WO2014075448A1 - 大景深三维纳米分辨成像方法、光学组件及成像系统 - Google Patents

大景深三维纳米分辨成像方法、光学组件及成像系统 Download PDF

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WO2014075448A1
WO2014075448A1 PCT/CN2013/078029 CN2013078029W WO2014075448A1 WO 2014075448 A1 WO2014075448 A1 WO 2014075448A1 CN 2013078029 W CN2013078029 W CN 2013078029W WO 2014075448 A1 WO2014075448 A1 WO 2014075448A1
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imaging
double
double helix
optical module
laguerre
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French (fr)
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牛憨笨
于斌
陈丹妮
李恒
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Shenzhen University
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Shenzhen University
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B5/00—Optical elements other than lenses
    • G02B5/18—Diffraction gratings
    • G02B5/1842—Gratings for image generation
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • 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
    • G01N21/6447—Fluorescence; Phosphorescence by visual observation
    • 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
    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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
    • G01N21/84—Systems specially adapted for particular applications
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B5/00—Optical elements other than lenses
    • G02B5/18—Diffraction gratings
    • G02B5/1876—Diffractive Fresnel lenses; Zone plates; Kinoforms
    • G02B5/189—Structurally combined with optical elements not having diffractive power

Definitions

  • the invention belongs to the field of microscopic imaging technology, and in particular relates to a three-dimensional nano-resolution imaging method, an optical component and an imaging system with a large depth of field. Background technique
  • Cells are the basic unit of organisms and life activities. In-depth study of cells is the key to uncovering the mystery of life, transforming life and conquering diseases. Molecular imaging under intact cells to obtain subcellular precision information has always been an important direction in cytological research. At the same time, nano-resolved three-dimensional structure and functional imaging of intact cells, understanding the relationship and regularity of subcellular structure and cell function changes at a higher level is an urgent need of life sciences, and also a major challenge to imaging science.
  • the Interferometric Light-Sensing Microscopy can increase the resolution of three-dimensional to within 20 nm, but the imaging range is limited to a depth of 500 nm below the coverslip. Therefore, the imaging depth of these methods is small.
  • Intracellular dynamic imaging is required to simultaneously track multiple molecules within a cell, which requires imaging to rapidly detect multiple target molecules in a range of depths of ten micrometers with nanopositioning accuracy in three dimensions.
  • the current single-molecule tracking (SPT) method can perform local detection on a region containing only target molecules in a sample to achieve fluorescence imaging with a precision of 1 nm (FIONA). It can also use a wide-field imaging method to simultaneously track multiple molecules. .
  • FIONA Fresnel particle tracking
  • a variety of axial resolution methods have been developed.
  • the nano-positioning, but the current imaging depth is only about 3 ⁇ , and the thickness of the intact cells is generally more than ten micrometers. Therefore, the existing methods still cannot meet the large depth of field requirements for multi-molecular tracking in cells. Summary of the invention
  • the present invention is achieved by a large depth of field three-dimensional nano-resolved imaging method comprising the steps of: creating an optical module having a double helix point spread function and a multi-level imaging property of an out-of-focus grating; Imaging, obtaining a double helix image of the molecule to be tested; determining a lateral position of the molecule to be tested by the position of the midpoint of the double helix side lobes on the imaging surface in the double helix image;
  • the axial position of the molecule to be detected is determined by the angle of rotation of the center line of the double helix side lobes in the double helix image and the position of the midpoint of the double helix side lobes on the imaging surface.
  • Another object of the present invention is to provide an optical component for three-dimensional nano-resolution imaging with large depth of field, including sequentially arranged along the optical path of transmission: a first lens for collimating fluorescence emitted by the molecule to be tested;
  • An optical module having a double helix point spread function and a multi-level imaging property of an out-of-focus grating for converting the fluorescence into an imaging beam having dual helix and multi-order imaging properties;
  • a second lens for outputting the imaging beam for imaging.
  • a filter for filtering the beam and outputting the fluorescence
  • a dichroic mirror for reflecting the fluorescence
  • a tube mirror for focusing and outputting the reflected fluorescence to the imaging assembly
  • An imaging assembly employing the optical assembly described above for converting the fluorescence into an imaging beam having dual helix and multi-level imaging properties
  • a detector for receiving the imaging beam and performing double helix and multi-level imaging.
  • the invention creates an optical module integrating the double helix point spread function and the double-effect imaging double-focus imaging effect, the multi-level imaging has a large depth of field, the double-helix imaging has a high resolution and a certain depth of field, and is imaged by the optical module.
  • the depth of field is greatly expanded by multi-stage imaging and double-helical imaging, and on the other hand, the resolution is significantly improved by double-helical imaging.
  • the imaging depth of field of the present invention can be up to ten micrometers, and can be used for any cell in a complete cell.
  • the dynamic range imaging of deep sub-cells, as well as the dynamic functional images of multiple motor molecules, is of great significance for understanding the relationship and laws of subcellular structure and cell function changes at a higher level.
  • FIG. 1 is a flow chart of a method for three-dimensional nano-resolution imaging of a large depth of field according to a first embodiment of the present invention
  • FIG. 2 is a comparison diagram of double-spiral point spread function and standard point spread function imaging at different depths
  • FIG. 3 is a double-spiral point spread. The intensity and phase distribution of the function image
  • Figure 4 is an imaging pattern of a double helix point spread function at different axial positions
  • Figure 5 is a graph showing the relationship between the rotation angle of the two side lobe center lines and the Z-axis position of the double helix image
  • Figure 6 is a schematic diagram of the imaging principle of the out-of-focus grating
  • FIG. 7 is a schematic diagram of a phase plate according to a first embodiment of the present invention.
