WO2013021615A1 - Dispositif d'éclairage structuré, procédé d'éclairage structuré et dispositif de microscope à éclairage structuré - Google Patents

Dispositif d'éclairage structuré, procédé d'éclairage structuré et dispositif de microscope à éclairage structuré Download PDF

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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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English (en)
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.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention vise à ajuster une longueur d'onde d'une onde sonore d'un élément audio-optique ayant une configuration simple. A cet effet, selon l'invention, un dispositif de microscope à éclairage structuré (10) comprend un ensemble d'éclairage structuré (11). L'ensemble d'éclairage structuré (11) comprend en outre une source lumineuse (15), un élément audio-optique unitaire (19), un objectif (26) et une unité de commande (29). La source lumineuse (15) émet une lumière d'une longueur d'onde prescrite. L'élément audio-optique (19) diffracte la lumière et forme un motif d'interférence sur une face d'échantillon (16a). La lentille optique (26) éclaire un échantillon (16) avec tout ou partie du faisceau lumineux qui est diffracté par l'élément audio-optique (19). L'unité de commande (29) commande l'élément audio-optique (19) de telle sorte que le pas de motif du motif d'interférence change selon la longueur d'onde de la lumière de la source lumineuse (15).
PCT/JP2012/004985 2011-08-11 2012-08-06 Dispositif d'éclairage structuré, procédé d'éclairage structuré et dispositif de microscope à éclairage structuré WO2013021615A1 (fr)

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Publication number Priority date Publication date Assignee Title
JP2013152304A (ja) * 2012-01-24 2013-08-08 Olympus Corp 顕微鏡システム
JP2015230439A (ja) * 2014-06-06 2015-12-21 株式会社ニコン 照明光学系、顕微鏡装置、及び照明方法
JP2016526185A (ja) * 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 構造化照明を用いた組織試料の顕微鏡観察
JPWO2015008415A1 (ja) * 2013-07-17 2017-03-02 株式会社ニコン 構造化照明装置及び構造化照明顕微鏡装置
JP2018106852A (ja) * 2016-12-22 2018-07-05 大日本印刷株式会社 照明装置

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JP2001023223A (ja) * 1999-07-05 2001-01-26 Nikon Corp 光情報再生装置
JP2003084206A (ja) * 2001-09-14 2003-03-19 Nikon Corp 格子照明顕微鏡
JP2007199572A (ja) * 2006-01-30 2007-08-09 Nikon Corp 顕微鏡装置
JP2007279287A (ja) * 2006-04-05 2007-10-25 Nikon Corp 構造化照明光学系、及びそれを備えた構造化照明顕微鏡
JP2009103958A (ja) * 2007-10-24 2009-05-14 Olympus Corp 走査型レーザ顕微鏡
JP2010175625A (ja) * 2009-01-27 2010-08-12 Nikon Corp 可変フィルタ装置及びコンフォーカル顕微鏡

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Publication number Priority date Publication date Assignee Title
JP2001023223A (ja) * 1999-07-05 2001-01-26 Nikon Corp 光情報再生装置
JP2003084206A (ja) * 2001-09-14 2003-03-19 Nikon Corp 格子照明顕微鏡
JP2007199572A (ja) * 2006-01-30 2007-08-09 Nikon Corp 顕微鏡装置
JP2007279287A (ja) * 2006-04-05 2007-10-25 Nikon Corp 構造化照明光学系、及びそれを備えた構造化照明顕微鏡
JP2009103958A (ja) * 2007-10-24 2009-05-14 Olympus Corp 走査型レーザ顕微鏡
JP2010175625A (ja) * 2009-01-27 2010-08-12 Nikon Corp 可変フィルタ装置及びコンフォーカル顕微鏡

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013152304A (ja) * 2012-01-24 2013-08-08 Olympus Corp 顕微鏡システム
JP2016526185A (ja) * 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 構造化照明を用いた組織試料の顕微鏡観察
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 (ja) * 2013-07-17 2017-03-02 株式会社ニコン 構造化照明装置及び構造化照明顕微鏡装置
JP2015230439A (ja) * 2014-06-06 2015-12-21 株式会社ニコン 照明光学系、顕微鏡装置、及び照明方法
JP2018106852A (ja) * 2016-12-22 2018-07-05 大日本印刷株式会社 照明装置

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