WO2024007674A1 - 一种实现超高速结构光照明显微成像的方法和装置 - Google Patents
一种实现超高速结构光照明显微成像的方法和装置 Download PDFInfo
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- 238000005286 illumination Methods 0.000 title claims abstract description 85
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- 238000003384 imaging method Methods 0.000 claims abstract description 48
- 230000003287 optical effect Effects 0.000 claims abstract description 48
- 238000007493 shaping process Methods 0.000 claims abstract description 4
- 230000010287 polarization Effects 0.000 claims description 44
- 230000008859 change Effects 0.000 claims description 13
- 229940125730 polarisation modulator Drugs 0.000 claims description 2
- 238000010586 diagram Methods 0.000 description 5
- 239000013307 optical fiber Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 102000014150 Interferons Human genes 0.000 description 2
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- 238000000399 optical microscopy Methods 0.000 description 2
- 210000001747 pupil Anatomy 0.000 description 2
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- 241001270131 Agaricus moelleri Species 0.000 description 1
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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
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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
- G02B21/0052—Optical details of the image generation
- G02B21/0068—Optical details of the image generation arrangements using polarisation
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- 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
- G02B26/12—Scanning systems using multifaceted mirrors
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B5/00—Optical elements other than lenses
- G02B5/30—Polarising elements
- G02B5/3083—Birefringent or phase retarding elements
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- 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
- G02F1/03—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 based on ceramics or electro-optical crystals, e.g. exhibiting Pockels effect or Kerr effect
Definitions
- the invention belongs to the field of optical engineering, and particularly relates to a method and device for realizing ultra-high-speed structured illumination microscopic imaging.
- Optical microscopy imaging technology is a means of detecting the microscopic world. It is widely used in biomedical research because of its non-contact and non-destructive characteristics. However, the resolution of traditional optical microscopy imaging methods cannot break through the diffraction limit and cannot be used to observe microstructures below 200nm.
- Structured illumination microscopy imaging is a super-resolution imaging technology that modulates the illumination light to obtain multiple sample images under different lighting, and finally reconstructs a super-resolution image. Its theoretical resolution limit can reach the diffraction limit. half of. Although structured illumination microscopy technology can improve resolution, it sacrifices imaging speed. The reconstruction of each super-resolution image requires several original pictures captured under illumination modulation. The final imaging speed not only depends on the camera's shooting and reading speed, but also is limited by the modulation speed of the illumination light.
- grating mechanical movement has the slowest modulation speed; spatial light modulator
- the modulation type can be divided into two types: liquid crystal spatial light modulator (SLM) and digital micromirror device (DMD).
- SLM liquid crystal spatial light modulator
- DMD digital micromirror device
- the former is slower and the latter has lower diffraction efficiency; the galvanometer scanning type is the fastest, but it still cannot meet the requirements. Imaging speed at KHz level.
- the patent application with publication number CN105487214A provides a fast three-dimensional super-resolution microscopy method, including: converting the laser beam into linearly polarized light after collimation, phase modulating the linearly polarized light and converting it into circularly polarized light and projecting it to the sample to be measured on the sample, and collect the signal light emitted from each scanning point of the sample to be tested; perform a three-dimensional scan on the sample, and the phase modulation includes primary phase modulation and secondary phase modulation; the primary phase modulation adopts phase modulation of the s-light component of the linearly polarized light
- the spatial light modulator, the secondary phase modulation adopts the spatial light modulation of phase modulating the p-light component of the linearly polarized light. controller, and finally obtain a three-dimensional super-resolution image based on the effective signal intensity.
- a spatial light modulator is used for illumination light modulation, and the imaging speed is slow.
- the patent application with publication number CN206848565U provides an optical focus enhancement system based on a digital micromirror device; the first optical fiber and the collimating lens are arranged after the laser, and the beam emitted by the laser is transmitted through the first optical fiber and then incident on the digital micromirror through the collimating lens.
- the digital micromirror device is equipped with a light barrier at the side exit end, a beam shrinking module is installed at the front exit end of the digital micromirror device, and a dichroic mirror is provided in front; the light beam enters through the scanning module after being reflected by the dichroic mirror
- the microscope objective is focused, and the experimental sample is located on the focal plane of the microscope objective; the fluorescence excited in the experimental sample passes through the microscope objective and the scanning module, and then passes through the dichroic mirror and is received by the light intensity detection module for light intensity detection.
- Using digital micromirror devices for illumination light modulation has the problem of low diffraction efficiency.
- the purpose of the present invention is to provide a method for realizing ultra-high-speed structured light microscopy imaging. This method can greatly increase the modulation speed of illumination light, thereby realizing ultra-high-speed (KHz level) structured light illumination imaging.
- a method to achieve ultra-high-speed structured illumination microscopy imaging including:
- Each interference sub-light path represents illumination interference fringes in different directions on the sample surface
- the beam of the central interference light path is combined with the p-polarized light and s-polarized light of each interference sub-light path and then illuminated on the sample to form a structured light illumination pattern;
- an image acquisition module based on a high-speed rotating mirror is used for imaging, and a single exposure can achieve Like multiple images, experimental images are captured at high speed to match the speed of lighting modulation.
