WO2013021615A1 - Structured illumination device, structured illumination method, and structured illumination microscope device - Google Patents

Structured illumination device, structured illumination method, and structured illumination microscope device Download PDF

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Publication number
WO2013021615A1
WO2013021615A1 PCT/JP2012/004985 JP2012004985W WO2013021615A1 WO 2013021615 A1 WO2013021615 A1 WO 2013021615A1 JP 2012004985 W JP2012004985 W JP 2012004985W WO 2013021615 A1 WO2013021615 A1 WO 2013021615A1
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structured illumination
light
wavelength
illumination
structured
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PCT/JP2012/004985
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French (fr)
Japanese (ja)
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達士 野村
大澤　日佐雄
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株式会社ニコン
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/14—Condensers affording illumination for phase-contrast observation

Definitions

  • the present invention relates to a structured illumination apparatus, a structured illumination method, and a structured illumination microscope apparatus.
  • the super-resolution microscope is used to illuminate the illumination beam that illuminates the specimen surface in order to contribute high spatial frequency information (large-angle diffracted light) that exceeds the resolution limit among the diffracted light emitted from the specimen.
  • Modulation is performed to demodulate the imaging light beam incident at a position substantially conjugate with the sample surface of the imaging optical system (see Patent Document 1).
  • Patent Document 1 discloses an example in which a structured illumination microscope is applied to fluorescence observation.
  • a light beam emitted from a coherent light source is divided into two light beams by a diffraction grating, and the two light beams are individually condensed at different positions on the pupil of the objective lens.
  • the two light beams are emitted from the objective lens as parallel light beams having different angles, and overlap on the sample surface to form interference fringes.
  • This provides structured illumination of the specimen surface.
  • a sample image is repeatedly acquired while changing the phase of structured illumination stepwise, and an operation corresponding to the above-described demodulation (demodulation) is performed on the plurality of acquired images. Calculation).
  • a wedge-shaped prism is inserted into one of the two light beams described above and moved stepwise in a direction perpendicular to the optical axis, or perpendicular to the grid lines.
  • a method of stepping the diffraction grating in a proper direction a method of stepping the specimen in the pitch direction of structured illumination, and the like.
  • the diffraction grating has to be replaced every time the wavelength of the light changes.
  • an object of the present invention is to provide a structured illumination apparatus, a structured illumination method, and a structured illumination microscope apparatus that can change the fringe pitch of interference fringes according to the wavelength of light emitted from a coherent light source. To do.
  • One aspect of the structured illumination device of the present invention is an optical modulator that is disposed in an emitted light beam having a predetermined wavelength from a light source and has a sound wave propagation path disposed in a direction across the emitted light beam, and the sound wave propagation path.
  • the interference fringes have a desired fringe pitch according to the illumination optical system that causes at least two diffracted lights of the emitted light flux that has passed therethrough to form interference fringes on the object to be observed, and the wavelength of the emitted light flux.
  • a control means for controlling the optical modulator for controlling the optical modulator.
  • the structured illumination apparatus described above and an observation light beam obtained from the observation object by being illuminated by the structured illumination apparatus are imaged on a photodetector. And an imaging optical system.
  • the emitted light beam that passes through a sound wave propagation path provided to traverse the emitted light beam in an optical modulator disposed in the emitted light beam having a predetermined wavelength from a light source.
  • the interference fringes are formed on the object to be observed by causing the illumination optical system to interfere with the at least two diffracted beams of the light, and the interference fringes have a desired fringe pitch according to the wavelength of the emitted light beam.
  • the modulator is controlled so that the object to be observed is structured and illuminated.
  • the optical modulator is controlled so that the interference fringes have a desired fringe pitch according to the wavelength of the emitted light beam, the wavelength of the sound wave of the acousto-optic device is adjusted with a simple configuration. can do.
  • FIG. (A) is a schematic diagram which shows the pattern of the ultrasonic standing wave which arises in the ultrasonic propagation path R of an acoustooptic device
  • (B) is the pattern of the structured illumination (bright part and dark part) corresponding to it.
  • FIG. (C) to (E) are diagrams for explaining changes in the number of stripes when the number of waves changes.
  • (A) is a figure explaining the relationship between length L and distance D
  • (B) is a conceptual diagram of structured illumination S 'corresponding to spot S
  • (C) is structured illumination. It is a figure explaining the shift
  • a structured illumination microscope apparatus 10 showing an embodiment of the present invention includes an illumination optical system (corresponding to “illumination optical apparatus” of the present invention) 11, an imaging optical system 12, and a photodetector 13. And image processing means 14.
  • the illumination optical system 11 guides illumination light from a light source (not shown) to the specimen 16, and includes an optical fiber 17, a collector lens 18, an acoustooptic device (light modulator) 19, a condenser lens 20, and a zero-order light cut mask 21. , Relay lens 22, field stop 23, illumination lens 24, dichroic mirror 25, objective lens 26, and control unit 27.
  • the specimen 16 is a biological specimen that is fluorescently stained, for example, and is placed on a stage (not shown).
  • Reference numeral 16a denotes an observation target surface (sample surface) of the sample 16.
  • the imaging optical system 12 includes an objective lens 26, a dichroic mirror 25, an imaging lens 28, and the like.
  • the photodetector is an imaging unit (CCD camera or the like) 29.
  • the imaging optical system 12 focuses the light from the sample 16 collected by the objective lens 26 and transmitted through the dichroic mirror 25 onto the imaging surface 29 a of the imaging unit 29 by the imaging lens 28.
  • the image processing means includes a control unit 27, an image storage / calculation unit (such as a computer) 30, and an image display unit 31.
  • the optical fiber 17 guides light from a coherent light source (not shown), and forms a secondary point light source (over-interfering secondary point light source) at the emission end.
  • the wavelength of a coherent light source (not shown) is set to the same wavelength as the excitation wavelength of the specimen 16.
  • the light emitted from the secondary point light source is converted into parallel light by the collector lens 18 and enters the acoustooptic device 19.
  • the acousto-optic element 19 is arranged on the optical axis of the illumination optical system 11 and at a position conjugate with the sample 16.
  • the incident light is diffracted by the acoustooptic device 19, and interference fringes are formed on the sample surface 16a using the diffracted light.
  • the acousto-optic element 19 is obtained by attaching a transducer such as a piezoelectric element to an acousto-optic medium. When a high-frequency AC voltage is applied to the transducer, the acousto-optic element 19 vibrates in the thickness direction and is Propagates ultrasonic waves into the optical medium.
  • the acoustooptic device 19 has an ultrasonic wave propagation path R for propagating ultrasonic waves into the acoustooptic medium in a direction across an emitted light beam having a predetermined wavelength from the light source.
  • an ultrasonic plane standing wave hereinafter referred to as “ultrasonic standing wave”
  • a sinusoidal refractive index distribution is imparted to the ultrasonic wave propagation path R.
  • Such an acoustooptic device 3 functions as a phase-type diffraction grating for incident light, and branches the light into diffracted light of each order.
  • the solid line indicates the 0th order diffracted light
  • the dotted line indicates the ⁇ 1st order diffracted light.
  • the diffracted light of each order emitted from the acoustooptic device 19 enters the 0th-order light cut mask 21 after passing through the condenser lens 20.
  • the 0th-order light cut mask 21 is disposed at or near the conjugate position of the pupil plane P of the objective lens 26, and among the diffracted light of each order incident on the conjugate position, the 0th-order diffracted light and the second and subsequent orders. It has a function of cutting high-order diffracted light and allowing only ⁇ first-order diffracted light (only the speed of two lights) to pass.
  • the 0th-order light cut mask 21 is a substrate in which a plurality of openings or transmission portions are formed. The position of the opening or transmission part when the optical path is inserted corresponds to a region through which ⁇ first-order diffracted light passes at a conjugate position.
  • the ⁇ 1st-order diffracted light After passing through the 0th-order light cut mask 21, the ⁇ 1st-order diffracted light enters the field stop 23 disposed at or near the conjugate position of the sample surface 16a after passing through the relay lens 22.
  • the field stop 23 has a function of controlling the size of the illumination area (observation area) on the sample surface 16a.
  • the conjugate position is a focal length (back focal position) of the lens 20, and the illumination lens 24, with respect to a pupil P (a position where ⁇ 1 next-order analysis light is condensed) of an objective lens 26 described later, And a conjugate position (pupil conjugate plane) via the relay lens 22 (note that the concept of “conjugate position” has been described by those skilled in the art in terms of the aberrations and vignetting of the objective lens 26, the relay lens 22, and the illumination lens 24. Etc., including positions determined in consideration of design requirements).
  • the ⁇ first-order diffracted light that has passed through the field stop 23 enters the dichroic mirror 25 after passing through the illumination lens 24 and is reflected by the dichroic mirror 25.
  • the ⁇ first-order diffracted lights reflected by the dichroic mirror 25 form spots at different positions on the pupil plane P of the objective lens 26, respectively. It should be noted that the formation positions of the two spots on the pupil plane P are approximately the outermost peripheral portion of the pupil plane P, and are symmetrical with respect to the optical axis of the objective lens 26.
  • the ⁇ first-order diffracted light emitted from the tip of the objective lens 26 irradiates the sample surface 16a from opposite directions at an angle corresponding to the NA (numerical aperture) of the objective lens 26.
  • NA numerical aperture
  • these ⁇ 1st order diffracted lights irradiated on the sample surface 16a are coherent lights emitted from a coherent light source. For this reason, striped interference fringes (hereinafter referred to as “fringes”) having a uniform fringe pitch are projected onto the specimen surface 16a.
  • the illumination pattern of the specimen surface 16a becomes an illumination pattern having a stripe structure by these ⁇ 1st order diffracted lights.
  • the illumination by the illumination pattern having the stripe structure in this way is structured illumination.
  • the fluorescent material is excited and emits fluorescence.
  • this structured illumination consists of only two light beams of ⁇ first-order diffracted light, it is structured in the in-plane direction of the specimen 16, but is structured in the depth direction (optical axis direction) of the specimen 16. It has not been.
  • Such structured illumination is referred to as two-beam structured illumination.
  • moire fringes having a difference between the spatial frequency of the structured illumination and the spatial frequency having the structure of the fluorescent region as a spatial frequency appear on the sample surface 16a.
  • the spatial frequency of the structure of the fluorescent region is modulated and shifted to a lower spatial frequency than actual. Therefore, according to structured illumination, even fluorescence that shows a component having a high spatial frequency in the structure of the fluorescent region, that is, fluorescence emitted at a large angle exceeding the resolution limit of the objective lens 26 may enter the objective lens 26. it can.
  • Fluorescence emitted from the specimen surface 16 a and incident on the objective lens 26 is converted into parallel light by the objective lens 26 and then incident on the dichroic mirror 25.
  • the fluorescence passes through the imaging lens 28 after passing through the dichroic mirror 25, thereby forming a fluorescence image of the sample surface 16 a on the imaging surface 29 a of the imaging unit 29.
  • This fluorescent image includes the structure information of the fluorescent region of the specimen surface 16a and the structure information of structured illumination.
  • the control unit 27 controls the acoustooptic device 19 so that the interference fringes projected on the sample surface 16a have a desired fringe pitch according to the wavelength of the light source. Specifically, the control unit 27 adjusts the wavelength of the sound wave generated by the acoustooptic device 19 by adjusting the frequency of the voltage (high frequency voltage) applied to the acoustooptic device 19 according to the wavelength of the light source. .
  • the resolution of the structured illumination microscope apparatus 10 is determined by the fringe pitch of interference fringes created by the illumination optical system 11.
  • the control unit 27 changes the frequency of the voltage applied to the acoustooptic device 19 to adjust the wavelength of the sound wave so that the fringe pitch becomes the finest, thereby increasing the resolution of the structured illumination microscope apparatus 10.
  • the fringe pitch is “P”
  • the wavelength of the light of the light source is “ ⁇ ”
  • the numerical aperture of the objective lens 26 is “NA”
  • the expression described in [Equation 1] is satisfied. That is, it can be seen that the fringe pitch P is proportional to the wavelength ⁇ of the light source.
  • the vibration proceeds in the acoustooptic medium as a sound wave, is reflected by the opposite surface of the acoustooptic medium, and overlaps.
  • the period of sound waves generated in the acousto-optic medium is substantially determined by the frequency of the applied voltage, but becomes a standing wave when there are an integer number of half-cycle sound waves in the acousto-optic medium.
  • the standing wave causes a periodic refractive index change in the acousto-optic medium due to the ultrasonic dense wave, acts as a diffraction grating, and branches light.
  • the standing wave does not have to be complete, and the frequency of the applied voltage (high-frequency voltage) may slightly deviate from the condition in which the standing wave stands in the acousto-optic medium.
  • the wavelength of the standing wave can be controlled by the frequency of the high frequency voltage. That is, the angle (branching angle) between the 0th order light and the 1st order light can be controlled.
  • the light of the light source that has selected the optimum excitation wavelength band for the fluorescent dye that is, the excitation light is branched into a plurality of lights when passing through the acoustooptic device 19, and then passes through the condenser lens 20 to make the direction of the light parallel,
  • the light is incident on the microscope observation optical path (including the imaging optical system 12) by the dichroic mirror 25, the direction of the light is bent inward by the objective lens 26, and the light is overlapped on the specimen 16 set on the specimen surface 16a (interference). It becomes excitation illumination light of a one-dimensional lattice pattern (stripe pattern).
  • the maximum resolution can be obtained when the illumination light is irradiated from the objective lens 26 to the specimen 16 at the maximum incident angle ⁇ .
  • the light traveling angles after passing through the objective lens 26 are configured such that the 0th-order light and the + 1st-order light have the maximum incident angle ⁇ from the objective lens 26 to the specimen 16, respectively. .
  • the frequency of power applied to the acoustooptic device 19 is changed so that the branching angle does not change.
  • the branch angle between the 0th order light and the + first order light is “ ⁇ 1”
  • the wavelength of the light source is “ ⁇ ”
  • the wavelength of the sound wave propagating through the acoustooptic medium is “ ⁇ ”
  • the frequency of the applied voltage is “f”
  • the velocity of the sound wave propagating through the optical medium is “v”
  • the equation described in [Equation 2] is satisfied. Note that the velocity “v” of the sound wave propagating through the acousto-optic medium is constant.
  • the branching angle “ ⁇ 1” In order to maintain the resolution, the branching angle “ ⁇ 1” must be maintained, and when the light wavelength “ ⁇ ” of the light source changes, the frequency “f” of the applied voltage may be changed.
  • the control unit 27 of the present embodiment changes the frequency of the voltage applied to the acoustooptic device 19 even when shifting the phase of structured illumination. Since the amount of change in frequency is sufficiently small, there is no significant effect on maintaining the maximum resolution.
