predetermined shift cycle via the liquid crystal drive circuit 15A, so that the phase of the interference fringes is N with the shift cycle φ = 2π / N. And N modulated images are acquired by driving the laser unit 100 and the image sensor 35 under these N phases.

Step S9: The control device 39 switches the direction V of the periodic structure of the phase diffraction grating to V ₂ by driving the SLM 13 via the liquid crystal drive circuit 15A, and at half wavelength via the wave plate drive circuit 17A. When the fast axis of the half-wave plate 17 is switched in the direction of the solid double arrow in FIG. 5B by driving the plate 17, step S8 is executed.

Note that the refractive index difference Δn and the structural period P are common between the phase diffraction grating displayed on the SLM 13 in step S9 and the phase diffraction grating displayed on the SLM 13 in step S7.

Step S10: the control device 39, by driving the SLM13 through the liquid crystal drive circuit 15A, it switches the direction V of the periodic structure of the phase grating to _{V 3,} 1/2 wavelength through a wavelength plate drive circuit 17A If the fast axis of the half-wave plate 17 is switched in the direction of the solid arrow in FIG. 5C by driving the plate 17, step S8 is executed.

The refractive index difference Δn and the structural period P are common between the phase diffraction grating displayed on the SLM 13 in step S10 and the phase diffraction grating displayed on the SLM 13 in step S7 (step S10).

As described above, the control device 39 of the present embodiment sets the refractive index difference Δn of the phase diffraction grating displayed on the SLM 13 to be larger as the light source wavelength λ is longer. Specifically, the control device 39 of the present embodiment sets the refractive index difference Δn to a large value Δn ₀₁ when the light source wavelength λ is the long wavelength λ ₁ and sets the refractive index when the light source wavelength λ is the short wavelength λ _2. setting the difference [Delta] n to a small value [Delta] n ₀₂ (see equation (2).).

Therefore, the control device 39 of the present embodiment can maintain the contrast of the demodulated image (super-resolution image) regardless of the switching of the light source wavelength λ.

Note that the image storage and computing unit 40 of this embodiment, by performing the demodulation operation of the 3D-SIM mode the modulated image acquired in step S3 ~ S5 (modulated image obtained at the wavelength lambda ₁₎ the generated super-resolution image of the first fluorescent region, applying demodulation operation 3D-SIM mode with respect to the steps S8 ~ S10 obtained at the modulation image (modulated image obtained at the wavelength lambda ₂₎ Thus, a super-resolution image of the second fluorescent region is generated.

As described above, the control device 39 of the present embodiment maintains the contrast of the demodulated image (super-resolution image) high before and after the switching of the light source wavelength λ, so that the modulated image acquired at the wavelength λ ₁ and the wavelength λ ₂ are used. The image quality of both the acquired modulated image is improved.

Therefore, the image storage arithmetic device 40 according to the present embodiment can acquire both the super-resolution image of the first fluorescent region and the super-resolution image of the second fluorescent region with high accuracy.

As a result, the user of this embodiment can accurately compare and evaluate different fluorescent regions (first fluorescent region and second fluorescent region) on the specimen 5.

[Supplement of embodiment]
In the above-described embodiment, the control device 39 adjusts the refractive index difference Δn of the phase diffraction grating according to the light source wavelength λ. However, it may be performed according to the type of the sample 5 or the type of the sample 5. And the light source wavelength λ.

This is because the intensity ratio of the diffracted light beam group necessary for optimizing the contrast of the demodulated image (super-resolution image) depends on the type of specimen 5 (mainly the thickness of specimen 5). 7: 1: 1).

Therefore, the structured illumination microscope apparatus 1 of the present embodiment may be equipped with the following automatic adjustment mode. The control device 39 in the automatic adjustment mode repeats a series of processes including acquisition of a modulated image and generation of a demodulated image (super-resolution image) while adjusting the refractive index difference Δn, and also includes a demodulated image (super-resolution image). When the contrast becomes optimal, the adjustment of the refractive index difference Δn is finished.

Note that it is desirable that this automatic adjustment mode appears at least when the sample 5 is replaced. Alternatively, this automatic adjustment mode may be developed continuously or periodically in order to cope with changes in the environment of the apparatus.

Further, the following manual adjustment mode may be mounted on the structured illumination microscope apparatus 1 of the present embodiment. The control device 39 and the image display device 45 in the manual adjustment mode adjust the refractive index difference Δn in accordance with an adjustment instruction from the user, and generate / demodulate a modulated image acquisition / demodulated image (super-resolution image). A series of processing consisting of display of a super-resolution image) is repeated. The user inputs an adjustment instruction while viewing the displayed reduced image (super-resolution image), and finishes inputting the adjustment instruction when the contrast of the demodulated image (super-resolution image) becomes optimal. .