  • Figure 8 is an effect diagram of imaging using the phase plate shown in Figure 7;
  • FIG. 9 is a schematic diagram of an optical component for three-dimensional nano-resolution imaging of a large depth of field according to a second embodiment of the present invention.
  • FIG. 10 is a schematic diagram of a large depth of field super-resolution fluorescence microscopic imaging detection system according to a third embodiment of the present invention.
  • FIG. 11 is a schematic diagram of another large depth of field super-resolution fluorescence microscopic imaging detection system according to a third embodiment of the present invention. detailed description
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • Fig. 1 is a flow chart showing a large depth of field three-dimensional nano-resolution imaging method according to a first embodiment of the present invention, and for convenience of explanation, only parts related to the present embodiment are shown.
  • the method mainly includes the following steps:
  • step S101 an optical module having a double helix point spread function and a multi-level imaging property of the out-of-focus grating is created;
  • step S102 the molecule to be tested is imaged by the optical module to obtain a double-spiral image of the molecule to be tested;
  • step S103 the position of the midpoint of the double helix side lobes in the double helix image on the imaging surface is confirmed Determining the lateral position of the molecule to be tested;
  • step S104 the axial position of the molecule to be tested is determined by the rotation angle of the center line of the double helix side lobes in the double helix image and the position of the midpoint of the double helix side lobes on the imaging surface.
  • DH-PSF double helix point spread function
  • the double helix point spread function consists of a linear superposition of LG beam patterns on a particular line on a Laguerre-Guss mode plane, which forms a self-imaged beam with rotation and scaling, and then The composite field in one cross section of the imaging beam serves as the optical transfer function of the optical module. Then, the transfer function of the optical module is the double helix point spread function.
  • the Laguerre-Gaussian beam pattern is:
  • the function is applied to the optical system as an optical transfer function, the point spread function of the optical system will become a double helix point spread function, and the double helix side lobes rotate with the change of the defocus amount.
  • the speed is proportional to the slope of the line selected on the LG mode plane, and the maximum speed in the focus area, as shown in Figure 4.
  • a DH-PSF system is to add a specially designed optical module to the Fourier plane of the standard imaging system.
  • the optical module has its transmittance function formed in the form of a double helix in the focus region of the Fourier transform, which is created in step S101.
  • the optical module has this characteristic, and the image formed by the optical module is two side lobes that rotate around the optical axis, one of which rotates clockwise around the optical axis and the other rotates counterclockwise.
  • the lateral positioning point of the molecule is estimated by the midpoint of the two side lobes, and the axial position is determined according to the rotation angle of the line connecting the two side lobes, and the positioning accuracy is extremely high.
  • the optical module also has multi-level imaging properties of the defocusing grating, and the defocusing grating is substantially an off-axis binary phase Fresnel zone plate.
  • the defocusing grating has the spectroscopic effect of a common grating, The incident light is split at different diffraction orders of the grating; on the other hand, it has the lens action of the Fresnel zone plate, introducing different lens effects at the unused diffraction orders.
  • the short focal length lens provides the main focusing ability.
  • the defocusing grating finely adjusts the focusing ability of the lens, so that the ⁇ 1 order diffracted light has different focal lengths, respectively. Slightly shorter and slightly longer than the focal length of the lens.
  • the focal plane of the short focal length lens on the ⁇ 1st order diffracted light is a symmetrical front and back symmetry plane, so the out-of-focus grating can image the same object in different image planes. At the same time, object points located on different object planes can be imaged on the same image plane. As shown in Fig.
  • objects at the three points of B, B, and C on different object planes can be imaged at A', B, and C on the same image plane, according to A', B, and C.
  • the distance ⁇ between the three points of A, B, and C can determine the axial relative distance ⁇ of the three points of A, B, and C.
  • the depth of field of the defocused grating is large, up to a dozen micrometers, which is almost the same as the size of the complete fine.
  • the phase function of the defocused grating is:
  • the present embodiment Based on the properties of the above-described double helix imaging and out-of-focus raster imaging, the present embodiment combines the double-helical point spread function with the defocusing grating to form a new optical module based on the wavefront coding method, and the wavefront coding is used for One or more specially designed phase masks to create a method of optical transfer function of an optical module such as a lens.
  • the optical module created by the method based on wavefront coding in this embodiment has the functions of multi-focal plane imaging and double-helical point spread function.
  • the phase function of the optical module can be expressed as:
  • the weights of the complex amplitudes are added and added, and the Laguerre-Gaussian beam modes may specifically be the above-mentioned ", (1, 1), (3) , 5), (5, 9), (7, 13), (9, 17) correspond to the five Laguerre-Gaussian beam modes.
  • This embodiment will be (1,1), (3,5), (5,9), (7,13), (9,17)
  • the five modes are equalized and added to form the phase form of the double-helical rotating beam as the initial value, and then optimized to obtain the pure phase distribution of the high efficiency double-helical beam.
  • Form having the formula (8) namely: wherein, R is the radius of the grating; 2. , indicating the defocusing ability of the optical module, which is the defocusing target Quasi-coefficient.
  • the optical module may specifically be a phase plate fabricated by microfabrication technology, or may be directly implemented by a spatial light modulator.
  • the lateral position of the molecule to be tested is specifically determined by the position of the midpoint of the double helix side lobes on the imaging surface in the double helix image; the axial position is the rotation angle of the center line of the double helix side lobes in the double helix image. And the position of the midpoint of the double helix side lobes on the imaging surface is determined.
