JP6127451B2 - Structured illumination device and structured illumination microscope device - Google Patents

Description

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

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

  In particular, in the structured illumination microscope disclosed in Patent Document 1 below, a light beam emitted from a light source is split into a plurality of light beams by a diffraction grating, and these light beams are interfered in the vicinity of a sample to form interference fringes. Is structured illumination light.

US-issued patent invention No. 6239909

  By the way, in the structured illumination microscope, as in other microscopes, there is a demand for switching the wavelength of the light source in order to observe a specimen stained with a plurality of dyes having different excitation wavelengths.

  However, when the light source wavelength is switched in the structured illumination microscope, the diffraction angle in the diffraction grating changes, so the spot height of the illumination light with respect to the pupil radius of the objective lens changes, and the desired super-resolution effect cannot be obtained. There are challenges.

  The present invention has been made in view of such circumstances, and an object thereof is to provide a structured illumination apparatus and a structured illumination microscope apparatus capable of obtaining a desired super-resolution effect at each of a plurality of wavelengths. To do.

According to one aspect of the structured illumination device of the present invention, a lens array including a pair of lenses that divides and collects a light beam emitted from a light source that emits light of a plurality of wavelengths simultaneously or sequentially, and the pair of lenses An objective lens that receives the pair of luminous fluxes divided in (1) at different positions on the pupil plane so that the pair of luminous fluxes interfere with each other on the object side, and forms interference fringes on the specimen arranged on the object side; Is provided. Each of the pair of lenses is disposed in a symmetric positional relationship with respect to the optical axis of the objective lens. The objective lens can be replaced with another objective lens having a different pupil diameter. In the lens array, the distance d from the optical axis of each of the pair of lenses to the optical axis of the objective lens is variable.
Another aspect of the structured illumination device of the present invention includes a lens array including a pair of lenses that divides and collects light beams emitted from a light source that emits light of a plurality of wavelengths simultaneously or sequentially, and the pair of lenses. An objective lens that receives a pair of light beams divided by the lens at different positions on the pupil plane, causes the pair of light beams to interfere with each other on the object side, and forms interference fringes on a specimen arranged on the object side With. Each of the pair of lenses is disposed in a symmetric positional relationship with respect to the optical axis of the objective lens. The objective lens can be replaced with another objective lens having a different pupil diameter. The lens array can be exchanged for another lens array having a different distance d. However, the distance d of the lens array is a distance from the optical axis of each of the pair of lenses included in the lens array to the optical axis of the objective lens.

One aspect of the structured illumination microscope apparatus of the present invention, the embodiment of one aspect or another of the structured illumination apparatus of the present invention, the observation light flux from the target book modulated by interference Watarushima imaged on the photodetector An imaging optical system.

  According to the present invention, a structured illumination apparatus and a structured illumination microscope apparatus capable of obtaining a desired super-resolution effect at each of a plurality of wavelengths are realized.

It is a block diagram of the structured illumination microscope apparatus 1 of 1st Embodiment. It is a figure explaining the condensing lens array. FIG. 6 is a diagram for explaining the function of a half-wave plate 19. It is a figure explaining the function of the light beam selection member. It is a figure explaining the function of the half-wave plate 19 and the light beam selection member 20. FIG. It is a figure explaining the rotation mechanism 18A provided in the light beam selection member 20. FIG. It is a figure explaining the phase plate. It is an example of the rotation pattern of the phase plate. FIG. 4 is a diagram showing a movable part of the condenser lens array 14. It is a figure which shows the space | interval adjustment mechanism 14A provided in the condensing lens array 14. FIG. It is an optical path figure of a condensing lens. It is lens data (1) of a condensing lens. It is lens data (2) of a condensing lens. It is a figure which shows the axial chromatic aberration of a condensing lens. It is a figure explaining the condensing lens array 14 '. It is a figure which shows the space | interval adjustment mechanism provided in the condensing lens array 14 '. It is a figure explaining condensing lens array 14 ''. It is a figure explaining the light beam selection member 20 '. It is a figure explaining the demodulation calculation in case of the number of phases _Nmax = 3 and the number of directions _Mmax = 3.

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

  FIG. 1 is a configuration diagram of the structured illumination microscope apparatus 1. Here, when the structured illumination microscope apparatus 1 is used as a total internal reflection fluorescence microscope (TIRFM) for observing a very thin layer on the surface of a fluorescent sample (specimen) 2, that is, A case where the structured illumination microscope apparatus 1 is used in the “TIRF-SIM mode” will be described.

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

  As shown in FIG. 1, the structured illumination microscope apparatus 1 includes a laser unit 100, an optical fiber 11, an illumination optical system 10, an imaging optical system 30, an imaging element 42, a control device 43, an image storage / An arithmetic device 44 and an image display device 45 are provided. The illumination optical system 10 is an epi-illumination type, and the sample 2 is illuminated using the objective lens 31 and the dichroic mirror 33 of the imaging optical system 30.

  The laser unit 100 includes a first laser light source 101, a second laser light source 102, shutters 103 and 104, a mirror 105, a dichroic mirror 106, and a lens 107. Each of the first laser light source 101 and the second laser light source 102 is a coherent light source, and the emission wavelengths thereof are different from each other. Here, it is assumed that the wavelength λ1 of the first laser light source 101 is longer than the wavelength λ2 of the second laser light source 102 (λ1> λ2). The first laser light source 101, the second laser light source 102, and the shutters 103 and 104 are driven by the control device 43, respectively.

  The optical fiber 11 is configured by, for example, a polarization-preserving single mode fiber in order to guide the laser light emitted from the laser unit 100. The position of the output end of the optical fiber 11 in the optical axis direction can be adjusted by the position adjusting mechanism 11A. This position adjustment mechanism 11 </ b> A is driven by the control device 43.

  The illumination optical system 10 includes a collector lens 12, a polarizing plate 13, a condenser lens array 14, a phase plate 16, a half-wave plate 19, and a light beam selection member in order from the emission end side of the optical fiber 11. 20, a lens 21, a field stop 22, a field lens 23, an excitation filter 24, a dichroic mirror 33, and an objective lens 31 are arranged.

  The condenser lens array 14 is provided with an interval adjusting mechanism 14A, the phase plate 16 is provided with a rotating mechanism 16A, and the light beam selecting member 20 and the half-wave plate 19 are provided with a rotating mechanism 18A. Each of the interval adjustment mechanism 14A, the rotation mechanism 16A, and the rotation mechanism 18A is driven by the control device 43.

  In the imaging optical system 30, an objective lens 31, a dichroic mirror 33, an absorption filter 34, and a second objective lens 35 are arranged in order from the sample 2 side.

  The objective lens 31 can be exchanged for another objective lens 31 'having a different magnification by the exchange mechanism 31A. The exchange mechanism 31A is driven by the control device 43.

  The specimen 2 is, for example, a culture solution dropped on a parallel plate-like glass surface, and fluorescent cells exist in the vicinity of the glass interface in the culture solution. In this cell, both the first fluorescent region excited by light of wavelength λ1 and the second fluorescent region excited by light of wavelength λ2 are expressed.

