WO2018205357A1 - 结构光照明显微成像系统 - Google Patents

结构光照明显微成像系统 Download PDF

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WO2018205357A1
WO2018205357A1 PCT/CN2017/089163 CN2017089163W WO2018205357A1 WO 2018205357 A1 WO2018205357 A1 WO 2018205357A1 CN 2017089163 W CN2017089163 W CN 2017089163W WO 2018205357 A1 WO2018205357 A1 WO 2018205357A1
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Prior art keywords
structured light
imaging system
dichroic mirror
microscopic imaging
light
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PCT/CN2017/089163
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English (en)
French (fr)
Inventor
熊大曦
杨西斌
王沛沛
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中国科学院苏州生物医学工程技术研究所
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Priority to JP2019549582A priority Critical patent/JP7000445B2/ja
Priority to US16/607,371 priority patent/US11681135B2/en
Publication of WO2018205357A1 publication Critical patent/WO2018205357A1/zh

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    • 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
    • G02B27/09—Beam shaping, e.g. changing the cross-sectional area, not otherwise provided for
    • G02B27/0938—Using specific optical elements
    • G02B27/095—Refractive optical elements
    • G02B27/0955—Lenses
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • 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
    • G02B21/006—Optical details of the image generation focusing arrangements; selection of the plane to be imaged
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • 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
    • G02B27/09—Beam shaping, e.g. changing the cross-sectional area, not otherwise provided for
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B5/00—Optical elements other than lenses
    • G02B5/08—Mirrors

Definitions

  • the present invention relates to the field of optics, and more particularly to a structured light illumination microscopic imaging system.
  • Structured Light Microscopy Imaging is a super-resolution microscopy imaging technique based on optical microscopy that uses a specially modulated structured light field illumination sample to extract focus plane information from modulated image data of different phases using a phase shift algorithm.
  • Obtaining image data of structured light illumination microscopic imaging can break through the limitation of optical diffraction limit of ordinary optical microscope to achieve higher microscopic imaging resolution.
  • Structured light illumination microscopy was first realized by adding a sinusoidal grating to the illumination path.
  • the grating pattern is projected onto the sample to form structured light illumination.
  • the grating must be moved to obtain Source images with different phase shifts, so this mechanical moving device reduces the stability of the system.
  • the invention patent 201110448980.8 proposes a high-speed structural illumination optical microscopy system and method based on digital micro-mirror device, which realizes high-speed imaging and high light energy utilization.
  • spatial light modulators have the disadvantages of high cost, limited imaging speed, and large system size.
  • a structured light illumination microscopy imaging system comprising:
  • a beam shaping lens, an excitation filter and a dichroic mirror disposed on the emission light path of the structured light source
  • An emission filter, a collimator, and a detector are disposed on the second optical path of the dichroic mirror in sequence.
  • the objective lens and the sample are sequentially disposed on a reflected light path of the dichroic mirror; the emission filter, the barrel mirror and the detector are sequentially disposed on the second The transmitted light path to the color mirror.
  • the objective lens and the sample are sequentially disposed on a transmitted light path of the dichroic mirror; the emission filter, the barrel mirror and the detector are sequentially disposed on the second Reflected light path to the color mirror.
  • the structured light source comprises a micro-sized illuminating light source for generating structured light having bright dark stripes.
  • the micro-sized illuminating light source comprises a substrate and an array of light-emitting units arranged on the substrate. Further, in the above structured light illumination microscopic imaging system, the size of each of the light emitting units does not exceed a square area of 500 ⁇ m ⁇ 500 ⁇ m.
  • the beam shaping lens is disposed on an emission light path of the structured light source
  • the excitation filter is disposed on an emission light path of the beam shaping lens
  • the dichroic mirror is configured to reflect structured light on the emission light path of the excitation filter to the objective lens.
  • the objective lens is configured to receive structured light reflected by the dichroic mirror and project the received structured light onto the sample;
  • the sample is configured to receive structured light projected by the objective lens to form structured light illumination and to excite fluorescence, the fluorescence being sequentially transmitted through the objective lens and the dichroic mirror.
  • the emission filter is configured to filter the fluorescence transmitted through the dichroic mirror
  • the barrel mirror is located on the transmitted light path of the emission filter for collecting and transmitting the filtered fluorescence of the emission filter;
  • the fluorescence detector is configured to receive fluorescence transmitted by the barrel mirror.
  • the surface of the structured light source and the sample, the surface of the sample and the imaging surface of the detector are both in a conjugate structure position.