  • the system when designing the optical system, the system is calibrated in advance, and the correspondence between the center position of the double-helical side lobes and the lateral position of the molecules to be tested, and the corresponding relationship between the molecules to be tested and the multi-level imaging object plane are established. And the correspondence between the rotation angle of the double helix side lobes and the defocus amount is established, and the information is pre-stored in the database to be called when actually measuring.
  • the lateral position of the object point can be determined according to the specific position of the midpoint of the two side lobes in the double helix image, and the object point is initially determined to be located near the multi-level imaging object surface, and further determined by the rotation angle of the two side lobes The distance from the point to the object surface to determine its axial position.
  • phase plate was designed based on the above method, as shown in Fig. 7, and the imaging method was simulated.
  • This phase plate was used in a three-dimensional imaging system to simulate the imaging of particles at different locations, as shown in Figure 8. Thus, it can be seen that the method can achieve an imaging range of 12 microns.
  • the optical module of the embodiment combines the dual effects of double-helical imaging and multi-level imaging of the out-of-focus grating, and the depth of field of the multi-level imaging is large, and the resolution of the double-helical imaging is high and has a certain depth of field.
  • multi-level imaging can greatly expand the depth of field, and on both sides of the object surface that can be clearly imaged, the range of axial positioning is further enlarged due to the double helix effect, thereby expanding the depth of field;
  • the resolution of the double helix imaging is high, and the object point on any object surface in the depth of field can achieve high-resolution axial positioning by the double helix effect, thereby improving the resolution of the three-dimensional imaging.
  • this embodiment combines the properties of the double-helix point spread function and the multi-level imaging of the out-of-focus grating Together for 3D imaging, it expands the depth of field and increases the resolution.
  • the depth of field can reach more than ten micrometers. It can be used for dynamic range imaging of sub-cells of any depth in intact cells, and the dynamic function of multiple motion molecules can be obtained. Images are important for understanding the relationship and regularity of subcellular structure and cellular function changes at a higher level.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • Fig. 9 is a view showing an optical component for a three-dimensional nano-resolution imaging of a large depth of field according to a second embodiment of the present invention, and for convenience of explanation, only parts related to the present embodiment are shown.
  • this embodiment further provides an optical component that can be used for three-dimensional nano-resolution imaging of large depth of field.
  • This component is primarily used in 3D imaging systems to achieve large depth of field and high resolution 3D imaging of cells.
  • the optical component mainly includes a first lens 901, an optical module 902, and a second lens 903 which are sequentially disposed in the optical path transmission direction.
  • the optical module 902 has the above-mentioned double-helix point spread function and the multi-step imaging property of the out-of-focus grating.
  • the optical module 902 is designed based on the above method, and has the functions described in the first embodiment, and details are not described herein again.
  • the optical system tracks the molecules by detecting the fluorescence emitted by the molecules to be tested.
  • the first lens 901 collimates the fluorescence emitted by the molecules to be detected and outputs to the optical module 902, and the optical module 902 will collimate.
  • the fluorescence is converted into an imaging beam having a double helix and multi-order imaging properties, which is then output by the second lens 903, focusing the imaging beam onto the image plane of the detector 904, and performing double helix and multi-level imaging on the detector.
  • the lateral position of the molecule to be tested can be determined by the position of the center of the double-helical side lobes on the imaging surface on the imaging surface, and the axis of the molecule to be tested is determined by the rotation angle of the line between the center of the double-helical side lobes and the line between the two side lobes To the location.
  • the optical module 902 may be a phase plate made by a lithography method, or a spatial light modulator may be directly used.
  • the phase function of the optical module 902 is as described in the first embodiment, and details are not described herein. .
  • Embodiment 3 is a diagrammatic representation of Embodiment 3
  • FIG. 10 is a schematic diagram showing a large depth of field super-resolution fluorescence microscopic imaging detection system according to a third embodiment of the present invention
  • FIG. 11 is a view showing another large depth of field super-resolution fluorescence microscopy according to a third embodiment of the present invention.
  • a schematic diagram of the imaging detection system for convenience of explanation, only the parts related to the present embodiment are shown.
  • Embodiments of the present invention provide a large depth of field super-resolution fluorescence microscopic imaging detection system based on the above imaging method and optical component, and the imaging method of the present invention and the super-resolution fluorescence microscopic imaging method (eg,
  • PALM, STORM combines to achieve three-dimensional nano-resolution fluorescence microscopic imaging detection of super-depth depth of field.
  • the large depth of field super-resolution fluorescence microscopic imaging detection system includes a detection objective lens 1, a filter 2, a dichroic mirror 3, a tube mirror 4, an imaging assembly 5 and a detector 6 which are arranged in the direction of the optical path.
  • the imaging assembly 5 employs the optical assembly of the second embodiment described above.
  • the optical module 53 in the optical component 5 may specifically be a phase plate for converting fluorescence into an imaging beam having dual helix and multi-order imaging properties.
  • the detecting objective lens 1 is located on the light emitting side of the object to be tested, and the object to be tested is excited by the excitation light to emit fluorescence, and the light beam containing the excitation light and the fluorescent light and other stray light is received by the detecting objective lens 1 and the light beam is filtered. After the filtering action of the sheet 2, the excitation light and the stray light are filtered out, and the fluorescence is transmitted. The fluorescence is reflected by the dichroic mirror 3 to the tube mirror 4, and is focused by the tube mirror 4 and transmitted to the first lens 51 of the optical component 5, the fluorescent beam. After the phase sheet is converted into a double helix and a multi-order imaging beam, finally, the second lens 52 is focused on the imaging surface of the detector 6, and a double helix image point is formed on the imaging surface.