  The objective lens 31 is configured as an immersion type (oil immersion type) objective lens in order to enable total reflection fluorescence observation. That is, the gap between the objective lens 31 and the glass of the sample 2 is filled with the immersion liquid (oil).

  The image sensor 42 is a two-dimensional image sensor composed of a CCD, a CMOS, or the like. When the image sensor 42 is driven by the control device 43, the image sensor 42 captures an image formed on the imaging surface 41 and generates an image. This image is taken into the image storage / arithmetic unit 44 via the control unit 43.

  The control device 43 drives and controls the laser unit 100, the position adjustment mechanism 11A, the interval adjustment mechanism 14A, the rotation mechanism 16A, the rotation mechanism 18A, the exchange mechanism 31A, and the image sensor 42.

  The image storage / arithmetic unit 44 performs a calculation on the image given via the control unit 43, stores the calculated image in an internal memory (not shown), and sends it to the image display unit 45.

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

  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 103, it reflects from the mirror 105 and enters the dichroic mirror 106. On the other hand, the laser light having the wavelength λ2 (second laser light) emitted from the second laser light source 102 enters the beam splitter 106 via the shutter 104 and is integrated with the first laser light. The first laser beam and the second laser beam emitted from the dichroic mirror 106 enter the incident end of the optical fiber 11 through the lens 107. When the control device 43 controls the laser unit 100, the wavelength of the laser light (= use wavelength λ) incident on the incident end of the optical fiber 11 is switched between the long wavelength λ1 and the short wavelength λ2.

  The laser light incident on the incident end of the optical fiber 11 propagates inside the optical fiber 11 and generates a point light source at the output end of the optical fiber 11. The laser light emitted from the point light source is converted into a parallel light beam by the collector lens 12 and enters the condenser lens array 14 via the polarizing plate 13, and by a plurality of pairs of condenser lenses included in the condenser lens array 14, Divided into multiple pairs of light fluxes. These plural pairs of light beams are condensed at different positions on the pupil conjugate plane 25 by the condensing lenses.

  Here, the pupil conjugate plane 25 is a focal position (rear focal position) of each condenser lens of the condenser lens array 14, and the lens 23 and the lens 21 are interposed with respect to the pupil plane 32 of the objective lens 31. (The concept of “conjugate position” is a position determined in consideration of design-related matters such as aberration and vignetting of the condenser lens array 14 and the lenses 21 and 23. Is also included.)

  Since the laser light emitted from the optical fiber 11 is basically linearly polarized, the polarizing plate 13 can be omitted, but it is effective for reliably cutting off the extra polarized component. Further, in order to increase the utilization efficiency of the laser light, it is desirable that the axis of the polarizing plate 13 coincides with the polarization direction of the laser light emitted from the optical fiber 11.

  A plurality of pairs of light beams directed toward the pupil conjugate surface 25 are incident on the phase plate 16, the half-wave plate 19, and the light beam selection member 20, which are disposed in the vicinity of the pupil conjugate surface 25.

  Here, since the structured illumination microscope apparatus 1 of this embodiment is used as a total reflection fluorescence microscope, the light beam selection member 20 selectively passes only one pair of light beams out of a plurality of incident light beams. Let

  Each of the pair of light beams that have passed through the light beam selection member 20 becomes a parallel light beam by the lens 21, passes through the field stop 22, becomes a condensed light beam by the field lens 23, passes through the excitation filter 24, and then is reflected by the dichroic mirror 33. Then, the light is condensed at different positions on the pupil plane 32 of the objective lens 31.

  Each of the pair of light beams collected on the pupil surface 32 becomes a parallel light beam when emitted from the tip of the objective lens 31 and interferes with each other on the surface of the sample 2 to form interference fringes. This interference fringe is used as structured illumination light.

  In addition, since the structured illumination microscope apparatus 1 of the present embodiment is used as a total reflection fluorescence microscope, the incident angle when entering the surface of the specimen 2 satisfies the conditions for generating an evanescent field. This condition is called a total reflection condition (TIRF condition) or the like. In order to satisfy this TIRF condition, the condensing point of a pair of light fluxes on the pupil plane 32 may be stored in a predetermined annular zone (TIRF area) located on the outermost periphery of the pupil plane 32 (details will be described later). ). As a result, an evanescent field caused by structured illumination light is generated near the surface of the specimen 2.

  When the specimen 2 is illuminated with such structured illumination light, moire fringes corresponding to the difference between the periodic structure of the structured illumination light and the periodic structure of the specimen 2 (of the fluorescent region) appear. In this moire fringe, Since the high frequency structure of the sample 2 is shifted to a lower frequency side than the original frequency, the light (fluorescence) indicating this structure is directed to the objective lens 31 at an angle smaller than the original angle. Therefore, when the specimen 2 is illuminated with structured illumination light, even the high-frequency structural information (of the fluorescent region) of the specimen 2 is transmitted by the objective lens 31.

  Fluorescence generated in the vicinity of the surface of the sample 2 (evanescent field) enters the objective lens 31 and is converted into parallel light by the objective lens 31, and then passes through the dichroic mirror 33 and the barrier filter 34. Then, a modulated image of the sample 2 is formed on the imaging surface 41 of the imaging element 42.

  This modulated image is imaged by the image sensor 42 and taken into the image storage / arithmetic device 44 via the control device 43. Further, the captured modulation image (modulation image) is subjected to a known demodulation calculation (details will be described later) in the image storage / arithmetic unit 44 to generate a demodulation image (super-resolution image). The super-resolution image is stored in an internal memory (not shown) of the image storage / arithmetic unit 44 and is sent to the image display unit 45.

  Next, the condenser lens array 14 will be described in detail.

  FIG. 2 is a diagram illustrating the condenser lens array 14. 2A is a view of the condensing lens array 14 as viewed from the optical axis direction, and FIG. 2B is a diagram illustrating that three pairs of light beams divided by the condensing lens array 14 are formed on the pupil conjugate plane. It is a figure which shows the positional relationship of 3 pairs of condensing points.

  As shown in FIG. 2A, the condenser lens array 14 is a multi-collector in which a pair of condenser lenses are arranged in each of the first direction V1, the second direction V2, and the third direction V3 perpendicular to the optical axis. It is a light lens. The first direction V1, the second direction V3, and the third direction V3 are three directions shifted by 120 °.

  In FIG. 2, reference numerals 14-1 and 14-1 ′ are assigned to a pair of condenser lenses arranged in the first direction V <b> 1, and reference numeral 14 − is assigned to the pair of condenser lenses arranged in the second direction V <b> 2. 2 and 14-2 ′, and a pair of condensing lenses arranged in the third direction V3 are denoted by reference numerals 14-3 and 14-3 ′.

  A configuration related to the first direction V1 (a pair of condensing lenses 14-1 and 14-1 ′), a configuration related to the second direction V2 (a pair of condensing lenses 14-2 and 14-2 ′), and a third Configurations in the direction V3 (a pair of condensing lenses 14-3 and 14-3 ′) are equal to each other. Here, as a representative, only the configuration related to the first direction V1 (a pair of condensing lenses 14-1 and 14-1 ') will be described.