  • control unit 27 of the present embodiment controls the ultrasonic standing wave generated in the ultrasonic wave propagation path R of the acoustooptic device 19 by changing the frequency of the voltage applied to the acoustooptic device 19.
  • the phase shift amount of the structured illumination is changed stepwise by 2 ⁇ / 3.
  • the control unit 27 drives the imaging unit 29 to obtain three types of image data I-1, I0, I + 1 when the structured illumination phase is in each state, and the image data I-1, I0 and I + 1 are sequentially sent to the image storage / arithmetic unit 30.
  • the image storage / calculation unit 30 performs a demodulation operation on the captured image data I-1, I0, I + 1, so that the image data I in which the spatial frequency of the structure information of the fluorescent region is returned to the actual spatial frequency. 'Is acquired and the image data I' is sent to the image display unit 31.
  • the method disclosed in US Pat. No. 8,115,806 can be used. As a result, a resolution image (super-resolution image) exceeding the resolution limit of the objective lens 26 is displayed on the image display unit 31.
  • FIG. 2A is a schematic diagram showing a pattern of an ultrasonic standing wave generated in the ultrasonic wave propagation path R
  • FIG. 2B shows a corresponding structured illumination pattern (bright part and dark part).
  • the number of ultrasonic standing waves generated in the ultrasonic wave propagation path R is set to “2”, which is smaller than the actual number.
  • the phase of the structured illumination corresponding to the focused portion is “ ⁇ ” by 3. It is changing on the street.
  • the phase of the structured illumination corresponding to the focused portion is “2 ⁇ / 3” by each. It has changed in three ways.
  • the structured illumination of the structured illumination can be obtained by changing the wave number of the ultrasonic standing wave by 1/2.
  • the phase can be changed by “ ⁇ ”.
  • the structured illumination of the structured illumination can be obtained by changing the number of ultrasonic standing waves by half.
  • the phase can be changed by “2 ⁇ / 3”.
  • the light is incident from the center of the spot (effective diameter) S of light incident on the ultrasonic wave propagation path R.
  • the number of ultrasonic standing waves generated in the ultrasonic wave propagation path R changes by 1 ⁇ 2
  • the number of ultrasonic standing waves generated in the spot S also slightly shifts.
  • the number of fringes of the two-beam structured illumination S ′ corresponding to the spot S also slightly deviates (however, the wave pattern and the fringe pattern shown in FIG. 3 are schematic diagrams, and the wave number and the fringe number are It does not always match the actual number.)
  • the length L of the ultrasonic wave propagation path R is set to be sufficiently larger than the diameter ⁇ of the spot S so that the deviation of the number of stripes of the two-beam structured illumination S ′ can be regarded as almost zero. .
  • the length L of the ultrasonic wave propagation path R and the diameter ⁇ of the spot S are ⁇ / L ⁇ with respect to the allowable amount ⁇ of the stripe number deviation of the two-beam structured illumination S ′. Is set to satisfy the relationship. For example, if it is necessary to suppress the deviation of the number of stripes of the two-beam structured illumination S ′ to 0.15 or less, the relational expression is ⁇ / L ⁇ 0.15.
  • the diameter ⁇ of the spot S on the ultrasonic wave propagation path R of the acoustooptic device 19 does not necessarily satisfy the relationship of ⁇ / L ⁇ .
  • the ⁇ first-order diffracted light emitted from the acoustooptic device 19 is
  • the length L of the ultrasonic wave propagation path R, the diameter ⁇ ′ of the illumination region (observation region, field region) on the sample surface 16a, and the optical magnification m from the sample surface 16a to the acoustooptic device 19 are obtained. May be set so as to satisfy the relationship of ⁇ ′ ⁇ m / L ⁇ with respect to the allowable amount ⁇ of the deviation of the number of stripes of the two-beam structured illumination S ′.
  • the diameter ⁇ of the spot S is assumed to be 4 mm.
  • the length L of the ultrasonic propagation path R is set to 30 mm, as shown in FIG. 3C, the deviation of the stripes at both ends of the two-beam structured illumination S ′ is about 0.068.
  • the dotted line indicates the ideal pattern of the two-beam structured illumination S ′ (pattern when the deviation of the number of stripes is zero), and the solid line indicates the two-beam structured. Although it is an actual pattern of the illumination S ′, the difference between the two is drawn for the sake of clarity.
  • FIG. 4 is a configuration diagram of the acoustooptic device 3.
  • FIG. 4A is a diagram of the acoustooptic device 19 viewed from the front (in the optical axis direction), and FIG. It is the figure which looked at from the side (direction perpendicular to the optical axis).
  • the acoustooptic device 19 of this embodiment has three ultrasonic propagation paths having a rib structure.
  • the acoustooptic medium 35 includes a single acoustooptic medium 35, and the acoustooptic medium 35 has a pair of side surfaces (a pair of side surfaces) 35a-35f, 35b-35g, 35c-35e that are parallel to each other.
  • the acoustooptic medium 35 has a pair of side surfaces (a pair of side surfaces) 35a-35f, 35b-35g, 35c-35e that are parallel to each other.
  • Transducers 36 to 38 are provided on one of the side surfaces 15a to 15c so as to project in a rib shape, thereby three ultrasonic propagation paths in the acoustooptic medium 35 between the other side surfaces 35f, 35g, and 35e.
  • Ra, Rb, and Rc are formed.
  • the ultrasonic wave propagation path Ra is a propagation path formed between the side surface 35a having the transducer 36 and the side surface 35f opposite to the side surface 35a, and the ultrasonic wave propagation path Rb is opposite to the side surface 35b having the transducer 37.
  • the propagation path is formed between the side face 35g, and the ultrasonic propagation path Rc is a propagation path formed between the side face 35c having the transducer 38 and the side face 35e facing the side face 35c.
  • the material of the acousto-optic medium 35 is a transparent optical material having a high refractive index, such as quartz glass, quartz, tellurite glass, heavy flint glass, flint glass, etc., and three side pairs 35a-35f, 35b- 35g, 35c-35e, and the two bottom surfaces 35d, 35h are each polished with sufficient accuracy.
  • the three ultrasonic propagation paths Ra, Rb, and Rc intersect at different angles every 60 degrees at a position separated by L / 3 from one end of each. The center of the spot S described above is located at the intersection position. Note that the angle of crossing each other is not limited to 60 degrees.
  • the number of ultrasonic propagation paths may be at least an odd number of 3 or more.
  • the structure is obtained each time image data for two-dimensional super-resolution observation of the specimen 16 is obtained by selectively switching and using a plurality of ultrasonic propagation paths crossing each other by a predetermined angle. It acts to change the direction of the illumination.
  • the transducer 36 is an ultrasonic transducer having a piezoelectric body 39 and two electrodes 40 individually formed on the upper and lower surfaces of the piezoelectric body 39, and is bonded to the side surface 35 a via the lower surface electrode 40.
  • a high-frequency AC voltage is applied between the control unit 27 and the upper and lower electrodes 40, the piezoelectric body 39 vibrates in the thickness direction, and planar ultrasonic waves reciprocate in the ultrasonic wave propagation path Ra.
  • the ultrasonic wave becomes a standing wave, so that the refractive index of the ultrasonic wave propagation path Ra is A sinusoidal distribution is given over the propagation direction of the ultrasonic wave.
  • the ultrasonic wave propagation path Ra becomes a phase type diffraction grating having a phase grating perpendicular to the ultrasonic wave propagation direction.
  • the propagation direction of the ultrasonic wave propagation path Ra is referred to as a “first direction”.
  • the transducer 37 has the same configuration as that of the transducer 36, and includes a piezoelectric body 41 and two electrodes 42 formed individually on the upper and lower surfaces of the piezoelectric body 41. It is joined to the side surface 35b.
  • the ultrasonic wave propagation path Rb has a propagation direction of the ultrasonic wave.
  • a phase type diffraction grating having a perpendicular phase grating.
  • the propagation direction of the ultrasonic wave propagation path Rb is referred to as a “second direction”.
  • the second direction forms an angle of 60 ° with the first direction.
  • the transducer 38 has the same configuration as that of the transducer 36, and includes a piezoelectric body 43 and two electrodes 44 formed individually on the upper and lower surfaces of the piezoelectric body 43. It is joined to the side surface 35c.
  • the ultrasonic wave propagation path Rc has the ultrasonic wave propagation direction.
  • a phase type diffraction grating having a perpendicular phase grating.
  • the propagation direction of the ultrasonic wave propagation path Rc is referred to as a “third direction”. This third direction forms an angle of ⁇ 60 ° with respect to the first direction.
  • the controller 27 includes a drive circuit 46 for the acoustooptic device 19 as shown in FIG.
  • the drive circuit 46 includes a high frequency AC power supply 47 and a changeover switch 48.
  • the high frequency AC power supply 47 generates an AC voltage to be supplied to the acoustooptic device 19.
  • the frequency of the AC voltage is controlled to an appropriate frequency (for example, any value within several tens of MHz to 100 MHz) by the CPU in the control unit 27.
  • the phase shift amount of the two-beam structured illumination S ′ described above is used to demodulate the image (modulated image) of the structured illumination sample 16 and obtain data of a plurality of modulated images having different phases.
  • the CPU slightly switches the frequency of the AC voltage between three appropriate frequencies f ⁇ 1, f0, and f + 1 having different frequencies.
  • the appropriate frequency f0 is for causing 100 ultrasonic standing waves (the number of stripes of structured illumination corresponding to 200) to be generated in the ultrasonic propagation paths Ra, Rb, and Rc having a length L of 30 mm.
  • Appropriate frequency 80 MHz.
  • the phase shift amount of the two-beam structured illumination S ′ is zero.
  • the proper frequency f-1 is (100-1 / 2) ultrasonic standing waves (corresponding to the structured illumination fringes) on the ultrasonic propagation paths Ra, Rb, Rc having a length L of 30 mm.
  • the number is an appropriate frequency (79.946 MHz) for generating 199).
  • the phase shift amount of the two-beam structured illumination S ′ is ⁇ 2 ⁇ / 3.
  • the appropriate frequency f + 1 is (100 + 1/2) ultrasonic standing waves (the number of fringes of structured illumination corresponding to 201) corresponding to ultrasonic propagation paths Ra, Rb, Rc having a length L of 30 mm. It becomes an appropriate frequency (80.54 MHz) for occurrence. According to this appropriate frequency f + 1, the phase shift amount of the two-beam structured illumination S ′ is + 2 ⁇ / 3.
  • the changeover switch 48 is disposed between the high-frequency AC power supply 47 and the acoustooptic element 19, and the connection destination on the acoustooptic element 19 side can be switched between the three transducers 36 to 38 of the acoustooptic element 19. It is.
  • the connection destination of the changeover switch 48 is appropriately changed over by the CPU in the control unit 27.
  • connection destination of the changeover switch 48 When the connection destination of the changeover switch 48 is on the transducer 36 side, an AC voltage is applied between the two electrodes 40 of the transducer 36, so that the ultrasonic wave propagation path among the three ultrasonic wave propagation paths Ra, Rb, Rc. Only Ra is valid.
  • connection destination of the changeover switch 48 is on the transducer 37 side, an AC voltage is applied between the two electrodes 42 of the transducer 37, so that the ultrasonic wave among the three ultrasonic wave propagation paths Ra, Rb, Rc. Only the propagation path Rb is effective.
  • connection destination of the changeover switch 48 is on the transducer 38 side, an AC voltage is applied between the two electrodes of the transducer 38, so that the ultrasonic wave propagation among the three ultrasonic wave propagation paths Ra, Rb, Rc. Only the path Rc is valid.
  • the direction of the two-beam structured illumination S ′ is set to the direction corresponding to the first direction, and the second direction. It is possible to quickly switch between a direction corresponding to the direction and a direction corresponding to the third direction.
  • FIG. 6 is an operational flowchart of the CPU. Hereinafter, each step will be described in order.
  • a light source having the same wavelength as the excitation wavelength of the specimen 16 is used.
  • the frequency of the drive signal (applied voltage) applied to the acoustooptic device 3 is adjusted according to the wavelength of the light from the light source.
  • control is performed so that the branch angle does not change by changing the frequency of the drive signal.
  • Step S11 The CPU sets the direction of the two-beam structured illumination S ′ to a direction corresponding to the first direction by setting the connection destination of the changeover switch 48 to the first transducer (transducer 36) side.
  • Step S12 The CPU sets the phase shift amount of the two-beam structured illumination S ′ to ⁇ 2 ⁇ / 3 by setting the frequency of the AC voltage generated by the high-frequency AC power supply 47 to an appropriate frequency f ⁇ 1.
  • Step S13 In this state, the CPU drives the imaging unit 29 to acquire the image data I-1.
  • Step S14 The CPU sets the frequency of the AC voltage generated by the high-frequency AC power supply 47 to an appropriate frequency f0, thereby setting the phase shift amount of the two-beam structured illumination S ′ to zero.
  • Step S15 The CPU drives the imaging unit 29 in this state to acquire the image data I0.
  • Step S16 The CPU sets the phase shift amount of the two-beam structured illumination S ′ to + 2 ⁇ / 3 by setting the frequency of the AC voltage generated by the high-frequency AC power supply 47 to an appropriate frequency f + 1.
  • Step S17 The CPU acquires the image data I + 1 by driving the imaging unit 29 in this state.
  • Step S18 The CPU determines whether or not the direction of the two-beam structured illumination S ′ has been set in all the three directions described above. If not, the CPU proceeds to step S19 and has been set. If so, the flow ends.
  • Step S19 The CPU switches the connection destination of the changeover switch 48 to switch the direction of the two-beam structured illumination S ′, and then proceeds to step S12.
  • These image data are taken into the image storage / calculation unit 30.
  • the image storage / calculation unit 30 obtains demodulated image data Ia ′ of the super-resolution image in the first direction by performing a demodulation operation on a series of three image data Ia ⁇ 1, Ia0, and Ia + 1.
  • the image storage / calculation unit 30 obtains demodulated image data Ib ′ of the super-resolution image in the second direction by performing a demodulation operation on the series of three image data Ib ⁇ 1, Ib0, and Ib + 1. To do.
  • the image storage / calculation unit 30 also obtains demodulated image data Ic ′ of a super-resolution image in the third direction by performing a demodulation operation on a series of three image data Ic ⁇ 1, Ic0, and Ic + 1. To do.
  • the image storage / calculation unit 30 combines the three demodulated image data Ia ′, Ib ′, and Ic ′ in the wave number space and then returns them to the real space again, whereby the first direction, the second direction, and the third direction
  • the image data I of the super-resolution image over the range is acquired, and the image data I is sent to the image display unit 31. Therefore, a super-resolution image showing in detail the structure of the fluorescent region of the specimen surface 16a is displayed on the image display unit 31.
  • interference fringes two-beam structured illumination
  • ⁇ first-order diffracted light are formed on the sample surface 16a (in the XY plane when the optical axis is in the Z direction).