- the illumination beam is reflected and transmitted after passing through the polarizing beam splitter, the reflected part of the light enters the central interference light path, and the transmitted light path is divided into multiple interference sub-light paths in sequence.
- each interference sub-optical path is selectively controlled.
- a picture is recorded.
- all the sub-optical paths are sequentially gated, a complete set of structured light illumination images is obtained.
- each interference sub-optical path is gated at the same time, and the light beams of each interference sub-optical path interfere together to generate a two-dimensional structured light illumination pattern.
- each optical path can be gated at the same time, and the energy of each sub-optical path can be distributed in any proportion, so that the light beams of each sub-optical path interfere together to produce a two-dimensional structured light illumination pattern without being limited to a one-dimensional grating type. Lighting patterns.
- the invention also provides a device for realizing ultra-high-speed structured illumination microscopic imaging, which includes a light source that emits an illumination beam, an image acquisition module that collects fluorescence emitted by the sample, and also includes:
- the first polarization beam splitter splits the illumination beam, the reflected light enters the central interference light path, and the transmitted light is divided into multiple interference sub-light paths in turn;
- a gating module corresponding to each of the multiple interference sub-optical paths is used to control the gating state of each interference sub-optical path;
- the second polarization beam splitter located on each interference sub-optical path divides the light in each interference sub-optical path into p-polarized light and s-polarized light;
- Deflection beam combining module used to combine the beam of the central interference optical path with the p-polarized light and s-polarized light of each interference sub-optical path;
- the beams After combining the beams, they are illuminated on the sample to form a structured light illumination pattern and excite fluorescence, which is imaged to the image acquisition module.
- the illumination laser generated by the light source is divided into two beams through the first polarization beam splitter, one beam enters the central interference optical path, and the other beam is controlled and gated by multiple sets of gating modules, so that multiple The interferon light paths are switched on and off in turn.
- the beam will pass through the high-speed phase control device and the optical path compensation system successively.
- the optical path compensation system compensates for the optical path difference between the central interference light path and the interference sub-light paths, thereby maintaining coherence between all four interference light paths.
- the high-speed phase control device converts the light beam into s-polarized light and p-polarized light with equal energy, and makes it have a specific phase difference.
- the polarizing beam splitter separates the two polarized lights and combines the reflector and the half-wave plate to change the spatial angle of the beam.
- the function of the half-wave plate is to rotate the polarization directions of the two interference beams so that their polarization directions become consistent, so that the interference result has maximum contrast.
- the light source and the first polarizing beam splitter are sequentially provided with: an acousto-optic modulator for high-speed on-off control of the light beam; a beam shaping module for generating collimated light with uniform spatial intensity distribution; and The beam shrinking module is used to change the beam diameter so that it matches the working aperture of the electro-optical modulator.
- the gating module includes a polarization electro-optical modulator and a polarization beam splitter, and the central interference optical path is provided with: a first phase electro-optical modulator, used to phase modulate the light beam; a first polarization modulator arranged sequentially along the optical path.
- the device is used to change the polarization direction of the beam; and the first beam expansion module is used to expand the beam.
- the electro-optical modulator is used to control the gating state of the sub-optical path at high speed, and the electro-optical modulator is used to perform high-speed phase modulation of the interference beam, control its phase difference, produce different lighting patterns, and break through the speed limit of ordinary phase modulation means;
- use Linearly polarized lighting uses electro-optical modulators to control the polarization direction of the beam at high speed, and cooperates with polarizing beam splitters to control the on and off of different sub-light paths at high speed.
- the polarization electro-optic modulator can change the polarization direction of light, thereby controlling its transmission or reflection at the polarization beam splitter.
- the reflected light will enter the first interference sub-optical path, and the transmitted light will enter the next set of gating modules and continue to be divided into the second Interference sub-light path and third interference sub-light path.
- each interference sub-optical path is provided with: a first half-wave plate, used to change the polarization direction of the light beam so that the s component and the p component of the light beam are equal; a second phase electro-optical modulator, It is used to phase modulate the beam, and the s component and p component of the beam produce a relative phase difference; the second beam expansion module is used to expand the beam, and the optical path compensation module is used to compensate for the optical path difference between the interference optical paths; and a third polarization beam splitter for dividing the light beam into p-polarized light and s-polarized light.
- a half-wave plate is used to change the polarization direction of the interference beam to obtain the maximum interference contrast, and all interference beams will be corrected for optical path difference through the optical path compensation module.
- the p-polarized light and s-polarized light enter the deflection beam combining module after passing through the second half-wave plate.
- the deflection beam combining module has a set of mirrors corresponding to the beam of the central interference light path, the p-polarized light and the s-polarized light. , the deflection beam combining module deflects all interference beams and combines them in space.
- all light paths are finally combined onto the same illumination path, and their interference areas overlap.
- the combined light beam is illuminated to the sample through the objective lens, and the fluorescence excited by the sample is imaged on the image acquisition module.
- the image acquisition module includes a rotating mirror and a camera. After the rotating mirror, there are multiple imaging light paths, and the fluorescence is sequentially Multiple imaging light paths circulate through each other and are ultimately imaged into different areas of the camera.
- the gating module, the high-speed phase control device and the high-speed camera are controlled in a coordinated manner.
- the high-speed phase control device of the sub-optical path completes a series of related phase controls.
