WO2015077926A1 - Procédé d'imagerie microscopique à très haute résolution et système pour éclairage de lumière structurée pouvant être continuellement réglée - Google Patents

Procédé d'imagerie microscopique à très haute résolution et système pour éclairage de lumière structurée pouvant être continuellement réglée Download PDF

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WO2015077926A1
WO2015077926A1 PCT/CN2013/087883 CN2013087883W WO2015077926A1 WO 2015077926 A1 WO2015077926 A1 WO 2015077926A1 CN 2013087883 W CN2013087883 W CN 2013087883W WO 2015077926 A1 WO2015077926 A1 WO 2015077926A1
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Prior art keywords
fourier transform
lens
structured light
phase
illumination
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PCT/CN2013/087883
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English (en)
Chinese (zh)
Inventor
叶燕
陈林森
楼益民
刘艳花
周云
申溯
魏国军
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苏州大学
苏州苏大维格光电科技股份有限公司
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Application filed by 苏州大学, 苏州苏大维格光电科技股份有限公司 filed Critical 苏州大学
Priority to CN201380081848.5A priority Critical patent/CN105814402B/zh
Priority to PCT/CN2013/087883 priority patent/WO2015077926A1/fr
Priority to KR1020167016974A priority patent/KR102074279B1/ko
Publication of WO2015077926A1 publication Critical patent/WO2015077926A1/fr

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    • 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
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/082—Condensers for incident illumination only
    • 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
    • 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
    • G02B26/0816—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the direction of light by means of one or more reflecting elements
    • G02B26/0833—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the direction of light by means of one or more reflecting elements the reflecting element being a micromechanical device, e.g. a MEMS mirror, DMD
    • 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
    • G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems

Definitions

  • the invention relates to a super-resolution microscopic imaging method and system, in particular to a real-time/dynamic three-dimensional topography and nano-scale imaging method and system, which are applied to laser confocal microscopic imaging, bio-fluorescence detection, micro-nanotopography detection and micro
  • the nano structure writes the tag.
  • SIM Super Resolution Microscopy
  • Optical system imaging resolution is very limited, and its optical resolution does not exceed ⁇ /2.
  • nanoscale detection and analysis is essentially how to achieve super-resolution microscopic imaging.
  • Electron microscopy SEM: It can reach lOnrn resolution, and it is necessary to increase the conductive layer to carry out structural detection. It is not suitable for non-destructive testing and cannot be used for the analysis of active biological samples. At the same time, the SEM serial operation mode has low detection efficiency and is difficult to use for full-field detection of large-sized samples. 2.
  • AFM probe In recent years, the rapid development of detection methods, micro-probe scanning, to obtain nano-scale structure distribution, but AFM detection efficiency is very low, detection time is long, for the sample to be tested The structure has many limitations. Rapid detection and dynamic analysis of samples are not possible. 3. Confocal scanning microscopy (CSIM): The three-dimensional shape of the sample is obtained by scanning the confocal detection on the surface by focusing the laser spot. Imaging resolution Depends on the system diffraction optical limit ⁇ /2. For example, at 405 nm blue light, the confocal scanning microscopic limit resolution is 200 nm, which cannot meet the detection requirements of smaller sizes. At the same time, the scanning method is not used for the detection and analysis of the dynamic evolution of the sample due to the delay of the timing.
  • SEM serial read/write
  • DMD digital micromirror array
  • a spatial light modulator for example, Chinese Patent, Application No. 200810071628.5, 201110448980.8, 201210402820.4
  • DMD digital micromirror array
  • the mirror-driven version controls the duty cycle of each micromirror switch state by generating a series of pulse width modulation signals, and produces 256 levels of different brightness grayscale patterns, which are convenient, easy to input and output.
  • DMD (1024X768, 10.68um as an example
  • the structural light change is digital, and there are also many shortcomings. 1.
  • the modulation of the digital micromirror DMD is based on the unit micro
  • the rotation of the mirror is achieved by the reflection of the light, so that the fringe space frequency of the structured light is changed according to the magnification (digitization) of the micromirror unit, and the continuously changing structured light field cannot be generated.
