WO2018190339A1 - 収差補正方法及び光学装置 - Google Patents
収差補正方法及び光学装置 Download PDFInfo
- Publication number
- WO2018190339A1 WO2018190339A1 PCT/JP2018/015080 JP2018015080W WO2018190339A1 WO 2018190339 A1 WO2018190339 A1 WO 2018190339A1 JP 2018015080 W JP2018015080 W JP 2018015080W WO 2018190339 A1 WO2018190339 A1 WO 2018190339A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- objective lens
- refractive index
- modulation
- index interface
- optical axis
- Prior art date
Links
Images
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/361—Optical details, e.g. image relay to the camera or image sensor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0056—Optical details of the image generation based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/02—Objectives
- G02B21/04—Objectives involving mirrors
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
- G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
- G02B27/0025—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for optical correction, e.g. distorsion, aberration
- G02B27/0068—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for optical correction, e.g. distorsion, aberration having means for controlling the degree of correction, e.g. using phase modulators, movable elements
-
- G—PHYSICS
- G02—OPTICS
- G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
- G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
- G02F1/01—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the intensity, phase, polarisation or colour
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0072—Optical details of the image generation details concerning resolution or correction, including general design of CSOM objectives
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B26/00—Optical devices or arrangements for the control of light using movable or deformable optical elements
- G02B26/08—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the direction of light
- G02B26/10—Scanning systems
- G02B26/101—Scanning systems with both horizontal and vertical deflecting means, e.g. raster or XY scanners
Definitions
- the present disclosure relates to an aberration correction method and an optical apparatus.
- Patent Documents 1 and 2 disclose a method of reducing the influence of aberration or the like at the refractive index interface using a spatial light modulator.
- the aberration of the laser beam is corrected so that the condensing point of the laser beam is positioned between the aberration range generated inside the medium.
- the refractive index of the condensing unit is determined from the incident surface of the medium when it is assumed that the refractive index of the medium is n and the refractive index n of the medium is equal to the refractive index of the atmospheric medium of the condensing unit.
- the condensing point of the laser light is within a range larger than n ⁇ d ⁇ s and smaller than n ⁇ d from the incident surface of the medium.
- the aberration of the laser beam is corrected so that is positioned. Thereby, the aberration of the laser beam can be corrected so that the focal point is located in a range where the longitudinal aberration exists in the medium when the aberration is not corrected.
- Non-Patent Document 1 describes a technique related to a two-photon excitation fluorescence microscope.
- an aberration correction pattern is displayed on the spatial light modulator, and the irradiation light to the observation object is modulated using this spatial light modulator, resulting in the surface shape of the observation object.
- the spherical aberration that occurs is corrected.
- spatial light having an aberration correction pattern for example, is used to correct aberration (for example, spherical aberration) caused by a refractive index interface existing on the surface or inside of an object.
- aberration for example, spherical aberration
- irradiation light or observation light
- SLM Spatial Light Modulator
- the aberration correction pattern is a pattern that spreads concentrically around the optical axis. Easy.
- the refractive index interface of the object may be inclined with respect to the optical axis.
- the calculation of the aberration correction pattern is complicated, and a long time is required for the calculation.
- the calculation of the aberration correction pattern itself may be difficult.
- Embodiments provide an aberration correction method and an optical apparatus capable of easily performing aberration correction in a short time even when a refractive index interface such as the surface of an object is inclined with respect to the optical axis.
- the purpose is to do.
- the aberration correction method includes a coupling step for optically coupling a modulation surface of a spatial light modulator and an object via an objective lens, and a correction pattern for correcting aberration caused by the refractive index interface of the object. And a control step of controlling the spatial light modulator based on the included modulation pattern.
- the position of the correction pattern in the modulation pattern is set based on the inclination information of the refractive index interface of the object with respect to the plane perpendicular to the optical axis of the objective lens.
- An optical device includes a spatial light modulator having a modulation surface, an objective lens disposed on an optical path between the modulation surface and an object, and a correction pattern for correcting aberration caused by a refractive index interface of the object.
- a control unit that controls the spatial light modulator based on a modulation pattern including: In the optical device, the position of the correction pattern in the modulation pattern is set based on the inclination information of the refractive index interface of the object with respect to the plane perpendicular to the optical axis of the objective lens.
- the inventor sets the position of the correction pattern in the modulation pattern according to the inclination information such as the inclination angle.
- the inventors have found that aberrations can be corrected appropriately.
- aberration correction can be easily performed in a short time without complicated calculations.
- the aberration correction method includes a coupling step for optically coupling a modulation surface of a spatial light modulator and an object via an objective lens, and a correction pattern for correcting aberration caused by the refractive index interface of the object. And a control step of controlling the spatial light modulator based on the included modulation pattern.
- the spatial light modulator and the objective lens are arranged based on inclination information of the refractive index interface of the object with respect to a plane perpendicular to the optical axis of the objective lens.
- An optical device has a modulation surface and modulates light based on a modulation pattern including a correction pattern for correcting an aberration caused by a refractive index interface of the object, the modulation surface and the object
- An objective lens disposed on an optical path between the objective lens and the spatial light modulator, a moving mechanism that moves at least one of the objective lens and the spatial light modulator in a direction intersecting the optical axis of the objective lens, and a control unit that controls the moving mechanism; .
- the control unit controls the moving mechanism based on the inclination information of the refractive index interface of the object with respect to the plane perpendicular to the optical axis of the objective lens.
- the aberration correction can be easily performed in a short time even when the refractive index interface of the object is inclined with respect to the optical axis.
- FIG. 1 is a diagram illustrating a configuration of a microscope apparatus as an optical apparatus according to an embodiment.
- FIG. 2 is a flowchart showing the operation of the microscope apparatus.
- FIG. 3 is a diagram conceptually showing the modulation pattern created in the creation steps (a) to (d).
- FIG. 4 is a schematic diagram showing a state where the refractive index interface is inclined with respect to a plane perpendicular to the optical axis of the objective lens.
- FIGS. 5A and 5B are images showing effects according to one embodiment, showing a state of blood vessels when a dry objective lens is used as an objective lens and the inside of a biological sample is observed as an object. Yes.
- FIG. 1 is a diagram illustrating a configuration of a microscope apparatus as an optical apparatus according to an embodiment.
- FIG. 2 is a flowchart showing the operation of the microscope apparatus.
- FIG. 3 is a diagram conceptually showing the modulation pattern created in the creation steps (a) to (d).
- FIG. 4 is a
- FIGS. 7A and 7B are diagrams conceptually showing one modified example.
- FIG. 8 is a flowchart showing the aberration correction method of the first modification.
- FIG. 9 is a diagram illustrating a configuration of a microscope apparatus that can realize an aberration correction method according to a modification.
- FIG. 10 is a diagram illustrating a case where it can be approximated that the refractive index interface of the object is constituted by two surfaces.
- FIG. 11 is a diagram illustrating a case where it can be approximated that the refractive index interface of an object is constituted by a large number of surfaces.
- FIG. 12 is a diagram illustrating a configuration of a microscope apparatus according to a third modification.
- 13A and 13B are mouse brain images obtained by (a) and (b) pre-scanning.
- FIG. 14 is a diagram illustrating a state where an object has a plurality of refractive index interfaces inside.
- FIG. 1 is a diagram illustrating a configuration of a microscope apparatus 1A as an optical apparatus according to an embodiment.
- 1 A of microscope apparatuses are apparatuses which acquire the enlarged image of the target object B, irradiating light with respect to the target object B, Comprising: As FIG. 1 shows, the microscope unit 10, the inclination measurement unit 20, and the image acquisition unit 30 and a control unit 40.
- the microscope unit 10 irradiates the object B with irradiation light P1 from an inclination measurement unit 20 and an image acquisition unit 30, which will be described later, and detects light P2 from the object B to the inclination measurement unit 20 and the image acquisition unit 30. Output each.