  The positional relationship between the pair of condenser lenses 14-1 and 14-1 ′ is set to a symmetrical positional relationship with respect to the optical axis of the illumination optical system 10. Among these, the lens characteristics (lens configuration, focal length, etc.) of one condenser lens 14-1 and the lens characteristics (lens configuration, focal length, etc.) of the other condenser lens 14-1 ′ are mutually different. equal.

  The parallel light flux incident on the condenser lens array 14 is split in the first direction V1, the second light flux pair divided in the second direction V2, and the third direction V3. It is converted into a third light flux pair.

  The first light beam pair, the second light beam pair, and the third light beam pair are respectively collected by the condensing lenses 14-1, 14-1 ′, 14-2, 14-2 ′, 14-3, and 14-3 ′. Light is condensed at different positions in the pupil conjugate plane.

  As shown in FIG. 2B, the condensing points 25d and 25g of the first light beam pair are symmetric with respect to the optical axis of the illumination optical system 10, and the arrangement direction of the condensing points 25d and 25g is the first direction. It corresponds to V1.

  The condensing points 25c and 25f of the second light beam pair are symmetric with respect to the optical axis of the illumination optical system 10, and the arrangement direction of the condensing points 25c and 25f corresponds to the second direction V2. In addition, the distance from the condensing points 25c and 25f of the second light beam pair to the optical axis of the illumination optical system 10 is the same as the distance from the condensing points 25d and 25g of the first light beam pair to the optical axis of the illumination optical system 10. It is.

  The condensing points 25b and 25e of the third light flux pair are symmetric with respect to the optical axis, and the arrangement direction of the condensing points 25b and 25e corresponds to the third direction V3. The distance from the condensing points 25b and 25e of the third light beam pair to the optical axis of the illumination optical system 10 is the same as the distance from the condensing points 25d and 25g of the first light beam pair to the optical axis of the illumination optical system 10. It is.

  Thus, in this embodiment, the condensing lens array 14 is used instead of the diffraction grating as means for generating a pair of light beams. Unlike a diffraction grating using diffraction, a lens using refraction is basically less dependent on the wavelength of the traveling direction of light. Therefore, in the present embodiment, the positional relationship between the condensing points formed on the pupil conjugate plane can be maintained regardless of the switching of the used wavelength λ.

  Next, the half-wave plate 19 and the light beam selection member 20 will be described in detail.

  FIG. 3 is a diagram illustrating the half-wave plate 19, and FIG. 4 is a diagram illustrating the light beam selection member 20. As shown in FIG. 3, the half-wave plate 19 sets the polarization directions of the incident first light beam pair, second light beam pair, and third light beam pair, and as shown in FIG. This is a mask that selectively allows only one of a pair of one beam, a second beam, and a third beam to pass through.

  The half-wave plate 19 and the light beam selection member 20 can be rotated around the optical axis of the illumination optical system 10 by a rotation mechanism 18A. When the light beam selection member 20 is rotated around the optical axis of the illumination optical system 10, the selected light beam pair can be switched among the first light beam pair, the second light beam pair, and the third light beam pair. When the half-wave plate 19 is rotated around the optical axis of the illumination optical system 10 in conjunction with the member 20, the polarization direction when the selected light beam pair is incident on the sample 2 can be maintained as S-polarized light. it can. That is, the half-wave plate 19 and the light beam selection member 20 can switch the direction of the interference fringes while maintaining the interference fringe state. Hereinafter, the conditions for maintaining the stripe state will be specifically described.

  First, the direction of the fast axis of the half-wave plate 19 is such that the polarization direction of the light beam pair is perpendicular to the split direction of the selected light beam pair (any one of the first direction V1 to the third direction V3). Need to be set to Here, the fast axis of the half-wave plate 19 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 19 is minimized. It is.

  The opening pattern of the light beam selection member 20 includes a first opening portion 20A and a second opening portion 20B through which one and the other of the light beams that make a pair are individually passed, and around the optical axis of the illumination optical system 10 The length of the first opening 20A and the length of the second opening 20B are set such that the light beam linearly polarized in the above-described direction can pass through. Therefore, the shape of each of the first opening 20A and the second opening 20B is a shape close to a partial ring shape.

  Here, as shown in FIG. 3A, the rotational position of the half-wave plate 19 when the direction of the fast axis of the half-wave plate 19 is parallel to the direction of the axis of the polarizing plate 13, The rotation position of the half-wave plate 19 is used as a reference (hereinafter referred to as “first reference position”).

  Further, the rotational position of the light beam selection member 20 when the light beam selection direction of the light beam selection member 20 (= the dividing direction of the selected light beam pair) is perpendicular to the axis direction of the polarizing plate 13 is The rotation position is used as a reference (hereinafter referred to as “second reference position”).

  At this time, as shown in FIG. 3B, the amount of rotation of the half-wave plate 19 from the first reference position is controlled to one half of the amount of rotation of the light beam selection member 20 from the second reference position. It should be.

  That is, when the rotation amount of the half-wave plate 19 from the first reference position is θ / 2, the rotation amount of the light beam selection member 20 from the second reference position is set to θ.

  Therefore, in order to select the first light beam pair (the dividing direction is the first direction V1), the light beam selecting direction of the light beam selecting member 20 is rotated to the right from the second reference position as shown in FIG. When rotated by the angle θ1, the direction of the fast axis of the half-wave plate 19 is rotated rightward from the first reference position by the rotation angle θ1 / 2.

  At this time, the polarization direction of the first light beam pair before passing through the half-wave plate 19 is parallel to the direction of the axis of the polarizing plate 13 as shown by the broken line double arrow in FIG. On the other hand, since the polarization direction of the first light beam pair after passing through the half-wave plate 19 is rotated to the right by the rotation angle θ1, the polarization direction of the selected first light beam pair is shown in FIG. As indicated by a solid double-pointed arrow in A), the first light beam pair is perpendicular to the dividing direction (first direction V1).

  In other words, the direction of the fast axis of the half-wave plate 19 is a direction according to the split direction (= first direction V1) of the first light beam pair selected by the light beam selection member 20, and is 1/2. The polarization direction (the axial direction of the polarizing plate 13) that the first beam pair incident on the wave plate 19 had and the polarization direction (the first direction V1) that the first beam pair emitted from the half-wave plate 19 should have. In the direction of the bisector of the angle formed by

  Further, in order to select the second light beam pair (the dividing direction is the second direction V2), as shown in FIG. 4B, the light beam selecting direction of the light beam selecting member 20 is rotated to the right from the second reference position. When rotated by the angle θ2, the direction of the fast axis of the half-wave plate 19 is rotated rightward from the first reference position by the rotation angle θ2 / 2.

  At this time, the polarization direction of the second light flux pair before passing through the half-wave plate 19 is parallel to the direction of the axis of the polarizing plate 13 as indicated by a broken line in FIG. 4B. On the other hand, since the polarization direction of the second light beam pair after passing through the half-wave plate 19 rotates to the right by the rotation angle θ2, the polarization direction of the selected second light beam pair is shown in FIG. As indicated by a solid double-pointed arrow in (B), the second light beam pair is perpendicular to the split direction (second direction V2).