  • a (0, pi/2, pi) phase change is produced, and a 500 lp/mm space-frequency DMD is best modulated.
  • DMD produces large digital errors in the rotation of the image, resulting in a significant degradation in phase modulation accuracy of structured light illumination.
  • the type of test sample is greatly limited, and the lateral resolution and detection accuracy need to be improved. 2.
  • Continuous light source illumination is used for detection and is greatly affected by the environment.
  • the dynamic factor makes the sample (system) vibration rate up to 2um/s-5um/s, and the CCD detection time is 40ms.
  • the sample instability range is 80 n m-200nm, and the final detection result will be superimposed with a random blur. the amount.
  • the CCD requires an integration acceptance time, and the detection time takes about several tens of milliseconds. Therefore, any system instability will result in uncertainty in the test data. This requires the detection system to have strict vibration isolation measures. 3.
  • the large NA objective optical system can improve the spatial frequency of the structured light field, but the focal depth (kl A /NA2) also decreases rapidly, such as NA1.49, 450nm wavelength, and the focal depth is only 200nm.
  • the system must have strict adaptive self-focusing measures.
  • the field of view of a single test is less than 20um. 4.
  • the breaking and focusing mode of DMD the samples are sampled by framing, and the 3D structure is synthesized. In the case of insufficient system stability, the detection accuracy of the 3D structure is not high.
  • the other is a modulator that uses a grid as a light source to project a fringe grid with a single spatial frequency to the sample.
  • the microscope can image the fringe of the fringe grille well, and the mark is sharp.
  • the stripe sample tomogram using a laterally moving or rotating grid (US Patent US8081378B2, US8160379B2, US6819415B2) changes its relative spatial position to achieve phase shifting of the structured illumination light, which requires a grid Position movement is strictly controlled and synchronized with the camera, imaging speed is slow and can only be used to observe static samples. Therefore, Chinese Patent 201210553557.9 uses three narrow-band lights or lasers with similar center wavelengths but non-overlapping spectra to form illumination light.
  • the three-way light passes through separate projection paths, and the grid is projected onto the sample to obtain illumination structure light, and then the split light is used.
  • the module projects the reflected light of samples of different wavelengths into different photosensitive areas of the imaging module and simultaneously images the system.
  • the system can only form three kinds of phase-shifted illumination structure light, and the system structure is complicated; Chinese patent 201210580743.1 is used to realize structured light
  • the illuminated disc divides the disc into nine sector-shaped regions, and each of the sector-shaped regions is provided with a grid, and the nine grids are sequentially divided into a first grid group including three grids, and a second grid.
  • each grid of the first grid group has a slope angle of minus 120 degrees
  • each grid of the second grid group has a slope angle of 0 degrees
  • the third grid group Each of the grids has a slope angle of 120 degrees
  • each set of grids is divided into three parallel grids, and When the grid in the same group is imaged, the stripe of each grid is laterally offset by one-third of a cycle. It is only necessary to rotate the disc to obtain different grids to meet the needs of the structured light illumination mode.
  • the system can obtain three groups of three.
  • the phase-shifted illumination structure light, phase shift amount and grid space frequency change are limited and discrete.
  • phase distribution is "unwrapped" to achieve structural detection that is less than the optical diffraction limit, ie, super-resolution imaging.
  • the structured light field reflected from the surface of the sample carries the phase change caused by the sample structure, and the structured light field acts as a "carrier frequency". Theoretically, the higher the “carrier frequency”, the greater the ability to change the phase; The smaller the phase shift of the fringes, the higher the accuracy of the constructed image.
  • the object of the present invention is to provide a super-resolution microscopic imaging method and system for continuously adjustable structured light illumination to obtain spatial super-resolution imaging, improve nano-detection reliability and detection speed, and simultaneously realize dynamic detection and analysis of samples.