  • the optical module 53 can also use a spatial light modulator to display the phase function of the phase plate to realize the function of the phase plate.
  • the imaging system further includes a polarizing plate 7 between the dichroic mirror 3 and the tube mirror 4 for converting the fluorescent light beam into linearly polarized light for use in a spatial light modulator.
  • the above imaging system performs double helix and multi-order imaging by the optical component provided by the present invention and based on the imaging method provided by the present invention, and utilizes the large depth of field effect of multi-level imaging and the high-precision axial positioning effect of double-helical imaging, and at the same time realizes super large Depth of field and high-resolution 3D nano-resolution imaging can realize dynamic range imaging of sub-cells at any depth, and obtain dynamic functional images of multiple motion molecules, suitable for 3D nano-resolution imaging of intact cells.
  • the large depth of field three-dimensional nano-resolution imaging system can be used alone for cell imaging, or can be built in cell imaging and other imaging devices. Therefore, an imaging device provided with the imaging system is also within the scope of the present invention.
  • the above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. Within the scope.

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Abstract

一种大景深三维纳米分辨成像方法，其适用于显微成像技术领域，包括下述步骤：创建具有双螺旋点扩散函数和离焦光栅多阶成像性质的光学模块；通过光学模块对待测分子进行成像，获得双螺旋图像；通过双螺旋图像中双螺旋旁瓣的中点的位置确定待测分子的横向位置；通过双螺旋图像中双螺旋旁瓣的中心连线的旋转角度及双螺旋旁瓣的中点位置确定待测分子的轴向位置。该方法将双螺旋点扩散函数及离焦光栅多阶成像的双重效应相结合，既扩大了景深，又提高了分辨率，该方法可用于完整细胞内任意深度亚细胞的动态范围成像，可获得多个运动分子的动态功能图像，对于在更高水平上认识亚细胞结构与细胞功能变化的关系和规律具有重要意义。

Description

大景深三维纳米分辨成像方法、 光学组件及成像系统
技术领域
本发明属于显微成像技术领域， 特别涉及一种大景深三维纳米分辨成像方 法、 光学组件及成像系统。 背景技术
细胞是生物体及生命活动的基本单元，对细胞的深入研究是揭开生命奥秘， 改造生命和征服疾病的关键。 在完整细胞下进行分子成像， 进而获取亚细胞精 信息一直是细胞学研究的重要方向。 同时， 对完整细胞进行纳米分辨三维结构 和功能成像， 在更高水平上认识亚细胞结构与细胞功能变化的关系和规律， 是 生命科学的迫切需求， 也是对成像科学的重大挑战。
近年来， 远场纳米分辨荧光显微成像技术取得了较大的发展。 目前最为突 出的方法有两种， 一种是基于缩小有效激发光斑， 通过直接减小点扩展函数的 半高宽来提高分辨率， 包括 STED, GSD等； 另一种则是基于单分子定位技术， 包括 STORM, PALM等。 前者是通过受激态或基态耗尽的方式， 压缩荧光有 效发射区域； 后者则是利用荧光标记本身的开关效应， 通过稀疏激发、 分时成 像、 质心定位以及图像合成来实现纳米分辨成像， 已经实现 20nm的横向空间 分辨率。