When the structured illumination microscope apparatus 1 is equipped with the manual adjustment mode, the structured illumination microscope apparatus 1 needs to be provided with a user interface (not shown) that accepts an adjustment instruction from the user.

Further, the control device 39 of the above-described embodiment makes the structural period P of the phase diffraction grating unchanged before and after the switching of the light source wavelength λ, but adjusts the structural period P of the phase diffraction grating before and after the switching of the light source wavelength λ. Also good. For example, the structural period P may be adjusted so that the super-resolution effect of the structured illumination microscope apparatus 1 remains unchanged before and after the switching of the light source wavelength λ. Specifically, the structural period P may be adjusted so that the distance from the condensing points 14b and 14c to the optical axis AZ is unchanged before and after the switching of the light source wavelength λ.

Further, in the structured illumination microscope apparatus 1 shown in FIG. 1, the attitude of the reflective spatial light modulator (SLM 13) is set such that the normal of the surface (incident surface) of the protective layer 13b is 45 with respect to the principal ray of the incident light beam. Although the angle is set so as to form an angle of 0 °, the normal line and the chief ray may be set so as to form an angle of 0 °, as indicated by reference numeral A in FIG. In that case, for example, the beam splitter 101 may be disposed at an angle of 45 ° between the lens 16 and the SLM 13. This beam splitter 101 reflects the light beam from the laser unit 100 side and makes it incident on the SLM 13 from the front, and transmits the light beam (diffracted light beam) reflected by the SLM 13 and makes it incident on the lens 16 from the front.

In the above-described embodiment, the structured illumination microscope apparatus 1 is used in the 3D-SIM mode (that is, the interference fringes projected on the specimen 5 with the 0th-order diffracted light beam turned on are set as the three-beam interference fringes). The structured illumination microscope apparatus 1 may be used in the 2D-SIM mode (that is, the interference fringes projected on the specimen 5 by turning off the 0th-order light diffraction light beam may be used as the two-beam interference fringes).

Further, the structured illumination microscope apparatus 1 may be used as a TIRFM (total reflection fluorescence microscope) which is a kind of 2D-SIM mode. When the structured illumination microscope apparatus 1 is used as TIRFM, 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 an immersion liquid (oil) (not shown).

Further, when the structured illumination microscope apparatus 1 is used as a TIRFM, the incident angle of the ± first-order diffracted light beam incident on the surface of the sample 5 needs to satisfy the total reflection condition (TIRF condition) that is the condition for generating the evanescent field. is there. In order to satisfy this 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 (TIRF area) 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.

Incidentally, since the 0th-order diffracted light beam is not used in the 2D-SIM mode, the intensity ratio of the diffracted light beam group contributing to the interference fringes does not basically change even when the light source wavelength λ is switched, but the 2D-SIM mode and the 3D- When the mode is switched between the SIM mode and the intensity ratio necessary for optimizing the contrast of the demodulated image (super-resolution image) changes, it is desirable to adjust the refractive index difference Δn. .

For example, the control device 39 in the 3D-SIM mode sets the refractive index difference Δn so that the intensity ratio of the 0th-order diffracted light beam, the + 1st-order diffracted light beam, and the −1st-order diffracted light beam is, for example, 0.7: 1: 1. The control device 39 in the 2D-SIM mode may set the refractive index difference Δn so that the intensity ratio of the 0th-order diffracted light beam, the + 1st-order diffracted light beam, and the −1st-order diffracted light beam is, for example, 0: 1: 1. .

The modulated image generated in the 3D-SIM mode has five components to be separated from each other, whereas the modulated image generated in the 2D-SIM mode has three components to be separated from each other. In the 3D-SIM mode, the number of phases N necessary for the phase shift of the interference fringes is, for example, “5”, whereas in the 2D-SIM mode, it is necessary for the phase shift of the interference fringes. The phase number N is, for example, “3”. In addition, since the number of modulated images is different between the 2D-SIM mode and the 3D-SIM mode, the contents of the demodulation operation to be executed by the image storage / arithmetic apparatus 40 are different.

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

In the embodiment described above, the ½ wavelength plate 17 capable of switching the direction of the fast axis is used in order to keep the diffracted light beam incident on the sample 5 as S-polarized light. Instead, two quarter-wave plates may be used and the direction of the fast axis of one quarter-wave plate may be switched.

In the above-described embodiment, a known calculation is performed using a series of modulated images in order to obtain a super-resolution image (demodulated image). However, the present invention is not limited to this. A demodulated image (super-resolution image) may be acquired by optical demodulation described in 80881378.