  • the technical solution adopted by the present invention is: a super-resolution microscopic imaging system with continuously adjustable structured light illumination, including a computer, a light source, a variable-scale Fourier transform optical path, a phase splitting device, and a double far a cardiac projection optical system, a large numerical aperture objective lens, a sample platform, and an area array camera, wherein the Fourier transform optical path includes a first Fourier transform lens or lens group and a second Fourier transform lens or lens group, and the phase splitting optical device is placed a Fourier transform lens or lens group and a second Fourier transform lens or lens group; the distance between the phase splitting device and the second Fourier transform lens or lens group is continuously adjustable; the phase splitting device has a Fourier transform The degree of freedom of motion of the optical axis of the optical path.
  • the light source may be a continuous laser source or a pulsed laser source, and the pulse source includes not limited to a nanosecond pulse laser source, a picosecond pulse laser source, a femtosecond pulse laser source, or the like.
  • the pulsed laser provides a stroboscopic source with a modulation frequency of up to 1 kHz and a pulse width of 5 ns to 20 ns.
  • the light source can be a laser diode source.
  • the system can write nanostructures; under nanosecond laser sources, nanoseconds Time-series stroboscopic framing illumination can not only achieve super-resolution microscopic imaging, improve nano-detection reliability and detection speed, but also perform dynamic detection and analysis of samples to realize detection of transient nanostructures. Fluorescence microscopy; when illuminated with diode light sources of different wavelengths, the interference fringes produced at the back focal plane of the second Fourier transform lens or lens group are different.
  • the area array camera can be a charge coupled device CCD camera, a complementary metal oxide semiconductor CMOS camera, or other camera.
  • Computer controlled light source phase splitting device, dual telecentric projection optics, and sample platform motion.
  • the phase splitting device is a one-dimensional grating structure.
  • the phase splitting device is a binary optical structure.
  • the phase splitting device forms nano interference fringes on the back focal plane of the second Fourier transform lens or lens group.
  • the phase splitting device by setting the phase splitting device, the positive and negative first-order diffracted spots are separated on both sides of the Fourier transform optical axis, and the zero-order light is eliminated.
  • the phase splitting device is located in the focal plane, the two diffracted light points are combined into one. .
  • interference fringes of continuously variable spatial frequencies can be generated at the back surface of the second Fourier transform lens or lens group.
  • interference fringes of different orientations can be produced at the back surface of the second Fourier transform lens or lens group.
  • interference fringes of different spatial frequencies and different orientations can be generated on the back focal plane of the second Fourier transform lens or lens group.
  • the system includes a spatial light modulator, and the spatial light modulator is located between the light source and the Fourier transform optical path.
  • the nano-interference strips on the back focal plane of the second Fourier transform lens or lens group, the sample on the platform, and the CCD are confocal.
  • a field stop and a iris diaphragm may be provided in the optical path as needed.
  • a super-resolution microscopic imaging method for continuously adjustable structured light illumination which is implemented by the above system, after the laser beam is expanded, after being irradiated on the first Fourier transform lens or lens group, The optical device generates positive and negative first-order diffracted light spots, passes through the second Fourier transform lens or lens group, forms an interference light field on the back focal plane, and then passes through the combination of the double telecentric projection optical system and the large numerical aperture objective lens, on the sample platform.
  • a nano-scale subdivided light field is formed on the surface; the Z-direction adjustment of the objective lens is performed to achieve nano-precision focusing, and the sample is formed with nano-scale structured light illumination to realize sample structure detection.
  • the red astigmatism path and the reflected green fluorescence are respectively subjected to autofocus optical detection (longitudinal) and alignment illumination (lateral) and detection, and the CCD and the sample are in a conjugate confocal position, and the reflected structure can be directly detected.
  • the phase distribution of the light field, the polarization splitting and the two-color beam splitter ensure that the wavelengths do not interfere with each other.
  • the beam is expanded, it is irradiated onto the first Fourier transform lens or lens group via a spatial light modulator (digital micro-mirror, DMD).
  • a spatial light modulator digital micro-mirror, DMD
  • the phase optical element can adopt a phase grating.
  • a phase grating is a grating that eliminates zero-order light.
  • the positive and negative first-order diffracted spots are on the focal plane of the first Fourier transform lens (group), on both sides of the discrete optical axes, the distance between the two spots and the phase grating to the focal plane. The distance is proportional to the distance of the phase grating from the focal plane, and the distance separating the spots is larger.