- the phase changes, in A picture is recorded on the high-speed camera; then another sub-light path is gated, and the phase control operation and camera shooting operation are repeated; when all sub-light paths are gated in sequence, a complete set of structured light illumination images are captured by the camera. Repeat the above steps to obtain multiple sets of structured light illumination images.
- the image high-speed module uses a high-speed rotating mirror to scan and divide several imaging areas on the camera target surface.
- the camera can capture multiple fluorescence images in a single exposure.
- a spatial filter can be set on the spectrum plane after the illumination light is combined to filter out the light of the required frequency order and improve the contrast of the final illumination pattern.
- the image high-speed acquisition module uses a high-speed rotating mirror to scan and record multiple images in a single exposure, which can further increase the acquisition speed several times based on the maximum imaging speed of the camera.
- Figure 1 is an optical path diagram of an ultra-high-speed structured light microscopy imaging device in an embodiment of the present invention
- Figure 2 is a schematic diagram of the deflection beam combining module in the embodiment of the present invention.
- Figure 3 is a schematic structural diagram of the high-speed image acquisition module of the present invention.
- the laser 1 emits an illumination beam, which is coupled into the single-mode optical fiber 4 after passing through the acousto-optic modulator 2 and the first lens 3.
- the acousto-optic modulator 2 can realize high-speed on-off control of the light beam.
- the laser is emitted from the end of the single-mode optical fiber 4 and becomes Gaussian collimated light after passing through the second lens 5.
- the collimated light becomes uniform flat-top collimated light after passing through the beam shaping module 6, and is then narrowed by the beam shrinking module 7 Finally, the beam diameter matches the light incident aperture of each electro-optical modulator.
- the polarization direction of the light beam is rotated by the first half-wave plate 8 , so that the light intensity is reflected and transmitted at a certain ratio after passing through the first polarizing beam splitter 9 . Reflected part of the light will enter the central interference light path.
- the light entering the central interference optical path passes through the second half-wave plate 10 and the first phase electro-optic modulator 11 and the first polarization electro-optic modulator 12 in sequence.
- the first phase electro-optic modulator 11 performs phase modulation on the light beam and enters the first polarization modulation.
- Device 12 changes the polarization direction of the beam.
- the first beam expansion module 13 then performs beam expansion.
- the light transmitted after passing through the first polarization beam splitter 9 enters the second polarization electro-optical modulator 14 and is selectively converted into p-polarized light (or s-polarized light).
- the s-polarized light will be reflected at the second polarization beam splitter 15, enter the first interference sub-optical path, and pass through the third half-wave plate 16, the second phase electro-optical modulator 17, the second beam expansion module 18 and The first optical path compensation module 19.
- the third half-wave plate 16 will change the polarization direction of the light beam so that the s component and the p component of the light beam are equal.
- the s component and the p component After passing through the second phase electro-optical modulator, the s component and the p component generate a relative phase difference, and then the second beam expansion module 18 performs beam expansion.
- the p-polarized light modulated by the second polarization electro-optic modulator 14 will enter the third polarization electro-optic modulator 20 and be selectively converted into p-polarized light (or s-polarized light) again.
- the s-polarized light will enter the second interference sub-optical path and pass through the fourth half-wave plate 22, the third phase electro-optical modulator 23, the third beam expansion module 24 and the second optical path compensation module 25 in sequence, so that the light beam
- the s component and p component of are equal in intensity and have a constant phase difference.
- the four beam expansion modules 29 and the third optical path compensation module 30 ensure that the s component and p component of the light beam have equal intensity and have a constant phase difference.
- the first optical path compensation module 19, the second optical path compensation module 25, and the third optical path compensation module 30 will compensate the optical path differences between the interference light paths, so that the optical path differences of all interference light beams are within the laser coherence length. Inside.
- the light beam in the central interference optical path is deflected by the second reflecting mirror 31 , the third reflecting mirror 32 and the fourth reflecting mirror 33 , and then is incident in parallel to the deflection beam combining module 56 .
- the light in the first interference sub-optical path is divided into p-polarized light and s-polarized light by the fourth polarization beam splitter 34.
- the p-polarized light passes through the sixth half-wave plate 35, the fifth reflecting mirror 36 and the sixth reflecting mirror 37.
- the deflection beam combining module 56 the s-polarized light enters the deflection beam combining module 56 through the seventh half-wave plate 38, the seventh reflecting mirror 39, the eighth reflecting mirror 40 and the ninth reflecting mirror 41.
- the light in the second interference sub-optical path is split into p-polarized light by the fifth polarization beam splitter 42
- the p-polarized light passes through the eighth half-wave plate 43, the tenth reflector 44 and the eleventh reflector 45, and enters the deflection beam combining module 56;
- the s-polarized light passes through the ninth half-wave plate 46.
- the twelfth reflector 47 and the thirteenth reflector 48 enter the deflection beam combining module 56.
- the light in the third interference sub-optical path is divided into p-polarized light and s-polarized light by the sixth polarization beam splitter 49, where the p-polarized light passes through the tenth half-wave plate 50, the fourteenth mirror 51 and the tenth
- the five reflecting mirrors 52 enter the deflection beam combining module 56; the s-polarized light passes through the eleventh half-wave plate 53, the sixteenth reflecting mirror 54 and the seventeenth reflecting mirror 55, and then enters the deflection beam combining module 56.