  • the present invention is not limited to this, and the case where interference fringes (three-beam structured illumination in which interference fringes are also formed in the optical axis direction) are formed on a sample by zero-order diffracted light and ⁇ first-order diffracted light.
  • interference fringes three-beam structured illumination in which interference fringes are also formed in the optical axis direction
  • it can also be applied. In this case, it is only necessary to make a hole in the center of the 0th-order light cut mask 21 to pass the 0th-order light.
  • three-beam interference caused by three diffracted lights with equal intervals between diffraction orders may be generated.
  • ⁇ 1st order diffracted light for example, a combination of 0th order diffracted light, 1st order diffracted light, 2nd order diffracted light, a combination of ⁇ 2nd order diffracted light and 0th order diffracted light, or a combination of ⁇ 3rd order diffracted light and 0th order diffracted light, etc.
  • ⁇ 1st order diffracted light for example, a combination of 0th order diffracted light, 1st order diffracted light, 2nd order diffracted light, a combination of ⁇ 2nd order diffracted light and 0th order diffracted light, or a combination of ⁇ 3rd order diffracted light and 0th order diffracted light, etc.
  • TIRF In TIRF, it is necessary to perform illumination using only a small area on the outer periphery of the pupil of the objective lens. At this time, if a conventionally known diffraction grating having the same pitch is used for excitation light having a different wavelength, the branch angle is changed and the requirement for TIRF illumination cannot be satisfied. Therefore, a diffraction grating with a different period is required for each wavelength of the excitation light, which takes time and labor.
  • a diffraction grating with a different period is required for each wavelength of the excitation light, which takes time and labor.
  • such a method is not necessary, and time can be reduced and labor can be reduced.
  • the fringe pitch is proportional to the wavelength of light from the light source.
  • the lower limit is determined.
  • an intermediate fringe pitch between the upper limit and the lower limit is most desirable.
  • the frequency of the voltage applied to the acoustooptic device 19 is changed so that the TIRF illumination conditions can be maintained. That is, even when the excitation light wavelength changes, the angle (branch angle) between the 0th order light and the 1st order light in the acoustooptic device 19 may be controlled so as not to change.
  • the branch angle is “ ⁇ 1”
  • the wavelength of the light of the light source is “ ⁇ ”
  • the wavelength of the sound wave propagating through the acoustooptic medium is “ ⁇ ”
  • the frequency of the applied voltage is “f”
  • the sound wave propagating through the acoustooptic medium When the speed is “v”, the equation described in [Equation 4] is satisfied.
  • the velocity “v” of the sound wave propagating through the acousto-optic medium is constant.
  • the branch angle “ ⁇ 1” may be maintained by changing the frequency “f” of the applied voltage.
  • the three ultrasonic propagation paths Ra, Rb, and Rc are arranged in an asymmetric relationship with respect to the center of the spot S (see FIG. 4).
  • the advantage of the example shown in FIG. 4 is that the contour of the outer shape of the acoustooptic medium 35 is small
  • the advantage of the example shown in FIG. 7 is that the environment of the three ultrasonic propagation paths Ra, Rb, Rc is completely There is a match.
  • the control unit 27 adjusts the frequency “f” of the applied voltage according to the wavelength “ ⁇ ” of the light from the light source, thereby changing the wavelength “ ⁇ ” of the acoustic wave of the acoustooptic device 19.
  • the present invention is not limited to this, and the control unit 27 may perform control so as to change the velocity “v” of the sound wave propagating through the acousto-optic medium in accordance with the drive signal.
  • a means for deforming the acousto-optic medium according to the drive signal may be provided.
  • the wavelength “ ⁇ ” of the sound wave may be changed by changing the temperature of the acousto-optic medium.
  • the three ultrasonic propagation paths Ra, Rb, and Rc crossing each other by a predetermined angle are provided.
  • the present invention is not limited to this.
  • only one ultrasonic propagation path may be provided, the arrangement direction of the ultrasonic propagation path may be rotated, and the phase may be shifted at each rotational position.
  • a phase plate having three or more different odd-numbered areas may be rotated to shift the phase at the rotation position where each area is set.

Abstract

[Problem] To adjust a wavelength of a sound wave of an audio-optical element with a simple configuration. [Solution] A structured illumination microscope device (10) comprises a structured illumination assembly (11). The structured illumination assembly (11) further comprises a light source (15), a unit audio-optical element (19), an objective lens (26), and a control unit (29). The light source (15) emits light of a prescribed wavelength. The audio-optical element (19) diffracts light and forms an interference pattern upon a specimen face (16a). The optical lens (26) illuminates a specimen (16) with some or all of the light beam which is diffracted with the audio-optical element (19). The control unit (29) controls the audio-optical element (19) such that the pattern pitch of the interference pattern changes according to the wavelength of the light of the light source (15).

Description

構造化照明装置及び構造化照明方法、並びに構造化照明顕微鏡装置Structured illumination apparatus, structured illumination method, and structured illumination microscope apparatus
　本発明は、構造化照明装置及び構造化照明方法、並びに構造化照明顕微鏡装置に関する。 The present invention relates to a structured illumination apparatus, a structured illumination method, and a structured illumination microscope apparatus.
　超解像顕微鏡は、標本から射出する回折光のうち解像限界を超える高い空間周波数の情報（大角度の回折光）を結像に寄与させるために、標本面を照明する照明光束に対して変調を施し、結像光学系の標本面と略共役な位置に入射する結像光束に対して復調を施すものである（特許文献１参照）。 The super-resolution microscope is used to illuminate the illumination beam that illuminates the specimen surface in order to contribute high spatial frequency information (large-angle diffracted light) that exceeds the resolution limit among the diffracted light emitted from the specimen. Modulation is performed to demodulate the imaging light beam incident at a position substantially conjugate with the sample surface of the imaging optical system (see Patent Document 1).
　特許文献１には、構造化照明顕微鏡を蛍光観察へ適用した例が開示されている。特許文献１に記載の発明では、可干渉光源から射出した光束を回折格子によって２つの光束に分割し、それら２つの光束を対物レンズの瞳上の互いに異なる位置へ個別に集光させる。このとき２つの光束は対物レンズから角度の異なる平行光束として射出し、標本面上で重なり合いストライプ状の干渉縞を形成する。これによって標本面が構造化照明される。そして、特許文献１に記載の発明では、構造化照明の位相をステップ状に変化させながら標本像の画像を繰り返し取得し、取得した複数の画像に対して、前述した復調に相当する演算（復調演算）を行っている。 Patent Document 1 discloses an example in which a structured illumination microscope is applied to fluorescence observation. In the invention described in Patent Document 1, a light beam emitted from a coherent light source is divided into two light beams by a diffraction grating, and the two light beams are individually condensed at different positions on the pupil of the objective lens. At this time, the two light beams are emitted from the objective lens as parallel light beams having different angles, and overlap on the sample surface to form interference fringes. This provides structured illumination of the specimen surface. In the invention described in Patent Document 1, a sample image is repeatedly acquired while changing the phase of structured illumination stepwise, and an operation corresponding to the above-described demodulation (demodulation) is performed on the plurality of acquired images. Calculation).
　ここで、構造化照明の位相をステップ状に変化させる方法としては、楔型プリズムを前述した２つの光束の一方に挿入して光軸と垂直な方向へステップ移動させる方法や、格子線と垂直な方向へ回折格子をステップ移動させる方法や、構造化照明のピッチ方向へ標本をステップ移動させる方法等が知られている。 Here, as a method of changing the phase of structured illumination stepwise, a wedge-shaped prism is inserted into one of the two light beams described above and moved stepwise in a direction perpendicular to the optical axis, or perpendicular to the grid lines. There are known a method of stepping the diffraction grating in a proper direction, a method of stepping the specimen in the pitch direction of structured illumination, and the like.
米国特許第６２３９９０９号明細書US Pat. No. 6,239,909
　しかしながら、可干渉光源から射出した光の波長に応じて干渉縞の縞ピッチを変えるためには、光の波長が変わる毎に回折格子を交換しなければいけなかった。 However, in order to change the fringe pitch of the interference fringes according to the wavelength of the light emitted from the coherent light source, the diffraction grating has to be replaced every time the wavelength of the light changes.
　そこで本発明は、可干渉光源から射出した光の波長に応じて干渉縞の縞ピッチを変えることができる構造化照明装置及び構造化照明方法、並びに構造化照明顕微鏡装置を提供することを目的とする。 Accordingly, an object of the present invention is to provide a structured illumination apparatus, a structured illumination method, and a structured illumination microscope apparatus that can change the fringe pitch of interference fringes according to the wavelength of light emitted from a coherent light source. To do.
　本発明の構造化照明装置の一態様は、光源からの所定の波長を有する射出光束中に配置され、その射出光束を横切る方向に音波伝搬路を配置した光変調器と、前記音波伝搬路を通過した前記射出光束の少なくとも２つの回折光を干渉させ、その干渉縞を被観察物に形成する照明光学系と、前記射出光束が有する波長に応じて前記干渉縞が所望の縞ピッチを持つように前記光変調器を制御する制御手段と、を備えるものである。 One aspect of the structured illumination device of the present invention is an optical modulator that is disposed in an emitted light beam having a predetermined wavelength from a light source and has a sound wave propagation path disposed in a direction across the emitted light beam, and the sound wave propagation path. The interference fringes have a desired fringe pitch according to the illumination optical system that causes at least two diffracted lights of the emitted light flux that has passed therethrough to form interference fringes on the object to be observed, and the wavelength of the emitted light flux. And a control means for controlling the optical modulator.
　また、本発明の構造化照明顕微鏡装置の一態様としては、前述した構造化照明装置と、構造化照明装置で照明されたことにより前記被観察物から得られる観察光束を光検出器に結像する結像光学系と、を備えるものである。 Further, as one aspect of the structured illumination microscope apparatus of the present invention, the structured illumination apparatus described above and an observation light beam obtained from the observation object by being illuminated by the structured illumination apparatus are imaged on a photodetector. And an imaging optical system.
　本発明の構造化照明方法の一観点によれば、光源からの所定の波長を有する射出光束中に配置した光変調器に前記射出光束を横切るように設けた音波伝搬路を通過する前記射出光束の少なくとも２つの回折光を照明光学系により干渉させることで前記干渉縞を被観察物に形成するとともに、前記射出光束が有する波長に応じて前記干渉縞が所望の縞ピッチを持つように前記光変調器を制御して前記被観察物を構造化照明するようにしたものである。 According to one aspect of the structured illumination method of the present invention, the emitted light beam that passes through a sound wave propagation path provided to traverse the emitted light beam in an optical modulator disposed in the emitted light beam having a predetermined wavelength from a light source. The interference fringes are formed on the object to be observed by causing the illumination optical system to interfere with the at least two diffracted beams of the light, and the interference fringes have a desired fringe pitch according to the wavelength of the emitted light beam. The modulator is controlled so that the object to be observed is structured and illuminated.
　本発明によれば、射出光束が有する波長に応じて干渉縞が所望の縞ピッチを持つように光変調器を制御する制御手段を備えたため、音響光学素子の音波の波長を簡単な構成で調整することができる。 According to the present invention, since the optical modulator is controlled so that the interference fringes have a desired fringe pitch according to the wavelength of the emitted light beam, the wavelength of the sound wave of the acousto-optic device is adjusted with a simple configuration. can do.
本発明の構造化照明顕微鏡装置の概略を示す説明図である。It is explanatory drawing which shows the outline of the structured illumination microscope apparatus of this invention. （Ａ）は、音響光学素子の超音波伝搬路Ｒ内に生起する超音波定在波のパターンを示す模式図であり、（Ｂ）は、それに対応する構造化照明のパターン（明部及び暗部の配置）を示す模式図である。（Ｃ）～（Ｅ）は、波本数が変化したときの縞本数の変化を説明する図である。(A) is a schematic diagram which shows the pattern of the ultrasonic standing wave which arises in the ultrasonic propagation path R of an acoustooptic device, (B) is the pattern of the structured illumination (bright part and dark part) corresponding to it. FIG. (C) to (E) are diagrams for explaining changes in the number of stripes when the number of waves changes. （Ａ）は、長さＬと距離Ｄとの関係を説明する図であり、（Ｂ）は、スポットＳに対応する構造化照明Ｓ’の概念図であり、（Ｃ）は、構造化照明Ｓ’の縞本数のずれを説明する図である。(A) is a figure explaining the relationship between length L and distance D, (B) is a conceptual diagram of structured illumination S 'corresponding to spot S, (C) is structured illumination. It is a figure explaining the shift | offset | difference of the stripe number of S '. 音響光学素子を示す正面図、及び側面図である。It is the front view and side view which show an acoustooptic device. 音響光学素子を駆動する駆動回路（制御手段）を説明する説明図である。It is explanatory drawing explaining the drive circuit (control means) which drives an acousto-optic device. ＣＰＵの動作手順を示すフローチャートである。It is a flowchart which shows the operation | movement procedure of CPU. 音響光学素子の他の例を示す正面図、及び側面図である。It is the front view and side view which show the other example of an acoustooptic device.
　本発明の一実施形態を示す構造化照明顕微鏡装置１０は、図１に示すように、照明光学系（本発明の「照明光学装置」に相当）１１、結像光学系１２、光検出器１３、及び画像処理手段１４で構成されている。 As shown in FIG. 1, a structured illumination microscope apparatus 10 showing an embodiment of the present invention includes an illumination optical system (corresponding to “illumination optical apparatus” of the present invention) 11, an imaging optical system 12, and a photodetector 13. And image processing means 14.
　照明光学系１１は、不図示の光源からの照明光を標本１６に導くものであり、光ファイバー１７、コレクタレンズ１８、音響光学素子（光変調器）１９、コンデンサレンズ２０、０次光カットマスク２１、リレーレンズ２２、視野絞り２３、照明レンズ２４、ダイクロイックミラー２５、対物レンズ２６、及び制御部２７等で構成される。標本１６は、例えば蛍光染色された生体標本であり、不図示のステージ上に載置されている。なお、符号１６ａは、標本１６の観察対象面（標本面）である。 The illumination optical system 11 guides illumination light from a light source (not shown) to the specimen 16, and includes an optical fiber 17, a collector lens 18, an acoustooptic device (light modulator) 19, a condenser lens 20, and a zero-order light cut mask 21. , Relay lens 22, field stop 23, illumination lens 24, dichroic mirror 25, objective lens 26, and control unit 27. The specimen 16 is a biological specimen that is fluorescently stained, for example, and is placed on a stage (not shown). Reference numeral 16a denotes an observation target surface (sample surface) of the sample 16.