In that case, the dichroic mirror 7 is replaced with a mirror, and the fluorescence (wavelengths λ ₁ ′, λ ₂ ′) generated according to the excitation light (wavelengths λ ₁ , λ ₂ ) in the optical path between the SLM 13 and the collector lens 12. ) Is separated from the excitation light, and an image sensor that receives the separated fluorescence may be disposed.

Moreover, although the illumination optical system 10 of the above-described embodiment is configured by the epi-illumination optical system by the objective lens 6, it is not limited to this. For example, the transmission illumination optical system by the condenser lens or the reflection by the condenser lens is used. You may comprise an illumination optical system. In this case, the condensing point is formed on the pupil plane of the condenser lens.

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

In any case, it is desirable that the control device 39 adjusts (controls) the intensity ratio of the diffracted light beam group contributing to the interference fringes so that the contrast of the demodulated image (super-resolution image) is optimized. .

[Effects of Embodiment]
As described above, the structured illumination device (illumination optical system 10) according to the present embodiment is an optical device that forms in the sample (5) the branch portion that branches the light beam emitted from the light source into a plurality of light beams and the interference fringes due to the plurality of light beams. A spatial light including a system ( lenses 16, 25, 27, 6) and a control unit (liquid crystal drive circuit 15A, control device 39) for controlling the branching unit, the branching unit including a member made up of a unit element group. The controller (SLM 13) includes a modulator (SLM 13), and the control unit (the liquid crystal driving circuit 15A and the control device 39) includes a first region for applying a first phase delay amount to the emitted light beam and a second phase delay amount. Is set in the spatial light modulator so that the intensity ratio of the plurality of light beams contributing to the interference fringes becomes a predetermined value. The first phase delay amount and the second position. A drive signal to impart a difference between the delay amount, and outputs the predetermined unit element of the unit element group.

The first phase delay amount and the second phase delay amount are constant values, and the first phase delay amount and the second phase delay amount are different from each other (for example, rectangular). Wave mode).

Alternatively, the first phase delay amount is a value distributed in a predetermined shape and includes a maximum value, and the second phase delay amount is a value distributed in a predetermined shape and has a minimum value. Including (for example, sine wave mode).

In addition, the control unit (the liquid crystal drive circuit 15A, the control device 39) may add the difference between the first region and the second region in order to give a phase delay amount difference between the first region and the second region. A drive signal that gives a difference in refractive index between the second region and the second region is output to the predetermined unit element.

Further, the control unit (the liquid crystal drive circuit 15A, the control device 39) switches the drive signal output to the predetermined unit element according to the wavelength of the emitted light beam.

Further, the control unit (the liquid crystal driving circuit 15A, the control device 39) determines the difference in the phase delay amount between the first region and the second region when the wavelength (λ) of the emitted light beam is long. Make it bigger.

Further, the control unit (the liquid crystal drive circuit 15A, the control device 39) is necessary for determining an appropriate drive signal to be output to the predetermined unit element according to the required intensity ratio and the wavelength. At least a part of the information is stored in advance as a table.

Further, the structured illumination device (illumination optical system 10) of the present embodiment has a phase shift unit (shifting the phase of the interference fringes by shifting the formation destination of the periodic region in the spatial light modulator (SLM 13) ( A liquid crystal driving circuit 15A and a control device 39) are further provided.

In addition, the structured illumination device (illumination optical system 10) of the present embodiment includes a direction switching unit (liquid crystal driving circuit 15A, 15A A control device 39) is further provided.

The spatial light modulator (SLM 13) is a spatial light modulator that changes the phase distribution of the emitted light beam while maintaining the polarization direction distribution and amplitude distribution of the emitted light beam.

Further, the structured illumination microscope apparatus (1) of the present embodiment is a modulation that is an image of the structured illumination apparatus (illumination optical system 10) of the present embodiment and the specimen (5) spatially modulated by the interference fringes. An imaging unit (image sensor 35, control device 39) for acquiring an image.

The structured illumination microscope apparatus (1) of the present embodiment further includes a calculation unit (image storage / calculation apparatus 40) that generates a demodulated image of the specimen (5) based on the modulated image.

Further, the control unit (the liquid crystal drive circuit 15A, the control device 39) performs the setting so that the contrast of the demodulated image is optimized.