- the detected light P2 is, for example, reflected light of the irradiation light P1, harmonics of the irradiation light P1, or fluorescence excited by the irradiation light P1.
- the microscope unit 10 includes a sample stage (stage) 11, an objective lens 12, an objective lens moving mechanism 13, and a beam splitter 14.
- the sample stage 11 is a plate-like member for supporting the object B (or a container that accommodates the object B).
- the sample stage 11 is made of, for example, glass.
- the irradiation light P ⁇ b> 1 is applied to the object B from the surface side of the sample table 11. Further, the detected light P ⁇ b> 2 from the object B is emitted to the surface side of the sample table 11.
- the objective lens 12 is disposed on the optical path of the irradiation light P1 between the SLM 33 described later and the surface Ba of the object B.
- the objective lens 12 is disposed on the optical path of the detected light P2 between the SLM 36, which will be described later, and the surface Ba of the object B.
- One surface of the objective lens 12 is disposed so as to face the sample stage 11, and the focal point on the one surface side of the objective lens 12 is located inside the object B.
- the objective lens 12 collects the irradiation light P ⁇ b> 1 at one point inside the object B.
- the objective lens 12 receives a part of the detected light P2 emitted from the one point of the object B and collimates the part.
- the objective lens 12 may be an immersion objective lens such as a dry objective lens, a water immersion objective lens, or an oil immersion objective lens.
- the objective lens for the irradiation light P1 and the objective lens for the detected light P2 are common, but the objective lens for the irradiation light P1 and the objective lens for the detected light P2 are common.
- the lens may be provided separately.
- an objective lens having a large pupil diameter may be used for the irradiation light P1, and the light may be locally focused by aberration correction described later. Further, an objective lens having a large pupil may be used for the detected light P2 so that more light can be extracted.
- the objective lens moving mechanism 13 is a mechanism for moving the objective lens 12 in the optical axis direction of the irradiation light P1.
- the objective lens 12 is supported by an objective lens moving mechanism 13 so as to be movable in the optical axis direction.
- the objective lens moving mechanism 13 is configured by, for example, a stepping motor or a piezo actuator.
- the beam splitter 14 divides and combines the optical path between the image acquisition unit 30 and the optical path between the tilt measurement unit 20. Specifically, the beam splitter 14 reflects the irradiation light P ⁇ b> 1 that has reached the microscope unit 10 from the image acquisition unit 30 toward the objective lens 12. Further, the beam splitter 14 reflects the detection light P ⁇ b> 2 collected by the objective lens 12 toward the image acquisition unit 30. On the other hand, the beam splitter 14 transmits the light P32 from the tilt measurement unit 20 and the reflected light of the light P32 on the object B.
- the beam splitter 14 is preferably configured by, for example, a half mirror or a dichroic mirror.
- the microscope unit 10 may further include a reflection mirror 15 that changes the optical axis direction of the light P32.
- the tilt measurement unit 20 is a measurement unit in the present embodiment, and measures the tilt angle of the refractive index interface of the object B with respect to a plane perpendicular to the optical axis of the objective lens 12.
- a refractive index interface between the surface Ba of the target object B and the surrounding medium will be described as an example as a refractive index interface of the target object B, but the refractive index interface of the target object B is not limited to this.
- the refractive index interface between the object B and the container that accommodates the object B may be used, or the refractive index interface in the internal structure of the object B may be used.
- the surrounding medium is, for example, air or immersion liquid.
- the tilt measurement unit 20 may be an interference light measurement unit that measures the surface shape of the object B using, for example, a Michelson interferometer.
- the inclination measurement unit 20 includes a coherent light source 21, a beam splitter 22, a reference light mirror 23, and a detector 24 as shown in FIG.
- the coherent light source 21 generates coherent light P3 irradiated on the object B.
- the coherent light source 21 is preferably configured by, for example, a semiconductor laser element.
- the beam splitter 22 branches the coherent light P3 from the coherent light source 21 into reference light P31 and light P32 to the microscope unit 10. Further, the beam splitter 22 reflects the reference light P31 reflected by the reference light mirror 23 and transmits the reflected light from the surface of the object B of the light P32, thereby synthesizing these lights and interfering light P4. Is generated.
- the interference light P4 enters the detector 24.
- the reference light mirror 23 may be configured to be movable with respect to the optical axis direction of the reference light P31, or may be fixed.
- the detector 24 detects the interference light P4 synthesized by the beam splitter 22 and outputs a detection signal S1.
- the detector 24 includes a two-dimensional light detection element such as a CCD image sensor or a CMOS image sensor.
- the tilt measurement unit is not limited to the above-described configuration.
- the tilt measurement unit may have an interference measurement method such as a mirrow type or a linique type.
- the tilt measurement unit may have a confocal reflectance microscope or a common path interferometer. According to such a microscope, it is possible to suitably measure the inclination angle of the surface Ba of the object B using the focusing information.
- the image acquisition unit 30 detects the detected light P2 from the object B and creates an enlarged image.
- the fluorescence optical system in the case where the detected light P2 is fluorescence from the object B will be described.
- the detected light P2 may be reflected light from the object B or a harmonic.
- the image acquisition unit 30 of this embodiment includes a laser light source 31, a beam expander 32, an SLM 33, a dichroic mirror 34, an optical scanner 35, an SLM 36, a detector 37, and a filter 38.
- the laser light source 31 is a light source for irradiating the object B with the irradiation light P ⁇ b> 1 through the objective lens 12.
- the laser light source 31 outputs light P5 that is the source of the irradiation light P1.
- the light P5 is laser light including the excitation wavelength of the object B, for example.
- the laser light source 31 includes, for example, a semiconductor laser element.
- the beam expander 32 includes, for example, a plurality of lenses 32a and 32b arranged side by side on the optical axis of the light P5, and adjusts the size of the cross section perpendicular to the optical axis of the light P5.
- the SLM 33 is a type of SLM that controls phase modulation for each of a plurality of pixels.
- the SLM 33 displays a modulation pattern (hologram) including a correction pattern for correcting an aberration caused by a difference in refractive index of the surface Ba of the object B on the modulation surface 33a.
- the SLM 33 modulates the light P5 from the laser light source 31 to generate light P1 that is irradiated to the object B.
- the objective lens 12 is disposed on the optical path of the light P1 between the modulation surface 33a of the SLM 33 and the surface Ba of the object B.
- the SLM 33 is not limited to the phase modulation type, and may be an amplitude (intensity) modulation type.
- the SLM 33 may be either a reflection type or a transmission type. Details of the modulation pattern including the correction pattern will be described later.
- the dichroic mirror 34 transmits one of the irradiation light P1 from the SLM 33 and the detected light P2 from the microscope unit 10, and reflects the other. In the example shown in FIG. 1, the dichroic mirror 34 transmits the irradiation light P1 and reflects the detection light P2.
- the optical scanner 35 scans the condensing position of the irradiation light P1 on the object B by moving the optical axis of the irradiation light P1 in a plane perpendicular to the optical axis of the irradiation light P1.
- the optical scanner 35 is configured by, for example, a galvanometer mirror, a resonance mirror, or a polygon mirror. Further, the detected light P ⁇ b> 2 from the object B is detected via the optical scanner 35. Thereby, the optical axis of irradiation light P1 and the optical axis of to-be-detected light P2 can be made mutually corresponded.
- the SLM 36 is a type of SLM that controls phase modulation for each of a plurality of pixels.
- the SLM 36 displays a modulation pattern including a correction pattern for correcting an aberration caused by the difference in refractive index of the surface Ba of the object B on the modulation surface 36a.
- the SLM 36 modulates the detected light P2 from the dichroic mirror 34.