- the deflection beam combining module 56 will deflect all the interference beams at appropriate angles, combine them in space, and finally emit them onto the eighteenth reflector 57. After reflection, they pass through the 4f composed of the third lens 58 and the fourth lens 62. system, the Fourier surface of the 4f system has multiple interference orders.
- the 10:90 beam splitter 59 reflects 10% of the light, and uses the fifth lens 63 to image the Fourier surface to the pupil surface monitoring camera 61 for monitoring the pupil surface. The remaining 90% of the energy will illuminate the sample through the field lens 63, the fluorescence module 64 and the objective lens 65, forming a structured light illumination pattern.
- the three-dimensional translation sample stage can control the three-dimensional translation of the sample to achieve lateral movement and axial scanning. The fluorescence excited by the sample will be reflected by the fluorescence module 64 and imaged onto the image high-speed acquisition module 68 through the sixth lens 67 .
- Figure 2 is a schematic structural diagram of the deflection beam combining module 56 of the present invention.
- the seven ramp reflection surfaces with specific angles can reflect the seven incident lights (one central interference light and three pairs of sub-interference lights) upward at certain angles.
- the upper reflector deflects the beam again to achieve beam combination.
- Figure 3 is a schematic structural diagram of the high-speed image acquisition module 68 of the present invention, including: a high-speed rotating mirror 69, a seventh lens 70, an eighth lens 71, a nineteenth mirror 72, a twentieth mirror 73, and a ninth lens 74.
- the sample image surface is conjugate with the reflective surface of the high-speed rotating mirror.
- the first imaging light path (the seventh lens 70, the eighth lens 71, the nineteenth mirror 72, and the twentieth mirror 73 ), the second imaging optical path (ninth lens 74, tenth lens 75), the third imaging optical path (eleventh lens 76, twelfth lens 77, twenty-first reflecting mirror 78, twenty-second reflecting mirror 79 ).
- the fluorescence will circulate through the three imaging light paths in sequence, and will eventually be imaged into different areas of the high-speed camera 80 .
- the acquisition speed can be further increased to three times based on the original maximum shooting speed of the camera.
- a method for achieving ultra-high-speed structured illumination microscopy imaging including:
- Each interference sub-light path has illumination interference fringes in different directions on the sample surface