  • the phase grating is in the focal plane, the two diffracted spots are combined into one. Therefore, moving the phase grating, the spot distance achieves a continuous change.
  • the interference fringes (light field) spatial frequencies formed on the back focal plane of the second Fourier transform lens (group) will continuously change.
  • the light field containing the interference fringes is further improved by the subsequent projection micro-deflection optical system.
  • the projection optical system have a micromultiplication of M, f is the focal length of the Fourier transform lens, and F is the phase grating grating frequency
  • ⁇ is the amount of change in the phase grating grating to the focal plane distance.
  • the focal length of the objective lens (NA0.95) is l mm
  • F 120 lp/mm
  • the structured light illumination can be used not only for one-dimensional interference fringes (grating structure), but also for replacing the phase grating with a binary phase element, and generating a two-dimensional structured light field for structural light illumination to obtain a two-dimensional space frequency. Adjustable structured light field.
  • the present invention has the following advantages over the prior art:
  • this scheme uses a pulsed laser source, which is illuminated with nanosecond timing, and the illumination time per frame is 5 nanoseconds to 20 nanoseconds. Assume that the sample amplitude is 5mm/s, and within 20 nanoseconds of illumination, the amount of blurring of the test results affected by vibration is less than 0.4nm. Therefore, the nanosecond timing illumination method overcomes the influence of environmental vibration, eliminates the detection unreliability caused by continuous light source modulation, and ensures the detection precision of finer phase change information.
  • positional triggering ensures a positioning accuracy of ⁇ 1 pulse.
  • crossover lighting can get more accurate 3D data detection.
  • the stroboscopic illumination of the repetition frequency such as 20 ns per illumination time and 80 um of light field size, can detect images of hundreds of frames or more during the detection period. In this way, not only the detection of the 3D sample can be detected and verified by the change of the space frequency, but also the dynamic evolution of the sample can be detected to obtain a more detailed 3D morphology.
  • the NA0.95 microscope objective has a focal depth of 200nm-300nm.
  • the detection is greatly affected by environmental vibration, and reliability is difficult to guarantee.
  • Optical interferometer using a continuous space-frequency lighting the size of the phase grating can be done 20mmx200m m, therefore, the size of the structured light field (field) up to Above 80um, in this way, the stripe box depth (depth of field) of the structured illumination reaches l Oum, and the large field of view 3D shape detection is realized by sampling and synthesizing the continuous sample.
  • this method allows for fluorescence microscopic examination of samples.
  • the system can write to the nanostructures.
  • Figure 1 Optical path system for continuous variable space-frequency structured light illumination
  • Figure 2 Continuously adjustable structured light illumination super-resolution microscopy imaging system
  • Figure 3 is a Fourier transform system when the phase element is close to the first Fourier transform lens
  • Figure 4 is a Fourier transform system when the phase element is remote from the first Fourier transform lens
  • Figure 7 Steps for dynamic detection of continuous frequency-divided structured light illumination
  • Fig. 8 Spatially modulated large-format continuously adjustable structured light illumination super-resolution microscopic imaging system; Figure 9 Different wavelength down-conversion optical path system;
  • Figure 10 Fourier transform system when the phase component is a two-dimensional orthogonal grating.
  • Embodiment 1 Continuously adjustable structured light illumination optical path system
  • FIG. 1 The schematic diagram of the propagation of two rays of the continuously adjustable structured light illumination optical path system in this embodiment is as shown in FIG. 1 , wherein the angle between the first-order diffracted light and the optical axis generated by the primary light passing through the phase grating grating is ⁇ , and then After the lens is collimated, the angle with the optical axis is ⁇ , and the phase grating grating period is ⁇ . After the zero-order light is eliminated, the positive and negative first-order diffracted lights are interfered in the light field of the output plane. (Grating) The period is PN.
  • the focal length of the 4f optical system is f
  • the distance between the phase grating grating and the front lens is dz
  • the angle between the chief ray and the focal plane is 0
  • the distance between the parallel light emitted by the O point and the optical axis is h
  • the parallel from the O point The light passing through the rear lens is parallel to the light passing through the rear lens.