  • the beam shaping lens comprises one or more lenses.
  • the present invention is sequentially disposed on the light beam of the structured light source by a structured light source, a beam shaping lens, an excitation filter and a dichroic mirror, which are sequentially disposed on the dichroic mirror.
  • the objective lens and the sample on the first optical path are sequentially disposed on the second optical path of the dichroic mirror, the emission filter, the barrel mirror and the detector, compared with the structured light microscopy imaging system provided with the fly-eye lens
  • the structured light microscopy imaging system provided with the fly-eye lens
  • the structured light microscopic imaging system is simple in structure, easy to adjust, and low in cost, and obtains high-resolution, high-stability, high-contrast structured light illumination microscopy.
  • Optical system Optical system.
  • FIG. 1 shows a schematic diagram of a structured light illumination microscopic imaging system in accordance with an embodiment of the present invention
  • FIG. 2 shows a schematic diagram of a structured light illumination microscopic imaging system in accordance with another embodiment of the present invention
  • FIG. 3 illustrates a front view of a circular array of light emitting cells arranged on a substrate in accordance with an embodiment of the present invention
  • FIG. 4 illustrates a front view of a square light emitting cell array arranged on a substrate in accordance with an embodiment of the present invention
  • Figure 5 shows a side view of Figure 4.
  • FIG. 6 is a schematic view showing the formation of vertical stripes by a square light emitting unit according to an embodiment of the invention.
  • FIG. 7 is a schematic view showing the formation of lateral stripes by a square light emitting unit according to an embodiment of the invention.
  • FIG. 8 is a schematic view showing the formation of diagonal stripes by a square light emitting unit according to an embodiment of the invention.
  • FIG. 9 is a schematic view showing the formation of vertical stripes by a circular light emitting unit according to an embodiment of the invention.
  • FIG. 10 is a schematic view showing the formation of lateral stripes by a circular light emitting unit according to an embodiment of the invention.
  • FIG. 11 is a schematic view showing the formation of diagonal stripes by a circular light emitting unit according to an embodiment of the invention.
  • Figure 12 illustrates a longitudinally structured light streak having a duty ratio of 1:2, in accordance with an embodiment of the present invention
  • Figure 13 illustrates a 1:3 longitudinal structured light stripe having a duty cycle in accordance with an embodiment of the present invention
  • FIG. 14 is a schematic diagram showing a driving mode of passive addressing of a micro-sized light emitting diode according to an embodiment of the invention.
  • the present invention provides a structured light illumination microscopic imaging system comprising:
  • the beam shaping lens 2, the excitation filter 3 and the dichroic mirror 4 may be disposed from the structured light source 1 in the near and far direction on the emission light path of the structured light source;
  • the objective lens 5 and the sample 6 are sequentially disposed on the first optical path of the dichroic mirror 4; as shown in FIGS. 1 and 2, the objective lens 5 and the sample 6 are in close proximity to the dichroic mirror 4 Provided on the first optical path of the dichroic mirror 4;
  • the emission filter 7, the cylindrical mirror 8, and the detector 9 are sequentially disposed on the second optical path of the dichroic mirror 4. As shown in FIGS. 1 and 2, the emission filter 7, the lens barrel 8 and the detector 9 may be disposed on the second optical path of the dichroic mirror in sequence from the dichroic mirror, wherein The first optical path and the second optical path of the dichroic mirror are parallel or perpendicular.
  • the structured light illumination microscopic imaging system of the present embodiment can obtain the signal-to-noise ratio and the premise of reducing the installation and processing precision requirements of the structured light illumination microscopic imaging system. Higher-resolution super-resolution microscopy images significantly reduce system cost and system stability compared to structured light microscopy imaging systems based on digital microlens arrays or gratings.
  • the first optical path of the dichroic mirror 4 is a reflected optical path of the dichroic mirror, and the dichroic mirror 4
  • the second optical path is a transmitted optical path of the dichroic mirror, such that the objective lens and the sample are sequentially disposed on the reflected light path of the dichroic mirror; the emission filter, the barrel mirror and the detector are sequentially disposed On the transmitted light path of the dichroic mirror.
  • the first optical path of the dichroic mirror 4 is the transmitted optical path of the dichroic mirror, and the dichroic mirror 4
  • the second optical path is a reflected light path of the dichroic mirror, such that the objective lens and the sample are sequentially disposed on the transmitted light path of the dichroic mirror; the emission filter, the barrel mirror and the detector are sequentially disposed On the reflected light path of the dichroic mirror.