- the objective lens 12 is disposed on the optical path of the detected light P2 between the modulation surface 36a of the SLM 36 and the surface Ba of the object B.
- the SLM 36 is not limited to the phase modulation type, and may be an amplitude (intensity) modulation type. Further, the SLM 36 may be either a reflection type or a transmission type.
- a pinhole is arranged in front of the detector 37, it is preferable to display a pattern for condensing the detected light P2 in the pinhole on the modulation surface 36a in addition to the correction pattern. Thereby, a confocal effect can be obtained.
- a pattern for condensing the detected light P2 on the detector 37 is a correction pattern.
- the confocal effect can be obtained by including the modulation pattern in the modulation pattern. Details of the modulation pattern including the correction pattern will be described later.
- the detector 37 detects the light intensity of the detected light P2 emitted from the object B through the objective lens 12, and outputs a detection signal S2.
- the detector 37 may be a point sensor such as a PMT (Photomultiplier Tube), a photodiode, or an avalanche photodiode.
- the detector 37 may be an area image sensor such as a CCD image sensor, a CMOS image sensor, a multi-anode PMT, or a photodiode array.
- a condensing lens 37a may be disposed immediately before the detector 37.
- the filter 38 is disposed on the optical axis between the dichroic mirror 34 and the detector 37.
- the filter 38 cuts the wavelength of the irradiation light P1 and the wavelength of fluorescence unnecessary for observation from the light incident on the detector 37. Note that the filter 38 may be disposed either before or after the condenser lens 37a.
- the image acquisition unit 30 of the present embodiment further includes a mirror 39a and a reflecting member 39b in addition to the above configuration.
- the mirror 39a bends the optical axes of the irradiation light P1 and the detected light P2 in order to optically couple the optical scanner 35 and the beam splitter 14 of the microscope unit 10.
- the reflecting member 39b is a prism having two reflecting surfaces, and is disposed to face the SLM 36.
- the reflection member 39b reflects the detection light P2 from the dichroic mirror 34 on one reflection surface toward the SLM 36, and reflects the detection light P2 from the SLM 36 toward the detector 37 on the other reflection surface.
- At least one 4f optical system may be provided on the optical axes of the irradiation light P1 and the detection light P2.
- two 4f optical systems 51 and 52 are shown in FIG.
- the 4f optical systems 51 and 52 have a role of transferring the wavefront of the irradiation light P1 generated in the SLM 33 to the rear focal point of the objective lens 12. If the objective lens 12 and the SLM 33 are extremely close to each other, the 4f optical system can be omitted.
- the control unit 40 is a control unit in the present embodiment.
- the control unit 40 controls the microscope unit 10, the tilt measurement unit 20, and the image acquisition unit 30.
- control unit 40 controls the position of the objective lens 12 in the optical axis direction using the objective lens moving mechanism 13 in the microscope unit 10. Further, the control unit 40 moves the sample table 11 that supports the object B in a direction that intersects the optical axis direction.
- the control unit 40 also controls the coherent light source 21, the detector 24, and the reference light mirror 23 of the tilt measurement unit 20.
- the control unit 40 also controls the laser light source 31, the optical scanner 35, and the detector 37 of the image acquisition unit 30. Further, the control unit 40 calculates a modulation pattern displayed on the SLMs 33 and 36 and causes the SLMs 33 and 36 to display the modulation pattern.
- the control unit 40 includes, for example, an input device 41 such as a mouse and a keyboard, a display device (display) 42, and a computer 43.
- the computer 43 is, for example, a personal computer, a microcomputer, a smart device, a cloud server, or the like.
- control unit 40 constitutes a part of the measurement unit in the present embodiment.
- the control unit 40 receives the detection signal S1 from the detector 24 of the tilt measurement unit 20, and based on the detection signal S1, the object B is detected using a method using Fourier transform or ⁇ / 4 phase shift interferometry.
- the information regarding the inclination angle of the surface Ba of is acquired.
- the control unit 40 creates modulation pattern data including a correction pattern for correcting an aberration caused by the refractive index difference of the surface Ba of the object B.
- the modulation pattern data is provided to the SLM 33 and the SLM 36.
- the control unit 40 creates an enlarged image related to the object B based on the detection signal S2 from the detector 37 and information on the light irradiation position by the optical scanner 35.
- the created image is displayed on the display device 42.
- FIG. 2 is a flowchart showing the operation of the above-described microscope apparatus 1A.
- the light irradiation method and the observation method including the aberration correction method according to the present embodiment will be described with reference to FIG.
- the modulation surfaces 33a, 36a of the SLMs 33, 36 and the surface Ba of the object B are optically coupled via the objective lens 12 (coupling step). S10).
- the light P3 is emitted from the light source 21 of the inclination measurement unit 20, and the interference light P4 between the reflected light from the surface of the object B and the reference light P31 is detected by the detector 24.
- interference fringes on the surface Ba of the object B are observed.
- the control unit 40 acquires the inclination angle of the surface Ba of the object B with respect to the plane perpendicular to the optical axis of the objective lens 12 (measurement step S11).
- the control unit 40 creates modulation pattern data including a correction pattern for correcting the aberration caused by the refractive index difference of the surface Ba of the object B ( Creating step S12).
- the SLM 33 and the SLM 36 are controlled based on the modulation pattern data, and the modulation pattern based on the modulation pattern data is displayed on the SLMs 33 and 36 (control step S13).
- the light P5 emitted from the laser light source 31 is modulated by the SLM 33, and the modulated irradiation light P1 is irradiated onto the object B through the objective lens 12 (irradiation step S14).
- the intensity of the detected light P2 generated in the object B is detected by the detector 37 (detection step S15).
- the detected light P ⁇ b> 2 is modulated by the SLM 36 and then enters the detector 37.
- the irradiation step S14 and the detection step S15 are repeated (or simultaneously performed continuously) while scanning the irradiation light P1 by the optical scanner 35.
- an enlarged image of the object B is created in the control unit 40 based on the detection information in the detection step S15 (image creation step S16).
- the “optical axis of the objective lens 12” refers to the optical axis of the objective lens 12. It means a straight line extending to the surface Ba.
- the "optical axis of the objective lens 12” means that the optical axis of the objective lens 12 is modulated by each of the SLMs 33 and 36.
- FIG. 3 is a diagram conceptually showing the modulation pattern created in creation step S12.
- 3A shows a state in which the optical axis 12a of the objective lens 12 and the surface Ba of the object B are perpendicular to each other, and FIG. 3B shows such a case on the modulation surfaces 33a and 36a.
- the modulation pattern D1 to be performed is shown.
- an XYZ orthogonal coordinate system in which the direction along the optical axis 12a is the Z direction is shown.
- the modulation pattern D1 includes a correction pattern D3 for correcting aberration caused by the refractive index difference of the surface Ba of the object B.
- the correction pattern D3 is a point-symmetrical pattern with respect to the optical axis 12a of the objective lens 12.
- the correction pattern D3 is concentrically centered on a point T where the optical axis 12a of the objective lens 12 intersects the modulation surfaces 33a and 36a of the SLMs 33 and 36. It is a spreading pattern. That is, the center O of the correction pattern D3 is located on the optical axis 12a of the objective lens 12.
- 3C shows a state where the surface Ba of the object B is inclined by an angle ⁇ with respect to the plane H perpendicular to the optical axis 12a of the objective lens 12, and FIG. In such a case, the modulation pattern D2 displayed on the modulation surfaces 33a and 36a is shown.
- the modulation pattern D2 includes the correction pattern D3 described above. However, the center O of the correction pattern D3 is deviated by a distance E in the inclination direction of the surface Ba with respect to the point T where the optical axis 12a of the objective lens 12 intersects the modulation surfaces 33a and 36a of the SLMs 33 and 36.