  • the continuous translation of the phase grating grating on the converging light behind the lens will cause the focal length of the positive and negative first-order diffracted lights to change continuously, and the interference fringes in the output light field realize the structured light.
  • the continuous change of the space frequency in this way, has the following relationship: ⁇ - ⁇ , tana" - ⁇ - ⁇ , P x sin - ⁇ tan
  • the light field containing the interference fringes is further improved by the subsequent projection micro-deflection optical system.
  • the projection optical system have a micromultiplication of M, f is the focal length of the Fourier transform lens, and F is the phase grating grating frequency
  • the space frequency of the interference fringes obtained by changing dz is [0, 2FM] lp/mm.
  • ⁇ is the amount of change in the phase grating grating to the focal plane distance.
  • the phase grating F 120 lp/mm
  • the focal length of the objective lens (NA0.95) is 1 mm
  • the interference fringe space frequency of the structured light field on the sample [0, 5000] lp/mm, where the maximum value is 5000 lp/mm (200 nm period) obtained at dz 0.
  • the structural illumination can be used not only for one-dimensional interference fringes (grating structure), but also for replacing the phase grating with a binary phase element, and generating a two-dimensional structured light field for structural light illumination to obtain a two-dimensional space frequency. Adjustable structured light field.
  • the Fourier transform system described in this embodiment includes a Fourier transform optical system of any form or combination.
  • Embodiment 2 Large-format continuous adjustable structured light illumination super-resolution microscopic imaging system.
  • the continuously adjustable structured light illumination super-resolution microscopic imaging system in this embodiment comprises a nanosecond pulse laser 12, a spatial filter 13, a first Fourier transform lens 8, and a diffracted light.
  • Grid 9 second Fourier transform lens 10, field stop 16, real-time detection optical path 17, half mirror 18, tubelensl9, micro objective 30, autofocus optical path 31, two-dimensional precision translation stage 32, motion controller 33
  • the control computer 34 detects the sample 35.
  • the laser light emitted by the nanosecond pulsed laser 12 is beam-aligned by the spatial filter 13 to form parallel light into the grating space frequency and angle formed by the first Fourier transform lens 8, the diffraction grating 9, and the second Fourier transform lens 10.
  • the optical path is continuously modulated, and raster fringe information specifying a spatial frequency and orientation is formed in a region after the second Fourier transform lens 10, and a field stop 16 is disposed on the back focal plane of the second Fourier transform lens for limiting interference fringes.
  • the grating stripe passing through the field stop passes through the half mirror 18, and the tubelens 19 and the micro objective lens 30 form a subdivided stripe having a high frequency structure on the surface of the test sample 35.
  • the autofocus optical path 31 monitors and adjusts the distance between the focusing objective and the surface of the detecting sample 35 in real time, ensures accurate imaging of the high frequency grating stripe on the surface of the photosensitive material, realizes nanometer precision focusing, and forms a structured light illumination with nanometer scale for the sample, Sample structure detection.
  • the real-time detection optical path 17 performs real-time imaging of the phase change of the surface stripe of the sample 35, where the CCD and the sample are in a conjugate confocal position, and the phase distribution of the reflected structured light field can be directly detected, and the polarization splitting and the two-color beam splitter ensure each The wavelengths do not interfere with each other.
  • the lateral detection of the surface morphology of the sample is realized, and the longitudinal detection of the surface topography of the sample is realized by combining the autofocus optical path to realize the three-dimensional shape detection of the sample 35.
  • the motion controller 33 controls the movement or rotation or simultaneous movement and rotation of the diffraction grating 9 under the program set by the control computer 34, thereby realizing the spatial frequency or orientation of the grating stripe or the continuous change of the spatial frequency and orientation, obtaining more than one type.
  • the phase distribution of the reflected structured light field realizes the super-resolution three-dimensional topography detection of the sample 35.
  • the motion controller 33 coordinates the pulse timing of the nanosecond pulsed laser 12, the movement and rotation of the diffraction grating 9, and the two-dimensional precision under the program control set by the control computer 34.