  In other words, the direction of the fast axis of the half-wave plate 19 is a direction corresponding to the split direction (= second direction V2) of the second light beam pair selected by the light beam selection member 20, and is 1/2. The polarization direction (the axial direction of the polarizing plate 13) that the second light beam pair incident on the wave plate 19 had and the polarization direction (second direction V2) that the second light beam pair emitted from the half-wave plate 19 should have. In the direction of the bisector of the angle formed by

  Further, in order to select the third light beam pair (the dividing direction is the third direction V3), as shown in FIG. 4C, the light beam selecting direction of the light beam selecting member 20 is set to the left (sample) from the second reference position. When viewed from the side, the same applies hereinafter), when the rotation angle θ3 is rotated, the direction of the fast axis of the half-wave plate 19 is rotated leftward from the first reference position by the rotation angle θ3 / 2.

  At this time, the polarization direction of the third light flux pair before passing through the half-wave plate 19 is parallel to the direction of the axis of the polarizing plate 13 as indicated by a broken line in FIG. 4C. On the other hand, since the polarization direction of the third light beam pair after passing through the half-wave plate 19 rotates to the left by the rotation angle θ3, the polarization direction of the selected light beam pair is shown in FIG. ) As indicated by the actual double-headed arrow, the direction is perpendicular to the direction of splitting the third light flux pair (third direction V3).

  In other words, the direction of the fast axis of the half-wave plate 19 is a direction according to the division direction (= third direction V3) of the third light flux pair selected by the light flux selection member 20, and is 1/2. The polarization direction (the axial direction of the polarizing plate 13) that the third light beam pair incident on the wave plate 19 had and the polarization direction (the third direction V3) that the third light beam pair emitted from the half-wave plate 19 should have. In the direction of the bisector of the angle formed by

  Therefore, the rotation mechanism 18A provided in the half-wave plate 19 and the light beam selection member 20 interlocks the half-wave plate 19 and the light beam selection member 20 with a gear ratio of 2: 1, thereby causing interference fringes. The direction of interference fringes is switched while maintaining

  FIG. 5 is a diagram illustrating the functions of the half-wave plate 19 and the light beam selection member 20 described above. In FIG. 5, a double arrow surrounded by a circular frame indicates the polarization direction of the light beam, and a double arrow surrounded by a square frame indicates the axial direction of the optical element.

  In the above description, the rotatable half-wave plate 19 is used to keep the pair of light beams incident on the specimen 2 as S-polarized light. However, the rotatable half-wave plate 19 is fixed instead of the rotatable half-wave plate 19. An arranged liquid crystal element may be used, and the liquid crystal element may function as the half-wave plate 19. If the orientation of the liquid crystal element is electrically controlled, the refractive index anisotropy of the liquid crystal element can be controlled. Therefore, the fast axis as a half-wave plate is rotated around the optical axis of the illumination optical system 10. be able to. Incidentally, there are other methods for keeping the pair of light beams incident on the specimen 2 to be S-polarized light.

  As shown in FIG. 6, a plurality of (six in the example shown in FIG. 6) notches 20C are formed on the outer peripheral portion of the light flux selecting member 20, and the rotation mechanism 18A includes these notches 20C. A timing sensor 20D for detecting the notch 20C is provided. As a result, the rotation mechanism 18A can detect the rotation position of the light beam selection member 20 and thus the rotation position of the half-wave plate 19.

  Next, the phase plate 16 will be described in detail.

  FIG. 7 is a diagram illustrating the phase plate 16.

  First, in order to enable the demodulation operation described above, three or more modulated images with the same direction of interference fringes and different phases are necessary for the same sample 2. This is because the modulated image generated by the structured illumination microscope apparatus 1 includes, in the structure of the sample 2, the 0th order modulation component, the + 1st order modulation component, which is the structure information whose spatial frequency is modulated by the structured illumination light, and −1. This is because the next modulation component is included, and in order to make these three unknown parameters known by demodulation calculation (details will be described later), the number of modulated images equal to or greater than the number of these unknown parameters is required.

  Therefore, as the phase plate 16 of the present embodiment, for example, a phase plate disclosed in US Pat. No. 7848017 is used. Here, an example of the phase plate 16 will be described.

  As shown in FIG. 7, the phase plate 16 is provided with two fan-shaped regions having different phase delay amounts around the optical axis (z-axis) of the illumination optical system 10, and the phase delay amount of one fan-shaped region 16a is set. If it is set to 0, the phase delay amount of the other fan-shaped region 16b is set to 2π / 3. The central angle of one fan-shaped region 16a is set to 120 °, and the central angle of the other fan-shaped region 16b is set to 240 °.

In order to provide a difference between the phase delay amount of the fan-shaped region 16a and the phase delay amount of the fan-shaped region 16b, for example, a SiO ₂ film having a predetermined thickness is deposited on only one of the fan-shaped regions 16a and 16b. do it. Alternatively, fan-shaped region 16a, a different SiO ₂ film thicknesses may be deposited against 16b.

  Further, the phase plate 16 can be rotated around the optical axis of the illumination optical system 10. If the rotation position is appropriately set, an appropriate phase difference Δφ can be given between the pair of light beams selected by the light beam selection member 20. Further, if the rotation position of the phase plate 16 is switched with an appropriate pattern, the phase difference Δφ of the pair of light beams can be switched with an appropriate pattern.

  FIG. 8 is a diagram showing a rotation pattern of the phase plate 16 necessary for switching the phase difference Δφ in three ways with the shift pitch “2π / 3”. The upper part of FIG. 8 shows a rotation pattern when the split direction of the selected pair of light beams is the first direction V1, and the middle part of FIG. 8 shows the second split direction of the selected pair of light beams. The rotation pattern in the case of the direction V2 is shown in the lower part of FIG. 8. The lower part of FIG. 8 is the rotation pattern in the case where the split direction of the selected pair of light beams is the third direction V3.

  Therefore, the rotation mechanism 16A provided on the phase plate 16 shifts the phase difference Δφ between the selected pair of light beams by 2π / 3 by switching the rotation position of the phase plate 16 by 120 °. Can do. Along with this, the phase of the interference fringes also shifts.

  Next, a configuration for satisfying the total reflection condition (TIRF condition) will be described in detail.

  In this embodiment, in order to satisfy the TIRF condition, the distance d from the optical axis of each condenser lens of the condenser lens array 14 to the optical axis of the illumination optical system 10 (= the optical axis of the condenser lens array 14) is The following conditional expression (1) is satisfied.

(Nw · fo) / β ≦ d ≦ (NA · fo) / β (1)
Here, nw is the refractive index of the specimen 2, fo is the focal length of the objective lens 31, β is the magnification from the condensing position of the condenser lens to the pupil plane 32, and NA is the numerical aperture of the objective lens 31. Incidentally, in this embodiment, since the cell is the specimen 2, nw is substantially equal to the refractive index of water.

  Hereinafter, the significance of conditional expression (1) will be described.

  First, the height Y from the condensing point formed by the pair of light beams on the pupil plane 32 to the optical axis of the illumination optical system 10 is expressed as follows.