  • the structured light source comprises a micro-sized light emitting diode source (Micro LED) for generating structured light having bright dark stripes.
  • the Micro LED technology that is, the LED miniaturization and matrixing technology, refers to a high-density and small-sized LED array integrated on one chip, and each of the light-emitting units can be addressed and individually driven to illuminate. It has the characteristics of high efficiency, high brightness, high reliability and fast response time, and has the characteristics of self-illumination without backlight, more energy saving, simple mechanism, small size and thin shape. As shown in FIG. 3 to FIG.
  • the micro-sized LED light source 1 is composed of a substrate 11 and an array of light-emitting units 12 arranged on the substrate, and each of the light-emitting units 12 can be a micro-sized light-emitting diode.
  • the structure light stripe required for the structured light illumination microscopic imaging system can be obtained by controlling the micro-sized LED array, and the high-resolution switching and phase shift of the structured light stripe can achieve the purpose of super-resolution microscopic imaging.
  • the structured light microscopy imaging system based on the micro-sized LED array provided by the embodiment has a simple structure, is easy to adjust, and has lower cost.
  • the structured light having bright and dark stripes is generated by the micro-sized light-emitting diode, and the structured light illumination is realized instead of the grating or the two-dimensional spatial light modulator used in the conventional structured light microscopic imaging system, thereby greatly simplifying the optical path structure of the illumination system and reducing the structure.
  • the cost of light microscopy imaging system at the same time, due to the high efficiency, high brightness, high reliability and short response time of micro-sized LEDs, the structured light illumination microscopic imaging system based on micro-sized LEDs has higher reliability and stability. Sex.
  • the structured light illumination microscopic imaging system of the embodiment obtains a structured light illumination micro-optical system with high resolution, high stability and high contrast.
  • each of the light emitting units has its own independent driving circuit, and each of the light emitting units simultaneously emits one or more different wavelengths of light to make the micro
  • the size LED light source produces a specific structured light.
  • the wavelength of the emitted light of a certain light-emitting unit is 488 nm.
  • the shape of the light emitting unit is square or circular.
  • FIG. 3 is a front view of a circular light emitting unit array arranged on a substrate.
  • 4 is a front view of a square light emitting cell array arranged on a substrate
  • FIG. 5 is a side view of FIG.
  • Each of the light emitting units has a size range of not more than 500 ⁇ m ⁇ 500 ⁇ m square area, for example, the shape of the light emitting unit is square or circular
  • the circular light emitting unit has a diameter of ⁇ 1 to 500 ⁇ m
  • the square light emitting unit has a size of 1 ⁇ m ⁇ 1 ⁇ m to 500 ⁇ m ⁇ 500 ⁇ m.
  • the circular light emitting unit may have a diameter of ⁇ 1 to 500 ⁇ m
  • the square light emitting unit may have a size of 1 ⁇ m ⁇ 1 ⁇ m to 500 ⁇ m ⁇ 500 ⁇ m.
  • the beam shaping lens is disposed on an emission light path of the structured light source
  • the excitation filter is disposed on an emission light path of the beam shaping lens
  • the dichroic mirror is configured to reflect structured light on the emission light path of the excitation filter to the objective lens.
  • the objective lens is configured to receive structured light reflected by the dichroic mirror and project the received structured light onto the sample;
  • the objective lens is configured to receive structured light reflected by the dichroic mirror and magnify the received structured light to a predetermined magnification to be projected onto the sample;
  • the sample is disposed on a stage for receiving structured light projected by the objective lens to form structured light illumination and exciting to generate fluorescence, the fluorescence being sequentially transmitted through the objective lens and the dichroic mirror.
  • the emission filter is configured to filter the fluorescence transmitted through the dichroic mirror
  • the barrel mirror is located on the transmitted light path of the emission filter for collecting and transmitting the filtered fluorescence of the emission filter;
  • the fluorescence detector is configured to receive fluorescence transmitted by the barrel mirror.
  • the structured light source and the surface of the sample, the surface of the sample, and the imaging surface of the detector are both in a conjugated structure position to ensure The images acquired by the detector have a higher contrast and signal to noise ratio.
  • the micro-sized LED light source is a resolution
  • the ratio is 512 ⁇ 512 light-emitting unit arrays, each of which is a square having a size of 20 ⁇ m ⁇ 20 ⁇ m, and a spacing between adjacent light-emitting units is 4 ⁇ m.