Therefore, the performance of the structured illumination microscope apparatus (1) of the present embodiment is maintained regardless of the use state of the structured illumination microscope apparatus (1), for example, the wavelength of the emitted light beam, the thickness of the sample, and the like. The

[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, 35 ... Imaging element, 39 ... Control apparatus, 40 ... Image storage and calculation apparatus, 45 DESCRIPTION OF SYMBOLS ... Image display apparatus, 12 ... Collector lens, 23 ... Polarizing plate, 15 ... Light beam branching part, 16 ... Condensing lens, 24 ... Light beam selection part, 25 ... Lens, 26 ... Field stop, 27 ... Field lens, 28 ... Excitation Filter, 7 ... Dichroic mirror, 6 ... Objective lens, 5 ... Sample, 17 ... Half-wave plate, 18 ... High-order light cut member, 17A ... Wave plate drive circuit, 200 ... Zero-order light shutter, 200A ... Turning mechanism, 15A ... Liquid crystal drive circuit, 13 ... SLM

Claims (15)

1. A spatial light modulator that branches light from a light source into a plurality of light fluxes;
   An optical system that forms an interference fringe with all or a part of the plurality of light beams, and illuminates the specimen with the interference fringe;
   A controller for controlling the spatial light modulator,
   The controller is
   By outputting a drive signal to the spatial light modulator, the first region of the spatial light modulator is set so that the intensity ratio of a plurality of light beams contributing to the interference fringes becomes a predetermined value. A structured lighting apparatus, wherein a first phase delay amount is set for the second region, and a second phase delay amount is set for the second region.
2. The structured lighting device according to claim 1,
   The spatial light modulator is
   A first substrate provided with a plurality of pixel electrodes, a second substrate facing the first substrate, and a liquid crystal member sandwiched between the first substrate and the second substrate,
   The controller is
   By outputting a drive signal to each of the pixel electrodes, the first region of the spatial light modulator is changed so that the intensity ratio of a plurality of light beams contributing to the interference fringes becomes a predetermined value. 1. A structured lighting apparatus, wherein a phase delay amount of 1 is set and a second phase delay amount is set for a second region.
3. The structured lighting device according to claim 1 or 2,
   The first phase delay amount and the second phase delay amount are constant values, and the first phase delay amount and the second phase delay amount are different from each other. Structured lighting device.
4. The structured lighting device according to claim 1 or 2,
   The first phase delay amount has a maximum value in the first region,
   The structured lighting device, wherein the second phase delay amount has a minimum value in the second region.
5. The structured lighting device according to any one of claims 1 to 4,
   The controller is
   A drive signal for providing a difference in refractive index between the first region and the second region in order to provide a difference in phase delay amount between the first region and the second region. Is output to the spatial light modulator.
6. The structured lighting device according to any one of claims 1 to 5,
   The controller is
   The structured illumination device, wherein the drive signal output to the spatial light modulator is switched according to the wavelength of the emitted light beam.
7. The structured lighting device according to claim 6.
   The controller is
   The structured illumination device characterized in that the difference in the phase delay amount between the first region and the second region is increased as the wavelength of the emitted light beam is longer.
8. The structured lighting device according to claim 7.
   The controller is
   At least a part of information necessary for determining an appropriate drive signal to be output to the spatial light modulator according to the required intensity ratio and the wavelength is stored in advance as a table. A structured lighting device.
9. The structured lighting device according to any one of claims 1 to 8,
   The structured illuminating device, further comprising: a phase shift unit that shifts a phase of the interference fringes by changing a distribution of the first region and the second region in the spatial light modulator.
10. The structured lighting device according to any one of claims 1 to 9,
    The structured illumination device further comprising: a direction switching unit that switches a direction of the interference fringes by changing a distribution of the first region and the second region in the spatial light modulator.
11. The structured lighting device according to any one of claims 1 to 10,
    The spatial light modulator is
    The structured illumination device, characterized in that the spatial light modulator changes the phase distribution of the emitted light beam while maintaining the polarization direction distribution and amplitude distribution of the emitted light beam.
12. The structured lighting device according to any one of claims 1 to 11,
    The plurality of light beams that contribute to the interference fringes are a zero-order diffracted light beam and a ± first-order diffracted light beam,
    The control unit makes the intensity of the 0th-order diffracted light beam lower than the intensity of the ± 1st-order diffracted light beam.
13. A structured lighting device according to any one of claims 1 to 12,
    An imaging unit that obtains a modulated image that is an image of the sample spatially modulated by the interference fringes;
    A structured illumination microscope apparatus comprising:
14. The structured illumination microscope apparatus according to claim 13,
    A structured illumination microscope apparatus, further comprising an arithmetic unit that generates a demodulated image of the sample based on the modulated image.
15. The structured illumination microscope apparatus according to claim 14,
    The controller is
    The structured illumination microscope apparatus, wherein the setting is performed so that the contrast of the demodulated image is maximized.
    

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