Y = d × β (2)
The pupil radius r of the objective lens 31 is expressed by the following formula.

r = fo × NA (3)
Therefore, if the following expression (4) is satisfied, the condensing point of a pair of light beams is within the TIRF region.

nw × fo ≦ Y ≦ r (4)
By substituting equations (2) and (3) into equation (4) and solving for d, conditional equation (1) is obtained. Therefore, it is clear that the conditional expression (1) is an expression for satisfying the TIRF condition.

  Next, the interval adjustment of the condenser lens array 14 will be described in detail.

  FIG. 9 is a diagram illustrating a movable portion of the condenser lens array 14, and FIG. 10 is a diagram illustrating a distance adjusting mechanism 14 </ b> A provided in the condenser lens array 14.

  First, as described above, in the present embodiment, it is assumed that the objective lens 31 is replaced with another objective lens 31 'having a different magnification. When the objective lens 31 is replaced with another objective lens 31 'having a different magnification, the pupil radius r (formula (3)) is likely to change (in most cases, the pupil radius r changes). Therefore, if the distance d from the optical axis of each condenser lens of the condenser lens array 14 to the optical axis of the illumination optical system 10 is fixed, the TIRF condition (formula (1)) is obtained by exchanging the objective lens. May collapse.

  Therefore, in the condensing lens array 14 of the present embodiment, as shown by the dotted lines in FIG. 9, the condensing lenses 14-1, 14-1 ′, 14-2, 14-2 ′, 14-3, 14-3 ′. The distance d from each optical axis to the optical axis of the illumination optical system 10 is variable.

  For this purpose, the interval adjusting mechanism 14A provided in the condenser lens array 14 includes a cam member 14E, a guide member 14D, and an elastic member 14F, as shown in FIG. The cam member 14E can rotate around the optical axis of the illumination optical system 10, and by the rotation, the condensing lenses 14-1, 14-1 ′, 14-2, 14-2 ′, 14-3. , 14-3 ′ and the distance d from the optical axis of the illumination optical system 10 are simultaneously changed. Further, the guide member 14D restricts the moving direction of the condensing lenses 14-1 and 14-1 ′ to the first direction V1, and the moving direction of the condensing lenses 14-2 and 14-2 ′ to the second direction V2. The movement direction of the condensing lenses 14-3 and 14-3 ′ is limited to the third direction V3. The elastic member 14F presses each of the condenser lenses 14-1, 14-1 ', 14-2, 14-2', 14-3, and 14-3 'against the cam surface of the cam member 14E.

  Therefore, the distance adjusting mechanism 14A is configured so that the optical axis of the illumination optical system 10 is from the optical axis of each of the condenser lenses 14-1, 14-1 ′, 14-2, 14-2 ′, 14-3, and 14-3 ′. , The symmetry of the condenser lenses 14-1 and 14-1 ′ with respect to the optical axis of the illumination optical system 10, and the condenser lens 14-2 with respect to the optical axis of the illumination optical system 10. The symmetry of 14-2 ′ and the symmetry of the condenser lenses 14-3 and 14-3 ′ with respect to the optical axis of the illumination optical system 10 can be maintained.

  10A shows a state when the distance d is set to be relatively narrow, and FIG. 10B shows a state when the distance d is set to be relatively wide. Comparing FIG. 10A and FIG. 10B, it can be seen that the distance d changes according to the rotation angle of the cam member 14E.

  Then, the control device 43 (see FIG. 1) of the present embodiment interlocks the interval adjusting mechanism 14A with the replacement mechanism 31A (see FIG. 1) so that the expression (1) is satisfied regardless of the replacement of the objective lens. Control the distance d. As a result, the TIRF condition can be maintained regardless of the replacement of the objective lens.

  However, when the pupil plane 32 is displaced in the optical axis direction by exchanging the objective lens, the control device 43 causes the position adjusting mechanism 11A to interlock with the exchanging mechanism 31A, so that the optical axis direction of the condensing point does not depend on the objective lens exchange. It is desirable to perform the focus adjustment so that the position of is the same as that of the pupil plane 32.

  Incidentally, the focus adjustment is performed by adjusting the position in the optical axis direction of the emission end of the optical fiber 11 by the position adjusting mechanism 11A, and adjusting the position of at least one of the condenser lens array 14, the lens 21, and the lens 23 in the optical axis direction. Can be done.

  In this embodiment, when it is desired to increase the adjustment range of the distance d, the position of the opening (in a direction perpendicular to the optical axis) is prevented in order to prevent the position of the condensing point from being removed from the opening of the light beam selection member 20. A plurality of light beam selection members having different positions) may be prepared, and the plurality of light beam selection members may be switched and used.

  In the present embodiment, since the distance d is variable, the amount of the evanescent field that leaks out can be arbitrarily finely adjusted. For example, the control device 43 according to the present embodiment finely adjusts the distance d within a range satisfying the expression (1) in accordance with an instruction from the user, thereby finely adjusting the amount of the evanescent field that leaks while maintaining the TIRF condition. Can be adjusted.

  Next, a design example of individual condenser lenses of the condenser lens array 14 will be described.

  Here, it is assumed that the focal length of the lens 21 is 160 mm, the focal length of the lens 23 is 160 mm, the magnification β is 1, and the wavelength range of the working wavelength λ is 400 nm to 700 nm. Further, it is assumed that NA = 0.11 is required for the NA of the light beam pair incident on the pupil plane 32 due to the size of the image sensor 42 and the like.

  According to this assumption, in order to make the above-described interference fringe pattern linear and maintain the above-described demodulation calculation accuracy, the amount of misalignment in the optical axis direction of the condensing point by switching the use wavelength λ is determined by the pupil plane 32. It is necessary to keep it within 0.1 mm above. In this case, the axial chromatic aberration of the condensing lens in the wavelength range (400 nm to 700 nm) of the operating wavelength λ needs to be suppressed to 0.1 mm or less. For this purpose, it is desirable that the condensing lens is a bonded lens (a cemented lens) having an achromatic function. Specifically, it is desirable that the condensing lens has a combination of a convex lens and a concave lens.

  11 is an optical path diagram of the condenser lens, and FIGS. 12 and 13 are lens data of the condenser lens. However, “nd” in FIG. 13 is the refractive index with respect to the d-line, and “νd” is the Abbe number. The unit of each numerical value in the table is “mm”.

  According to the above lens data, the axial chromatic aberration of the condensing lens within the wavelength range of about 400 nm to about 700 nm is suppressed within the range of −0.02351 mm to 0.07329 mm as shown in FIG.

  Therefore, if the lens data described above is adopted for the condenser lens of the present embodiment, it is possible to omit focus adjustment when switching the use wavelength λ.

[Modification]
In the present embodiment, the number of wavelengths of laser light that can be emitted from the laser unit 100 is two, but may be three or more. Regardless of the number of wavelengths, when the lens data described above is employed, it is desirable to set the wavelength emitted by the laser unit 100 to any wavelength within the wavelength range of about 400 nm to about 700 nm.

  In this embodiment, in order to perform total reflection fluorescence observation (TIRF), the value of the distance d is controlled so that the focal point falls within the TIRF region. However, when the TIRF is not necessary (that is, structured illumination). When the microscope apparatus 1 is used in the SIM mode instead of the TIRF-SIM mode), the condensing point may not be within the TIRF region. Incidentally, if the structured illumination microscope apparatus 1 according to the present embodiment is used in the SIM mode, the position of the condensing point can be made unchanged when the operating wavelength λ is switched, so that the super-resolution effect is common among the wavelengths. Can be.