  • 6-8 are micro-sized LED arrays of square illumination units, each of which is independently controllable for obtaining desired structural light strips, including longitudinal stripes (Fig. 6) and lateral stripes (e.g. Figure 7) and oblique 45° stripes ( Figure 8).
  • the light-emitting unit of the micro-sized light-emitting diode may also be a circular shape having a diameter of ⁇ 20 ⁇ m.
  • the beam shaping lens comprises one or more lenses, the beam shaping lens may have a focal length of 270 mm, the objective lens magnification may be 100, and the objective lens focal length is 1.8 mm. Since the spacing between the light-emitting diodes of the micro-sized LEDs is only 4 ⁇ m, after the beam shaping lens, the dichroic mirror, and the objective lens are projected onto the surface of the sample, the distance between the images of the light-emitting unit on the surface of the sample is not more than 200 nm, which is smaller than the wavelength of the emitted light.
  • Half of (488 nm) reaches the diffraction limit, so continuous stripe light is obtained on the surface of the sample, and it is not necessary to provide a fly-eye lens to achieve double the number of stripes so that the distance of the light-emitting unit is smaller than the diffraction limit to obtain continuous stripe light.
  • the elimination of the compound eye lens can reduce the difficulty of system adjustment and processing, and improve the contrast and signal-to-noise ratio of the microscopic image to make the imaging quality higher.
  • the duty ratio of the stripe light that is, the ratio of light and dark stripes can be appropriately adjusted, and the contrast of the stripe light can be improved to improve the image quality.
  • 12 to 13 show that the duty ratios of the light and dark stripes are 1:2, 1:3 longitudinal structure light stripes, respectively.
  • a passive addressing driving mode of a micro-sized LED connecting the anode of the light-emitting unit of each column of the micro-sized LED array to the column scan line, and connecting the cathode of each row of the light-emitting unit to the row scan line .
  • a particular Y-th column scan line and X-th line scan line are gated, the LED pixels at the intersection (X, Y) are illuminated.
  • the entire micro-scale LED array is scanned at high speed in a high-speed, point-by-point manner to achieve the desired structural light streaks.
  • micro-sized LEDs can also be in an active location, each of which has its own independent drive circuit, and the drive current is provided by the drive transistor.
  • the control mode of the active site selection is more complicated, but the connection between each light-emitting unit can be simplified, and the defect that the strobe signal is prone to crosstalk is improved.
  • the present invention is sequentially disposed on the emission of the structured light source by a structured light source.
  • a beam shaping lens, an excitation filter and a dichroic mirror on the optical path, and an objective lens and a sample which are sequentially disposed on the first optical path of the dichroic mirror, and are sequentially disposed on the second optical path of the dichroic mirror.
  • the emission filter, the barrel mirror and the detector compared with the structured light microscopy imaging system provided with the fly-eye lens, can obtain the signal-to-noise ratio under the premise of reducing the installation and processing accuracy requirements of the structured light illumination microscopic imaging system.
  • Super-resolution microscopy images with higher contrast ratios significantly reduce system cost and system stability compared to structured light microscopy imaging systems based on digital microlens arrays or gratings.
  • the structured light microscopic imaging system is simple in structure, easy to adjust, and low in cost, and obtains high-resolution, high-stability, high-contrast structured light illumination microscopy.
  • Optical system Optical system.