　結像光学系１２は、対物レンズ２６、ダイクロイックミラー２５、及び結像レンズ２８等で構成されている。また、光検出器は、撮像部（ＣＣＤカメラ等）２９になっている。結像光学系１２は、対物レンズ２６で集光され、ダイクロイックミラー２５を透過した標本１６からの光を結像レンズ２８により撮像部２９の撮像面２９ａに結像する。また、画像処理手段は、制御部２７、画像記憶・演算部（コンピュータ等）３０、及び画像表示部３１等で構成される。 The imaging optical system 12 includes an objective lens 26, a dichroic mirror 25, an imaging lens 28, and the like. The photodetector is an imaging unit (CCD camera or the like) 29. The imaging optical system 12 focuses the light from the sample 16 collected by the objective lens 26 and transmitted through the dichroic mirror 25 onto the imaging surface 29 a of the imaging unit 29 by the imaging lens 28. The image processing means includes a control unit 27, an image storage / calculation unit (such as a computer) 30, and an image display unit 31.
　光ファイバー１７は、不図示の可干渉光源からの光を導光し、その出射端に二次点光源（過干渉な二次点光源）を形成する。なお、不図示の可干渉光源の波長は、標本１６の励起波長と同じ波長に設定されている。その二次点光源から射出した光は、コレクタレンズ１８によって平行光に変換され、音響光学素子１９へ入射する。 The optical fiber 17 guides light from a coherent light source (not shown), and forms a secondary point light source (over-interfering secondary point light source) at the emission end. The wavelength of a coherent light source (not shown) is set to the same wavelength as the excitation wavelength of the specimen 16. The light emitted from the secondary point light source is converted into parallel light by the collector lens 18 and enters the acoustooptic device 19.
　音響光学素子１９は、照明光学系１１の光軸上で、かつ標本１６と共役な位置に配置されている。音響光学素子１９で入射光を回折し、その回折光を用いて標本面１６ａ上に干渉縞を形成する。詳しく説明すると、音響光学素子１９は、圧電素子等のトランスデューサを音響光学媒体に貼り付けたものであり、トランスデューサに高周波の交流電圧が印加されると厚み方向に振動し、トランスデューサの形成面から音響光学媒体中に超音波を伝搬する。 The acousto-optic element 19 is arranged on the optical axis of the illumination optical system 11 and at a position conjugate with the sample 16. The incident light is diffracted by the acoustooptic device 19, and interference fringes are formed on the sample surface 16a using the diffracted light. More specifically, the acousto-optic element 19 is obtained by attaching a transducer such as a piezoelectric element to an acousto-optic medium. When a high-frequency AC voltage is applied to the transducer, the acousto-optic element 19 vibrates in the thickness direction and is Propagates ultrasonic waves into the optical medium.
　音響光学素子１９は、超音波を音響光学媒体の中に伝搬する超音波伝搬路Ｒを、光源からの所定の波長を有する射出光束を横切る方向に有しており、その超音波伝搬路Ｒに超音波の平面定在波（以下、「超音波定在波」という。）を生起させることにより、超音波伝搬路Ｒに正弦波状の屈折率分布を付与する。このような音響光学素子３は、入射光に対して位相型回折格子の働きをし、その光を各次数の回折光に分岐する。図１において実線で示したのは０次回折光であり、点線で示したのは±１次回折光である。 The acoustooptic device 19 has an ultrasonic wave propagation path R for propagating ultrasonic waves into the acoustooptic medium in a direction across an emitted light beam having a predetermined wavelength from the light source. By generating an ultrasonic plane standing wave (hereinafter referred to as “ultrasonic standing wave”), a sinusoidal refractive index distribution is imparted to the ultrasonic wave propagation path R. Such an acoustooptic device 3 functions as a phase-type diffraction grating for incident light, and branches the light into diffracted light of each order. In FIG. 1, the solid line indicates the 0th order diffracted light, and the dotted line indicates the ± 1st order diffracted light.
　音響光学素子１９から出射した各次数の回折光は、コンデンサレンズ２０を通過した後に、０次光カットマスク２１に入射する。０次光カットマスク２１は、対物レンズ２６の瞳面Ｐの共役位置、又はその近傍に配置されており、共役位置へ入射した各次数の回折光のうち、０次回折光、及び２次以降の高次回折光をカットし、かつ±１次回折光のみ（２光速のみ）を通過させる機能を有する。０次光カットマスク２１は、基板に複数の開口部、又は透過部を形成したものである。光路挿入時における開口部、又は透過部の位置は、共役な位置において±１次回折光が通過する領域に対応する。 The diffracted light of each order emitted from the acoustooptic device 19 enters the 0th-order light cut mask 21 after passing through the condenser lens 20. The 0th-order light cut mask 21 is disposed at or near the conjugate position of the pupil plane P of the objective lens 26, and among the diffracted light of each order incident on the conjugate position, the 0th-order diffracted light and the second and subsequent orders. It has a function of cutting high-order diffracted light and allowing only ± first-order diffracted light (only the speed of two lights) to pass. The 0th-order light cut mask 21 is a substrate in which a plurality of openings or transmission portions are formed. The position of the opening or transmission part when the optical path is inserted corresponds to a region through which ± first-order diffracted light passes at a conjugate position.
　０次光カットマスク２１を通過した±１次回折光は、リレーレンズ２２を通過した後に標本面１６ａの共役位置、又はその近傍に配置された視野絞り２３に入射する。視野絞り２３は、標本面１６ａ上の照明領域（観察領域）のサイズを制御する機能を有している。
　ここで、共役位置とは、レンズ２０の焦点距離（後ろ側焦点位置）であって、後述する対物レンズ２６の瞳Ｐ（±１次回析光が集光する位置）に対して照明レンズ２４、及びリレーレンズ２２を介して共役な位置（瞳共役面）をいう（なお、「共役な位置」の概念には、当業者が対物レンズ２６、リレーレンズ２２、及び照明レンズ２４の収差、ビネッティング等、設計上必要な事項を考慮して決定した位置も含まれる。）。 After passing through the 0th-order light cut mask 21, the ± 1st-order diffracted light enters the field stop 23 disposed at or near the conjugate position of the sample surface 16a after passing through the relay lens 22. The field stop 23 has a function of controlling the size of the illumination area (observation area) on the sample surface 16a.
Here, the conjugate position is a focal length (back focal position) of the lens 20, and the illumination lens 24, with respect to a pupil P (a position where ± 1 next-order analysis light is condensed) of an objective lens 26 described later, And a conjugate position (pupil conjugate plane) via the relay lens 22 (note that the concept of “conjugate position” has been described by those skilled in the art in terms of the aberrations and vignetting of the objective lens 26, the relay lens 22, and the illumination lens 24. Etc., including positions determined in consideration of design requirements).
　視野絞り２３を通過した±１次回折光は、照明レンズ２４を通過した後にダイクロイックミラー２５へ入射し、そのダイクロイックミラー２５を反射する。ダイクロイックミラー２５を反射した±１次回折光は、対物レンズ２６の瞳面Ｐ上の互いに異なる位置にそれぞれスポットを形成する。なお、瞳面Ｐにおける２つのスポットの形成位置は、瞳面Ｐの概ね最外周部であって、対物レンズ２６の光軸に関して互いに対称な位置である。 The ± first-order diffracted light that has passed through the field stop 23 enters the dichroic mirror 25 after passing through the illumination lens 24 and is reflected by the dichroic mirror 25. The ± first-order diffracted lights reflected by the dichroic mirror 25 form spots at different positions on the pupil plane P of the objective lens 26, respectively. It should be noted that the formation positions of the two spots on the pupil plane P are approximately the outermost peripheral portion of the pupil plane P, and are symmetrical with respect to the optical axis of the objective lens 26.
　したがって、対物レンズ２６の先端から射出する±１次回折光は、対物レンズ２６のＮＡ（開口数）に相当する角度で互いに反対の方向から標本面１６ａを照射する。なお、後述するように印加電圧の周波数を微少に変更させた結果、回折格子ピッチ（１周期）が微少に変化した場合には、２つのスポットの形成位置が極めて微少に変化する。ここで、標本面１６ａに照射されるこれらの±１次回折光は、可干渉光源から射出した互いに可干渉な光である。このため、標本面１６ａには、縞ピッチが一様なストライプ状の干渉縞（以下「縞」）が投影される。よって、これらの±１次回折光により標本面１６ａの照明パターンは、縞構造を持った照明パターンとなる。このように縞構造を持った照明パターンによる照明が、構造化照明である。構造化照明された標本面１６ａの蛍光領域（前述した蛍光染色された領域）では蛍光物質が励起され、蛍光を発する。 Therefore, the ± first-order diffracted light emitted from the tip of the objective lens 26 irradiates the sample surface 16a from opposite directions at an angle corresponding to the NA (numerical aperture) of the objective lens 26. As will be described later, when the diffraction grating pitch (one cycle) changes slightly as a result of changing the frequency of the applied voltage slightly, the formation positions of the two spots change extremely slightly. Here, these ± 1st order diffracted lights irradiated on the sample surface 16a are coherent lights emitted from a coherent light source. For this reason, striped interference fringes (hereinafter referred to as “fringes”) having a uniform fringe pitch are projected onto the specimen surface 16a. Therefore, the illumination pattern of the specimen surface 16a becomes an illumination pattern having a stripe structure by these ± 1st order diffracted lights. The illumination by the illumination pattern having the stripe structure in this way is structured illumination. In the fluorescent region of the structured-illuminated specimen surface 16a (the fluorescently stained region described above), the fluorescent material is excited and emits fluorescence.
　なお、この構造化照明は、±１次回折光の２光束のみからなるので、標本１６の面内方向にかけては構造化されているが、標本１６の深さ方向（光軸方向）にかけては構造化されていない。このような構造化照明を２光束構造化照明と称す。 Since this structured illumination consists of only two light beams of ± first-order diffracted light, it is structured in the in-plane direction of the specimen 16, but is structured in the depth direction (optical axis direction) of the specimen 16. It has not been. Such structured illumination is referred to as two-beam structured illumination.
　ここで、２光束構造化照明によると、構造化照明の空間周波数と蛍光領域の構造を持つ空間周波数との差を空間周波数として持つモアレ縞が標本面１６ａに現れる。このモアレ縞上では、蛍光領域の構造の空間周波数が変調されており、実際よりも低い空間周波数にシフトしている。したがって、構造化照明によると、蛍光領域の構造のうち空間周波数の高い成分を示す蛍光、すなわち対物レンズ２６の解像限界を超える大角度で射出した蛍光までもが対物レンズ２６へ入射することができる。 Here, according to the two-beam structured illumination, moire fringes having a difference between the spatial frequency of the structured illumination and the spatial frequency having the structure of the fluorescent region as a spatial frequency appear on the sample surface 16a. On this moire fringe, the spatial frequency of the structure of the fluorescent region is modulated and shifted to a lower spatial frequency than actual. Therefore, according to structured illumination, even fluorescence that shows a component having a high spatial frequency in the structure of the fluorescent region, that is, fluorescence emitted at a large angle exceeding the resolution limit of the objective lens 26 may enter the objective lens 26. it can.
　標本面１６ａから出射して対物レンズ２６へ入射した蛍光は、対物レンズ２６により平行光に変換された後にダイクロイックミラー２５へ入射する。その蛍光は、ダイクロイックミラー２５を透過した後、結像レンズ２８を通過することにより撮像部２９の撮像面２９ａ上に標本面１６ａの蛍光像を形成する。この蛍光像には、標本面１６ａの蛍光領域の構造情報と構造化照明の構造情報とが含まれている。 Fluorescence emitted from the specimen surface 16 a and incident on the objective lens 26 is converted into parallel light by the objective lens 26 and then incident on the dichroic mirror 25. The fluorescence passes through the imaging lens 28 after passing through the dichroic mirror 25, thereby forming a fluorescence image of the sample surface 16 a on the imaging surface 29 a of the imaging unit 29. This fluorescent image includes the structure information of the fluorescent region of the specimen surface 16a and the structure information of structured illumination.
　制御部２７は、光源の波長に応じて標本面１６ａに投影される干渉縞が所望の縞ピッチを持つように音響光学素子１９を制御する。具体的には、制御部２７は、音響光学素子１９に印加する電圧（高周波電圧）の周波数を、光源の波長に応じて調整することで、音響光学素子１９が発生する音波の波長を調整する。構造化照明顕微鏡装置１０の分解能は、照明光学系１１が作る干渉縞の縞ピッチで決まる。制御部２７は、音響光学素子１９に印加する電圧の周波数を変えることで、縞ピッチが最も細かくなるように音波の波長を調整して、構造化照明顕微鏡装置１０の分解能を高める。 The control unit 27 controls the acoustooptic device 19 so that the interference fringes projected on the sample surface 16a have a desired fringe pitch according to the wavelength of the light source. Specifically, the control unit 27 adjusts the wavelength of the sound wave generated by the acoustooptic device 19 by adjusting the frequency of the voltage (high frequency voltage) applied to the acoustooptic device 19 according to the wavelength of the light source. . The resolution of the structured illumination microscope apparatus 10 is determined by the fringe pitch of interference fringes created by the illumination optical system 11. The control unit 27 changes the frequency of the voltage applied to the acoustooptic device 19 to adjust the wavelength of the sound wave so that the fringe pitch becomes the finest, thereby increasing the resolution of the structured illumination microscope apparatus 10.
　ここで、縞ピッチを「Ｐ」、光源の光の波長を「λ」、対物レンズ２６の開口数を「ＮＡ」とすると、［数１］に記載の式を満足することを特徴とする。つまり、縞ピッチＰは、光源の波長λに比例することが分かる。 Here, when the fringe pitch is “P”, the wavelength of the light of the light source is “λ”, and the numerical aperture of the objective lens 26 is “NA”, the expression described in [Equation 1] is satisfied. That is, it can be seen that the fringe pitch P is proportional to the wavelength λ of the light source.
［数１］
Ｐ≧λ／（２＊ＮＡ） [Equation 1]
P ≧ λ / (2 * NA)
　制御部２７は、音響光学素子１９に電圧を印加すると、音響光学媒質内をその振動が音波として進行し、音響光学媒質の反対面で反射して戻り、それらが重なり合う。音響光学媒質内に発生する音波の周期は、印加電圧の周波数によってほぼ決まるが、音響光学媒質内において半周期の音波が整数個存在する場合に定在波となる。定在波は、超音波の疎密波により音響光学媒質中に周期的な屈折率の変化が生じて、回折格子として作用し、光を分岐する。 When the control unit 27 applies a voltage to the acoustooptic device 19, the vibration proceeds in the acoustooptic medium as a sound wave, is reflected by the opposite surface of the acoustooptic medium, and overlaps. The period of sound waves generated in the acousto-optic medium is substantially determined by the frequency of the applied voltage, but becomes a standing wave when there are an integer number of half-cycle sound waves in the acousto-optic medium. The standing wave causes a periodic refractive index change in the acousto-optic medium due to the ultrasonic dense wave, acts as a diffraction grating, and branches light.