- the distance E is proportional to the inclination angle ⁇ of the surface Ba with respect to the plane H, and is preferably obtained based on the inclination angle ⁇ .
- the distance E is determined based on the inclination angle ⁇ (decision step S12a).
- the diameter d3 of the display area of the correction pattern D3 on the modulation surfaces 33a and 36a may be larger than the pupil diameter of the objective lens 12.
- the region where the correction pattern D3 exists is biased with respect to the point T. Therefore, when the display area of the correction pattern D3 is small, there is a possibility that a part of light does not pass through the correction pattern D3 and the aberration correction is not performed. Since the diameter d3 of the display area of the correction pattern D3 is larger than the pupil diameter of the objective lens 12, such a concern can be reduced.
- the correction patterns displayed on the SLMs 33 and 36 have the same shape, and the center thereof coincides with the optical axis of the objective lens 12.
- the pixel position in the modulation surfaces 33a, 36a of the SLMs 33, 36 is (x, y)
- the pixel pitch is p
- the position where the optical axis of the objective lens 12 and the modulation surfaces 33a, 36a intersect is T (x 0 , y 0 )
- the spherical aberration correction pattern ⁇ in such a case is obtained by, for example, the following formula (1).
- ⁇ is the wavelength of light
- n 1 is the refractive index of the surrounding medium
- n 2 is the refractive index of the object B
- NA is The numerical aperture, ⁇ , of the objective lens 12 is a defocus parameter that moves the actual light collection position back and forth on the optical axis.
- the light P5 output as a plane wave from the laser light source 31 is modulated by the SLM 33.
- the modulated light P1 is diffracted on the surface Ba of the object B, but is condensed at one point of a predetermined depth in the object B.
- FIG. 4 is a schematic diagram showing a state where the refractive index interface Ba is inclined with respect to a plane H perpendicular to the optical axis A1 of the objective lens 12.
- the position on the wavefront C of the light P1 corresponding to the pixel position (x, y) in the modulation surfaces 33a and 36a of the SLMs 33 and 36 is (x ′, y ′). That is, the position T ′ on the wavefront C of the light P1 corresponding to the position T (x 0 , y 0 ) where the optical axis of the objective lens 12 and the modulation surfaces 33a and 36a intersect is (x ′ 0 , y ′ 0 ). It becomes.
- the inclination angle of the refractive index interface Ba with respect to the plane H is ⁇ .
- a straight line perpendicular to the refractive index interface Ba becomes the new optical axis A2, and the wavefront of the optical axis A2 and the light P1 emitted from the objective lens 12 (or the light P2 incident on the objective lens 12)
- the intersection point Q ′ 1 (x ′ 0 + x ′ 1 , y ′ 0 + y ′ 1 ) with the paraboloid C is the central axis of the new aberration correction pattern. That is, the aberration correction pattern ⁇ when the refractive index interface Ba is tilted can be obtained by the following formula (2).
- the center of the new correction pattern is used as the original correction pattern (the correction pattern when the refractive index interface Ba is perpendicular to the optical axis A 1 of the objective lens 12. ) How many pixels should be moved.
- the tilt azimuth angle ⁇ and the tilt angle ⁇ are measured based on the surface shape of the object B obtained by the tilt measurement unit 20.
- the obtained surface shape is approximated by a polynomial
- the tilt azimuth angle ⁇ and the tilt angle ⁇ are derived from the first-order terms (ax + by, a and b are coefficients) of the obtained polynomial.
- the derivation of the tilt azimuth angle ⁇ and the tilt angle ⁇ is not limited to polynomial approximation.
- approximation other than polynomial approximation or interpolation such as spline interpolation may be used.
- creation step S12 the influence of the tilt angle ⁇ on the aberration correction is suppressed based on the position of the aberration correction pattern when the surface Ba of the object B is perpendicular to the optical axis of the objective lens 12.
- the movement distance (inclination information) of the aberration correction pattern for determination is determined (decision step S12a). This moving distance is suitably obtained from the numerical aperture NA, the focal length f, and the imaging magnification M of the objective lens 12 as shown below.
- the radius L1 of the pupil of the objective lens 12 is obtained by the following formula (4) based on the numerical aperture NA and the focal length f of the objective lens 12.
- L1 NA ⁇ f (4)
- the imaging magnification M of the objective lens 12 with respect to the SLMs 33 and 36 (the objective lens 12 and the SLMs 33 and 36 have an M: 1 enlarged imaging relationship (the light diameter on the SLMs 33 and 36 side is larger than the light diameter on the objective lens 12 side).
- the light radius L2 in the SLMs 33 and 36 is obtained by the following equation (5).
- L2 L1 / M (5)
- the number R of pixels of the SLMs 33 and 36 included in the radius L2 is obtained by the following equation (6).
- R L2 / p (6)
- the number r of pixels per angle of 1 ° formed by the optical axis of the objective lens 12 and the light beam is obtained by the following formula (7).
- the moving distance (number of pixels) of the aberration correction pattern is obtained by the product of the inclination angle ⁇ and the number of pixels r.
- the tilt angle component ⁇ x in the x-axis direction and the tilt angle component ⁇ y in the y-axis direction are calculated based on the tilt azimuth angle ⁇ and the tilt angle ⁇ .
- x 1 ⁇ x ⁇ r (8)
- y 1 ⁇ y ⁇ r (9)
- the inventor determines the correction pattern D3 when these are substantially vertical as the tilt angle. It has been found that aberrations can be suitably corrected by moving in the tilt direction by a distance corresponding to ⁇ . That is, in the present embodiment, the modulation pattern D2 including the pattern obtained by moving the correction pattern D3 in the modulation surfaces 33a and 36a when the optical axis 12a of the objective lens 12 and the surface Ba of the object B are substantially perpendicular is used. And displayed on the modulation surfaces 33a and 36a. Then, the moving distance of the correction pattern D3 is determined based on the inclination angle ⁇ of the surface Ba of the object B. Thus, aberration correction can be easily performed in a short time without complicated calculations.
- FIG. 5 (a) and 5 (b) are images showing the effects of the present embodiment.
- a dry objective lens is used as the objective lens 12, and a blood vessel when the inside of the biological sample is observed as the object B is shown. It shows a state.
- the vertical axis in the figure is the depth from the surface of the biological sample.
- FIG. 5A is an image obtained by applying the aberration correction method of the present embodiment
- FIG. 5B is an image obtained without correcting the aberration.
- the blood vessel appears to be blurred due to the influence of the aberration when the depth exceeds 500 ⁇ m.
- FIG. 5A when the aberration correction method of this embodiment is applied, blood vessels can be clearly seen up to a depth exceeding 1000 ⁇ m.
- the objective lens 12 is a dry objective lens and the aberration due to the surface Ba of the object B is large, the aberration is preferably corrected to a deeper portion. A clear image can be obtained. Further, since the objective lens 12 is a dry objective lens or an immersion objective lens, simple measurement can be performed in a non-contact and minimally invasive manner.
- the SLMs 33 and 36 of this embodiment may be any type of SLM that performs phase modulation for each of a plurality of pixels.
- an LCOS (Liquid Crystal On On Silicon) type SLM or a deformable mirror may be applied.
- the deformable mirror may be either a membrane type or a segment type.
- the LCOS type SLM has a larger number of pixels than the deformable mirror and can correct aberrations with high accuracy. Further, the deformable mirror can operate at a higher speed than the LCOS type SLM, and the working time can be shortened.
- the microscope apparatus 1A of the present embodiment may be a laser scanning two-photon excitation fluorescence microscope (TPFLM).
- TPFLM laser scanning two-photon excitation fluorescence microscope
- fluorescence is generated only in a portion where the photon density is extremely high due to the two-photon absorption process (portion where the excitation light is collected by the objective lens 12).