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Abstract

一种结构光照明显微成像系统，通过依次设置于结构光光源（1）的发射光路上的光束整形透镜（2）、激发滤光片（3）和二向色镜（4），依次设置于二向色镜（4）第一光路上的物镜（5）和样品（6），依次设置于二向色镜（4）的第二光路上的发射滤光片（7）、筒镜（8）和探测器（9），与设置有复眼透镜的结构光显微成像系统相比，在降低结构光照明显微成像系统的安装和加工精度要求前提下，可以得到信噪比和对比度更高的超分辨显微图像；与基于数字微透镜阵列或光栅的结构光显微成像系统相比，大幅降低系统成本，且系统稳定性更高。

Description

结构光照明显微成像系统 技术领域
本发明涉及光学领域，尤其涉及一种结构光照明显微成像系统。
背景技术
结构光显微成像技术是一种基于光学显微镜的超分辨显微成像技术，该技术使用特殊调制的结构光场照明样品，通过运用相移算法从不同相位的调制图像数据中提取聚焦平面的信息，得到结构光照明显微成像的图像数据，可以突破普通光学显微镜受光学衍射极限的限制，达到更高的显微成像分辨率。
结构光照明显微最早是通过将照明光路中加入一个正弦光栅来实现的，光栅图案被投影到样品上形成结构光照明，但在这些用光栅获得结构光照明的系统中,必须移动光栅来获得不同相移下的源图像，因此这种机械移动装置会减低系统的稳定性。
发明专利201110448980.8提出了一种基于数字微镜器件的高速结构照明光学显微系统及方法，实现了高速成像和高光能量利用率。但是空间光调制器存在成本高、成像速度仍然受到限制、系统体积大等缺点。
另外，也有通过阵列排布的LED光源结合复眼透镜实现结构光照明的显微系统，该系统中需要引入复眼透镜，而且对复眼透镜的加工和装调有极高的难度，安装和加工精度要求非常高。当复眼透镜在安装和加工精度没有达到要求时，通过安装有该复眼透镜的结构光照明显微成像系统，无法得到高对比度、高信噪比的显微图像。
发明内容
本发明的一个目的是提供一种结构光照明显微成像系统，能够解决已 有结构光照明显微成像系统成本高、装调困难、稳定性差等问题。
根据本发明的一个方面，提供了一种结构光照明显微成像系统，包括：
结构光光源；
依次设置于所述结构光光源的发射光路上的光束整形透镜、激发滤光片和二向色镜；
依次设置于所述二向色镜的第一光路上的物镜和样品；
依次设置于所述二向色镜的第二光路上的发射滤光片、筒镜和探测器。
进一步的，上述结构光照明显微成像系统中，所述物镜和样品依次设置于所述二向色镜的反射光路上；所述发射滤光片、筒镜和探测器依次设置于所述二向色镜的透射光路上。
进一步的，上述结构光照明显微成像系统中，所述物镜和样品依次设置于所述二向色镜的透射光路上；所述发射滤光片、筒镜和探测器依次设置于所述二向色镜的反射光路上。
进一步的，上述结构光照明显微成像系统中，所述结构光光源包括微尺寸发光光源，用于产生具有亮暗条纹的结构光。
进一步的，上述结构光照明显微成像系统中，所述微尺寸发光光源包括基板和排布于所述基板上的发光单元阵列。进一步的，上述结构光照明显微成像系统中，所述每个发光单元的尺寸范围不超过500μm×500μm方形区域。
进一步的，上述结构光照明显微成像系统中，所述光束整形透镜，设置于所述结构光光源的发射光路上；
所述激发滤光片，设置于所述光束整形透镜的发射光路上；
所述二向色镜，用于反射所述激发滤光片的发射光路上的结构光至所述物镜。
进一步的，上述结构光照明显微成像系统中，所述物镜，用于接收所述二向色镜反射的结构光并将所述接收的结构光投影至所述样品；
所述样品，用于接收所述物镜投影的结构光后形成结构光照明并激发产生荧光，所述荧光依次透射过所述物镜和二向色镜。
进一步的，上述结构光照明显微成像系统中，所述发射滤光片，用于对透射过所述二向色镜的所述荧光进行滤波；
所述筒镜位于所述发射滤光片的透射光路上，用于收集和透射所述经所述发射滤光片滤波后的荧光；
所述荧光探测器，用于接收所述筒镜透射后的荧光。
进一步的，上述结构光照明显微成像系统中，所述结构光光源与所述样品的表面、所述样品的表面与所述探测器的成像面均处于共轭结构位置。
进一步的，上述结构光照明显微成像系统中，所述光束整形透镜包括一个或多个透镜。