- the beam of the central interference light path is combined with the p-polarized light and s-polarized light of each interference sub-light path and then illuminated on the sample to form a structured light illumination pattern;
- the method can be implemented based on the above device embodiment, or can be implemented using optical paths with other structures.
- an image acquisition module based on a high-speed rotating mirror is used for imaging. Multiple images can be imaged with a single exposure, and experimental images can be captured at high speed to match the speed of illumination modulation.
- the illumination beam is reflected and transmitted after passing through the polarization beam splitter.
- the reflected part of the light enters the central interference optical path, and the transmitted optical path is divided into multiple interference sub-optical paths in turn.
- each interference sub-optical path is selectively controlled. Each time an interference sub-optical path is gated, a picture is recorded. When all the sub-optical paths are gated in sequence, a complete set of structured light illumination images is obtained. Or each interference sub-optical path is gated at the same time, and the light beams of each interference sub-optical path interfere together to produce a two-dimensional structured light illumination pattern.
- each optical path can be gated at the same time, and the energy of each sub-optical path can be distributed in any proportion, so that the light beams of each sub-optical path interfere together to produce a two-dimensional structured light illumination pattern without being limited to a one-dimensional grating type. Lighting patterns.
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Abstract
一种实现超高速结构光照明显微成像的方法，包括：1)对光源发出的照明光束进行整形，形成光强均匀分布的照明光束；2)将照明光束分光，分束为一个中心干涉光路和多个干涉子光路，每个干涉子光路在样品面具有不同方向的照明干涉条纹；3)对各干涉子光路进行光程补偿，使所有子光路的光程相同，且每个干涉子光路的光均被分为p偏振光和s偏振光；4)中心干涉光路的光束与各干涉子光路的p偏振光和s偏振光合束后照明到样品上，形成结构光照明图案；5)采集样品激发的荧光进行显微成像。还公开一种实现超高速结构光照明显微成像的装置，可以大幅提高对照明光的调制速度，从而实现超高速的结构光照明成像。
Description
本发明属于光学工程领域，特别涉及一种实现超高速结构光照明显微成像的方法和装置。
光学显微成像技术是一种对微观世界的探测手段，因其非接触、无损伤的特点，被广泛应用于生物医学研究。但是传统光学显微成像方法的分辨率无法突破衍射极限，不能用于观察200nm以下的微观结构。
结构光照明显微成像是一种超分辨成像技术，通过对照明光进行调制，获得多张不同照明下的样品图，并最终重建出一幅超分辨图像，其理论分辨率极限可以达到衍射极限的一半。结构光照明显微技术虽然能够提升分辨率，但是牺牲了成像速度，每一张超分辨图像的重建，需要若干张照明调制下拍摄到的原始图片。最终的成像速度，不仅取决于相机的拍摄、读取速度，还受限于照明光的调制速度。
在结构光显微成像领域，传统的照明光调制方式有光栅机械移动式、空间光调制器调制式与振镜扫描式等几种，其中光栅机械移动式的调制速度最慢；空间光调制器调制式可分为液晶空间光调制器(SLM)与数字微镜器件(DMD)两类，前者的速度较慢，后者衍射效率较低；振镜扫描式的速度最快，但仍无法满足KHz级别的成像速度。
公开号CN105487214A的专利申请提供一种快速三维超分辨率显微方法，包括：激光光束在准直后转换为线偏振光，对线偏振光进行相位调制并转换为圆偏振光投射到待测样品上，以及收集待测样品各扫描点发出的信号光；对样品进行三维扫描，所述的相位调制包括一次相位调制和二次相位调制；一次相位调制采用对线偏振光s光分量进行相位调制的空间光调制器，二次相位调制采用对线偏振光p光分量进行相位调制的空间光调