　なお、定在波は、完全なものである必要はなく、印加電圧（高周波電圧）の周波数が音響光学媒質内で定在波の立つ条件から多少ずれがあってもよい。定在波の波長は、高周波電圧の周波数で制御することができる。すなわち、０次光と１次光との間の角度（分岐角）を制御することができる。 Note that the standing wave does not have to be complete, and the frequency of the applied voltage (high-frequency voltage) may slightly deviate from the condition in which the standing wave stands in the acousto-optic medium. The wavelength of the standing wave can be controlled by the frequency of the high frequency voltage. That is, the angle (branching angle) between the 0th order light and the 1st order light can be controlled.
　蛍光色素に最適な励起波長帯を選択した光源の光、つまり励起光は、音響光学素子１９を通ると複数の光に分岐されるが、その後、コンデンサレンズ２０を通り光の向きを平行にし、ダイクロイックミラー２５により顕微鏡観察光学経路（結像光学系１２を含む）内に入射され、対物レンズ２６により光の向きを内側に曲げ、標本面１６ａにセットされた標本１６上で光が重なり（干渉して）一次元格子模様（縞模様）の励起照明光となる。その際、対物レンズ２６から標本１６への最大入射角度θで照明光を照射すると最大分解能が得られることが知られている。これを利用し、対物レンズ２６を透過した後の光の進む角度は、０次光と+１次光がそれぞれ、対物レンズ２６から標本１６への最大入射角度θとなるように構成されている。 The light of the light source that has selected the optimum excitation wavelength band for the fluorescent dye, that is, the excitation light is branched into a plurality of lights when passing through the acoustooptic device 19, and then passes through the condenser lens 20 to make the direction of the light parallel, The light is incident on the microscope observation optical path (including the imaging optical system 12) by the dichroic mirror 25, the direction of the light is bent inward by the objective lens 26, and the light is overlapped on the specimen 16 set on the specimen surface 16a (interference). It becomes excitation illumination light of a one-dimensional lattice pattern (stripe pattern). At this time, it is known that the maximum resolution can be obtained when the illumination light is irradiated from the objective lens 26 to the specimen 16 at the maximum incident angle θ. Utilizing this, the light traveling angles after passing through the objective lens 26 are configured such that the 0th-order light and the + 1st-order light have the maximum incident angle θ from the objective lens 26 to the specimen 16, respectively. .
　標本１６の励起波長に応じて光源の光の波長を変えたい場合には、音響光学素子１９に印加される電力の周波数を変えて分岐角が変わらないように制御する。０次光と+１次光の間の分岐角を「θ１」、光源の波長を「λ」、音響光学媒質を伝搬する音波の波長を「Λ」、印加電圧の周波数を「ｆ」、音響光学媒体を伝播する音波の速度を「ｖ」とすると、［数２］に記載の式を満足する。なお、音響光学媒体を伝搬する音波の速度「ｖ」は一定である。 When it is desired to change the light wavelength of the light source in accordance with the excitation wavelength of the specimen 16, the frequency of power applied to the acoustooptic device 19 is changed so that the branching angle does not change. The branch angle between the 0th order light and the + first order light is “θ1”, the wavelength of the light source is “λ”, the wavelength of the sound wave propagating through the acoustooptic medium is “Λ”, the frequency of the applied voltage is “f”, When the velocity of the sound wave propagating through the optical medium is “v”, the equation described in [Equation 2] is satisfied. Note that the velocity “v” of the sound wave propagating through the acousto-optic medium is constant.
［数２］
Ｓｉｎθ１＝λ／Λ／２＝λ＊ｆ／ｖ／２ [Equation 2]
Sin θ1 = λ / Λ / 2 = λ * f / v / 2
　分解能を維持するためには、分岐角「θ１」を維持しなければならず、光源の光の波長「λ」が変わったときには、印加電圧の周波数「ｆ」を変えて対応すればよい。 In order to maintain the resolution, the branching angle “θ1” must be maintained, and when the light wavelength “λ” of the light source changes, the frequency “f” of the applied voltage may be changed.
　本実施形態の制御部２７は、構造化照明の位相をシフトする際にも音響光学素子１９に印加される電圧の周波数を変える。なお、この周波数の変化量は十分少ないため、最大分解能を維持するのに大きな影響はない。 The control unit 27 of the present embodiment changes the frequency of the voltage applied to the acoustooptic device 19 even when shifting the phase of structured illumination. Since the amount of change in frequency is sufficiently small, there is no significant effect on maintaining the maximum resolution.
　具体的には、本実施形態の制御部２７は、音響光学素子１９への印加電圧の周波数を変えて、音響光学素子１９の超音波伝搬路Ｒに生起する超音波定在波を制御することにより、構造化照明の位相シフト量を２π／３ずつステップ状に変化させる。そして、制御部２７は、構造化照明の位相が各状態にあるときに撮像部２９を駆動して３種類の画像データＩ－１、Ｉ０、Ｉ＋１を取得し、それらの画像データＩ－１、Ｉ０、Ｉ＋１を順次に画像記憶・演算部３０へ送出する。 Specifically, the control unit 27 of the present embodiment controls the ultrasonic standing wave generated in the ultrasonic wave propagation path R of the acoustooptic device 19 by changing the frequency of the voltage applied to the acoustooptic device 19. Thus, the phase shift amount of the structured illumination is changed stepwise by 2π / 3. Then, the control unit 27 drives the imaging unit 29 to obtain three types of image data I-1, I0, I + 1 when the structured illumination phase is in each state, and the image data I-1, I0 and I + 1 are sequentially sent to the image storage / arithmetic unit 30.
　画像記憶・演算部３０は、取り込まれた画像データＩ－１、Ｉ０、Ｉ＋１に対して復調演算を施すことにより、蛍光領域の構造情報の空間周波数が実際の空間周波数に戻された画像データＩ’を取得し、その画像データＩ’を画像表示部３１へ送出する。なお、具体的な演算には、例えば米国特許第８１１５８０６号明細書に開示された方法を用いることができる。これによって、画像表示部３１には、対物レンズ２６の解像限界を超えた解像画像（超解像画像）が表示される。 The image storage / calculation unit 30 performs a demodulation operation on the captured image data I-1, I0, I + 1, so that the image data I in which the spatial frequency of the structure information of the fluorescent region is returned to the actual spatial frequency. 'Is acquired and the image data I' is sent to the image display unit 31. For the specific calculation, for example, the method disclosed in US Pat. No. 8,115,806 can be used. As a result, a resolution image (super-resolution image) exceeding the resolution limit of the objective lens 26 is displayed on the image display unit 31.
　図２（Ａ）は、超音波伝搬路Ｒ内に生起する超音波定在波のパターンを示す模式図であり、同図（Ｂ）は、それに対応する構造化照明のパターン（明部及び暗部の配置）を示す模式図である（但し、超音波伝搬路Ｒのパターンのうち、実際の構造化照明に反映されるのは、有効な光束が通過する部分のパターンのみである）。また、図２（Ａ）では、説明をわかりやすくするため、超音波伝搬路Ｒに生起する超音波定在波の波本数を実際よりも少ない「２」とした。 FIG. 2A is a schematic diagram showing a pattern of an ultrasonic standing wave generated in the ultrasonic wave propagation path R, and FIG. 2B shows a corresponding structured illumination pattern (bright part and dark part). (However, in the pattern of the ultrasonic wave propagation path R, only the pattern of the portion through which the effective light beam passes is reflected in the actual structured illumination). Further, in FIG. 2A, in order to make the explanation easy to understand, the number of ultrasonic standing waves generated in the ultrasonic wave propagation path R is set to “2”, which is smaller than the actual number.
　図２（Ａ）に示すとおり、超音波定在波の波本数（位相変化２πで波本数１本とカウントする）が「２」であるときには、図２（Ｂ）に示すとおり、±１次光の干渉による構造化照明の縞本数（明部又は暗部の本数）は「４」となる。つまり、構造化照明の縞本数は、それに対応する超音波定在波の波本数の２倍となる。 As shown in FIG. 2 (A), when the number of ultrasonic standing waves (counting as one wave number with a phase change of 2π) is “2”, as shown in FIG. The number of stripes of structured illumination due to light interference (the number of bright portions or dark portions) is “4”. That is, the number of stripes of structured illumination is twice the number of ultrasonic standing waves corresponding thereto.
　したがって、図２（Ｃ）、（Ｄ）、（Ｅ）に示すとおり超音波定在波の波本数を２、（２＋１／２）、３のように１／２ずつ３通りに変化させたならば（即ち、超音波定在波の波長を変化させたならば）、それに対応する構造化照明の縞本数は４、５、６のように１ずつ３通りに変化する。 Therefore, as shown in FIGS. 2C, 2D, and 2E, if the number of ultrasonic standing waves is changed in three increments of 1/2, such as 2, (2 + 1/2), and 3, If the wavelength of the ultrasonic standing wave is changed, the number of fringes of the structured illumination corresponding to it changes in three ways one by one, like 4, 5, and 6.
　ここで、図２中に白矢印で示すとおり超音波伝搬路Ｒの一端から１／２だけずれた部分のみに着目すると、その着目部分に対応する構造化照明の位相は、「π」ずつ３通りに変化している。 Here, when attention is paid only to a portion shifted by ½ from one end of the ultrasonic wave propagation path R as indicated by a white arrow in FIG. 2, the phase of the structured illumination corresponding to the focused portion is “π” by 3. It is changing on the street.
　また、図２中に黒矢印で示すとおり超音波伝搬路Ｒの一端から１／３だけずれた部分のみに着目すると、その着目部分に対応する構造化照明の位相は、「２π／３」ずつ３通りに変化している。 Further, when attention is paid only to a portion shifted by 1/3 from one end of the ultrasonic wave propagation path R as indicated by a black arrow in FIG. 2, the phase of the structured illumination corresponding to the focused portion is “2π / 3” by each. It has changed in three ways.
　よって、仮に、超音波伝搬路Ｒに対する光の入射領域を白矢印で示した位置のみに制限したならば、超音波定在波の波本数を１／２ずつ変化させるだけで、構造化照明の位相を「π」ずつ変化させることができる。 Therefore, if the incident region of the light with respect to the ultrasonic wave propagation path R is limited to only the position indicated by the white arrow, the structured illumination of the structured illumination can be obtained by changing the wave number of the ultrasonic standing wave by 1/2. The phase can be changed by “π”.
　また、仮に、超音波伝搬路Ｒに対する光の入射領域を黒矢印で示した位置のみに制限したならば、超音波定在波の波本数を１／２ずつ変化させるだけで、構造化照明の位相を「２π／３」ずつ変化させることができる。 Also, if the incident area of light with respect to the ultrasonic wave propagation path R is limited to only the position indicated by the black arrow, the structured illumination of the structured illumination can be obtained by changing the number of ultrasonic standing waves by half. The phase can be changed by “2π / 3”.
　そこで本実施形態では、１ステップ当たりの位相シフト量を２π／３とするべく、図３（Ａ）に示すとおり、超音波伝搬路Ｒへ入射する光のスポット（有効径）Ｓの中心から超音波伝搬路Ｒの一端までの距離Ｄは、超音波伝搬路Ｒの伝搬方向の長さＬの１／３倍に設定される（Ｄ＝Ｌ／３）。 Therefore, in the present embodiment, in order to set the phase shift amount per step to 2π / 3, as shown in FIG. 3A, the light is incident from the center of the spot (effective diameter) S of light incident on the ultrasonic wave propagation path R. The distance D to one end of the sound wave propagation path R is set to 1/3 times the length L in the propagation direction of the ultrasonic wave propagation path R (D = L / 3).
　但し、超音波伝搬路Ｒに生起する超音波定在波の波本数が１／２だけ変化すると、スポットＳの内部に生起する超音波定在波の波本数も少しずれるので、図３（Ｂ）に示すとおりスポットＳに対応する２光束構造化照明Ｓ’の縞本数も少しずれてしまう（但し、図３に示した波パターン及び縞パターンは模式図であって、波本数及び縞本数は実際の本数に一致しているとは限らない。）。 However, if the number of ultrasonic standing waves generated in the ultrasonic wave propagation path R changes by ½, the number of ultrasonic standing waves generated in the spot S also slightly shifts. ), The number of fringes of the two-beam structured illumination S ′ corresponding to the spot S also slightly deviates (however, the wave pattern and the fringe pattern shown in FIG. 3 are schematic diagrams, and the wave number and the fringe number are It does not always match the actual number.)
　そこで本実施形態では、超音波伝搬路Ｒの長さＬは、２光束構造化照明Ｓ’の縞本数のズレがほぼゼロとみなせるよう、スポットＳの径φに比べて十分に大きく設定される。 Therefore, in the present embodiment, the length L of the ultrasonic wave propagation path R is set to be sufficiently larger than the diameter φ of the spot S so that the deviation of the number of stripes of the two-beam structured illumination S ′ can be regarded as almost zero. .
　具体的には、超音波伝搬路Ｒの長さＬと、スポットＳの径φとは、２光束構造化照明Ｓ’の縞本数のズレの許容量δとに対して、φ／Ｌ＜δの関係を満たすように設定される。例えば、２光束構造化照明Ｓ’の縞本数のズレを０．１５本以下に抑える必要があったならば、その関係式は、φ／Ｌ≦０．１５となる。 Specifically, the length L of the ultrasonic wave propagation path R and the diameter φ of the spot S are φ / L <δ with respect to the allowable amount δ of the stripe number deviation of the two-beam structured illumination S ′. Is set to satisfy the relationship. For example, if it is necessary to suppress the deviation of the number of stripes of the two-beam structured illumination S ′ to 0.15 or less, the relational expression is φ / L ≦ 0.15.
　また、音響光学素子１９の超音波伝搬路Ｒ上でスポットＳの径φがφ／Ｌ＜δの関係を必ずしも満たしてなくてもよく、例えば、音響光学素子１９から射出した±１次回折光を視野絞り２３で絞った場合は、超音波伝搬路Ｒの長さＬ、標本面１６ａにおける照明領域（観察領域、視野領域）の直径φ’、標本面１６ａから音響光学素子１９への光学倍率ｍが、２光束構造化照明Ｓ’の縞本数のズレの許容量δに対して、φ’×ｍ／Ｌ＜δの関係を満たすように設定されればよい。 Further, the diameter φ of the spot S on the ultrasonic wave propagation path R of the acoustooptic device 19 does not necessarily satisfy the relationship of φ / L <δ. For example, the ± first-order diffracted light emitted from the acoustooptic device 19 is When the field stop 23 is used, the length L of the ultrasonic wave propagation path R, the diameter φ ′ of the illumination region (observation region, field region) on the sample surface 16a, and the optical magnification m from the sample surface 16a to the acoustooptic device 19 are obtained. May be set so as to satisfy the relationship of φ ′ × m / L <δ with respect to the allowable amount δ of the deviation of the number of stripes of the two-beam structured illumination S ′.