  • the two-dimensional movement of the translation stage 32 detects the three-dimensional shape of the large-format sample by detecting the three-dimensional shape of different areas on the surface of the sample.
  • Embodiment 3 Detecting method of framing structure light field illumination
  • the system shown in FIG. 2 is used, and the framing structure light field illumination detection method with different space frequencies can be adopted.
  • the phase element 9 is translated along the optical axis of the optical path system under the control of the computer, thereby causing the phase element to be separated from the Fourier transform lens 8
  • the spatial frequencies of the resulting structured light illumination are also different, as shown in Figure 3 and Figure 4.
  • the phase grating F 120 lp/mm
  • the focal length of the objective lens (NA0.95) is 1 mm.
  • the resulting structured light illumination space frequency is 45001 p/mm; when the phase grating 9 and the lens 8 have a distance dz of 80 mm, as shown in FIG. 4, the resulting structured light illumination space frequency is 30001p. /mm.
  • the spatial frequency of the structured light projected by the system on the surface of the sample is different.
  • the system shown in Fig. 2 is used, and the framing illumination detection mode of different orientations can also be adopted.
  • the phase element 9 is rotated about the optical axis under the control of the computer, and is rotated from the P1 direction to the P2 direction, as shown in Fig. 5, when the phase element 9 is at the P1 position.
  • the grating stripe is perpendicular to the P1 direction.
  • the phase component is at the P2 position
  • the illumination grating is in the xy plane, and the grating stripe is perpendicular to the P2 direction, between the two grating strips.
  • the included angle is ⁇ , that is, when the phase element is rotated about the optical axis, the grating orientation formed on the back focal plane of the Fourier transform lens is different, so that structured light illumination of different orientations can be realized by rotating the phase element.
  • the phase element 9 is in the x ly l plane.
  • the phase element is at the P 1 position (ie, the yl direction)
  • the grating formed on the back focal plane is in the xy plane, and the grating stripe is perpendicular to P 1 .
  • Direction ie y direction parallel to yl
  • the phase element is at the P2 position (ie, at a 20 degree angle to the yl direction)
  • the grating formed on the back focal plane is in the xy plane, and the grating stripe is perpendicular to the P2 direction ( That is, it is 60 degrees from the y direction).
  • the system shown in Fig. 2 is used, and a framing illumination detection method of a specific space frequency and a specific orientation can also be employed.
  • the phase element 9 is translated along the optical axis or rotated around the optical axis under the control of a computer, and the gratings formed on the back focal plane of the Fourier transform lens have different spatial frequencies and orientations, thereby being translated and rotated.
  • the phase elements enable structured light illumination in different orientations.
  • the phase grating 9 When it is desired to obtain a 60-degree angle between the stripe orientation of the structured light illumination and the clockwise direction of the P1 direction, the phase grating 9 is rotated clockwise by an angle of 60 degrees in the direction of the optical axis; when it is desired to obtain the stripe orientation of the structured light illumination When the P1 direction is clockwise at a 90 degree angle, the phase grating 9 is rotated clockwise by 90 degrees in the direction of the optical axis; when the stripe orientation of the structured light illumination is desired to be 30 degrees from the counterclockwise direction in the P1 direction, Then, the phase grating 9 is rotated counterclockwise by an angle of 30 degrees around the optical axis.
  • the system can adopt different spatial frequency structured optical framing illumination methods; structured optical framing illumination methods with the same spatial frequency and different orientations, and structured optical framing illumination with specific variable spatial frequencies and orientations. the way.
  • Embodiment 4 Structured light illumination using DMD and continuous variable space frequency mutual modulation
  • a structured light illumination system using DMD and continuous variable spatial frequency modulation includes a nanosecond pulse laser 12, a spatial filter 13, a mirror 14, a DMD spatial light modulator 15, First Fourier transform lens 8, diffraction grating 9, second Fourier transform lens 10, field stop 16, real-time detection optical path 17, half mirror 18, tubelensl9, micro objective 30, autofocus optical path 31, two-dimensional precision Translation stage 32, motion controller 33, control computer 34, and sample 35 are detected.