然而， 利用单分子定位技术对直径为 ΙΟμηι以上的细胞进行纳米分辨三维 成像仍然存在很多问题。 首先， 单分子定位对于轴向分辨率并没有提高， 常常 需要结合某些改进轴向分辨率的方法， 如柱面镜像散法、 双螺旋点扩散函数法 ( DH-PSF )、 双层平面探测法、 虚拟空间超分辨显微术（ VVSRM )等， 可实现 横向空间分辨率达 20-30nm左右， 轴向分辨率达 40-70nm的三维成像， 目前， 这些方法的成像深度只有 2μηι。 另外， 干涉光敏定位显微技术（iPALM)可将三 维的分辨率提高到 20nm以内， 但成像范围仅被限制在盖玻片以下 500nm的深 度范围内， 因此， 这些方法的成像深度均较小。
细胞内动态成像要实现细胞内同时追踪多个分子， 这要求成像手段在三维 空间以纳米定位精度快速探测十几微米景深范围内的多个目标分子。 目前的单 分子追踪 (SPT)方法， 既可以对样品中只包含目标分子的区域进行局部探测， 实 现 lnm精度的荧光成像（FIONA ); 也可以采用宽场成像的方法， 实现同时追 踪多个分子。 虽然宽场探测的 SPT方法已经发展了图像堆栈、 离焦成像、 用聚 焦激光光束环绕粒子运动、 Fresnel粒子追踪 (FPT), 以及柱面镜像散等多种轴 向分辨的方法， 已经可以实现三维的纳米定位， 但目前实现的成像深度仅仅为 3μηι左右， 而一般完整细胞的厚度有十几微米， 因此， 现有的方法仍不能满足 细胞内多分子追踪的大景深要求。 发明内容
本发明的目的在于提供一种大景深三维纳米分辨成像方法， 旨在解决传统 方法成像深度小， 难以满足分子定位的大景深要求的问题。
本发明是这样实现的，一种大景深三维纳米分辨成像方法， 包括下述步骤： 创建具有双螺旋点扩散函数和离焦光栅多阶成像性质的光学模块； 通过所述光学模块对待测分子进行成像， 获得待测分子的双螺旋图像； 通过所述双螺旋图像中双螺旋旁瓣的中点在成像面上的位置确定待测分子 的横向位置；
通过所述双螺旋图像中双螺旋旁瓣的中心连线的旋转角度及所述双螺旋旁 瓣的中点在成像面上的位置确定待测分子的轴向位置。
本发明的另一目的在于提供一种用于大景深三维纳米分辨成像的光学组 件， 包括沿光路传输方向依次设置的： 第一透镜， 用于将待测分子发出的荧光进行准直；
光学模块， 具有双螺旋点扩散函数和离焦光栅多阶成像性质， 用于将所述 荧光转换为具有双螺旋及多阶成像性质的成像光束；
第二透镜， 用于将所述成像光束输出以用于成像。
本发明的又一目的在于提供一种大景深超分辨荧光显微成像探测系统， 包 括沿光路传输方向依次设置的：
探测物镜， 用于接收含有待测分子发出的荧光的光束；
滤光片， 用于对所述光束进行滤波并输出所述荧光；
双色镜， 用于对所述荧光进行反射；
管镜， 用于将反射的荧光聚焦并向成像组件输出；
成像组件， 采用上述的光学组件， 用于将所述荧光转换为具有双螺旋及多 阶成像性质的成像光束；
探测器， 用于接收所述成像光束并进行双螺旋及多阶成像。
本发明创建了综合双螺旋点扩散函数及离焦光栅多阶成像双重效应的光学 模块， 多阶成像的景深较大， 双螺旋成像的分辨率较高且具有一定的景深， 通 过上述光学模块成像时，一方面通过多阶成像和双螺旋成像大幅度扩大了景深， 另一方面通过双螺旋成像显著的提高了分辨率， 本发明的成像景深可达十几微 米， 既可用于完整细胞内任意深度亚细胞的动态范围成像， 又可获得多个运动 分子的动态功能图像， 对于在更高水平上认识亚细胞结构与细胞功能变化的关 系和规律具有重要意义。 附图说明
图 1是本发明第一实施例提供的大景深三维纳米分辨成像方法的流程图； 图 2是不同深度的双螺旋点扩散函数和标准点扩散函数成像的对照图； 图 3是双螺旋点扩散函数成像的强度与相位分布；
图 4是双螺旋点扩散函数在不同轴向位置处的成像图形； 图 5 是双螺旋图像两个旁瓣中心连线的旋转角度与 Z 轴位置的关系曲线 图；
图 6是离焦光栅成像原理示意图；
图 7是本发明第一实施例提供的相位片的示意图；
图 8是利用图 7所示相位片进行成像的效果图；
图 9是本发明第二实施例提供的用于大景深三维纳米分辨成像的光学组件 示意图；
图 10 是本发明第三实施例提供的一种大景深超分辨荧光显微成像探测系 统示意图；
图 11 是本发明第三实施例提供的另一种大景深超分辨荧光显微成像探测 系统示意图。 具体实施方式
为了使本发明的目的、 技术方案及优点更加清楚明白， 以下结合附图及实 施例， 对本发明进行进一步详细说明。 应当理解， 此处所描述的具体实施例仅 仅用以解释本发明， 并不用于限定本发明。
以下结合具体实施例对本发明的具体实现进行更加详细的描述：
实施例一：
图 1示出了本发明第一实施例提供的大景深三维纳米分辨成像方法的流程 图， 为了便于说明， 仅示出了与本实施例相关的部分。
参考附图 1 , 该方法主要包括下述步骤：
在步骤 S101中，创建具有双螺旋点扩散函数和离焦光栅多阶成像性质的光 学模块；
在步骤 S102中，通过光学模块对待测分子进行成像，获得待测分子的双螺 旋图像；