  The super-resolution effect here is the ratio of the resolution R of the structured illumination microscope apparatus when the specimen is not modulated to the resolution (R + K) of the structured illumination microscope apparatus when the specimen is modulated (R + K) / It is R.

  In the present embodiment, it is assumed that a plurality of light beams having different wavelengths are sequentially irradiated onto the sample (a plurality of types of fluorescent regions are sequentially excited), but a plurality of light beams having different wavelengths are simultaneously irradiated onto the sample. (A plurality of types of fluorescent regions may be excited simultaneously). In this case, it is desirable that the structured illumination microscope apparatus 1 is equipped with a function of separating and detecting a plurality of fluorescence having different wavelengths.

  In the present embodiment, the half-wave plate 19 that can rotate around the optical axis of the illumination optical system 10 is used to keep the pair of light beams incident on the sample 2 as S-polarized light. You may use the quarter wavelength plate and the quarter wavelength plate which can rotate the optical axis of the illumination optical system 10. However, in that case, the rotation position of the quarter-wave plate with the first reference position as a reference is set to be the same as the rotation position of the light beam selection member 20 with the second reference position as a reference.

  In this embodiment, the distance d from the optical axis of the condenser lens of the condenser lens array 14 to the optical axis of the illumination optical system 10 is variable in order to maintain the TIRF condition regardless of replacement of the objective lens. However, instead of making the distance d variable, a plurality of condensing lens arrays having different distances d may be prepared, and the plurality of condensing lens arrays may be switched and used.

  In the present embodiment, as means for dividing the light flux emitted from the light source, the condenser lens array 14 in which the number of directions in which the condenser lens pairs are arranged is two or more, that is, a plurality of light flux pairs having different division directions. The condensing lens array 14 (see FIG. 2A) generated at the same time was used, but as shown in FIG. 15, the condensing lens array 14 ′ in which the number of directions in which the condensing lens pairs are arranged is only 1, That is, a condensing lens array 14 ′ that generates only a pair of light beams divided in a predetermined direction may be used.

  However, in that case, the condenser lens array 14 ′ may be rotated around the optical axis of the illumination optical system 10 in order to switch the direction of the interference fringes. If the condensing lens array 14 ′ is rotated by 3 steps by 120 °, a modulated image similar to the case where the condensing lens array 14 is used can be acquired. Further, when the condensing lens array 14 'is used, only a pair of light beams are generated at the same time, so that the light beam selection member 20 is unnecessary. Note that the spacing adjustment mechanism when the condenser lens 14 'is used has a configuration as shown in FIG. 16, for example.

  In the present embodiment, the interference fringes projected onto the specimen 2 are two-beam interference fringes (that is, the 2D-SIM mode), but the SIM fringes where the interference fringes projected onto the specimen 2 are three-beam interference fringes (that is, the two-beam interference fringes). (3D-SIM mode) may be mounted on the structured illumination microscope apparatus 1.

  In the 3D-SIM mode, a condensing lens array 14 ″ as shown in FIG. 17 may be used instead of the condensing lens array 14 shown in FIG. 2. This condensing lens array 14 ″ is shown in FIG. In the condenser lens array 14 shown, one condenser lens 14-0 is added. The condenser lens 14-0 is disposed near the optical axis of the illumination optical system 10, and the optical axis of the condenser lens 14-0 is the optical axis of the illumination optical system 10 (= the condenser lens array 14). The optical axis). Further, the lens characteristics (lens configuration, focal length, etc.) of the condenser lens 14-0 are the same as those of the other condenser lenses 14-1, 14-1 ′, 14-2, 14-2 ′, 14-3, 14. −3 ′ has the same lens characteristics (lens configuration, focal length, etc.). According to such a condensing lens array 14 ″, the above-described pair of condensing points by the pair of light beams and 1 by the light beam that has passed through the condensing lens 14-0 (hereinafter referred to as “center light beam”). Three condensing points with one condensing point are formed on the pupil conjugate plane 25.

  In the 3D-SIM mode, a light beam selection member 20 ′ as shown in FIG. 18 may be used instead of the light beam selection member 20 shown in FIG. 6. This light beam selection member 20 'is the same as the light beam selection member 20 shown in FIG. 6, but further provided with an opening 20E for allowing the above-described central light beam to pass therethrough. The opening 20E is formed in the vicinity of the optical axis of the illumination optical system 10, and the shape of the opening 20E is, for example, a circle. According to such a light beam selection member 20 ′, the light beams from each of the three condensing points formed on the pupil conjugate plane 25 can contribute to the interference fringes.

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

  In the 3D-SIM mode, the number of phases of the modulated image necessary for the demodulation operation described above is 4 or more, so the phase plate 16 also needs to be deformed. An example of a phase plate for increasing the number of phases to 4 or more is disclosed in US Pat. No. 7848017.

  In this embodiment, a relay system (lenses 21 and 23) is inserted between the condenser lens array and the objective lens. However, this relay system can be omitted.

  Further, in the present embodiment, a cemented lens of a convex lens and a concave lens is described as an example of the individual condensing lens of the condensing lens array (condensing lens arrays 14, 14 ′, 14 ″) for dividing the light. However, it may be constituted by a plurality of lens groups, and even if constituted by a plurality of lens groups, it is included in the concept of a condenser lens (lens) and the concept of a condenser lens array (a lens array including a pair of lenses) .

[Effect of the embodiment]
As described above, the illumination optical system (10) of the present embodiment includes a lens including at least one pair of lenses that divides and collects light beams emitted from the light sources (100, 11) that emit light of a plurality of wavelengths simultaneously or sequentially. By receiving a pair of light beams divided by the array (14) and the pair of lenses at different positions on the pupil plane (32), the pair of light beams interfere with each other on the object side, And an objective lens (31) for forming interference fringes on the arranged specimen (2).

  Thus, if a lens array is used instead of a diffraction grating to generate a pair of light beams, there is no problem that the traveling direction changes between a plurality of wavelengths. Therefore, if a lens array is used, the incident positions of a pair of light beams on the pupil plane (32) can be made to coincide among a plurality of wavelengths.

  Therefore, according to the illumination optical system 10 of the present embodiment, a desired super-resolution effect can be obtained at each of a plurality of wavelengths.

  In the illumination optical system (10) of the present embodiment, the positional relationship between the pair of lenses is set to a symmetric positional relationship with respect to the optical axis of the objective lens (31).

  Therefore, according to the illumination optical system (10) of this embodiment, the pattern and contrast of the interference fringes formed on the specimen (2) can be improved.

  In the illumination optical system (10) of the present embodiment, the distance from the optical axis of each of the pair of lenses to the optical axis of the objective lens (31) is such that the pair of luminous fluxes are near the surface of the specimen (2). It has been adjusted to generate an evanescent field. Specifically, the distance d satisfies the conditional expression (1).

  Therefore, the structured illumination microscope apparatus (1) of this embodiment can observe an extremely thin layer on the surface of the specimen (2) at each of a plurality of wavelengths.