  • the laser light emitted by the nanosecond pulsed laser 12 is expanded and collimated by the spatial filter 13 to form parallel light incident on the DMD spatial light modulator 15, and the DMD spatial light modulator is used as an iris diaphragm for controlling the parallel beam.
  • the spot size and shape, the light reflected by the DMD spatial light modulator enters the grating space frequency and the angle continuous modulation optical path formed by the first Fourier transform lens 8, the diffraction grating 9, and the second Fourier transform lens 10, in the second Fourier transform
  • a grating fringe specifying a spatial frequency and orientation is formed on the back focal plane of the lens 10
  • a field stop 16 is disposed on the back focal plane, and a grating stripe passing through the field stop passes through the half mirror 18, tubelensl9 and
  • the miniature objective lens 30 forms a high frequency grating stripe on the sample 35.
  • the lateral detection of the surface morphology of the sample is realized, and the longitudinal detection of the surface topography of the sample is realized by combining the autofocus optical path to realize the three-dimensional shape detection of the sample 35.
  • Motion controller 33 is provided at control computer 34 Under the fixed program, the movement or rotation or simultaneous movement and rotation of the diffraction grating 9 is controlled to realize the continuous change of the space frequency or orientation or the spatial frequency and orientation of the grating stripe, and the phase distribution of the light field of more than one reflective structure is obtained.
  • Sample 35 super-resolution three-dimensional topography detection.
  • Embodiment 5 Structured light illumination with continuous frequency division, timing for dynamic sample detection.
  • the step of continuously detecting the dynamic illumination of the continuous frequency division structure is as shown in FIG. 7. First, the sample platform is pre-scanned, and the position sensor is passed.
  • the microscopic objective lens with a numerical aperture of NA0.95 has a focal depth of generally 200 nm to 300 nm, and the detection is greatly affected by environmental vibration, and reliability is difficult to ensure.
  • the size of the phase grating can be 20mmx200mm, therefore, the size of the structured light field
  • the framing illumination that is, the stroboscopic illumination of the repetition frequency, such as 20 ns per illumination time and 80 um of the optical field size, can detect images of hundreds of frames or more during the detection period. In this way, not only the detection of the 3D sample can be detected and verified by the change of the space frequency, but also the dynamic evolution of the sample can be detected to obtain a more detailed 3D morphology; in addition, by sampling and synthesizing the continuous sample, Large field of view 3D shape detection.
  • Embodiment 6 Large-area framing structure light field illumination detection method
  • the continuous-frequency structured light illumination is used for dynamic sample detection.
  • the detection system can also be as shown in FIG. Show.
  • the automatic optical path 31 uses two different wavelengths of illumination light sources 311 and 312, wherein the 311 and 18, 30 and 313 units constitute an autofocus detection optical path to achieve an autofocus function perpendicular to the motion platform; 312 and 18, 30 and 313 units
  • the automatic positioning optical path is formed to realize seamless splicing of the detection light field parallel to the direction of the motion platform.
  • high-precision large-format dynamic detection is realized.
  • Automatic light in this embodiment The road 31 can be applied to a detection system as shown in Fig. 2 or Fig. 6.
  • Embodiment 7 3D structure detection using structured light field illumination according to Fourier series
  • the low frequency part reflects the low gradient part of the surface topography, That is, the contour or shape of the shape
  • the high frequency part reflects the high gradient characteristic of the surface topography, that is, the detail or the mutation
  • the higher the frequency the higher the level of detail reflected. Therefore, continuous variable frequency structured light illumination enables high-resolution 3D structure detection of objects.
  • Frequency is [0,
  • , ⁇ !j Az AFnf/(2FM), where ⁇ is the phase element 9
  • ⁇ is the phase element 9
  • the amount of change to the focal plane distance, the positive and negative signs indicate the direction in which the phase element 9 translates along the optical axis.
  • the focal length of the objective lens (NA0.95) is l mm
  • the spatial frequency of the structured light illumination is [0,
  • Example 8 Using different wavelengths
  • various types of light sources such as a continuous laser light source, a pulsed laser light source, and a laser diode light source may be employed, and various wavelengths or different colors may be employed.
  • the source of the wavelength may be employed.