在步骤 S103中，通过双螺旋图像中双螺旋旁瓣的中点在成像面上的位置确 定待测分子的横向位置；
在步骤 S104中，通过双螺旋图像中双螺旋旁瓣的中心连线的旋转角度及双 螺旋旁瓣的中点在成像面上的位置确定待测分子的轴向位置。
通过双螺旋点扩散函数（ DH-PSF )实现三维纳米定位是基于一种被称为自 成像的现象。 DH-PSF是一种三维光学响应,具有随离焦量不断旋转的圓形不对 称横截面轮廓， 如图 2 所示。 双螺旋点扩散函数主要通过位于拉盖尔 -高斯 (Laguerre- Gauss , 筒记为 LG)模式平面上特定直线上的 LG光束模式的线性叠 加构成带有旋转和缩放的自成像光束， 然后将自成像光束的一个横截面中的复 合场作为光学模块的光学传递函数， 那么， 光学模块的传递函数就是双螺旋点 扩散函数。 该拉盖尔 -高斯光束模式为：
Figure imgf000007_0001
其中， r = (A^，z;»为空间点的柱坐标， = O为高斯光斑的径向坐标， ^) = «。 s=z/z。为纵向坐标， =^2A为瑞利长度，
"«，m( 的组成为：
G{p,z) =
Figure imgf000007_0002
Φ exp(im^)
(4)
Ζπ (I) = exp[-m^(z
( 5 ) 其中， ^£;> = arctan^;)为古伊相位， II：_HV2为广义的拉盖尔多项式， n,m为 丈 = |m|,|m| + 2,|m| + 4,|m| + 6,.... 当"， 取下列五组数值： （1, 1 ) , (3, 5 ) , ( 5, 9 ) , (7, 13 ) , ( 9, 17 ) , 可获得五种拉盖尔-高斯光束模式。 将这五种拉盖尔-高斯光束模式 进行等权重叠加， 可形成带有旋转和缩放的自成像光束， 即形成一个新的光场 分布函数一双螺旋旋转光束， 如图 3。 基于 LG函数的傅立叶变换不变特性，该 函数如作为光学传递函数应用到光学系统中，光学系统的点扩散函数将变为双 螺旋点扩散函数,且双螺旋旁瓣随离焦量变化而旋转的速度与 LG模式平面上所 选取的直线斜率成正比,在聚焦区速度最大， 如图 4。
一个 DH-PSF系统就是在标准成像系统的傅里叶平面加入一个特殊设计的 光学模块， 此光学模块使其透射率函数在傅里叶变化的聚焦区形成双螺旋的形 式，步骤 S101中所创建的光学模块即具有该特性，通过该光学模块所成的像是 两个围绕着光轴旋转的旁瓣， 其中一个绕着光轴顺时针旋转， 而另一个则逆时 针旋转。 用 DH-PSF进行三维纳米定位时， 分子的横向定位点通过两个旁瓣的 中点来估计， 而其轴向位置则根据两个旁瓣中心连线的旋转角度确定，且定位 精度极高， 具体可参考图 5所示的 DH-PSF两个旁瓣中心连线的旋转角度与 Z 轴位置的关系曲线。
另一方面， 该光学模块还具有离焦光栅的多阶成像性质， 离焦光栅实质上 是一个离轴的二元相位菲涅耳波带片， 一方面， 它具有普通光栅的分光作用， 将入射光在光栅的不同衍射级上分束； 另一方面， 它具有菲涅耳波带片的透镜 作用， 在不用的衍射级上引入不同的透镜效应。 该光栅与短焦距透镜密接使用 时， 短焦距透镜提供主要的聚焦能力， 在 ±1级衍射光轴上， 离焦光栅对透镜聚 焦能力进行微调， 使得 ±1级衍射光有不同的焦距， 分别稍短于和稍长于透镜焦 距。 短焦距透镜的焦平面在 ±1级衍射光上的截面是前后对称的离焦面， 因此离 焦光栅能够使同一个物体成像在不同的像平面。 同时可以使位于不同物平面上 的物点成像在同一像平面。 如图 6所示， 位于不同物面的八、 B、 C三点的物可 以在像方同一个像平面上的 A'、 B，、 C，处成像， 根据 A'、 B，、 C，之间的间距 Δύί 可确定 A、 B、 C三点的轴向相对距离 Δζ , 离焦光栅的景深较大， 可达十几微 米， 几乎与完整细 的大小相应。 离焦光栅的相位函数为:
2πτηΑχ(Χ,Υ)
d
(6)
Figure imgf000009_0001
20
m- (x2 + y2)
R (8)
R
式中， R为光栅半径； 2 , 表示离焦光栅的散焦能力， 是散焦的标 准系数。
基于上述双螺旋成像及离焦光栅成像的性质， 本实施例基于波前编码的方 式， 将双螺旋点扩散函数与离焦光栅相组合， 形成一个新的光学模块， 波前编 码是用于使用一个或多个专门设计的相位掩模来创建诸如透镜之类光学模块的 光学传递函数的方法。 本实施例基于波前编码的方式创建的光学模块同时具有 多焦面成像和双螺旋点扩散函数的功能。 基于上述说明， 该光学模块的相位函 数可表示为：
其中， 为几种拉盖尔-高斯光束模式等权重叠加后形成的复振幅的相位， 这几种拉盖尔 -高斯光束模式具体可以是当上述的"， 取 （ 1, 1 ) , (3, 5) , (5, 9) , (7, 13) , (9, 17) 时对应的五种拉盖尔-高斯光束模式。 本实施 例将 ( 1,1 ) , (3,5) , (5,9) , ( 7,13 ) , ( 9,17 )五种模式等权重叠加形成双螺旋 旋转光束的相位形式作为初始值， 然后通过优化， 获得高效率的双螺旋光束纯 相位分布。
2π 。 。
Φ Φ.