  In the illumination optical system (10) of the present embodiment, the objective lens (31) can be replaced with another objective lens (31 ′) having a different pupil diameter, and the lens array (14) has a pair of lenses. The distance d from each optical axis of the lens to the optical axis of the objective lens (31) is variable.

  Alternatively, in the illumination optical system (10) of the present embodiment, the objective lens (31) can be replaced with another objective lens (31 ′) having a different pupil diameter, and the lens array (14) has a distance d. It can be replaced with a different lens array.

  Therefore, the illumination optical system (10) of the present embodiment can cope with a change in pupil diameter due to replacement of the objective lens.

  In the illumination optical system (10) of the present embodiment, the lens array (14) has a plurality of the pair of lenses, and a pair of light beams divided by the plurality of pairs of lenses. Further provided is a light beam selection means (20) for selecting only the light beam.

  Alternatively, in the illumination optical system (10) of the present embodiment, the lens array (14) can be rotated around the optical axis of the objective lens (31).

  Therefore, the illumination optical system (10) of the present embodiment can switch the direction of the interference fringes. Therefore, it is possible to obtain the super-resolution effect in a plurality of directions within the sample surface.

  In the illumination optical system (10) of the present embodiment, the focal lengths of one and the other of the pair of lenses are equal to each other.

  Therefore, in the illumination optical system (10) of the present embodiment, the condensing position in the optical axis direction of the pair of light beams can be made common. Therefore, the interference fringe pattern and contrast can be improved.

  In the illumination optical system (10) of the present embodiment, one and the other of the pair of lenses are cemented lenses, and the axial chromatic aberration of the pair of lenses is such that the condensing position of the pair of lenses has a plurality of wavelengths. Are corrected so as to substantially coincide with each other.

  Therefore, according to the illumination optical system (10) of this embodiment, the condensing position of the pair of light beams in the optical axis direction can be made common among a plurality of wavelengths. Therefore, it is possible to save the trouble of focus adjustment when switching the used wavelength.

  In the illumination optical system (10) of the present embodiment, the lens array (14) further includes a lens disposed on the optical axis of the objective lens (31) in addition to the pair of lenses.

  Therefore, the structured illumination microscope apparatus (1) of the present embodiment can operate not only in the 2D-SIM mode but also in the 3D-SIM mode.

  The illumination optical system (10) of the present embodiment further includes position adjusting means (11A) for adjusting the condensing position of the pair of light beams in the optical axis direction. For example, the lens array (14) of the illumination optical system (10) of the present embodiment has a variable position in the optical axis direction.

  Therefore, the illumination optical system (10) of the present embodiment can cope with the displacement of the pupil position due to the exchange of the objective lens.

  The illumination optical system (10) of the present embodiment further includes phase shift means (18B) for shifting the phase of the interference fringes.

  Therefore, the structured illumination microscope apparatus 1 of the present embodiment can reliably acquire a series of modulated images necessary for demodulation calculation.

  Further, in the illumination optical system (10) of the present embodiment, the plurality of wavelengths are contained within a wavelength range of about 400 nm to about 700 nm.

  If the wavelength is within this range, it is possible to design the lens array (14) so that the condensing points of a pair of light beams substantially coincide between a plurality of wavelengths.

[Explanation of demodulation operation]
Hereinafter, an example of demodulation operation will be described.

Here, a 2D-SIM mode is assumed, and first, it is assumed that the number of interference fringe directions M _max is 1 and the number of phases N _max is 3.

The coordinate in the modulation direction on the sample is set as x, the point image intensity distribution of the imaging optical system is set as P _r (x), the structure of the fluorescent region of the sample is set as O _r (x), and the spatial frequency of the interference fringes (modulation frequency) the K Distant, interference fringes of the phase (modulation phase) phi Distant, amplitude of the interference fringes (modulation amplitude) putting a _{m l} (where, l is the modulation order -1,0,1 The interference fringe pattern (modulation waveform) is expressed by m _l exp (ilKx + φ), and the modulated image I _r (x) of the sample is expressed by the following equation.

  However, the symbol “*” in the expression represents a convolution integral. Hereinafter, in order to distinguish between the quantity in the real space and the quantity in the wave number space, the subscript “r” is given to the quantity in the real space, and the subscript “k” is given to the quantity in the wave number space.

Therefore, the Fourier transform of the modulated image I _r (x) (= I _k (k)) is expressed as the following equation.

Note that the Fourier transform (= P _k (k)) of the point image intensity distribution P _r (x) of the imaging optical system corresponds to an optical transfer function (OTF) of the imaging optical system.

Here, O _k (k + lK) (l = −1, 0, 1) in the equation is a modulation component of each order superimposed on the modulation image I _k (k). In the primary modulation component O _k (k + K) and the −1st order modulation component (k−K), the actual spatial frequency component of the sample 15 is shifted by K (to the low frequency side). The greater the amount of shift, the greater the super-resolution effect. For this reason, it is desirable to set the spatial frequency of the interference fringes as high as possible within the range in which the imaging optical system can form an image.

Now, among a series of modulated images, ie, modulated images of (N _max × M _max ) frames, among three frames of modulated images having different phases of interference fringes, only φ is different and _ml and K are common. is there. Therefore, if the phase of the interference fringes reflected in the modulation image of the j-th frame among these three frames is set to φ _j , the modulation image I _kj (k) of the j-th frame is expressed by the following equation.

Therefore, in the demodulation operation, the modulated image of 3 frames is applied to the above equation, and the three equations obtained thereby are simultaneously solved to solve each order of the modulation component O _k (k + lK) (l = −1, 0). , 1) is known (separated from each other). Incidentally, since P _k (k) in this equation is unique to the imaging optical system, it can be measured in advance.

When the modulation components O _k (k + lK) (l = −1, 0, 1) of the respective orders are separated from each other, O _k (k + lK) P _k (k) (l = −1, 0, 1) Each of these may be known and then divided by the value of P _k (k). However, instead of performing simple division, a known method that is less susceptible to noise, such as a Wiener filter, may be used.

In the demodulation operation, the ± first-order modulation components O _k (k + K) and O _k (k−K) are shifted (rearranged) in the x direction by the modulation frequency K, and then the modulation components O of the respective orders. _k (k + lK) (l = −1, 0, 1) is weighted and synthesized in the wave number space to generate a demodulated image O _k (x) having a wide frequency range.

A super-resolution image O _r (x) of the sample 15 is obtained by inverse Fourier transforming the demodulated image O _k (x). This super-resolution image O _r (x) has a high super-resolution in the modulation direction of interference fringes (x direction).

Next, a case where the number of interference fringes direction M _max is assumed to be 3 and the number of phases N _max in each direction is assumed to be 3 will be described.

FIG. 19 is a diagram for explaining a demodulation operation when the number of phases N _max = 3 and the number of directions M _max = 3. In FIG. 19, symbols I _r (1,1), I _r (2,1),..., I _r (3,3) indicate modulated images having different combinations of phases and directions of interference fringes. , "N" of the modulated image I _{r (N,} M) is the phase number of the interference fringes contribute to its modulated image, "M" in the modulated image I _{r (N,} M) is the contribution to the modulated image The direction number of the interference fringe.