- Near-infrared light is used as the excitation light, but this excitation light is less absorbed and scattered by the living body than visible light.
- a point where the generation of fluorescence is local and a point where absorption and scattering are small are suitable for deep observation of a biological sample.
- the aberration correction method of this embodiment which can perform aberration correction easily in a short time when observing the deep part of a biological sample is very useful. According to the aberration correction method of the present embodiment, it is possible to improve the resolution of the deep portion of the biological sample, and facilitate observation at a depth that is difficult to observe with the conventional TPFLM.
- excitation light is scanned by a biaxial galvano scanner in a plane perpendicular to the optical axis 12a of the objective lens 12, and fluorescence generated at the focal point position is detected by a detector such as PMT.
- a detector such as PMT.
- the above operation was repeated while moving the objective lens 12 or the sample stage 11 in the optical axis direction, and a plurality of images having different depths were obtained.
- the distance between the objective lens 12 and the sample stage 11 was changed from 600 ⁇ m to 800 ⁇ m.
- FIG. 6A to FIG. 6C are two-dimensional images obtained by cutting the three-dimensional image obtained in the present embodiment in the depth direction.
- FIG. 6A is an image when the aberration correction method of the above embodiment is applied
- FIG. 6B is a conventional parallel light without correcting the aberration (that is, the wavefront is perpendicular to the optical axis).
- 6C is an image obtained without moving the correction pattern (see FIG. 3A).
- the moving distance (number of pixels) is 65 pixels.
- the surrounding medium is air, there is a difference in refractive index between the surrounding medium and the model sample (epoxy resin). Accordingly, aberration occurs on the surface of the model sample. Further, since the surface of the model sample is inclined, aberrations other than spherical aberration also occur.
- the outline of the fluorescent beads in the image extends in the depth direction under the influence of the aberration described above. Further, when the correction pattern is not moved (FIG. 6C), the maximum fluorescence intensity of the fluorescent beads is 0.4 times that when the correction of aberration is not performed (FIG. 6B). As a result, the image was unclear compared to when no aberration correction was performed. This is considered to be due to the fact that other aberrations (for example, astigmatism) remain in the correction pattern that corrects only spherical aberration, and that other aberrations have become larger because the wavefront is not appropriate.
- other aberrations for example, astigmatism
- FIG. 6A when the aberration correction method of the above embodiment is applied, other aberrations other than spherical aberration are also corrected well, so that the contour shape of the fluorescent beads in the image is improved. It can be seen that it is close to a sphere and is remarkably improved.
- the maximum fluorescence intensity of the fluorescent beads was 6.5 times that of the case where the aberration was not corrected (FIG. 6B), which was much brighter. That is, according to the present example, it was shown that when the surface Ba of the object B is inclined with respect to the optical axis 12a, the aberration can be corrected with high accuracy by applying the aberration correction method of the above embodiment.
- FIG. 7 is a diagram conceptually showing a modification of the above embodiment.
- the SLMs 33 and 36 are exemplified as the SLMs that control the modulation for each of a plurality of pixels.
- the SLMs 33 and 36 are not limited to this, for example, the modulation patterns are fixed on the modulation surfaces 33a and 36a. It may be an SLM.
- the moving distance E is determined based on the inclination angle ⁇ of the surface Ba with respect to the plane H.
- the aberration correction can be easily performed in a short time without performing a complicated calculation as in the above embodiment.
- the above-described mathematical formulas (8) and (9) are replaced by the following mathematical formulas (10) and (11), respectively.
- x 1 ⁇ x ⁇ L2 / ⁇ max (10)
- y 1 ⁇ y ⁇ L2 / ⁇ max (11)
- the modulation surface 33a, a 36a may be moved respectively in the x-axis direction to x 1, y-axis direction by y 1.
- such a method can be applied to an SLM of a type that controls modulation for each of a plurality of pixels.
- the movement distance E is determined based on the inclination angle ⁇ of the surface Ba with respect to the plane H.
- the correction pattern D3 is displayed on the SLMs 33 and 36 so that the center O of the correction pattern D3 is positioned on the optical axis 12a of the objective lens 12, and the SLMs 33 and 36 themselves are relatively distance E from the optical axis 12a. Just move. Even with such a method, it is possible to obtain the same effect as in the above embodiment.
- FIG. 8 is a flowchart showing the aberration correction method of this modification.
- this modification first, the inclination angle of the surface Ba of the object B with respect to a plane perpendicular to the optical axis of the objective lens 12 is acquired (measurement step S11).
- the determination step S21 the moving distance E of the modulation surfaces 33a and 36a is determined based on the inclination angle ⁇ of the surface Ba with respect to the plane H.
- the modulation surfaces 33a and 36a having the aberration correction pattern D3 are moved by a distance E relative to the optical axis 12a of the objective lens 12 in the direction intersecting the optical axis 12a.
- FIG. 9 is a diagram showing a configuration of a microscope apparatus 1B that can realize the aberration correction method of the present modification.
- the microscope apparatus 1B further includes moving mechanisms 33b and 36b in addition to the configuration of the microscope apparatus 1A shown in FIG.
- the moving mechanism 33b supports the SLM 33 and moves the modulation surface 33a relative to the optical axis 12a of the objective lens 12 in a direction intersecting the optical axis 12a.
- the moving mechanism 36b supports the SLM 36 and moves the modulation surface 36a relative to the optical axis 12a of the objective lens 12 in a direction intersecting the optical axis 12a.
- the moving mechanisms 33 b and 36 b are controlled by the computer 43 of the control unit 40.
- the computer 43 determines the moving distance of the modulation surfaces 33a and 36a based on the inclination angle ⁇ of the surface Ba with respect to the plane H.
- the microscope apparatus 1B may include a mechanism for moving the objective lens 12 in a direction intersecting the optical axis 12a instead of moving the SLMs 33 and 36 or together with the movement of the SLMs 33 and 36.
- the surface shape of the object B is approximated by a polynomial, and a method of deriving the tilt azimuth angle ⁇ and the tilt angle ⁇ from the first-order terms of the obtained polynomial (in other words, the surface Ba of the object B is represented by one A method for approximating a flat surface) was exemplified.
- the optical axis 12a of the objective lens 12 may be divided into a plurality of regions, and the movement distance of the correction pattern D3 corresponding to each region may be determined based on the inclination angle ⁇ of each region.
- the inclination azimuth ⁇ 1 and the inclination angle ⁇ 1 related to the surface Ba1 and the surface Ba2 are related.
- the tilt azimuth angle ⁇ 2 and the tilt angle ⁇ 2 can each be measured.
- decision step S12a on the basis of the tilt azimuth angle alpha 1 and the inclination angle beta 1, and calculates the moving direction and moving distance of the partial correction pattern for correcting aberration caused by surface Ba1.
- step S12a on the basis of the tilt azimuth angle alpha 2 and the inclination angle beta 2, and calculates the moving direction and moving distance of the partial correction pattern for correcting aberration caused by surface Ba2. Then, by superimposing the partial correction patterns after movement, a modulation pattern for correcting aberrations can be created. This is the same even when it can be approximated that the surface Ba of the object B is constituted by a large number of surfaces Baa, as shown in FIG. 11, for example.
- the complex calculation is performed as in the above embodiment.
- aberration correction can be easily performed in a short time.
- FIG. 12 is a diagram showing a configuration of a microscope apparatus 1C according to a third modification of the embodiment.
- the microscope apparatus 1C is different from the microscope apparatus 1A shown in FIG. 1 in that it does not include the tilt measurement unit 20 but includes a shape memory unit 60 instead.