具有式（8) 的形式， 即: 其中， R为光栅半径； 2。 , 表示光学模块的散焦能力， 是散焦的标 准系数。
进一步的， 该光学模块具体可以是通过微细加工技术制作的相位片， 也可 以直接采用空间光调制器实现。
进一步的， 待测分子的横向位置具体通过双螺旋图像中双螺旋旁瓣的中点 在成像面上的位置确定； 轴向位置则通过双螺旋图像中双螺旋旁瓣的中心连线 的旋转角度及双螺旋旁瓣的中点在成像面上的位置确定。
可以理解， 在进行光学系统设计时， 会预先对系统进行标定， 建立双螺旋 旁瓣的中心位置与待测分子的横向位置的对应关系， 以及待测分子与多阶成像 物平面的对应关系， 并建立双螺旋旁瓣的旋转角度和离焦量的对应关系等， 将 这些信息预存入数据库待实际测量时调用。 在实际测量时， 可根据双螺旋图像 中两旁瓣的中点的具体位置确定物点的横向位置， 并初步确定物点位于多阶成 像某物面附近， 再进一步通过两旁瓣的旋转角度确定物点与该物面的距离， 进 而确定其轴向位置。
为了验证该方法， 进行了初步的计算机模拟验证， 基于上述方法设计了衍 射相位片，如图 7所示，并模拟了该成像方法。该相位片的通光直径： D = mm, 像素大小： = l¾ n, 像素数： ：33 X 33 , 波长： =δ：? ¾i 。 将此相位版用于三维成像系统中， 模拟了粒子处于不同位置处的成像， 如 图 8所示。 由此， 可看出该方法可以实现 12微米的成像范围。
综上所述， 本实施例的光学模块综合了双螺旋成像及离焦光栅多阶成像的 双重效应， 多阶成像的景深较大，双螺旋成像的分辨率较高且具有一定的景深， 通过该光学模块成像时， 一方面， 多阶成像可以大幅度扩大景深， 并且在可清 晰成像的物面两侧， 还会由于双螺旋效应进一步扩大轴向定位的范围， 进而扩 大景深； 另一方面， 双螺旋成像的分辨率较高， 在景深范围内任意物面上的物 点都可通过双螺旋效应实现高分辨率的轴向定位， 进而提高了三维成像的分辨 率。 因此， 本实施例通过将双螺旋点扩散函数和离焦光栅多阶成像的性质结合 在一起来进行三维成像， 即扩大了景深又提高了分辨率，其景深可达十几微米， 可以用于完整细胞内任意深度亚细胞的动态范围成像， 又可获得多个运动分子 的动态功能图像， 对于在更高水平上认识亚细胞结构与细胞功能变化的关系和 规律具有重要意义。
实施例二：
图 9示出了本发明第二实施例提供的用于大景深三维纳米分辨成像的光学 组件示意图， 为了便于说明， 仅示出了与本实施例相关的部分。
基于上述实施例提供的大景深三维纳米分辨成像方法， 本实施例进一步提 供一种可用于大景深三维纳米分辨成像的光学组件。 该组件主要用于三维成像 系统中， 以实现细胞的超大景深及高分辨率的三维成像。
该光学组件主要包括沿光路传输方向依次设置的第一透镜 901、 光学模块 902和第二透镜 903。其中， 光学模块 902具有上述的双螺旋点扩散函数和离焦 光栅多阶成像性质， 是基于上述方法设计的光学模块 902 , 其具有上述实施例 一中所述的功能， 此处不再赘述。 通常， 光学系统通过探测待测分子发出的荧 光对分子进行定位追踪， 该系统中， 第一透镜 901将待测分子发出的荧光进行 准直并向光学模块 902输出， 光学模块 902将准直后的荧光转换为具有双螺旋 及多阶成像性质的成像光束， 然后由第二透镜 903输出， 将成像光束聚焦于探 测器 904的像平面上， 在探测器上实现双螺旋及多阶成像。 通过成像面上的双 螺旋旁瓣的中心在成像面上的位置可以确定待测分子的横向位置， 通过双螺旋 旁瓣的中心及两旁瓣之间的连线的旋转角度确定待测分子的轴向位置。
在本实施例中， 光学模块 902具体可以是通过光刻的方法制作的相位版， 也可以直接采用空间光调制器， 该光学模块 902的相位函数如实施例一所述， 此处不再赘述。
实施例三：
图 10 示出了本发明第三实施例提供的大景深超分辨荧光显微成像探测系 统示意图，图 11示出了本发明第三实施例提供的另一种大景深超分辨荧光显微 成像探测系统示意图， 为了便于说明， 仅示出了与本实施例相关的部分。
本发明实施例提供了一种基于上述成像方法和光学组件的大景深超分辨荧 光显微成像探测系统， 将本发明的成像方法与超分辨荧光显微成像方法 （如
PALM, STORM )相结合， 实现超大景深三维纳米分辨荧光显微成像探测。
如图 10, 该大景深超分辨荧光显微成像探测系统包括沿光路的传输方向依 次设置的探测物镜 1、 滤光片 2、 双色镜 3、 管镜 4、 成像组件 5及探测器 6。 其中， 成像组件 5采用上述实施例二中的光学组件。 作为一种实现方式， 该光 学组件 5中的光学模块 53具体可以是一相位片，用于将荧光转换为具有双螺旋 及多阶成像性质的成像光束。
在该系统中， 探测物镜 1位于待测物的出光侧， 待测物被激发光激发后可 发出荧光， 含有激发光和荧光及其他杂散光的光束由探测物镜 1接收， 该光束 经过滤光片 2的滤光作用后滤除激发光及杂散光， 透过荧光， 荧光经双色镜 3 反射至管镜 4, 通过管镜 4进行聚焦并传输至光学组件 5的第一透镜 51 , 荧光 光束通过相位片后转变为双螺旋及多阶成像光束，最后通过第二透镜 52聚焦于 探测器 6的成像面上， 在成像面上形成双螺旋的像点。
作为另一种实现方式， 如图 11 , 光学模块 53还可以采用空间光调制器来 显示相位片的相位函数， 实现相位片的功能。 此时， 该成像系统还包括位于双 色镜 3和管镜 4之间的偏振片 7, 用于将荧光光束转换为线偏振光， 以适用于 空间光调制器。