制器，最后根据有效信号光强得到三维超分辨图像。采用空间光调制器进行照明光调制，成像速度较慢。
公开号CN206848565U的专利申请提供一种基于数字微镜器件的光学聚焦增强系统；第一光纤和准直透镜布置在激光器之后，激光器发射出光束经第一光纤传输之后通过准直透镜入射到数字微镜器件，数字微镜器件旁侧出射端置有光挡，数字微镜器件前侧出射端置有缩束模块，前方设有二向色镜；光束经二向色镜反射后经过扫描模块进入显微物镜聚焦，实验样品位于显微物镜焦平面上；实验样品内激发出的荧光经过显微物镜与扫描模块后透过二向色镜被光强探测模块接收进行光强探测。采用数字微镜器件进行照明光调制，存在衍射效率较低的问题。
可见，现有的方案都各自具有局限性。想要实现超高速的结构光照明显微成像，需要一种全新的设计与方法，来满足高速成像的要求。
发明内容
本发明的目的为提供一种实现超高速结构光照明显微成像的方法，利用该方法可以大幅提高对照明光的调制速度，从而实现超高速(KHz级别)的结构光照明成像。
一种实现超高速结构光照明显微成像的方法，包括：
1)对光源发出的照明光束进行整形，形成光强均匀分布的照明光束；
2)将所述照明光束分光，分束为一个中心干涉光路和多个干涉子光路，每个干涉子光路在样品面代表不同方向的照明干涉条纹；
3)对各干涉子光路进行光程补偿，使所有子光路的光程相同，且每个干涉子光路的光均被分为p偏振光和s偏振光；
4)通过电光调制器对每个干涉子光路的p，s偏振光进行调制，使其维持相位差，然后用半波片改变其偏振方向，使其偏振方向一致；
5)中心干涉光路的光束与各干涉子光路的p偏振光和s偏振光合束后照明到样品上，形成结构光照明图案；
6)采集样品激发的荧光进行显微成像。
本发明中，使用基于高速转镜的图像采集模块成像，单次曝光即可成
像多幅图像，高速拍摄实验图，匹配照明调制的速度。
优选的，所述照明光束通过偏振分束器后反射和透射，反射部分的光进入中心干涉光路，透射的光路再依次分为多个干涉子光路。
在具体的实施例中，可以是三个干涉子光路，或可根据需要拓展为多路，以实现更为特殊的结构光照明图案，如特殊晶格式照明。
优选的，选择地控制各干涉子光路的选通，每当选通一路干涉子光路时，记录一幅图片，当所有子光路依次选通后，得到一组完整的结构光照明图像。
优选的，各干涉子光路同时选通，各干涉子光路的光束共同干涉，产生二维结构光照明图案。
本发明中，各光路可以同时选通，并且各子光路的能量可按任意比例分配，从而使各个子光路的光束共同干涉，产生二维结构光照明图案，而不必局限于一维的光栅型照明图案。
本发明还提供一种实现超高速结构光照明显微成像的装置，包括发出照明光束的光源，采集样品发出荧光的图像采集模块；还包括布置在照明光束光路上的：
第一偏振分束器，将所述照明光束分束，反射的光进入中心干涉光路，透射的光依次分为多个干涉子光路；
与各多个干涉子光路对应的选通模块，用于控制各干涉子光路的选通状态；
位于各干涉子光路上的第二偏振分束器，将各干涉子光路的光分为p偏振光和s偏振光；
偏转合束模块，用于将中心干涉光路的光束与各干涉子光路的p偏振光和s偏振光合束；
合束后照明到样品上，形成结构光照明图案并激发荧光，成像至所述的图像采集模块。
本发明中，光源产生的照明激光，通过第一偏振分束器，将光分为两束，其中一束进入中心干涉光路，另一束由多组选通模块控制选通，使多
个干涉子光路依次通断。在每一路干涉子光路中，光束会先后通过高速相位调控装置与光程补偿系统。光程补偿系统会补偿中心干涉光路与干涉子光路之间的光程差，从而保持全部四个干涉光路之间的相干性。高速相位调控装置将光束变成能量等分的s偏振光与p偏振光，并使其具备特定相位差。偏振分束镜会将这两种偏振光分开，结合反射镜与半波片，改变光束的空间角。半波片作用是旋转这两束干涉光的偏振方向，从而将其偏振方向变成一致，使其干涉结果具有最大对比度。合束后所有光经合束成像系统，照亮样品面，在物镜焦面处形成结构光照明图案。激发的荧光会被二向色镜反射，并最终被图像高速采集模块捕捉。同时，10:90分光器还会反射10％的能量，反射的光会进入频谱观测路，经过透镜在相机上成干涉面的频谱像，用于监控干涉光束。
优选的，所述光源与第一偏振分束器间依次设置有：声光调制器，用于对光束的高速通断控制；光束整形模块，用于产生空间强度均匀分布的准直光；和缩束模块，用来改变光束直径，使其符合电光调制器的工作口径。
优选的，所述选通模块包括偏振电光调制器和偏振分束器，所述中心干涉光路上具有沿依次设置的：第一相位电光调制器，用于对光束进行相位调制；第一偏振调制器，用于改变光束的偏振方向；和第一扩束模块，用于光束扩束。
本发明中，基于电光调制器高速地控制子光路的选通状态，使用电光调制器对干涉光束进行高速相位调制，控制其相位差，产生不同照明图案，突破普通相位调制手段的速度限制；使用线偏光照明，利用电光调制器高速地调控光束偏振方向，并配合偏振分束器，高速控制不同子光路的通断。
偏振电光调制器可以改变光的偏振方向，从而控制其在偏振分束器处透射或反射，反射光会进入第一干涉子光路，透射光将进入下一组选通模块，继续分为第二干涉子光路和第三干涉子光路。
优选的，各干涉子光路上具有依次设置的：第一半波片，用于改变光束的偏振方向，使得光束的s分量与p分量相等；第二相位电光调制器，
用于对光束进行相位调制，光束的s分量与p分量产生相对相位差；第二扩束模块，用于光束扩束，光程补偿模块，用于补偿各干涉光路之间的光程差；和第三偏振分束器，用于将光束分为p偏振光与s偏振光。
结合干涉光束的空间角度，使用半波片改变干涉光束的偏振方向，以获得最大的干涉对比度，且所有干涉光束将通过光程补偿模块进行光程差校正。
优选的，所述p偏振光和s偏振光通过第二半波片后进入偏转合束模块，偏转合束模块上具有与中心干涉光路的光束、p偏振光和s偏振光对应的反射镜组，偏转合束模块偏转所有干涉光束，并将其在空间上合束。
本发明中所有光路最终合束到同一照明路上，且其干涉区域重合。使用固定的反射镜组，来控制干涉光束的空间角。
进一步优选的，合束后的光束通过物镜照明到样品，样品激发出的荧光成像到图像采集模块上，所述图像采集模块包括转镜和相机，转镜后有多个成像光路，荧光依次在多条成像光路上循环通过，最终成像到相机的不同区域。