　本実施形態では、スポットＳの径φを４ｍｍと仮定する。この場合、超音波伝搬路Ｒの長さＬを３０ｍｍに設定すれば、図３（Ｃ）に示すとおり、２光束構造化照明Ｓ’の両端における縞のズレは、０．０６８本分程度に抑えられ、２光束構造化照明Ｓ’の全域における縞本数のズレは０．６８＋０．６８＝０．１３本程度に抑えられる。なお、図３（Ｃ）において点線で示したのは２光束構造化照明Ｓ’の理想パターン（縞本数のズレがゼロである場合のパターン）であり、実線で示したのは２光束構造化照明Ｓ’の実際のパターンであるが、わかりやすくするために両者のずれを強調して描いた。 In this embodiment, the diameter φ of the spot S is assumed to be 4 mm. In this case, if the length L of the ultrasonic propagation path R is set to 30 mm, as shown in FIG. 3C, the deviation of the stripes at both ends of the two-beam structured illumination S ′ is about 0.068. The deviation of the number of stripes in the entire area of the two-beam structured illumination S ′ is suppressed to about 0.68 + 0.68 = 0.13. In FIG. 3C, the dotted line indicates the ideal pattern of the two-beam structured illumination S ′ (pattern when the deviation of the number of stripes is zero), and the solid line indicates the two-beam structured. Although it is an actual pattern of the illumination S ′, the difference between the two is drawn for the sake of clarity.
　図４は、音響光学素子３の構成図であり、同図（Ａ）は、音響光学素子１９を正面（光軸方向）から見た図であり、同図（Ｂ）は、音響光学素子１９を側面（光軸に垂直な方向）から見た図である。 FIG. 4 is a configuration diagram of the acoustooptic device 3. FIG. 4A is a diagram of the acoustooptic device 19 viewed from the front (in the optical axis direction), and FIG. It is the figure which looked at from the side (direction perpendicular to the optical axis).
　本実施形態の音響光学素子１９は、リブ構造をもつ超音波伝搬路を３個もっている。具体的には、図４に示すように、単一の音響光学媒体３５を備え、音響光学媒体３５は、互いに平行をなす一対の側面（側面対）３５ａ－３５ｆ，３５ｂ－３５ｇ，３５ｃ－３５ｅを３対備えた正面視八角形のブロック状に形成されている。一方の側面１５ａ～１５ｃには、トランスデューサ３６～３８がリブ状に突出して設けられており、これによって他方の側面３５ｆ，３５ｇ，３５ｅとの間の音響光学媒体３５内に３つの超音波伝搬路Ｒａ，Ｒｂ，Ｒｃがそれぞれ形成される。 The acoustooptic device 19 of this embodiment has three ultrasonic propagation paths having a rib structure. Specifically, as shown in FIG. 4, the acoustooptic medium 35 includes a single acoustooptic medium 35, and the acoustooptic medium 35 has a pair of side surfaces (a pair of side surfaces) 35a-35f, 35b-35g, 35c-35e that are parallel to each other. Are formed in an octagonal block shape with three pairs. Transducers 36 to 38 are provided on one of the side surfaces 15a to 15c so as to project in a rib shape, thereby three ultrasonic propagation paths in the acoustooptic medium 35 between the other side surfaces 35f, 35g, and 35e. Ra, Rb, and Rc are formed.
　超音波伝搬路Ｒａは、トランスデューサ３６をもつ側面３５ａとそれに対向する側面３５ｆとの間に形成される伝搬路であり、また、超音波伝搬路Ｒｂは、トランスデューサ３７をもつ側面３５ｂとそれに対向する側面３５ｇとの間に形成される伝搬路であり、さらに、超音波伝搬路Ｒｃは、トランスデューサ３８をもつ側面３５ｃとそれに対向する側面３５ｅとの間に形成される伝搬路である。 The ultrasonic wave propagation path Ra is a propagation path formed between the side surface 35a having the transducer 36 and the side surface 35f opposite to the side surface 35a, and the ultrasonic wave propagation path Rb is opposite to the side surface 35b having the transducer 37. The propagation path is formed between the side face 35g, and the ultrasonic propagation path Rc is a propagation path formed between the side face 35c having the transducer 38 and the side face 35e facing the side face 35c.
　なお、音響光学媒体３５の材質は、屈折率の高い透明な光学材料、例えば石英ガラス、水晶、テルライトガラス、重フリントガラス、及びフリントガラス等であり、３つの側面対３５ａ－３５ｆ，３５ｂ－３５ｇ，３５ｃ－３５ｅ、及び２つの底面３５ｄ，３５ｈは、それぞれ十分な精度で研磨されている。 The material of the acousto-optic medium 35 is a transparent optical material having a high refractive index, such as quartz glass, quartz, tellurite glass, heavy flint glass, flint glass, etc., and three side pairs 35a-35f, 35b- 35g, 35c-35e, and the two bottom surfaces 35d, 35h are each polished with sufficient accuracy.
　ここで、３個の超音波伝搬路Ｒａ，Ｒｂ，Ｒｃの各々の長さＬは共通であり（Ｌ＝３０ｍｍ）、その長さＬは前述したスポットＳの径φに対して前述した条件を満たしている。また、３個の超音波伝搬路Ｒａ，Ｒｂ，Ｒｃは、各々の一端からＬ／３だけ離れた位置において６０度毎に異なる角度で交差している。その交差位置に前述したスポットＳの中心が位置する。なお、互いの交差させる角度としては６０度に限ることはない。また、超音波伝搬路の個数としては、少なくとも３個以上の奇数個あればよい。このように、所定角度ずつ交差させた複数の超音波伝搬路を択一的に切り換えて使用することで、標本１６の二次元の超解像観察をするための画像データを取得する度に構造化照明の方向を変化させる作用をなす。 Here, the length L of each of the three ultrasonic propagation paths Ra, Rb, and Rc is common (L = 30 mm), and the length L satisfies the above-described conditions for the diameter φ of the spot S described above. Satisfies. Further, the three ultrasonic propagation paths Ra, Rb, and Rc intersect at different angles every 60 degrees at a position separated by L / 3 from one end of each. The center of the spot S described above is located at the intersection position. Note that the angle of crossing each other is not limited to 60 degrees. The number of ultrasonic propagation paths may be at least an odd number of 3 or more. In this way, the structure is obtained each time image data for two-dimensional super-resolution observation of the specimen 16 is obtained by selectively switching and using a plurality of ultrasonic propagation paths crossing each other by a predetermined angle. It acts to change the direction of the illumination.
　トランスデューサ３６は、圧電体３９と、圧電体３９の上下面に個別に形成された２つの電極４０とを有した超音波トランスデューサであり、そのうち下面の電極４０を介して側面３５ａに接合されている。制御部２７から上下面の電極４０の間に高周波の交流電圧を印加すると、圧電体３９が厚み方向に振動し、超音波伝搬路Ｒａ内を平面超音波が往復する。上下面の電極４０の間に印加される交流電圧の周波数が特定の周波数（適正周波数）に設定された場合、その超音波は定在波となるので、超音波伝搬路Ｒａの屈折率には、超音波の伝搬方向にかけて正弦波状の分布が付与される。これによって、超音波伝搬路Ｒａは、超音波の伝搬方向と垂直な位相格子を持った位相型回折格子となる。以下、この超音波伝搬路Ｒａの伝搬方向を、「第１方向」と称す。 The transducer 36 is an ultrasonic transducer having a piezoelectric body 39 and two electrodes 40 individually formed on the upper and lower surfaces of the piezoelectric body 39, and is bonded to the side surface 35 a via the lower surface electrode 40. . When a high-frequency AC voltage is applied between the control unit 27 and the upper and lower electrodes 40, the piezoelectric body 39 vibrates in the thickness direction, and planar ultrasonic waves reciprocate in the ultrasonic wave propagation path Ra. When the frequency of the AC voltage applied between the upper and lower electrodes 40 is set to a specific frequency (appropriate frequency), the ultrasonic wave becomes a standing wave, so that the refractive index of the ultrasonic wave propagation path Ra is A sinusoidal distribution is given over the propagation direction of the ultrasonic wave. Thus, the ultrasonic wave propagation path Ra becomes a phase type diffraction grating having a phase grating perpendicular to the ultrasonic wave propagation direction. Hereinafter, the propagation direction of the ultrasonic wave propagation path Ra is referred to as a “first direction”.
　また、トランスデューサ３７も、トランスデューサ３６と同じ構成をしており、圧電体４１と、圧電体４１の上下面に個別に形成された２つの電極４２とを有し、そのうち下面の電極４２を介して側面３５ｂに接合されている。 The transducer 37 has the same configuration as that of the transducer 36, and includes a piezoelectric body 41 and two electrodes 42 formed individually on the upper and lower surfaces of the piezoelectric body 41. It is joined to the side surface 35b.
　したがって、トランスデューサ３７の２つの電極４２の間に適正周波数の交流電圧が印加されると、超音波伝搬路Ｒｂ内を平面超音波が伝搬するので、超音波伝搬路Ｒｂは、超音波の伝搬方向と垂直な位相格子を持った位相型回折格子となる。以下、この超音波伝搬路Ｒｂの伝搬方向を、「第２方向」と称す。この第２方向は、第１方向に対して６０°の角度を成す。 Therefore, when an alternating voltage having an appropriate frequency is applied between the two electrodes 42 of the transducer 37, the plane ultrasonic wave propagates in the ultrasonic wave propagation path Rb. Therefore, the ultrasonic wave propagation path Rb has a propagation direction of the ultrasonic wave. And a phase type diffraction grating having a perpendicular phase grating. Hereinafter, the propagation direction of the ultrasonic wave propagation path Rb is referred to as a “second direction”. The second direction forms an angle of 60 ° with the first direction.
　また、トランスデューサ３８も、トランスデューサ３６と同じ構成をしており、圧電体４３と、圧電体４３の上下面に個別に形成された２つの電極４４とを有し、そのうち下面の電極４４を介して側面３５ｃに接合されている。 The transducer 38 has the same configuration as that of the transducer 36, and includes a piezoelectric body 43 and two electrodes 44 formed individually on the upper and lower surfaces of the piezoelectric body 43. It is joined to the side surface 35c.
　したがって、トランスデューサ３８の２つの電極４４の間に適正周波数の交流電圧が印加されると、超音波伝搬路Ｒｃ内を平面超音波が伝搬するので、超音波伝搬路Ｒｃは、超音波の伝搬方向と垂直な位相格子を持った位相型回折格子となる。以下、この超音波伝搬路Ｒｃの伝搬方向を、「第３方向」と称す。この第３方向は、第１方向に対して－６０°の角度を成す。 Therefore, when an alternating voltage having an appropriate frequency is applied between the two electrodes 44 of the transducer 38, the plane ultrasonic wave propagates in the ultrasonic wave propagation path Rc. Therefore, the ultrasonic wave propagation path Rc has the ultrasonic wave propagation direction. And a phase type diffraction grating having a perpendicular phase grating. Hereinafter, the propagation direction of the ultrasonic wave propagation path Rc is referred to as a “third direction”. This third direction forms an angle of −60 ° with respect to the first direction.
　制御部２７には、図５に示すように、音響光学素子１９の駆動回路４６を備えている。駆動回路４６は、高周波交流電源４７と切り換えスイッチ４８とを備える。高周波交流電源４７は、音響光学素子１９へ供給されるべき交流電圧を生成する。その交流電圧の周波数は、制御部２７内のＣＰＵによって適正周波数（例えば、数十ＭＨｚ～１００ＭＨｚ内の何れかの値）に制御される。 The controller 27 includes a drive circuit 46 for the acoustooptic device 19 as shown in FIG. The drive circuit 46 includes a high frequency AC power supply 47 and a changeover switch 48. The high frequency AC power supply 47 generates an AC voltage to be supplied to the acoustooptic device 19. The frequency of the AC voltage is controlled to an appropriate frequency (for example, any value within several tens of MHz to 100 MHz) by the CPU in the control unit 27.
　本実施形態では、構造化照明された標本１６の像（変調像）を復調して位相の異なる複数の変調像のデータを取得するために、前述した２光束構造化照明Ｓ’の位相シフト量を－２π／３、０、＋２π／３の３通りにステップ状に変化させている。このため、ＣＰＵは、その交流電圧の周波数を、周波数の異なる３通りの適正周波数ｆ－１、ｆ０、ｆ＋１の間で僅かに切り換える。 In the present embodiment, the phase shift amount of the two-beam structured illumination S ′ described above is used to demodulate the image (modulated image) of the structured illumination sample 16 and obtain data of a plurality of modulated images having different phases. Are changed stepwise in three ways: -2π / 3, 0, and + 2π / 3. For this reason, the CPU slightly switches the frequency of the AC voltage between three appropriate frequencies f−1, f0, and f + 1 having different frequencies.
　例えば、適正周波数ｆ０は、長さＬが３０ｍｍである超音波伝搬路Ｒａ，Ｒｂ，Ｒｃに１００本の超音波定在波（それに対応する構造化照明の縞本数は２００）を生起させるための適正周波数（８０ＭＨｚ）である。この適正周波数ｆ０によると、２光束構造化照明Ｓ’の位相シフト量はゼロとなる。 For example, the appropriate frequency f0 is for causing 100 ultrasonic standing waves (the number of stripes of structured illumination corresponding to 200) to be generated in the ultrasonic propagation paths Ra, Rb, and Rc having a length L of 30 mm. Appropriate frequency (80 MHz). According to the appropriate frequency f0, the phase shift amount of the two-beam structured illumination S ′ is zero.
　この場合、適正周波数ｆ－１は、長さＬが３０ｍｍである超音波伝搬路Ｒａ，Ｒｂ，Ｒｃに（１００－１／２）本の超音波定在波（それに対応する構造化照明の縞本数は１９９）を生起させるための適正周波数（７９．９４６ＭＨｚ）となる。この適正周波数ｆ－１によると、２光束構造化照明Ｓ’の位相シフト量は－２π／３となる。 In this case, the proper frequency f-1 is (100-1 / 2) ultrasonic standing waves (corresponding to the structured illumination fringes) on the ultrasonic propagation paths Ra, Rb, Rc having a length L of 30 mm. The number is an appropriate frequency (79.946 MHz) for generating 199). According to the appropriate frequency f−1, the phase shift amount of the two-beam structured illumination S ′ is −2π / 3.
　また、適正周波数ｆ＋１は、長さＬが３０ｍｍである超音波伝搬路Ｒａ，Ｒｂ，Ｒｃに（１００＋１／２）本の超音波定在波（それに対応する構造化照明の縞本数は２０１）を生起させるための適正周波数（８０．０５４ＭＨｚ）となる。この適正周波数ｆ＋１によると、２光束構造化照明Ｓ’の位相シフト量は＋２π／３となる。 The appropriate frequency f + 1 is (100 + 1/2) ultrasonic standing waves (the number of fringes of structured illumination corresponding to 201) corresponding to ultrasonic propagation paths Ra, Rb, Rc having a length L of 30 mm. It becomes an appropriate frequency (80.54 MHz) for occurrence. According to this appropriate frequency f + 1, the phase shift amount of the two-beam structured illumination S ′ is + 2π / 3.