与现有技术相比，本发明通过结构光光源，依次设置于所述结构光光源的发射光路上的光束整形透镜、激发滤光片和二向色镜，依次设置于所述二向色镜的第一光路上的物镜和样品，依次设置于所述二向色镜的第二光路上的发射滤光片、筒镜和探测器，与设置有复眼透镜的结构光显微成像系统相比，在降低结构光照明显微成像系统的安装和加工精度要求前提下，可以得到信噪比和对比度更高的超分辨显微图像；与基于数字微透镜阵列或光栅的结构光显微成像系统相比，大幅降低系统成本，且系统稳定性更高。另外，通过采用微尺寸发光二极管光源作为结构光光源，使结构光显微成像系统结构简单，易于装调，且成本更低，获得高分辨率、高稳定性、高对比度的结构光照明显微光学系统。
附图说明
通过阅读参照以下附图所作的对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1示出根据本发明一实施例的结构光照明显微成像系统示意图；
图2示出根据本发明另一实施例的结构光照明显微成像系统示意图；
图3示出根据本发明一实施例的排布于基板上的圆形发光单元阵列的正视图；
图4示出根据本发明一实施例的排布于基板上的方形发光单元阵列的正视图；
图5示出图4的侧视图；
图6示出根据本发明一实施例的正方形发光单元形成纵向条纹的示意图；
图7示出根据本发明一实施例的正方形发光单元形成横向条纹的示意图；
图8示出根据本发明一实施例的正方形发光单元形成斜向条纹的示意图；
图9示出根据本发明一实施例的圆形发光单元形成纵向条纹的示意图；
图10示出根据本发明一实施例的圆形发光单元形成横向条纹的示意图；
图11示出根据本发明一实施例的圆形发光单元形成斜向条纹的示意图；
图12示出根据本发明一实施例的占空比为1:2纵向结构光条纹；
图13示出根据本发明一实施例的占空比为1:3纵向结构光条纹；
图14示出根据本发明一实施例的微尺寸发光二极管的无源选址的驱动模式示意图。
附图中相同或相似的附图标记代表相同或相似的部件。
具体实施方式
下面结合附图对本发明作进一步详细描述。
如图1和2所示，本发明提供一种结构光照明显微成像系统，包括：
结构光光源1；
依次设置于所述结构光光源1的发射光路上的光束整形透镜2、激发滤光片3和二向色镜4；如图1和2所示，所述光束整形透镜2、激发滤光片3和二向色镜4可以离所述结构光光源1由近及远依次设置于所述结构光光源的发射光路上；
依次设置于所述二向色镜4的第一光路上的物镜5和样品6；如图1和2所示，物镜5和样品可6以离所述二向色镜4由近及远依次设置于所述二向色镜4的第一光路上；
依次设置于所述二向色镜4的第二光路上的发射滤光片7、筒镜8和探测器9。如图1和2所示，所述发射滤光片7、筒镜8和探测器9可以离所述所述二向色镜依次设置于所述二向色镜的第二光路上，其中，所述二向色镜的第一光路和第二光路平行或垂直。本实施例的结构光照明显微成像系统与设置有复眼透镜的结构光显微成像系统相比，在降低结构光照明显微成像系统的安装和加工精度要求前提下，可以得到信噪比和对比度更高的超分辨显微图像，与基于数字微透镜阵列或光栅的结构光显微成像系统相比，大幅降低系统成本，且系统稳定性更高。
本发明的结构光照明显微成像系统一实施例中，如图1所示，所述二向色镜4的第一光路为所述二向色镜的反射光路，所述二向色镜4的第二光路为所述二向色镜的透射光路，如此，所述物镜和样品依次设置于所述二向色镜的反射光路上；所述发射滤光片、筒镜和探测器依次设置于所述二向色镜的透射光路上。
本发明的结构光照明显微成像系统一实施例中，如图2所示，所述二向色镜4的第一光路为所述二向色镜的透射光路，所述二向色镜4的第二光路为所述二向色镜的反射光路，如此，所述物镜和样品依次设置于所述二向色镜的透射光路上；所述发射滤光片、筒镜和探测器依次设置于所述二向色镜的反射光路上。
本发明的结构光照明显微成像系统一实施例中，所述结构光光源包括微尺寸发光二极管光源(Micro LED)，用于产生具有亮暗条纹的结构光。在此，Micro LED技术，即LED微缩化和矩阵化技术，指的是在一个芯片上集成的高密度微小尺寸的LED阵列，每一个发光单元可定址、单独驱动点亮。具有高效率、高亮度、高可靠度及反应时间快等特点，并且具自发光无需背光源的特性，更具节能、机构简易、体积小、薄型等优势。如图3～5所示，所述微尺寸发光二极管光源1由基板11和排布于所述基板上的发光单元12阵列组成，每个发光单元12可以为一个微尺寸发光二极管，本实施例中，可以通过控制微尺寸发光二极管阵列，得到结构光照明显微成像系统所需的结构光条纹，通过结构光条纹的高速切换与相移，实现超分辨显微成像的目的。相比传统的基于DMD或光栅的结构光显微成像系统，本实施例提供的基于微尺寸发光二极管阵列的结构光显微成像系统结构简单，易于装调，且成本更低。