上述成像系统通过本发明提供的光学组件并基于本发明提供的成像方法进 行双螺旋及多阶成像， 利用多阶成像的大景深效应及双螺旋成像的高精度轴向 定位效应， 同时实现了超大景深及高分辨率的三维纳米分辨成像， 可实现任意 深度亚细胞的动态范围成像， 又可获得多个运动分子的动态功能图像， 适用于 完整细胞的三维纳米分辨成像。 该大景深三维纳米分辨成像系统既可以单独用 于细胞成像， 也可内置于细胞成像以及其它成像设备中， 因此， 设有该成像系 统的成像设备也在本发明的保护范围内。 以上所述仅为本发明的较佳实施例而已， 并不用以限制本发明， 凡在本发 明的精神和原则之内所作的任何修改、 等同替换和改进等， 均应包含在本发明 的保护范围之内。

Claims

权 利 要 求 书
1、 一种大景深三维纳米分辨成像方法， 其特征在于， 包括下述步骤： 创建具有双螺旋点扩散函数和离焦光栅多阶成像性质的光学模块； 通过所述光学模块对待测分子进行成像， 获得待测分子的双螺旋图像； 通过所述双螺旋图像中双螺旋旁瓣的中点在成像面上的位置确定待测分子 的横向位置；
通过所述双螺旋图像中双螺旋旁瓣的中心连线的旋转角度及所述双螺旋旁 瓣的中点在成像面上的位置确定待测分子的轴向位置。
2、 如权利要求 1所述的方法， 其特征在于， 所述光学模块的双螺旋点扩散 函数通过下述方法实现：
通过位于拉盖尔-高斯模式平面上特定直线上的拉盖尔 -高斯光束模式的线 性叠加构成带有旋转和缩放的自成像光束；
将自成像光束的一个横截面中的复合光场作为所述光学模块的光学传递函 数， 使所述光学模块具有双螺旋点扩散函数。
3、 如权利要求 2所述的方法， 其特征在于， 所述拉盖尔 -高斯光束模式为：
其中， r = (A^，z;»为空间点的柱坐标， = M 为高斯光斑的径向坐标， ( = «。[i+ S2] , ω。为束腰半径，
S = z/z。为纵向坐标， =^2Α为瑞利长度， 的组成为：
G(Az) = -yi7exp(- 2)exp( 2z)exp[-i (z)]
ω ζ)
Figure imgf000014_0001
Φη) (φ) = &ΧΌ(ίηιφ) Zn ( ) = exp[-m^( )] 其中， ^ arctan ;)为古伊相位， 442为广义的拉盖尔多项式， "， 为 整数， 且取下列五组数值： （1, 1) , (3, 5) , (5, 9) , (7, 13) , (9, 17) , 获得五种拉盖尔-高斯光束模式；
所述五种拉盖尔 -高斯光束模式进行等权重叠加，形成所述带有旋转和缩放 的自成像光束。
4、 如权利要求 3所述的方法， 其特征在于， 所述光学模块的相位函数为： 其中， 为所述五种拉盖尔-高斯光束模式等权重叠加后形成的复振幅的 相位；
Φ8 =ΦΜ(Χ,Υ) = 1^(χ2 + /) 其中， R为光栅半径； 2。= ,表示光学模块的散焦能力， 是散焦的标 准系数。
5、 如权利要求 4所述的方法， 其特征在于， 所述光学模块为采用微细加工 技术制作的相位片或采用空间光调制器实现。
6、 一种用于大景深三维纳米分辨成像的光学组件， 其特征在于， 包括沿光 路传输方向依次设置的：
第一透镜， 用于将待测分子发出的光束进行准直；
光学模块， 具有双螺旋点扩散函数和离焦光栅多阶成像性质， 用于将所述 光束转换为具有双螺旋及多阶成像性质的成像光束；
第二透镜， 用于将所述成像光束输出以用于成像。
7、 如权利要求 6所述的光学组件， 其特征在于， 所述光学模块的相位函数 为： 其中， , = m(X,F) = m¾ 2 + )
R为光栅半径； 2。= ,表示光学模块的散焦能力， 是散焦的标准系数, db 为五种拉盖尔-高斯光束模式等权重叠加后形成的复振幅的相位； 所述拉盖尔 -高斯光束模式为：
其中， = (Α^)为空间点的柱坐标， = w(0为高斯光斑的径向坐标， ζ) = ω0[ΐ + ζ2 , %为束腰半径， 2 = ζ/ 为纵向坐标， 扁。 7/1为瑞利长度, 的组成为：
G( z) = -7iTexp(- 2)exp(i 2z)exp[-iy(z)] υ )=( ¾)Μ 1Η)/2(2 2)
®m(^) = exp 'm^)
Zn( ) = exp[-m^( 其中， W^Harctan^;)为古伊相位， （《'_H 2为广义的拉盖尔多项式， "， 为 整数；
所述五种拉盖尔-高斯光束模式分别为当"， 取 （1, 1) , (3, 5) , (5, 9) , (7, 13) , (9, 17) 时对应的模式。
8、一种大景深超分辨荧光显微成像探测系统， 其特征在于， 包括沿光路传 输方向依次设置的：
探测物镜， 用于接收含有待测分子发出的荧光的光束；
滤光片， 用于对所述光束进行滤波并输出所述荧光；
双色镜， 用于对所述荧光进行反射；
管镜， 用于将反射的荧光聚焦并向成像组件输出； 成像组件， 采用权利要求 6或 7所述的光学组件， 用于将所述荧光转换为 具有双螺旋及多阶成像性质的成像光束；
探测器， 用于接收所述成像光束并进行双螺旋及多阶成像。
9、 如权利要求 8所述的系统， 其特征在于， 所述光学组件中的光学模块为 通过微细加工技术制作的相位片。
10、 如权利要求 8所述的系统， 其特征在于， 所述光学组件中的光学模块 为空间光调制器， 所述系统还包括：
偏振片， 位于所述双色镜和管镜之间， 用于将所述荧光转换为适用于所述 空间光调制器的线偏振光。
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