　切り換えスイッチ４８は、高周波交流電源４７と音響光学素子１９との間に配置され、音響光学素子１９の側の接続先を、音響光学素子１９の３つのトランスデューサ３６～３８の間で切り換えることが可能である。切り換えスイッチ４８の接続先は、制御部２７内のＣＰＵによって適宜に切り換えられる。 The changeover switch 48 is disposed between the high-frequency AC power supply 47 and the acoustooptic element 19, and the connection destination on the acoustooptic element 19 side can be switched between the three transducers 36 to 38 of the acoustooptic element 19. It is. The connection destination of the changeover switch 48 is appropriately changed over by the CPU in the control unit 27.
　切り換えスイッチ４８の接続先がトランスデューサ３６の側であるとき、交流電圧はトランスデューサ３６の２つの電極４０の間に印加されるので、３つの超音波伝搬路Ｒａ，Ｒｂ，Ｒｃのうち超音波伝搬路Ｒａのみが有効となる。 When the connection destination of the changeover switch 48 is on the transducer 36 side, an AC voltage is applied between the two electrodes 40 of the transducer 36, so that the ultrasonic wave propagation path among the three ultrasonic wave propagation paths Ra, Rb, Rc. Only Ra is valid.
　また、切り換えスイッチ４８の接続先がトランスデューサ３７の側であるとき、交流電圧はトランスデューサ３７の２つの電極４２の間に印加されるので、３つの超音波伝搬路Ｒａ，Ｒｂ，Ｒｃのうち超音波伝搬路Ｒｂのみが有効となる。 Further, when the connection destination of the changeover switch 48 is on the transducer 37 side, an AC voltage is applied between the two electrodes 42 of the transducer 37, so that the ultrasonic wave among the three ultrasonic wave propagation paths Ra, Rb, Rc. Only the propagation path Rb is effective.
　さらに、切り換えスイッチ４８の接続先がトランスデューサ３８の側であるとき、交流電圧はトランスデューサ３８の２つの電極の間に印加されるので、３つの超音波伝搬路Ｒａ，Ｒｂ，Ｒｃのうち超音波伝搬路Ｒｃのみが有効となる。 Furthermore, when the connection destination of the changeover switch 48 is on the transducer 38 side, an AC voltage is applied between the two electrodes of the transducer 38, so that the ultrasonic wave propagation among the three ultrasonic wave propagation paths Ra, Rb, Rc. Only the path Rc is valid.
　このように、有効な超音波伝搬路を３つの超音波伝搬路Ｒａ，Ｒｂ，Ｒｃの間で切り換えれば、２光束構造化照明Ｓ’の方向を第１方向に対応する方向と、第２方向に対応する方向と、第３方向に対応する方向との間で迅速に切り換えることができる。 As described above, when the effective ultrasonic wave propagation path is switched among the three ultrasonic wave propagation paths Ra, Rb, and Rc, the direction of the two-beam structured illumination S ′ is set to the direction corresponding to the first direction, and the second direction. It is possible to quickly switch between a direction corresponding to the direction and a direction corresponding to the third direction.
　図６は、ＣＰＵの動作フローチャートである。以下、各ステップを順に説明する。ここで、光源としては、標本１６の励起波長と同じ波長のものが使用される。音響光学素子３へ与える駆動信号（印加電圧）の周波数は、光源の光の波長に応じて調整されている。励起光波長を変える場合には、駆動信号の周波数を変えて分岐角が変わらないように制御する。これにより、構造化照明顕微鏡装置１０の分解能を最大に維持することができる。 FIG. 6 is an operational flowchart of the CPU. Hereinafter, each step will be described in order. Here, a light source having the same wavelength as the excitation wavelength of the specimen 16 is used. The frequency of the drive signal (applied voltage) applied to the acoustooptic device 3 is adjusted according to the wavelength of the light from the light source. When the pumping light wavelength is changed, control is performed so that the branch angle does not change by changing the frequency of the drive signal. Thereby, the resolution of the structured illumination microscope apparatus 10 can be maintained at the maximum.
　ステップＳ１１：ＣＰＵは、切り換えスイッチ４８の接続先を１番目のトランスデューサ（トランスデューサ３６）の側に設定することにより、２光束構造化照明Ｓ’の方向を第１方向に対応する方向に設定する。 Step S11: The CPU sets the direction of the two-beam structured illumination S ′ to a direction corresponding to the first direction by setting the connection destination of the changeover switch 48 to the first transducer (transducer 36) side.
　ステップＳ１２：ＣＰＵは、高周波交流電源４７が生成する交流電圧の周波数を、適正周波数ｆ－１に設定することにより、２光束構造化照明Ｓ’の位相シフト量を－２π／３に設定する。 Step S12: The CPU sets the phase shift amount of the two-beam structured illumination S ′ to −2π / 3 by setting the frequency of the AC voltage generated by the high-frequency AC power supply 47 to an appropriate frequency f−1.
　ステップＳ１３：ＣＰＵは、この状態で撮像部２９を駆動して画像データＩ－１を取得する。 Step S13: In this state, the CPU drives the imaging unit 29 to acquire the image data I-1.
　ステップＳ１４：ＣＰＵは、高周波交流電源４７が生成する交流電圧の周波数を、適正周波数ｆ０に設定することにより、２光束構造化照明Ｓ’の位相シフト量をゼロに設定する。 Step S14: The CPU sets the frequency of the AC voltage generated by the high-frequency AC power supply 47 to an appropriate frequency f0, thereby setting the phase shift amount of the two-beam structured illumination S ′ to zero.
　ステップＳ１５：ＣＰＵは、この状態で撮像部２９を駆動して画像データＩ０を取得する。 Step S15: The CPU drives the imaging unit 29 in this state to acquire the image data I0.
　ステップＳ１６：ＣＰＵは、高周波交流電源４７が生成する交流電圧の周波数を、適正周波数ｆ＋１に設定することにより、２光束構造化照明Ｓ’の位相シフト量を＋２π／３に設定する。 Step S16: The CPU sets the phase shift amount of the two-beam structured illumination S ′ to + 2π / 3 by setting the frequency of the AC voltage generated by the high-frequency AC power supply 47 to an appropriate frequency f + 1.
　ステップＳ１７：ＣＰＵは、この状態で撮像部２９を駆動して画像データＩ＋１を取得する。 Step S17: The CPU acquires the image data I + 1 by driving the imaging unit 29 in this state.
　ステップＳ１８：ＣＰＵは、２光束構造化照明Ｓ’の方向が前述した３方向の全てに設定済みであるか否かを判別し、設定済みで無い場合はステップＳ１９へ移行し、設定済みであった場合はフローを終了する。 Step S18: The CPU determines whether or not the direction of the two-beam structured illumination S ′ has been set in all the three directions described above. If not, the CPU proceeds to step S19 and has been set. If so, the flow ends.
　ステップＳ１９：ＣＰＵは、切り換えスイッチ４８の接続先を切り換えることにより２光束構造化照明Ｓ’の方向を切り換えてから、ステップＳ１２へ移行する。 Step S19: The CPU switches the connection destination of the changeover switch 48 to switch the direction of the two-beam structured illumination S ′, and then proceeds to step S12.
　以上のフローによると、第１方向に関する画像データＩａ－１、Ｉａ０、Ｉａ＋１と、第２方向に関する画像データＩｂ－１、Ｉｂ０、Ｉｂ＋１と、第３方向に関する画像データＩｃ－１、Ｉｃ０、Ｉｃ＋１とが取得される。これらの画像データは、画像記憶・演算部３０へ取り込まれる。 According to the above flow, the image data Ia-1, Ia0, Ia + 1 for the first direction, the image data Ib-1, Ib0, Ib + 1 for the second direction, the image data Ic-1, Ic0, Ic + 1 for the third direction, Is acquired. These image data are taken into the image storage / calculation unit 30.
　画像記憶・演算部３０は、一連の３つの画像データＩａ－１、Ｉａ０、Ｉａ＋１に対して復調演算を施すことにより、第１方向に亘る超解像画像の復調画像データＩａ’を取得する。 The image storage / calculation unit 30 obtains demodulated image data Ia ′ of the super-resolution image in the first direction by performing a demodulation operation on a series of three image data Ia−1, Ia0, and Ia + 1.
　また、画像記憶・演算部３０は、一連の３つの画像データＩｂ－１、Ｉｂ０、Ｉｂ＋１に対して復調演算を施すことにより、第２方向に亘る超解像画像の復調画像データＩｂ’を取得する。 In addition, the image storage / calculation unit 30 obtains demodulated image data Ib ′ of the super-resolution image in the second direction by performing a demodulation operation on the series of three image data Ib−1, Ib0, and Ib + 1. To do.
　また、画像記憶・演算部３０は、一連の３つの画像データＩｃ－１、Ｉｃ０、Ｉｃ＋１に対して復調演算を施すことにより、第３方向に亘る超解像画像の復調画像データＩｃ’を取得する。 The image storage / calculation unit 30 also obtains demodulated image data Ic ′ of a super-resolution image in the third direction by performing a demodulation operation on a series of three image data Ic−1, Ic0, and Ic + 1. To do.
　そして画像記憶・演算部３０は、３つの復調画像データＩａ’、Ｉｂ’、Ｉｃ’を波数空間上で合成してから再び実空間に戻すことにより、第１方向、第２方向、第３方向に亘る超解像画像の画像データＩを取得し、その画像データＩを画像表示部３１へ送出する。したがって、画像表示部３１には、標本面１６ａの蛍光領域の構造を詳細に示す超解像画像が表示される。 Then, the image storage / calculation unit 30 combines the three demodulated image data Ia ′, Ib ′, and Ic ′ in the wave number space and then returns them to the real space again, whereby the first direction, the second direction, and the third direction The image data I of the super-resolution image over the range is acquired, and the image data I is sent to the image display unit 31. Therefore, a super-resolution image showing in detail the structure of the fluorescent region of the specimen surface 16a is displayed on the image display unit 31.
　上記実施形態の構造化照明顕微鏡装置１０では、±１次回折光による干渉縞（２光束構造化照明）を標本面１６ａ（光軸をＺ方向とするとＸ-Ｙ面内）に形成する例を用いて説明しているが、本発明はこれに限らず、０次回折光及び±１次回折光による干渉縞（光軸方向にも干渉縞が形成される３光束構造化照明）を標本に形成する場合にも当然適用することができる。この場合には、０次光カットマスク２１の中央に穴を開けて０次光を通過させればよい。
　また、３光束構造化照明としては、３光束干渉縞を形成するために、回折次数の間隔が等間隔な３つの回折光による３光束干渉を生起させればよいので、前述した０次回折光及び±１次回折光の組み合わせに限らず、例えば０次回折光、１次回折光、２次回折光の組み合わせ、又は±２次回折光及び０次回折光の組み合わせ、あるいは±３次回折光及び０次回折光の組み合わせ、等を用いることが可能である。 In the structured illumination microscope apparatus 10 of the above embodiment, an example is used in which interference fringes (two-beam structured illumination) by ± first-order diffracted light are formed on the sample surface 16a (in the XY plane when the optical axis is in the Z direction). However, the present invention is not limited to this, and the case where interference fringes (three-beam structured illumination in which interference fringes are also formed in the optical axis direction) are formed on a sample by zero-order diffracted light and ± first-order diffracted light. Of course it can also be applied. In this case, it is only necessary to make a hole in the center of the 0th-order light cut mask 21 to pass the 0th-order light.
In addition, in the three-beam structured illumination, in order to form a three-beam interference fringe, three-beam interference caused by three diffracted lights with equal intervals between diffraction orders may be generated. Not limited to a combination of ± 1st order diffracted light, for example, a combination of 0th order diffracted light, 1st order diffracted light, 2nd order diffracted light, a combination of ± 2nd order diffracted light and 0th order diffracted light, or a combination of ± 3rd order diffracted light and 0th order diffracted light, etc. Can be used.
　ところで、開口数ＮＡが標本の屈折率よりも高い対物レンズを利用すると、標本の屈折率よりも大きなＮＡ領域を通った照明光は標本とカバーガラスの界面で全反射し、前記界面においてエバネッセント場を形成し標本内部には入っていかない。このエバネセント場を利用しカバーガラス界面のみ蛍光励起を行うことでバックグラウンドの極めて少ない蛍光観察を行うことができる全反射照明蛍光顕微鏡(ＴＩＲＦ(Total Internal Reflection Fluorescence))が知られている。 By the way, when an objective lens having a numerical aperture NA higher than the refractive index of the sample is used, the illumination light passing through the NA region larger than the refractive index of the sample is totally reflected at the interface between the sample and the cover glass, and the evanescent field at the interface. And does not enter the specimen. A total reflection illumination fluorescence microscope (TIRF (Total Internal Reflection Fluorescence)) capable of performing fluorescence observation with very little background by performing fluorescence excitation only on the cover glass interface using this evanescent field is known.
　ＴＩＲＦにおいては対物レンズの瞳外周部のわずかな領域のみを使った照明を行う必要がある。その際、従来から知られている同一ピッチの回折格子を異なる波長の励起光に使うと分岐角が変わってしまいＴＩＲＦ照明の要求を満たせなくなる。そのため、励起光の波長ごとに異なる周期の回折格子が必要となり、時間や手間がかかっていた。しかしながら、このようなＴＩＲＦにも本発明を採用することで、そのような手法が必要なくなり、時間の短縮、及び手間を少なくすることができる。 In TIRF, it is necessary to perform illumination using only a small area on the outer periphery of the pupil of the objective lens. At this time, if a conventionally known diffraction grating having the same pitch is used for excitation light having a different wavelength, the branch angle is changed and the requirement for TIRF illumination cannot be satisfied. Therefore, a diffraction grating with a different period is required for each wavelength of the excitation light, which takes time and labor. However, by adopting the present invention for such a TIRF, such a method is not necessary, and time can be reduced and labor can be reduced.
　ここで、ＴＩＲＦの場合、縞ピッチを「Ｐ」、光源の光の波長を「λ」、標本を光が通過する時の屈折率を「ｎ」、対物レンズの開口数を「ＮＡ」とすると、［数３］に記載の式を満足することを特徴とする。 Here, in the case of TIRF, when the stripe pitch is “P”, the wavelength of the light from the light source is “λ”, the refractive index when the light passes through the sample is “n”, and the numerical aperture of the objective lens is “NA”. , [Equation 3] is satisfied.