  • the lens has different refractive powers for incident light of different wavelengths, in the same optical path system, as shown in FIG. 9, incident light of different wavelengths After passing through the first Fourier transform lens, the position of the illumination on the phase element and the incident angle are different ( ⁇ ⁇ ⁇ 3).
  • different wavelengths can also be used, combined with the translation and rotation of the phase components, and structured light illumination detection of different spatial frequency variation ranges can be realized according to the detection requirements.
  • Embodiment 9 Using a two-dimensional phase modulation component to form a two-dimensional structured light illumination
  • a two-dimensional structured light illumination method in which the grating space frequency and the orientation are continuously variable includes a first Fourier transform lens 8 having a focal length f1 and a 4F optical lens formed by a second Fourier transform lens 10 having a focal length f2.
  • the diffraction grating 9 is located between the focal length of the first Fourier transform lens and the first Fourier transform lens, and the diffraction grating is a two-dimensional grating structure of various shapes.
  • the diffraction grating can be moved along the optical axis 11 or can be rotated about the optical axis 11.
  • the continuous modulation of the grating null frequency parameter is achieved by moving the diffraction grating 9 to change the distance dz between the diffraction grating and the first Fourier transform lens.
  • Continuous modulation of the grating orientation angle parameter is achieved by rotating the diffraction grating 9. As shown in Figure 1.
  • the diffraction grating 9 may be a two-dimensional grating structure of various forms.
  • the phase grating 9 is a rectangular orthogonal two-dimensional grating, as shown in FIG. 10, it is on the back focal plane of the Fourier transform lens. Form an orthogonal two-dimensional lattice.
  • carrier frequency 500 lp/mm 5000 lp/mm
  • Phase modulation 0, 0.5pi, lpi (stepped) 0 - pi, 0.05pi (continuous change)
  • Structural light field rotation has digital error.
  • the rotation of the structured light field has no digital error.
  • the interference fringe (grating) formation method in continuous space-frequency modulation, phase change is more precise, higher, nanosecond timing illumination with phase sampling, detectable dynamic evolution process and 3D structure.

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Abstract

La présente invention porte sur un procédé d'imagerie microscopique à très haute résolution et sur un système pour un éclairage par une lumière structurée pouvant être continuellement réglée. Le système comprend un ordinateur (34), une source lumineuse (12), un trajet optique de transformée de Fourier ayant une échelle variable, un dispositif de division de lumière de phase (9), un système optique de projection télécentrique double (19), une lentille de focalisation (30) ayant une ouverture numérique large, une plate-forme d'échantillon (32) et une caméra à réseau matriciel. La présente invention est caractérisée par le fait que le trajet optique de transformée de Fourier comporte une première lentille de transformée de Fourier ou un premier groupe de lentilles (8) et une seconde lentille de transformée de Fourier ou un second groupe de lentilles (10), et le dispositif de division de lumière de phase (9) est disposé entre les deux, présente une distance pouvant être continuellement réglée depuis la seconde lentille de transformée de Fourier ou le second groupe de lentilles (10) et possède un degré de liberté de mouvement pour tourner autour de l'axe optique du trajet optique de transformée de Fourier. Le procédé d'imagerie microscopique à très haute résolution et le système pour un éclairage par une lumière structurée pouvant être continuellement réglée peuvent réaliser de façon flexible les franges d'interférence d'une fréquence spatiale continuellement variable et sont utilisés pour un éclairage de champ de lumière structurée d'un système optique microscopique confocal de manière à obtenir une imagerie de très haute résolution spatiale. En un mode d'éclairage de trame stroboscopique nanoseconde, non seulement l'imagerie microscopique à très haute résolution peut être effectuée pour améliorer la fiabilité et la vitesse de détection nanométrique, mais l'analyse de détection dynamique d'un échantillon peut encore être effectuée afin d'obtenir la détection d'une nanostructure transitoire.
PCT/CN2013/087883 2013-11-27 2013-11-27 Procédé d'imagerie microscopique à très haute résolution et système pour éclairage de lumière structurée pouvant être continuellement réglée WO2015077926A1 (fr)

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