本发明中，选通模块、高速相位调控装置与高速相机联动控制，每当选通一路子光路时，该路子光路的高速相位调控装置完成一系列相关的相位调控，每当相位发生一次改变，在高速相机上记录一幅图片；随后选通另一路子光路，重复相位控制操作与相机拍摄操作；当所有子光路依次选通后，一组完整的结构光照明图像被相机捕获完成。重复上述步骤，以获取多组结构光照明图像。
图像高速模块使用高速转镜进行扫描，在相机靶面划分若干成像区域，相机单次曝光即可拍摄多幅荧光图像。
本发明中，在照明光合束后的频谱面，可以设置一个空间滤波器，用以筛选出所需的频率级次的光，提高最终照明图案的对比度。
与现有技术相比，本发明的优点在于：
1)使用电光调制器进行偏振调制，控制各个子光路的通断，选通速度最高可达数百kHz；
2)使用电光调制器进行相位调制，实现对干涉光束的超高速相位调制，调制速度可达数百kHz，远高于已有技术方案，整个系统的成像速度将不再受限于照明调制；
3)使用固定的反射镜控制干涉光束角度，具有更高的稳定性；
4)利用半波片改变干涉光束的偏振方向，干涉图像的对比度提高。
5)所有照明光束合束，共用同一照明成像光路；
6)图像高速采集模块使用高速转镜进行扫描，单次曝光记录多幅图像，可以在相机最大成像速度的基础上，进一步提升数倍采集速度。
图1为本发明实施例中超高速结构光照明显微成像装置的光路图；
图2为本发明实施例中偏转合束模块示意图；
图3为本发明图像高速采集模块结构示意图。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是，本发明还可以采用其他不同于在此描述的其他方式来实施，因此，本发明并不限于下面公开的具体实施例的限制。
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。
采用图1所示的装置，实现高速结构光照明显微成像的方法如下：
激光器1发出照明光束，经过声光调制器2与第一透镜3后被耦合到单模光纤4中，声光调制器2可以实现对光束的高速通断控制。激光从单模光纤4末端出射，经过第二透镜5后变为高斯型准直光，准直光通过光束整形模块6后变为均匀的平顶准直光，然后被缩束模块7缩束后，光束直径与各电光调制器的入光口径匹配。
在进入第一偏振分束器9之前，光束的偏振方向被第一半波片8旋转，使得光强经过第一偏振分束器9后以一定比例反射及透射。反射部分的光
将进入中心干涉光路。进入中心干涉光路的光依次经过第二半波片10与第一相位电光调制器11和第一偏振电光调制器12，由第一相位电光调制器11对光束进行相位调制，进入第一偏振调制器12，改变光束的偏振方向。随后由第一扩束模块13进行光束扩束。经第一偏振分束器9后透射的光进入第二偏振电光调制器14，选择性地转换为p偏振光(或s偏振光)。其中的s偏振光将会在第二偏振分束器15处被反射，进入第一干涉子光路，依次经过第三半波片16、第二相位电光调制器17、第二扩束模块18和第一光程补偿模块19。第三半波片16将改变光束的偏振方向，使得光束的s分量与p分量相等。经过第二相位电光调制器后，s分量与p分量产生相对相位差，随后由第二扩束模块18进行扩束。经过第二偏振电光调制器14调制后的p偏振光，将会进入第三偏振电光调制器20，再次被选择性地转换为p偏振光(或s偏振光)。类似地，其中的s偏振光会进入第二干涉子光路，依次经过第四半波片22、第三相位电光调制器23、第三扩束模块24和第二光程补偿模块25，从而光束的s分量和p分量强度相等并且具有恒定相位差。而经过第三偏振电光调制器20调制后的p偏振光，经第一反射镜26反射后，进入第三干涉子光路，依次经过第五半波片27，第四相位电光调制器28，第四扩束模块29，第三光程补偿模块30，保证光束的s分量和p分量强度相等并且具有恒定相位差。
第一光程补偿模块19、第二光程补偿模块25、第三光程补偿模块30，将补偿各干涉光路之间的光程差，以使得所有干涉光束的光程差处于激光相干长度之内。
中心干涉光路的光束经第二反射镜31、第三反射镜32和第四反射镜33偏转后，平行入射到偏转合束模块56。
第一干涉子光路的光被第四偏振分束器34分为了p偏振光与s偏振光，其中的p偏振光经过第六半波片35、第五反射镜36和第六反射镜37进到偏转合束模块56；其中的s偏振光经过第七半波片38、第七反射镜39、第八反射镜40和第九反射镜41进到偏转合束模块56。
类似地，第二干涉子光路的光被第五偏振分束器42分为了p偏振光
与s偏振光，其中的p偏振光经过第八半波片43、第十反射镜44和第十一反射镜45，进到偏转合束模块56；其中的s偏振光经过第九半波片46、第十二反射镜47和第十三反射镜48，进到偏转合束模块56。
类似地，第三干涉子光路的光被第六偏振分束器49分为了p偏振光与s偏振光，其中的p偏振光经过第十半波片50、第十四反射镜51和第十五反射镜52，进到偏转合束模块56；其中的s偏振光经过第十一半波片53、第十六反射镜54和第十七反射镜55，进到偏转合束模块56。
偏转合束模块56会以适当的角度偏转所有干涉光束，并将其在空间上合束，最后出射到第十八反射镜57上，反射后经过第三透镜58和第四透镜62组成的4f系统，在该4f系统的傅里叶面具有多个干涉级次。10:90分光片59将10％的光反射，并用第五透镜63将傅里叶面成像至瞳面监测相机61，用于监测瞳面。剩余的90％能量将通过场镜63，荧光模块64与物镜65，照明到样品上，形成结构光照明图案。三维平移样品台可控制样品进行三维平移，实现横向移动和轴向扫描。样品激发出的荧光将会通过荧光模块64反射，经过第六透镜67成像到图像高速采集模块68上。
图2是本发明偏转合束模块56的结构示意图，七个特定角度的斜台反射面，能将入射的七束光(一束中心干涉光与三对子干涉光)以一定角度向上反射，并由上方的反射镜再次偏折光束，实现合束。
图3是本发明图像高速采集模块68的结构示意图，包括：高速转镜69，第七透镜70，第八透镜71，第十九反射镜72，第二十反射镜73，第九透镜74，第十透镜75，第十一透镜76，第十二透镜77，第二十一反射镜78，第二十二反射镜79和高速相机80。样品像面与高速转镜的反射面共轭，高速转镜后有三条成像光路：第一成像光路(第七透镜70，第八透镜71，第十九反射镜72，第二十反射镜73)，第二成像光路(第九透镜74，第十透镜75)，第三成像光路(第十一透镜76，第十二透镜77，第二十一反射镜78，第二十二反射镜79)。在高速转镜工作时，荧光将依次在这三条成像光路上循环通过，最终成像到高速相机80的不同区域。在高速相机80单次曝光的时间内，完成一个完整的扫描，同时拍摄三张实验