［数３］
λ／（２＊ｎ）（但しｎ＜ＮＡ）≧Ｐ≧λ／（２＊ＮＡ）（但しｎ≧ＮＡ） [Equation 3]
λ / (2 * n) (where n <NA) ≧ P ≧ λ / (2 * NA) (where n ≧ NA)
　このようにＴＩＲＦの場合も縞ピッチは、光源の光の波長に比例する。また、対物レンズの開口数で決まる縞ピッチよりも細かいものは作れないので、下限が決められる。そして、ＴＩＲＦの場合、標本の奥の方に光が行かないように全反射させるので、できれば上限と下限との中間の縞ピッチが最も望ましい。 Thus, also in the case of TIRF, the fringe pitch is proportional to the wavelength of light from the light source. In addition, since a finer pitch than the fringe pitch determined by the numerical aperture of the objective lens cannot be made, the lower limit is determined. In the case of TIRF, since total reflection is performed so that light does not go to the back of the specimen, an intermediate fringe pitch between the upper limit and the lower limit is most desirable.
　また、ＴＩＲＦ観察時に励起光波長を変えたい場合には、音響光学素子１９に印加される電圧の周波数を変えて、ＴＩＲＦ照明条件を維持できるようにする。すなわち、励起光波長が変化した場合でも音響光学素子１９における０次光と１次光との間の角度（分岐角）が変わらないように制御すればよい。すなわち、分岐角を「θ１」、光源の光の波長を「λ」、音響光学媒質を伝搬する音波の波長を「Λ」、印加電圧の周波数を「ｆ」、音響光学媒体を伝播する音波の速度を「ｖ」とすると、［数４］に記載の式を満足する。なお、音響光学媒体を伝播する音波の速度「ｖ」は一定である。 Further, when it is desired to change the excitation light wavelength during TIRF observation, the frequency of the voltage applied to the acoustooptic device 19 is changed so that the TIRF illumination conditions can be maintained. That is, even when the excitation light wavelength changes, the angle (branch angle) between the 0th order light and the 1st order light in the acoustooptic device 19 may be controlled so as not to change. That is, the branch angle is “θ1”, the wavelength of the light of the light source is “λ”, the wavelength of the sound wave propagating through the acoustooptic medium is “Λ”, the frequency of the applied voltage is “f”, and the sound wave propagating through the acoustooptic medium When the speed is “v”, the equation described in [Equation 4] is satisfied. The velocity “v” of the sound wave propagating through the acousto-optic medium is constant.
［数４］
Ｓｉｎθ１＝λ／Λ／２＝λ＊ｆ／ｖ／２ [Equation 4]
Sin θ1 = λ / Λ / 2 = λ * f / v / 2
　このように、ＴＩＲＦ照明条件を維持するために、光源の光の波長「λ」が変わったときには、印加電圧の周波数「ｆ」を変えることで分岐角「θ１」を維持すればよい。 Thus, in order to maintain the TIRF illumination condition, when the wavelength “λ” of the light source is changed, the branch angle “θ1” may be maintained by changing the frequency “f” of the applied voltage.
　上記実施形態の音響光学媒体３５は、３つの超音波伝搬路Ｒａ，Ｒｂ，ＲｃをスポットＳの中心に関して非対称な関係で配置していたが（図４参照）、例えば図７に示すとおり対称な関係で配置してもよい。因みに、図４に示す例の利点は、音響光学媒体３５の外形の凹凸が少ないところにあり、図７に示す例の利点は、３つの超音波伝搬路Ｒａ，Ｒｂ，Ｒｃの環境が完全に一致するところにある。 In the acoustooptic medium 35 of the above embodiment, the three ultrasonic propagation paths Ra, Rb, and Rc are arranged in an asymmetric relationship with respect to the center of the spot S (see FIG. 4). For example, as shown in FIG. You may arrange in relation. Incidentally, the advantage of the example shown in FIG. 4 is that the contour of the outer shape of the acoustooptic medium 35 is small, and the advantage of the example shown in FIG. 7 is that the environment of the three ultrasonic propagation paths Ra, Rb, Rc is completely There is a match.
　上記各実施形態では、光源の光の波長「λ」に応じて制御部２７が印加電圧の周波数「ｆ」を調整することで音響光学素子１９の音波の波長「Λ」を変えているが、本発明ではこれに限らず、制御部２７から駆動信号に応じて音響光学媒体を伝搬する音波の速度「ｖ」を変えるように制御してもよい。この場合には、駆動信号に応じて音響光学媒体を変形させる手段を設ければよい。また、音響光学媒体の温度を変えることで音波の波長「Λ」を変えるようにしてもよい。 In each of the above embodiments, the control unit 27 adjusts the frequency “f” of the applied voltage according to the wavelength “λ” of the light from the light source, thereby changing the wavelength “Λ” of the acoustic wave of the acoustooptic device 19. The present invention is not limited to this, and the control unit 27 may perform control so as to change the velocity “v” of the sound wave propagating through the acousto-optic medium in accordance with the drive signal. In this case, a means for deforming the acousto-optic medium according to the drive signal may be provided. Further, the wavelength “Λ” of the sound wave may be changed by changing the temperature of the acousto-optic medium.
　上記各実施形態では、超解像効果を標本面１６ａ上の各方向に亘り得るために、所定角度ずつ交差させた３個の超音波伝搬路Ｒａ，Ｒｂ，Ｒｃを設けているが、本発明ではこれに限らず、例えば１個の超音波伝搬路のみを設け、その超音波伝搬路の配置方向を回転させ、各々の回転位置において位相をシフトするようにしてもよいし、また、位相の異なる３個以上の奇数個の領域をもつ位相板を回転させて、各領域がセットされる回転位置で位相をそれぞれシフトするようにしてもよい。 In each of the above embodiments, in order to obtain the super-resolution effect in each direction on the sample surface 16a, the three ultrasonic propagation paths Ra, Rb, and Rc crossing each other by a predetermined angle are provided. However, the present invention is not limited to this. For example, only one ultrasonic propagation path may be provided, the arrangement direction of the ultrasonic propagation path may be rotated, and the phase may be shifted at each rotational position. A phase plate having three or more different odd-numbered areas may be rotated to shift the phase at the rotation position where each area is set.
　なお、本明細書において開示した以下の全ての文献について参照引用する（incorporated by reference）。
　１）米国特許第６２３９９０９号明細書
　２）米国特許第８１１５８０６号明細書 In addition, all the following documents disclosed in this specification are incorporated by reference.
1) US Pat. No. 6,239,909 2) US Pat. No. 8,115,806
　１０　構造化照明顕微鏡装置
　１１　照明光学系
　１６　標本
　１９　音響光学素子
　２７　制御部
　２６　対物レンズ DESCRIPTION OF SYMBOLS 10 Structured illumination microscope apparatus 11 Illumination optical system 16 Sample 19 Acousto-optic element 27 Control part 26 Objective lens

Claims (7)

  1. 　光源からの所定の波長を有する射出光束中に配置され、その射出光束を横切る方向に音波伝搬路を配置した光変調器と、
    　前記音波伝搬路を通過した前記射出光束の少なくとも２つの回折光を干渉させ、その干渉縞を被観察物に形成する照明光学系と、
    　前記射出光束が有する波長に応じて前記干渉縞が所望の縞ピッチを持つように前記光変調器を制御する制御手段と、
    　を備えたことを特徴とする構造化照明装置。     An optical modulator disposed in an emitted light beam having a predetermined wavelength from a light source, and a sound wave propagation path disposed in a direction crossing the emitted light beam;
    An illumination optical system that interferes with at least two diffracted lights of the emitted light flux that has passed through the sound wave propagation path, and forms interference fringes on the object to be observed;
    Control means for controlling the optical modulator so that the interference fringes have a desired fringe pitch according to the wavelength of the emitted light beam;
    A structured lighting apparatus comprising:
  2. 　請求項１に記載の構造化照明装置において、
    　前記制御手段は、
    　駆動信号を前記光変調器へ与えることにより前記音波伝搬路の媒体を振動させて前記音波伝搬路内に音波定在波を生起させる駆動手段を有し、
    　前記駆動手段は、
    　前記駆動信号を変化させることで、前記音波定在波の周波数を変化させて前記干渉縞の縞ピッチを調節する
    　ことを特徴とする構造化照明装置。     The structured lighting device according to claim 1,
    The control means includes
    Drive means for generating a sound wave standing wave in the sound wave propagation path by vibrating the medium of the sound wave propagation path by applying a drive signal to the optical modulator;
    The driving means includes
    By changing the drive signal, the fringe pitch of the interference fringe is adjusted by changing the frequency of the sound wave standing wave.
  3. 　請求項１又は２に記載の構造化照明装置において、
    　前記照明光学系は対物レンズを有し、
    　前記縞ピッチを「Ｐ」、前記光源の光の波長を「λ」、前記対物レンズの開口数を「ＮＡ」とすると、
    　Ｐ≧λ／（２＊ＮＡ）
    　の関係を満たす
    　ことを特徴とする構造化照明装置。     The structured lighting device according to claim 1 or 2,
    The illumination optical system has an objective lens,
    When the fringe pitch is “P”, the light wavelength of the light source is “λ”, and the numerical aperture of the objective lens is “NA”,
    P ≧ λ / (2 * NA)
    A structured lighting device characterized by satisfying the above relationship.
  4. 　請求項１又は２に記載の構造化照明装置において、
    　前記照明光学系は対物レンズを有し、
    　前記縞ピッチを「Ｐ」、前記光源の光の波長を「λ」、前記標本を光が通過する時の屈折率を「ｎ」、前記対物レンズの開口数を「ＮＡ」とすると、
    　λ／（２＊ｎ）（但しｎ＜ＮＡ）≧Ｐ≧λ／（２＊ＮＡ）（但しｎ≧ＮＡ）
    　の関係を満たす
    　ことを特徴とする構造化照明装置。     The structured lighting device according to claim 1 or 2,
    The illumination optical system has an objective lens,
    When the fringe pitch is “P”, the light wavelength of the light source is “λ”, the refractive index when light passes through the sample is “n”, and the numerical aperture of the objective lens is “NA”,
    λ / (2 * n) (where n <NA) ≧ P ≧ λ / (2 * NA) (where n ≧ NA)
    A structured lighting device characterized by satisfying the above relationship.
  5. 　請求項１から４のいずれか１項に記載の構造化照明装置と、
    　前記構造化照明装置で照明されたことにより前記被観察物から得られる観察光束を光検出器に結像する結像光学系と、
    　を備えたことを特徴とする構造化照明顕微鏡装置。     The structured lighting device according to any one of claims 1 to 4,
    An imaging optical system that forms an image of an observation light beam obtained from the observation object by being illuminated by the structured illumination device on a photodetector;
    A structured illumination microscope apparatus comprising:
  6. 　請求項５に記載の構造化照明顕微鏡装置において、
    　前記観察光束は、蛍光光束である
    　ことを特徴とする構造化照明顕微鏡装置。     The structured illumination microscope apparatus according to claim 5,
    The structured illumination microscope apparatus, wherein the observation light beam is a fluorescent light beam.
  7. 　光源からの所定の波長を有する射出光束中に配置した光変調器に前記射出光束を横切るように設けた音波伝搬路を通過する前記射出光束の少なくとも２つの回折光を照明光学系により干渉させることで前記干渉縞を被観察物に形成するとともに、前記射出光束が有する波長に応じて前記干渉縞が所望の縞ピッチを持つように前記光変調器を制御して前記被観察物を構造化照明することを特徴とする構造化照明方法。 An illumination optical system interferes at least two diffracted lights of the emitted light beam passing through a sound wave propagation path provided so as to cross the emitted light beam in an optical modulator disposed in the emitted light beam having a predetermined wavelength from a light source. The interference fringes are formed on the object under observation, and the light modulator is controlled so that the interference fringes have a desired fringe pitch according to the wavelength of the emitted light beam, thereby structuring the object under observation. A structured illumination method characterized by:
PCT/JP2012/004985 2011-08-11 2012-08-06 Structured illumination device, structured illumination method, and structured illumination microscope device WO2013021615A1 (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013152304A (en) * 2012-01-24 2013-08-08 Olympus Corp Microscope system
JP2015230439A (en) * 2014-06-06 2015-12-21 株式会社ニコン Illumination optical system, microscope device and illumination method
JP2016526185A (en) * 2013-05-15 2016-09-01 ジ アドミニストレーターズ オブ ザ テュレーン エデュケーショナル ファンド Microscopic observation of tissue samples using structured illumination
JPWO2015008415A1 (en) * 2013-07-17 2017-03-02 株式会社ニコン Structured illumination device and structured illumination microscope device
JP2018106852A (en) * 2016-12-22 2018-07-05 大日本印刷株式会社 Luminaire

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001023223A (en) * 1999-07-05 2001-01-26 Nikon Corp Optical information reproducing device
JP2003084206A (en) * 2001-09-14 2003-03-19 Nikon Corp Grid lighting microscope
JP2007199572A (en) * 2006-01-30 2007-08-09 Nikon Corp Microscope
JP2007279287A (en) * 2006-04-05 2007-10-25 Nikon Corp Structured illuminating optical system and structured illuminating microscope having the same
JP2009103958A (en) * 2007-10-24 2009-05-14 Olympus Corp Scanning laser microscope
JP2010175625A (en) * 2009-01-27 2010-08-12 Nikon Corp Variable filter device and confocal microscope

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001023223A (en) * 1999-07-05 2001-01-26 Nikon Corp Optical information reproducing device
JP2003084206A (en) * 2001-09-14 2003-03-19 Nikon Corp Grid lighting microscope
JP2007199572A (en) * 2006-01-30 2007-08-09 Nikon Corp Microscope
JP2007279287A (en) * 2006-04-05 2007-10-25 Nikon Corp Structured illuminating optical system and structured illuminating microscope having the same
JP2009103958A (en) * 2007-10-24 2009-05-14 Olympus Corp Scanning laser microscope
JP2010175625A (en) * 2009-01-27 2010-08-12 Nikon Corp Variable filter device and confocal microscope

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013152304A (en) * 2012-01-24 2013-08-08 Olympus Corp Microscope system
JP2016526185A (en) * 2013-05-15 2016-09-01 ジ アドミニストレーターズ オブ ザ テュレーン エデュケーショナル ファンド Microscopic observation of tissue samples using structured illumination
US10042150B2 (en) 2013-05-15 2018-08-07 The Administrators Of The Tulane Educational Fund Microscopy of a tissue sample using structured illumination
US10768402B2 (en) 2013-05-15 2020-09-08 The Administrators Of The Tulane Educational Fund Microscopy of a tissue sample using structured illumination
JPWO2015008415A1 (en) * 2013-07-17 2017-03-02 株式会社ニコン Structured illumination device and structured illumination microscope device
JP2015230439A (en) * 2014-06-06 2015-12-21 株式会社ニコン Illumination optical system, microscope device and illumination method
JP2018106852A (en) * 2016-12-22 2018-07-05 大日本印刷株式会社 Luminaire

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