- the shape storage unit 60 is a storage unit in the present embodiment, and stores in advance information related to the inclination angle ⁇ of the surface Ba of the object B. 2, the computer 43 determines the moving distance of the correction pattern D3 based on the information stored in the shape storage unit 60. According to such an aberration correction method and microscope apparatus 1C, the measurement time S11 shown in FIG. 2 can be omitted, and the working time can be further shortened. In particular, it is suitable when the tilt azimuth angle ⁇ and tilt angle ⁇ of the surface Ba are known, such as a semiconductor device.
- the microscope unit 10A does not include the reflecting mirror 15 (see FIG. 1), and instead of the beam splitter 14 (see FIG. 1). A reflection mirror 16.
- the configuration of the other microscope unit 10A is the same as that of the microscope unit 10 of the above embodiment.
- the inclination measurement unit 20 measures the surface shape of the object B using a Michelson interferometer.
- a Michelson interferometer There are various other methods for measuring the tilt azimuth angle ⁇ and the tilt angle ⁇ of the surface Ba of the object B.
- the surface shape of the object B can be measured from the contours of the object B included in a plurality of images with different depths obtained by prescanning.
- the surface shape of the object B can be measured by irradiating the surface Ba of the object B with ultrasonic waves and measuring the reflected waves.
- a light cutting method can be used.
- FIG. 13 (a) and FIG. 13 (b) are mouse brain images obtained by pre-scanning.
- FIG. 13A is an image at a depth position of 120 ⁇ m from the top of the object B in the optical axis direction
- FIG. 13B is an image at a depth position of 560 ⁇ m from the top of the object B in the optical axis direction. is there.
- the outline Bc of the object B due to autofluorescence appears clearly in the image.
- the tilt azimuth angle ⁇ and the tilt angle ⁇ of the surface Ba can be suitably obtained.
- FIG. 14 is a diagram illustrating a state where the object B has a plurality of refractive index interfaces B12 and B23 therein.
- the refractive index interface B12 with respect to a plane perpendicular H12 to the optical axis A1 of the objective lens 12 is inclined by an angle beta 12
- the refractive index interface B23 with respect to a plane perpendicular H23 to the optical axis A1 of the objective lens 12 is It is inclined by an angle ⁇ 23.
- a straight line perpendicular to the refractive index interface B12 becomes a new optical axis A12, and the wavefront of the optical axis A12 and the light P1 emitted from the objective lens 12 (or the light P2 incident on the objective lens 12) ( The intersection point Q ′ 2 (x ′ 0 + x ′ 2 , y ′ 0 + y ′ 2 ) with the paraboloid C is obtained.
- a straight line perpendicular to the refractive index interface B23 becomes a new optical axis A23, and an intersection point Q ′ 3 (x ′ 0 + x ′ 3 , y ′ 0 + y ′ 3 ) between the optical axis A23 and the wavefront C is obtained. can get.
- the aberration correction pattern ⁇ when the refractive index interfaces B12 and B23 are inclined is obtained by the following equation (12).
- the aberration correction is performed using one spatial light modulator. It can be performed.
- the aberration correction pattern ⁇ taking into account the inclinations at the respective refractive index interfaces B12 and B23 may be obtained, and the respective spatial light modulators may be controlled.
- d 2 is a distance between the refractive index boundary B12 on the optical axis A1 and the refractive index interface B23
- d 3 denotes a distance between the refractive index interface B23 and the focusing point on the optical axis
- n 1 is from the refractive index interface B12 refractive index of the portion positioned on the objective lens 12 side
- n 2 is the side opposite to the refractive index
- n 3 objective lens 12 than the refractive index interface B23 is between the refractive index interface B12 of the object B and the refractive index interface B23 Is the refractive index of the portion located at.
- x 2 and y 2 can be obtained from the inclination of the refractive index interface B12
- x 3 and y 3 can be obtained from the inclination of the refractive index interface B23.
- the aberration correction method and the optical apparatus are not limited to the above-described embodiments, and various other modifications are possible.
- the above-described embodiments and modifications may be combined with each other according to the necessary purpose and effect.
- the third modification has been described as a modification of the embodiment shown in FIGS. 1 and 2, but in the first modification as well, the shape memory is used instead of the inclination measurement unit 20 shown in FIG.
- the unit 60 may be provided, and the computer 43 may determine the movement distances of the modulation surfaces 33a and 36a based on the information stored in the shape memory unit 60 during the determination step S21 shown in FIG.
- the storage unit may expand the correction pattern into a polynomial such as a Zernike polynomial and store the coefficient of each term of the polynomial.
- the modulation pattern displayed on the SLM need not be the correction pattern itself.
- it may be a modulation pattern in which another pattern such as a pattern for controlling the condensing shape or condensing position of the irradiation light P1 irradiated to the object B and a correction pattern are superimposed.
- a microscope apparatus is illustrated as an optical apparatus.
- the aberration correction method can be applied to various optical apparatuses such as a laser processing apparatus.
- the tilt measurement unit 20 may be a measurement unit based on an optical cutting method that performs measurement by combining a line laser and a camera, or moves the object B in the optical axis direction of the objective lens 12 to measure the in-focus position. It may be a measurement unit. Two or more detectors may be built in the optical system.
- the microscope apparatuses 1A, 1B, and 1C have a configuration of an upright microscope, they are not limited to this, and may have a configuration of an inverted microscope.
- the glass surface serves as a reference surface, and the object B is pressed, but a portion that is not pressed and does not become a flat surface can be measured by the measuring unit 20.
- the detected light P2 is descanned by the optical scanner 35 and detected by the detector 37.
- the present invention is not limited to this, and the detected light P2 is descanned by the optical scanner 35. Instead, it may be detected by the detector 37. Further, the tilt information may be the tilt angle itself.
- the position of the correction pattern in the modulation pattern is set based on the inclination information of the refractive index interface of the object with respect to the plane perpendicular to the optical axis of the objective lens.
- the spatial light modulator having the modulation surface, the objective lens disposed on the optical path between the modulation surface and the object, and the aberration caused by the refractive index interface of the object are reduced.
- the position of the correction pattern in the modulation pattern is set based on the inclination information of the refractive index interface of the object with respect to the plane perpendicular to the optical axis of the objective lens.
- the spatial light modulator and the objective lens are arranged based on inclination information of the refractive index interface of the object with respect to a plane perpendicular to the optical axis of the objective lens.
- the spatial light modulator has a modulation surface and modulates light based on a modulation pattern including a correction pattern for correcting an aberration caused by a refractive index interface of an object.
- An objective lens disposed on the optical path between the modulation surface and the object, a moving mechanism for moving at least one of the objective lens and the spatial light modulator in a direction crossing the optical axis of the objective lens, and a moving mechanism
- a control unit for controlling.
- the control unit controls the moving mechanism based on the inclination information of the refractive index interface of the object with respect to the plane perpendicular to the optical axis of the objective lens.
- the aberration correction method may further include a measurement step of measuring the tilt angle of the refractive index interface of the object, and the tilt information may be determined based on the tilt angle measured by the measurement step.
- the optical device may further include a measurement unit that measures the inclination angle of the refractive index interface of the object, and the inclination information may be determined based on the inclination angle measured by the measurement unit.
- a plurality of images of the object having different depths from the refractive index interface of the object may be acquired, and the tilt angle may be obtained based on the plurality of images.
- the measurement unit of the optical device may acquire a plurality of images of the object having different depths from the refractive index interface of the object, and obtain the inclination angle based on the plurality of images. Thereby, an inclination angle can be measured easily.
- the tilt information may be determined based on information relating to the tilt angle of the refractive index interface of the object stored in advance.
- the optical device may further include a storage unit that stores in advance information regarding the inclination angle of the refractive index interface of the object, and the inclination information may be determined based on information stored in the storage unit.
- the diameter of the correction pattern region on the modulation surface may be larger than the pupil diameter of the objective lens.