As shown in FIG. 19, in the demodulation operation when the number of phases N _max = 3 and the number of directions M _max = 3, the 3 × 3 = 9 frames of the modulated images are individually Fourier transformed (FIG. 19 (a)), Modulation components are separated from each other in the modulation images having the same interference fringe direction (FIG. 19B), and each of the nine modulation components obtained thereby is rearranged (FIG. 19C). ) And weighted and synthesized in the same wave number space (FIG. 19 (d)) to generate a demodulated image _Ok having a wide frequency range in three directions. That the inverse Fourier transform the demodulated image O _k is a super-resolution image O _r of the sample 15. The super-resolution image O _r has a high resolution over the respective three directions.

As apparent in FIG. _19, the case of _{N max = 3, M max =} 3, N max × N max = 9 frame super-resolution image _{O r} of 1 frame from the modulation image is generated.

Here, since the phase number N _max is assumed to be 3, the separation of the modulation components is performed by solving simultaneous equations. However, when the phase number N _max is greater than 3, International Publication No. 2006/109448 You may carry out by the method disclosed by the pamphlet.

Although the 2D-SIM mode has been described here, in the 3D-SIM mode, the modulation components to be superimposed on the modulated image are the −2nd order modulation component, the −1st order modulation component, the 0th order modulation component, and the + 1st order. Since there are five types of modulation components and + second order modulation components (that is, l = −2, −1, 0, +1, +2), these modulation components may be separated from each other. Therefore, in the 3D-SIM mode, for example, the number of phases N _max may be set to 5 or more to increase the number of frames of a series of modulated images.

  Further, here, the rearrangement (FIG. 19C) and the synthesis (FIG. 19D) processing in the demodulation operation are sequentially performed, but it is also possible to perform them in a batch operation expression. As the calculation formula, Formula (1) in Online Methods of Non-Patent Document 1 below can be applied.

Non-Patent Document 1: “Super-Resolution Video Microscopy of Live Cells by Structured Illumination”, Peter Kner, Bryant B. Chhun, Eric R. Griffis, Lukman Winoto, and Mats GL Gustafsson, NATURE METHODS Vol.6 NO.5, pp .339-342, (2009)
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, 42 ... Imaging device, 43 ... Control apparatus, 44 ... Image storage and calculation apparatus, 45 DESCRIPTION OF SYMBOLS ... Image display apparatus, 12 ... Collector lens, 13 ... Polarizing plate, 14 ... Condensing lens array, 16 ... Phase plate, 19 ... 1/2 wavelength plate, 20 ... Light beam selection member, 21 ... Lens, 22 ... Field stop, DESCRIPTION OF SYMBOLS 23 ... Field lens, 24 ... Excitation filter, 33 ... Dichroic mirror, 31 ... Objective lens, 34 ... Absorption filter, 35 ... 2nd objective lens, 2 ... Sample

Claims (17)

A lens array including at least one pair of lenses that divides and collects light beams emitted from a light source that emits light of a plurality of wavelengths simultaneously or sequentially;
By receiving the pair of light beams divided by the pair of lenses at different positions on the pupil plane, the pair of light beams interfere with each other on the object side, and interference fringes are formed on the specimen arranged on the object side. An objective lens to be formed;
Equipped with a,
Each of the pair of lenses is disposed in a symmetric positional relationship with respect to the optical axis of the objective lens,
The objective lens can be replaced with another objective lens having a different pupil diameter.
The lens array is a structured illumination device in which a distance d from an optical axis of each of the pair of lenses to an optical axis of the objective lens is variable . A lens array including at least one pair of lenses that divides and collects light beams emitted from a light source that emits light of a plurality of wavelengths simultaneously or sequentially;
  By receiving the pair of light beams divided by the pair of lenses at different positions on the pupil plane, the pair of light beams interfere with each other on the object side, and interference fringes are formed on the specimen arranged on the object side. An objective lens to be formed;
With
  Each of the pair of lenses is disposed in a symmetric positional relationship with respect to the optical axis of the objective lens,
  The objective lens can be replaced with another objective lens having a different pupil diameter.
  The structured illumination device, wherein the lens array can be replaced with another lens array having a different distance d.
  However, the distance d of the lens array is a distance from the optical axis of each of the pair of lenses included in the lens array to the optical axis of the objective lens. The structured lighting device according to claim 1 or 2 ,
The distance from the optical axis of each of the pair of lenses to the optical axis of the objective lens is:
It said pair of structured illumination device that have been adapted generate an evanescent field near the surface of the light beam the specimen. The structured lighting device according to claim 3.
The first distance from the optical axis of each lens of the pair to the optical axis of the objective lens d, the following conditional expression satisfy be structured illumination device.
(Nw · fo) / β ≦ d ≦ (NA · fo) / β
However was, nw is the refractive index of the specimen, fo is the focal length of the objective lens, beta magnification from each of the condensing positions of the pair of lens to the pupil plane, NA is a numerical aperture of said objective lens is there. In structured illumination apparatus according to any one of claims 1 to 4,
The lens array is structured illumination device that have a plurality of the pair of lenses. The structured lighting device according to claim 5 , wherein
A plurality of further comprising Structured illumination device light selecting means for selecting only the light flux of the pair of light fluxes of the plurality of pairs divided by a pair of lenses. In structured illumination apparatus according to any one of claims 1 to 4,
The lens array is structured illumination device Ru pivotally der around the optical axis of the objective lens. In structured illumination apparatus according to any one of claims 1 to 7,
One and the other focal length of the pair of lenses, each other equal I構 Zoka lighting device. In structured illumination apparatus according to any one of claims 1 to 8,
One and the other of said pair of lenses, a cemented lens der Ru structured illumination device. The structured lighting device according to claim 9 .
Axial chromatic aberration of the pair of lenses, the pair of lenses of the condensing positions corrected have that structured illumination device to match substantially between said plurality of wavelengths. In structured illumination apparatus according to any one of claims 1 to 10,
The lens array, the 1 in addition to the pair of lenses, further structured illumination device that have a lens disposed on the optical axis. In the structured lighting device according to any one of claims 1 to 11 ,
Further comprising Ru Structured illumination device position adjustment means for adjusting the focusing position of the light flux of the pair in the direction of the optical axis of the objective lens. The structured lighting device according to claim 12 ,
The position of the lens array in the optical axis direction of the objective lens is variable der Ru structured illumination device. A lighting device according to any one of claims 1 to 13,
Further comprising Ru Structured illumination device a phase shifting means for shifting the phase of the interference fringes. In the structured lighting device according to any one of claims 1 to 14 ,
The plurality of wavelengths is structured illumination device that contained from 400nm to the wavelength range of 700 nm. The structured lighting device according to any one of claims 1 to 15 ,
An imaging optical system that forms an image on the light beam of the observation light beam from the sample modulated by the interference fringes;
Structured illumination microscope equipped with. The structured illumination microscope apparatus according to claim 16 ,
Further structured illumination microscope apparatus having an arithmetic means for calculating a demodulated image of the specimen based on the image of the photodetector is produced.

Images