本实施例通过微尺寸发光二极管产生具有亮暗条纹的结构光，代替传统结构光显微成像系统中使用的光栅或二维空间光调制器实现结构光照明，大幅简化照明系统光路结构，降低结构光显微成像系统成本；同时因微尺寸发光二极管效率高、亮度高、可靠性强及响应时间短的特性，基于微尺寸发光二极管的结构光照明显微成像系统有更高的可靠性和稳定性。本实施例的结构光照明显微成像系统，获得高分辨率、高稳定性、高对比度的结构光照明显微光学系统。
本发明的结构光照明显微成像系统一实施例中，所述每个发光单元有其对应的独立驱动电路，每个发光单元同时出射一种或多种不同波长的光，以使所述微尺寸LED光源产生特定的结构光。例如，某个发光单元的出射光波长为488nm。
本发明的结构光照明显微成像系统一实施例中，所述发光单元的形状为方形或圆形，例如,图3所示为排布于基板上的圆形发光单元阵列的正视图, 图4所示为排布于基板上的方形发光单元阵列的正视图,图5为图4的侧视图。所述每个发光单元的尺寸范围在不超过500μm×500μm方形区域，例如，所述发光单元的形状为方形或圆形，圆形发光单元直径尺寸为φ1～500μm，方形发光单元尺寸为1μm×1μm～500μm×500μm。进一步的，圆形发光单元直径尺寸可以为φ1～500μm，方形发光单元尺寸可以为1μm×1μm～500μm×500μm。
本发明的结构光照明显微成像系统一实施例中，所述光束整形透镜，设置于所述结构光光源的发射光路上；
所述激发滤光片，设置于所述光束整形透镜的发射光路上；
所述二向色镜，用于反射所述激发滤光片的发射光路上的结构光至所述物镜。
本发明的结构光照明显微成像系统一实施例中，所述物镜，用于接收所述二向色镜反射的结构光并将所述接收的结构光投影至所述样品；在此，所述物镜，可用于接收所述二向色镜反射的结构光并将所述接收的结构光按照预设倍率放大后投影至所述样品；
所述样品设置于载物台上，所述样品用于接收所述物镜投影的结构光后形成结构光照明并激发产生荧光，所述荧光依次透射过所述物镜和二向色镜。
本发明的结构光照明显微成像系统一实施例中，所述发射滤光片，用于对透射过所述二向色镜的所述荧光进行滤波；
所述筒镜位于所述发射滤光片的透射光路上，用于收集和透射所述经所述发射滤光片滤波后的荧光；
所述荧光探测器，用于接收所述筒镜透射后的荧光。
本发明的结构光照明显微成像系统一实施例中，所述结构光光源与所述样品的表面、所述样品的表面与所述探测器的成像面均处于共轭结构位置，以保证所述探测器采集的图像具有较高的对比度和信噪比。
本发明一实施例中，所述微尺寸发光二极管光源(micro LED)为一分辨 率为512×512的发光单元阵列，每个发光单元为尺寸20μm×20μm的正方形，相邻发光单元之间间距为4μm。图6～8为正方形发光单元的微尺寸发光二极管阵列，阵列中的每个发光单元均独立可控，用于得到所需的结构光条纹，包括纵向条纹(如图6)、横向条纹(如图7)和斜向45°条纹(如图8)。
同理，如图9～11所示，所述微尺寸发光二极管的发光单元也可以是直径为φ20μm的圆形。所述光束整形透镜包括一个或多个透镜，所述光束整形透镜的焦距可以是270mm，所述物镜放大倍数可以为100，物镜焦距1.8mm。由于微尺寸发光二极管发光单元之间间距仅4μm，经光束整形透镜、二向色镜、物镜作用后投影到样品表面后，发光单元在样品表面的像之间间距不超过200nm，小于出射光波长(488nm)的一半，达到衍射极限，故在样品表面得到的是连续的条纹光，不需要设置复眼透镜实现条纹数目倍增使发光单元间距小于衍射极限而得到连续条纹光。复眼透镜的取消可以降低系统的装调和加工难度，并提高显微图像的对比度和信噪比，使成像质量更高。
为进一步提高结构光显微成像系统的图像质量，可以适当调整条纹光的占空比，即明暗条纹的比例，可以提高条纹光的对比度，达到改善成像质量的目的。图12～13为亮暗条纹的占空比分别为1:2、1:3纵向结构光条纹。
图14为微尺寸发光二极管的无源选址的驱动模式，把微尺寸发光二极管阵列中每一列的发光单元的阳极连接到列扫描线，同时把每一行的发光单元的阴极连接到行扫描线。当某一特定的第Y列扫描线和第X行扫描线被选通的时候，其交叉点(X，Y)的LED像素即会被点亮。整个微尺寸发光二极管阵列以这种方式进行高速逐点扫描即可实现所需的结构光条纹。