- the correction pattern or modulation surface
- the region where the correction pattern exists is biased with respect to the optical axis. Therefore, when the area of the correction pattern is small, there is a possibility that a part of the light does not pass through the correction pattern and the aberration correction is not performed. Such a fear can be reduced by making the diameter of the region of the correction pattern larger than the pupil diameter of the objective lens.
- the refractive index interface of the object is divided into a plurality of regions, the inclination angle in each region is measured, and the inclination information is determined based on the inclination angle in each region. May be.
- the measurement unit of the optical device divides the refractive index interface of the object into a plurality of regions, measures the tilt angle in each region, and the tilt information is determined based on the tilt angle in each region. Also good. As a result, even when the refractive index interface of the object has a complicated shape in which the inclination angle varies from region to region, the functions and effects of the aberration correction method and the optical device described above can be suitably obtained.
- the embodiment can be used as an aberration correction method and an optical apparatus.
- SYMBOLS 1A, 1B, 1C DESCRIPTION OF SYMBOLS 1A, 1B, 1C ... Microscope apparatus, 10, 10A ... Microscope unit, 11 ... Sample stand, 12 ... Objective lens, 12a ... Optical axis, 13 ... Objective lens moving mechanism, 14 ... Beam splitter, 15, 16 ... Reflection mirror, DESCRIPTION OF SYMBOLS 20 ... Inclination measurement unit, 21 ... Coherent light source, 22 ... Beam splitter, 23 ... Reference light mirror, 24 ... Detector, 30 ... Image acquisition unit, 31 ... Laser light source, 32 ... Beam expander, 33, 36 ... SLM , 33a, 36a ... modulation surface, 33b, 36b ... moving mechanism, 34 ...
- dichroic mirror 35 ... optical scanner, 37 ... detector, 37a ... condensing lens, 38 ... filter, 39a ... mirror, 39b ... reflecting member, 40 ... Control unit, 41 ... Input device, 42 ... Display device, 43 ... Computer, 51 ... 4f optical system, 60 ... Shape memory unit G, A1, A2 ... optical axis, B ... object, Ba ... surface, Bc ... contour, D1, D2 ... modulation pattern, D3 ... aberration correction pattern, H ... plane, P1 ... irradiation light, P2 ... detected light, P3: coherent light, P4: interference light, P5: light, O: center of correction pattern.
Landscapes
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Multimedia (AREA)
- Engineering & Computer Science (AREA)
- Nonlinear Science (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Computer Vision & Pattern Recognition (AREA)
- Microscoopes, Condenser (AREA)
- Optical Modulation, Optical Deflection, Nonlinear Optics, Optical Demodulation, Optical Logic Elements (AREA)
Abstract
Description
θmax=asin(NA/n1) ・・・(3)
L1=NA・f ・・・(4)
また、SLM33,36に対する対物レンズ12の結像倍率M(対物レンズ12とSLM33,36とがM:1の拡大結像関係(SLM33,36側の光径が対物レンズ12側の光径よりも小さい)にある)に基づいて、SLM33,36における光の半径L2は次の数式(5)により求められる。
L2=L1/M ・・・(5)
R=L2/p ・・・(6)
上記の数式(3)及び数式(6)から、対物レンズ12の光軸と光線とのなす角度1°あたりの画素数rは、次の数式(7)により求められる。
r=R/θmax=(NA・f)/(M・p) ・・・(7)
x1=βx・r ・・・(8)
y1=βy・r ・・・(9)
これらを数式(2)に適用することにより、SLM33,36に表示すべき収差補正パターンが導出される。
x1=βx・L2/θmax ・・・(10)
y1=βy・L2/θmax ・・・(11)
そして、変調面33a,36aを、x軸方向にx1、y軸方向にy1だけそれぞれ移動させるとよい。
Claims (14)
- 空間光変調器の変調面と対象物とを、対物レンズを介して光学的に結合する結合ステップと、
前記対象物の屈折率界面に起因する収差を補正するための補正パターンを含む変調パターンに基づいて前記空間光変調器を制御する制御ステップと、
を含み、
前記変調パターンにおける前記補正パターンの位置は、前記対物レンズの光軸に垂直な平面に対する前記対象物の前記屈折率界面の傾斜情報に基づいて設定される、収差補正方法。 - 空間光変調器の変調面と対象物とを、対物レンズを介して光学的に結合する結合ステップと、
前記対象物の屈折率界面に起因する収差を補正するための補正パターンを含む変調パターンに基づいて前記空間光変調器を制御する制御ステップと、
を含み、
前記空間光変調器及び前記対物レンズは、前記対物レンズの光軸に垂直な平面に対する前記対象物の前記屈折率界面の傾斜情報に基づいて配置される、収差補正方法。 - 前記対象物の前記屈折率界面の傾斜角度を計測する計測ステップを更に含み、
前記傾斜情報は、前記計測ステップにより計測された前記傾斜角度に基づいて決定される、請求項1または2に記載の収差補正方法。 - 前記計測ステップでは、前記対象物の前記屈折率界面からの深さがそれぞれ異なる前記対象物の複数の画像を取得し、前記複数の画像に基づいて前記傾斜角度を求める、請求項3に記載の収差補正方法。
- 前記傾斜情報は、予め記憶された前記対象物の前記屈折率界面の傾斜角度に関する情報に基づいて決定される、請求項1または2に記載の収差補正方法。
- 前記変調面における前記補正パターンの領域の直径が、前記対物レンズの瞳径より大きい、請求項1~5のいずれか一項に記載の収差補正方法。
- 前記計測ステップでは、前記対象物の前記屈折率界面を複数の領域に分割し、各領域における前記傾斜角度を計測し、
前記傾斜情報は、前記各領域における前記傾斜角度に基づいて決定される、請求項3に記載の収差補正方法。 - 変調面を有する空間光変調器と、
前記変調面と対象物との間の光路上に配置された対物レンズと、
前記対象物の屈折率界面に起因する収差を補正するための補正パターンを含む変調パターンに基づいて前記空間光変調器を制御する制御部と、
を備え、
前記変調パターンにおける前記補正パターンの位置は、前記対物レンズの光軸に垂直な平面に対する前記対象物の前記屈折率界面の傾斜情報に基づいて設定される、光学装置。 - 変調面を有し、対象物の屈折率界面に起因する収差を補正するための補正パターンを含む変調パターンに基づいて光を変調する空間光変調器と、
前記変調面と前記対象物との間の光路上に配置された対物レンズと、
前記対物レンズ及び前記空間光変調器の少なくともいずれか一方を前記対物レンズの光軸と交差する方向に移動させる移動機構と、
前記移動機構を制御する制御部と、
を備え、
前記制御部は、前記対物レンズの前記光軸に垂直な平面に対する前記対象物の前記屈折率界面の傾斜情報に基づいて、移動機構を制御する、光学装置。 - 前記対象物の前記屈折率界面の傾斜角度を計測する計測部を更に備え、
前記傾斜情報は、前記計測部により計測された前記傾斜角度に基づいて決定される、請求項8または9に記載の光学装置。 - 前記計測部は、前記対象物の前記屈折率界面からの深さがそれぞれ異なる前記対象物の複数の画像を取得し、前記複数の画像に基づいて前記傾斜角度を求める、請求項10に記載の光学装置。
- 前記対象物の前記屈折率界面の傾斜角度に関する情報を予め記憶する記憶部を更に備え、
前記傾斜情報は、前記記憶部に記憶された前記情報に基づいて決定される、請求項8または9に記載の光学装置。 - 前記変調面における前記補正パターンの領域の直径が、前記対物レンズの瞳径より大きい、請求項8~12のいずれか一項に記載の光学装置。
- 前記計測部は、前記対象物の前記屈折率界面を複数の領域に分割し、各領域における前記傾斜角度を計測し、
前記傾斜情報は、前記各領域における前記傾斜角度に基づいて決定される、請求項10に記載の光学装置。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020197020383A KR102490763B1 (ko) | 2017-04-14 | 2018-04-10 | 수차 보정 방법 및 광학 장치 |
US16/604,312 US11454793B2 (en) | 2017-04-14 | 2018-04-10 | Aberration correction method and optical device |