图，能够在相机原有最高拍摄速度基础上，采集速度进一步提升至三倍。
在另一个实施例中，还提供一种实现超高速结构光照明显微成像的方法，包括：
1)对光源发出的照明光束进行整形，形成光强均匀分布的照明光束；
2)将所述照明光束分光，分束为一个中心干涉光路和多个干涉子光路，每个干涉子光路在样品面具有不同方向的照明干涉条纹；
3)对各干涉子光路进行光程补偿，使所有子光路的光程相同，且每个干涉子光路的光均被分为p偏振光和s偏振光；
4)中心干涉光路的光束与各干涉子光路的p偏振光和s偏振光合束后照明到样品上，形成结构光照明图案；
5)采集样品激发的荧光进行显微成像。
本发明中，该方法可以基于上述的装置实施例实现，也可以采用其他结构的光路来实施。具体地，使用基于高速转镜的图像采集模块成像，单次曝光即可成像多幅图像，高速拍摄实验图，匹配照明调制的速度。
所述照明光束通过偏振分束器后反射和透射，反射部分的光进入中心干涉光路，透射的光路再依次分为多个干涉子光路。
在另一些优选的实施例中，可以是三个干涉子光路，或可根据需要拓展为多路，以实现更为特殊的结构光照明图案，如特殊晶格式照明。
本实施例中，选择地控制各干涉子光路的选通，每当选通一路干涉子光路时，记录一幅图片，当所有子光路依次选通后，得到一组完整的结构光照明图像。或者各干涉子光路同时选通，各干涉子光路的光束共同干涉，产生二维结构光照明图案。
本发明中，各光路可以同时选通，并且各子光路的能量可按任意比例分配，从而使各个子光路的光束共同干涉，产生二维结构光照明图案，而不必局限于一维的光栅型照明图案。
以上所述仅为本发明的较佳实施举例，并不用于限制本发明，凡在本发明精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
- 一种实现超高速结构光照明显微成像的方法，其特征在于，包括：1)对光源发出的照明光束进行整形，形成光强均匀分布的照明光束；2)将所述照明光束分光，分束为一个中心干涉光路和多个干涉子光路，每个干涉子光路在样品面代表不同方向的照明干涉条纹；3)对各干涉子光路进行光程补偿，使所有子光路的光程相同，且每个干涉子光路的光均被分为p偏振光和s偏振光；4)通过电光调制器对每个干涉子光路的p、s偏振光进行调制，使其维持相位差，然后用半波片改变其偏振方向，使其偏振方向一致；5)中心干涉光路的光束与各干涉子光路的p偏振光和s偏振光合束后照明到样品上，形成结构光照明图案；6)采集样品激发的荧光进行显微成像。
- 根据权利要求1所述的实现超高速结构光照明显微成像的方法，其特征在于，所述照明光束通过偏振分束器后反射和透射，反射部分的光进入中心干涉光路，透射的光路再依次分为多个干涉子光路。
- 根据权利要求1所述的实现超高速结构光照明显微成像的方法，其特征在于，选择地控制各干涉子光路的通断，每当选通一路干涉子光路时，记录一幅图片，当所有子光路依次选通后，得到一组完整的结构光照明图像。
- 根据权利要求1所述的实现超高速结构光照明显微成像的方法，其特征在于，各干涉子光路同时选通，各干涉子光路的光束共同干涉，产生二维结构光照明图案。
- 一种实现超高速结构光照明显微成像的装置，包括发出照明光束的光源，采集样品发出荧光的图像采集模块；其特征在于，还包括布置在照明光束光路上的：第一偏振分束器，将所述照明光束分束，反射的光进入中心干涉光路，透射的光依次分为多个干涉子光路；与各多个干涉子光路对应的选通模块，用于控制各干涉子光路的选通状态；位于各干涉子光路上的第二偏振分束器，将各干涉子光路的光分为p偏振光和s偏振光；偏转合束模块，用于将中心干涉光路的光束与各干涉子光路的p偏振光和s偏振光合束；合束后照明到样品上，形成结构光照明图案并激发荧光，成像至所述的图像采集模块。
- 根据权利要求5所述的实现超高速结构光照明显微成像的装置，其特征在于，所述光源与第一偏振分束器间依次设置有：声光调制器，用于对光束的高速通断控制；光束整形模块，用于产生空间强度均匀分布的准直光；和缩束模块，用来改变光束直径，使其符合电光调制器的工作口径。
- 根据权利要求5所述的实现超高速结构光照明显微成像的装置，其特征在于，所述中心干涉光路上具有沿依次设置的：第一相位电光调制器，用于对光束进行相位调制；第一偏振调制器，用于改变光束的偏振方向；和第一扩束模块，用于光束扩束。
- 根据权利要求1所述的实现超高速结构光照明显微成像的装置，其特征在于，所述选通模块包括偏振电光调制器和偏振分束器，各干涉子光路上具有依次设置的：第一半波片，用于改变光束的偏振方向，使得光束的s分量与p分量相等；第二相位电光调制器，用于对光束进行相位调制，光束的s分量与p分量产生相对相位差；第二扩束模块，用于光束扩束，光程补偿模块，用于补偿各干涉光路之间的光程差；和第三偏振分束器，用于将光束分为p偏振光与s偏振光。
- 根据权利要求8所述的实现超高速结构光照明显微成像的装置，其特征在于，所述p偏振光和s偏振光通过第二半波片后进入偏转合束模块，偏转合束模块上具有与中心干涉光路的光束、p偏振光和s偏振光对应的反射镜组，偏转合束模块偏转所有干涉光束，并将其在空间上合束。
- 根据权利要求5所述的实现超高速结构光照明显微成像的装置，其特征在于，合束后的光束通过物镜照明到样品，样品激发出的荧光成像到图像采集模块上，所述图像采集模块包括转镜和相机，转镜后有多个成像光路，荧光依次在多条成像光路上循环通过，最终成像到相机的不同区域。
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| DE102018110072A1 (de) * | 2018-04-26 | 2019-10-31 | Carl Zeiss Microscopy Gmbh | Optikanordnung zur strukturierten Beleuchtung für ein Lichtmikroskop und Verfahren hierzu |
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