此外，微尺寸发光二极管也可以采用有源选址的方式，每个发光单元有其对应的独立驱动电路，驱动电流由驱动晶体管提供。相比图14中无源选址的方式，有源选址的控制方式结构更复杂，但可以简化每个发光单元间的连接，改善选通信号易发生串扰的缺陷。
综上所述，本发明通过结构光光源，依次设置于所述结构光光源的发射 光路上的光束整形透镜、激发滤光片和二向色镜，依次设置于所述二向色镜的第一光路上的物镜和样品，依次设置于所述二向色镜的第二光路上的发射滤光片、筒镜和探测器，与设置有复眼透镜的结构光显微成像系统相比，在降低结构光照明显微成像系统的安装和加工精度要求前提下，可以得到信噪比和对比度更高的超分辨显微图像，与基于数字微透镜阵列或光栅的结构光显微成像系统相比，大幅降低系统成本，且系统稳定性更高。另外，通过采用微尺寸发光二极管光源作为结构光光源，使结构光显微成像系统结构简单，易于装调，且成本更低，获得高分辨率、高稳定性、高对比度的结构光照明显微光学系统。
显然，本领域的技术人员可以对本申请进行各种改动和变型而不脱离本申请的精神和范围。这样，倘若本申请的这些修改和变型属于本申请权利要求及其等同技术的范围之内，则本申请也意图包含这些改动和变型在内。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化涵括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。此外，显然“包括”一词不排除其他单元或步骤，单数不排除复数。装置权利要求中陈述的多个单元或装置也可以由一个单元或装置通过软件或者硬件来实现。第一，第二等词语用来表示名称，而并不表示任何特定的顺序。

Claims (11)

  1. 一种结构光照明显微成像系统，其中，该系统包括：
    结构光光源；依次设置于所述结构光光源的发射光路上的光束整形透镜、激发滤光片和二向色镜；依次设置于所述二向色镜的第一光路上的物镜和样品；及依次设置于所述二向色镜的第二光路上的发射滤光片、筒镜和探测器。
  2. 根据权利要求1所述的结构光照明显微成像系统，其中，所述物镜和样品依次设置于所述二向色镜的反射光路上；所述发射滤光片、筒镜和探测器依次设置于所述二向色镜的透射光路上。
  3. 根据权利要求1所述的结构光照明显微成像系统，其中，所述物镜和样品依次设置于所述二向色镜的透射光路上；所述发射滤光片、筒镜和探测器依次设置于所述二向色镜的反射光路上。
  4. 根据权利要求1所述的结构光照明显微成像系统，其中，所述结构光光源包括微尺寸发光光源，用于产生具有亮暗条纹的结构光。
  5. 根据权利要求5所述的结构光照明显微成像系统，其中，所述微尺寸发光光源包括基板和排布于所述基板上的发光单元阵列。
  6. 根据权利要求5所述的结构光照明显微成像系统，其中，所述每个发光单元的尺寸范围不超过500μm×500μm方形区域。
  7. 根据权利要求1所述的结构光照明显微成像系统，其中，所述光束整形透镜，设置于所述结构光光源的发射光路上；
    所述激发滤光片，设置于所述光束整形透镜的发射光路上；
    所述二向色镜，用于反射所述激发滤光片的发射光路上的结构光至所述物镜。
  8. 根据权利要求7所述的结构光照明显微成像系统，其中，所述物镜，用于接收所述二向色镜反射的结构光并将所述接收的结构光投影至所述样品；
    所述样品，用于接收所述物镜投影的结构光后形成结构光照明并激发产生荧光，所述荧光依次透射过所述物镜和二向色镜。
  9. 根据权利要求8所述的结构光照明显微成像系统，其中，所述发射滤光片，用于对透射过所述二向色镜的所述荧光进行滤波；
    所述筒镜位于所述发射滤光片的透射光路上，用于收集和透射所述经所述发射滤光片滤波后的荧光；
    所述荧光探测器，用于接收所述筒镜透射后的荧光。
  10. 根据权利要求1所述的结构光照明显微成像系统，其中，所述结构光光源与所述样品的表面、所述样品的表面与所述探测器的成像面均处于共轭结构位置。
  11. 根据权利要求1所述的结构光照明显微成像系统，其中，所述光束整形透镜包括一个或多个透镜。
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