CN201880024335.3A CN110520779B (zh) | 2017-04-14 | 2018-04-10 | 像差校正方法和光学装置 |
JP2019512527A JP7033123B2 (ja) | 2017-04-14 | 2018-04-10 | 収差補正方法及び光学装置 |
EP18783927.9A EP3611549B1 (en) | 2017-04-14 | 2018-04-10 | Aberration correction method and optical device |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2017-080749 | 2017-04-14 | ||
JP2017080749 | 2017-04-14 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2018190339A1 true WO2018190339A1 (ja) | 2018-10-18 |
Family
ID=63793391
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2018/015080 WO2018190339A1 (ja) | 2017-04-14 | 2018-04-10 | 収差補正方法及び光学装置 |
Country Status (6)
Country | Link |
---|---|
US (1) | US11454793B2 (ja) |
EP (1) | EP3611549B1 (ja) |
JP (1) | JP7033123B2 (ja) |
KR (1) | KR102490763B1 (ja) |
CN (1) | CN110520779B (ja) |
WO (1) | WO2018190339A1 (ja) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20220252512A1 (en) * | 2021-02-08 | 2022-08-11 | Kla Corporation | Three-dimensional imaging with enhanced resolution |
CN113985539B (zh) * | 2021-11-04 | 2022-09-23 | 中国人民解放军国防科技大学 | 阵列光束倾斜像差校正系统 |
CN114894712B (zh) * | 2022-03-25 | 2023-08-25 | 业成科技(成都)有限公司 | 光学量测设备及其校正方法 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2002054909A (ja) * | 2000-08-11 | 2002-02-20 | Dainippon Screen Mfg Co Ltd | 画像取得装置 |
JP2010075997A (ja) | 2008-09-01 | 2010-04-08 | Hamamatsu Photonics Kk | 収差補正方法、この収差補正方法を用いたレーザ加工方法、この収差補正方法を用いたレーザ照射方法、収差補正装置、及び、収差補正プログラム |
JP2011180290A (ja) | 2010-02-26 | 2011-09-15 | Hamamatsu Photonics Kk | 収差補正方法、この収差補正方法を用いた顕微鏡観察方法、この収差補正方法を用いたレーザ照射方法、収差補正装置、及び、収差補正プログラム |
JP2014521122A (ja) * | 2011-07-14 | 2014-08-25 | ホワルド フグヘス メドイクアル インストイトウテ | 適応光学系を有する顕微鏡検査法 |
JP2015219502A (ja) * | 2014-05-21 | 2015-12-07 | 浜松ホトニクス株式会社 | 光刺激装置及び光刺激方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6265898B2 (ja) * | 2012-08-16 | 2018-01-24 | シチズン時計株式会社 | 収差補正光学ユニット及びレーザー顕微鏡 |
JP6772442B2 (ja) | 2015-09-14 | 2020-10-21 | 株式会社ニコン | 顕微鏡装置および観察方法 |
-
2018
- 2018-04-10 US US16/604,312 patent/US11454793B2/en active Active
- 2018-04-10 EP EP18783927.9A patent/EP3611549B1/en active Active
- 2018-04-10 JP JP2019512527A patent/JP7033123B2/ja active Active
- 2018-04-10 KR KR1020197020383A patent/KR102490763B1/ko active IP Right Grant
- 2018-04-10 CN CN201880024335.3A patent/CN110520779B/zh active Active
- 2018-04-10 WO PCT/JP2018/015080 patent/WO2018190339A1/ja unknown
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2002054909A (ja) * | 2000-08-11 | 2002-02-20 | Dainippon Screen Mfg Co Ltd | 画像取得装置 |
JP2010075997A (ja) | 2008-09-01 | 2010-04-08 | Hamamatsu Photonics Kk | 収差補正方法、この収差補正方法を用いたレーザ加工方法、この収差補正方法を用いたレーザ照射方法、収差補正装置、及び、収差補正プログラム |
JP2011180290A (ja) | 2010-02-26 | 2011-09-15 | Hamamatsu Photonics Kk | 収差補正方法、この収差補正方法を用いた顕微鏡観察方法、この収差補正方法を用いたレーザ照射方法、収差補正装置、及び、収差補正プログラム |
JP2014521122A (ja) * | 2011-07-14 | 2014-08-25 | ホワルド フグヘス メドイクアル インストイトウテ | 適応光学系を有する顕微鏡検査法 |
JP2015219502A (ja) * | 2014-05-21 | 2015-12-07 | 浜松ホトニクス株式会社 | 光刺激装置及び光刺激方法 |
Non-Patent Citations (2)
Title |
---|
NAOYA MATSUMOTOTAKASHI INOUEAKIYUKI MATSUMOTOSHIGETOSHI OKAZAKI: "Correction of depth-induced spherical aberration for deep observation using two-photon excitation fluorescence microscopy with spatial light modulator", BIOMEDICAL OPTICS EXPRESS, vol. 6, no. 7, 2015, pages 2575 - 2587 |
See also references of EP3611549A4 |
Also Published As
Publication number | Publication date |
---|---|
JPWO2018190339A1 (ja) | 2020-02-27 |
EP3611549A1 (en) | 2020-02-19 |
KR20190133145A (ko) | 2019-12-02 |
US11454793B2 (en) | 2022-09-27 |
US20200150423A1 (en) | 2020-05-14 |
EP3611549B1 (en) | 2024-05-29 |
EP3611549A4 (en) | 2021-01-20 |
CN110520779A (zh) | 2019-11-29 |
JP7033123B2 (ja) | 2022-03-09 |
CN110520779B (zh) | 2021-10-29 |
KR102490763B1 (ko) | 2023-01-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10302569B2 (en) | Microscope device and image acquisition method | |
CN106461925B (zh) | 用于具有自适应光学系统的拉曼散射光学显微镜的系统和方法 | |
US8731272B2 (en) | Computational adaptive optics for interferometric synthetic aperture microscopy and other interferometric imaging | |
JP6518041B2 (ja) | 光刺激装置及び光刺激方法 | |
US20110267663A1 (en) | Holographic image projection method and holographic image projection system | |
JP7033123B2 (ja) | 収差補正方法及び光学装置 | |
JP6850684B2 (ja) | 光計測装置 | |
JP2014098835A (ja) | 顕微鏡用照明光学系およびこれを用いた顕微鏡 | |
Kuś | Illumination-related errors in limited-angle optical diffraction tomography | |
JP6539391B2 (ja) | 顕微鏡装置及び画像取得方法 | |
JP6300739B2 (ja) | 画像取得装置および画像取得方法 | |
JP2011128572A (ja) | ホログラム像投影方法およびホログラム像投影装置 | |
Booth et al. | Adaptive optics for fluorescence microscopy | |
EP3853651B1 (en) | Confocal laser scanning microscope configured for generating line foci | |
CN107209359B (zh) | 图像取得装置以及图像取得方法 | |
US20210373307A1 (en) | Method for digitally correcting an optical image of a sample by means of a microscope, and microscope | |
JP6030180B2 (ja) | 収差補正方法、この収差補正方法を用いた顕微鏡観察方法、この収差補正方法を用いたレーザ照射方法、収差補正装置、及び、収差補正プログラム | |
KR20240041450A (ko) | 다중 배율 렌즈 기반 광 회절 단층촬영장치 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 18783927 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2019512527 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 20197020383 Country of ref document: KR Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2018783927 Country of ref document: EP Effective date: 20191114 |