WO2017000701A1 - 一种荧光显微成像方法和装置 - Google Patents

一种荧光显微成像方法和装置 Download PDF

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WO2017000701A1
WO2017000701A1 PCT/CN2016/082768 CN2016082768W WO2017000701A1 WO 2017000701 A1 WO2017000701 A1 WO 2017000701A1 CN 2016082768 W CN2016082768 W CN 2016082768W WO 2017000701 A1 WO2017000701 A1 WO 2017000701A1
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light source
excitation light
monochromatic
fluorescent excitation
fluorescence
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PCT/CN2016/082768
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English (en)
French (fr)
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罗浦文
夏浩涵
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上海睿钰生物科技有限公司
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Priority to JP2017566638A priority Critical patent/JP2018519550A/ja
Priority to EP16817065.2A priority patent/EP3318865A4/en
Priority to US15/738,296 priority patent/US10161873B2/en
Publication of WO2017000701A1 publication Critical patent/WO2017000701A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • 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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • 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
    • G01N2021/6463—Optics
    • G01N2021/6471—Special filters, filter wheel
    • 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
    • G01N2021/6463—Optics
    • G01N2021/6478—Special lenses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/061—Sources
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/062—LED's
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements

Definitions

  • the present invention relates to the field of fluorescence microscopy imaging, and more particularly to a fluorescence microscopy imaging method and apparatus.
  • a conventional fluorescence microscopic imaging device comprising: a fluorescence excitation light source 1 , a dichroic phase mirror 2 disposed at an angle of 45 degrees to the illumination direction of the monochromatic fluorescence excitation light source 1 , and an objective lens 3 .
  • the monochromatic fluorescent excitation light source 1 emits excitation light
  • the dichroic mirror 2 is reflected by the dichroic mirror 2 to the objective lens 3, and then irradiated onto the sample plate to be tested of the sample placement table 4, and the particles in the sample plate to be tested are excited to emit fluorescence, and pass through the second After the hue mirror 2 and the emission filter 5, the camera 6 is imaged.
  • Existing fluorescence microscopy imaging devices require dichroic mirror splitting, making the structure complex and costly.
  • the present invention provides a fluorescence microscopic imaging method and apparatus, which eliminates the need for dichroic mirror splitting, so that the device is not only simple in structure and low in cost, but also reduces monochromatic fluorescent excitation light entering the objective lens, so that the acquired fluorescent image is obtained. More precise.
  • a fluorescence microscopic imaging method characterized in that, after the sample plate to be tested is placed, the method comprises:
  • the fluorescent excitation light source is a target light source, wherein the monochromatic fluorescent excitation light emitted by each of the monochromatic fluorescent excitation light sources is obliquely incident into the preset detection area of the sample plate to be tested;
  • the detection area is enlarged to a preset multiple
  • the method further includes:
  • the monochromatic fluorescent excitation light emitted by each of the monochromatic fluorescent excitation light sources in the target light source is condensed.
  • the present invention also provides a fluorescence microscopic imaging apparatus, comprising:
  • a light source device a sample placement table, an objective lens, an emission filter module, and an image acquisition device;
  • the light source device includes: a plurality of monochromatic fluorescent excitation light sources and a control system electrically connected to the plurality of monochromatic fluorescent excitation light sources, wherein the plurality of monochromatic fluorescent excitation light sources surround the objective lens and the image acquisition device Forming an imaging optical path in which the central axis is disposed, and the monochromatic fluorescent excitation light emitted by each of the monochromatic fluorescent excitation light sources intersects the central axis of the imaging optical path at a preset position of the sample placement stage, and the control system is in accordance with experimental requirements. Illuminating at least one monochromatic fluorescent excitation light source of the same color in the plurality of monochromatic fluorescent excitation light sources as a target light source;
  • the sample placement stage is disposed at a intersection of the monochromatic fluorescent excitation light emitted by the plurality of monochromatic fluorescent excitation light sources for placing the sample plate to be tested, and the preset detection area of the sample plate to be tested is set in the The preset position of the sample placement table;
  • the objective lens is disposed on a side of the sample placing table facing away from the light source device;
  • the emission filter module is disposed on a side of the objective lens facing away from the sample placement table;
  • the image acquiring device is disposed on a side of the emission filter module facing away from the objective lens.
  • the light source device further includes:
  • a bright field light source the full-band white light emitted by the bright field light source is directed toward the sample placement stage and coincides with an axis of the imaging optical path.
  • the monochromatic fluorescent excitation source is a monochromatic LED fluorescent excitation source.
  • the light source device further includes:
  • an excitation filter disposed between the monochromatic LED fluorescence excitation light source and the sample placement stage disposed in an illumination direction of the monochromatic LED fluorescence excitation light source.
  • the monochromatic fluorescent excitation light source comprises:
  • an excitation filter disposed in an irradiation direction of the white light excitation light source and disposed between the white light excitation light source and the sample placement stage.
  • the emission filter module is an emission filter turntable, and a plurality of emission filter regions are disposed around the emission filter disk, and a pass band of each emission filter region is inside the sample plate to be tested.
  • the light source device further includes:
  • a concentrating module disposed in an irradiation direction of the monochromatic fluorescent excitation light source and disposed between the monochromatic fluorescent excitation light source and the sample placing stage.
  • the concentrating module is a concentrating lens group composed of a condensing lens or a plurality of lenses.
  • the image acquisition device is an eyepiece or a camera.
  • the technical solution provided by the present invention has at least the following advantages:
  • the present invention provides a fluorescence microscopic imaging method and apparatus, comprising: illuminating at least one monochromatic fluorescent excitation light source of the same color as a target light source in a plurality of monochromatic fluorescent excitation light sources according to an experimental requirement, wherein each single color
  • the monochromatic fluorescent excitation light emitted by the fluorescent excitation light source is obliquely incident on the predetermined detection area of the sample plate to be tested; and the predetermined detection area is collected on the side of the sample plate to be tested facing away from the target light source
  • the particles are excited by the illumination of the monochromatic fluorescent excitation light emitted by the target light source, and the predetermined detection area is enlarged to a preset multiple; filtering the fluorescence excited by the particles in the preset detection area Light processing; acquiring a fluorescent image of the preset detection area.
  • the technical solution provided by the present invention eliminates the need to provide a dichroic mirror to split the monochromatic fluorescence excitation light and fluorescence, so that the fluorescence microscopic device has a simple structure and low cost, and avoids fluorescence when passing through the dichroic mirror.
  • the resulting fluorescence image is brighter and clearer.
  • the monochromatic fluorescent excitation light is transmitted into the objective lens through the sample plate to be tested, thereby reducing the influence of the monochromatic fluorescent excitation light in subsequent imaging.
  • the acquired fluorescence image is more accurate.
  • FIG. 1 is a schematic structural view of a conventional fluorescence microscopic imaging device
  • FIG. 2 is a flow chart of a fluorescence microscopic imaging method according to an embodiment of the present application
  • FIG. 3 is a schematic structural view of a fluorescence microscopic imaging device according to an embodiment of the present application.
  • FIG. 4 is a schematic structural diagram of another fluorescence microscopic imaging apparatus according to an embodiment of the present application.
  • the existing fluorescence microscopic imaging apparatus requires dichroic mirror splitting, which makes the structure complicated and costly.
  • the embodiment of the present application provides a fluorescence microscopic imaging method and device, which eliminates the need for dichroic mirror splitting, so that the device is not only simple in structure and low in cost, but also reduces monochromatic fluorescent excitation light entering the objective lens, so that the acquired fluorescence is obtained.
  • the image is more precise.
  • FIG. 2 a flowchart of a fluorescence microscopic imaging method provided by an embodiment of the present application, wherein after the sample plate to be tested is placed, the method includes:
  • the plurality of monochromatic fluorescent excitation light sources provided by the embodiments of the present application may include a monochromatic fluorescent excitation light source having different colors (or bands), or a monochromatic fluorescent excitation light source having all the same colors (or bands).
  • a plurality of monochromatic fluorescent excitation light sources are different color monochromatic fluorescent excitation light sources, there are various cases, that is, all the monochrome fluorescent excitation light sources provided by the embodiments of the present application have different colors; or, all monochrome
  • the fluorescent excitation light source is divided into multiple groups, and the color of the monochromatic fluorescent excitation light source in each group is the same, and the colors between the groups are different, thereby improving the color of each color by illuminating a plurality of monochromatic fluorescent excitation light sources of the same color.
  • the brightness of the fluorescent excitation light is not specifically limited in this application.
  • the technical solution provided by the embodiment of the present invention does not need to provide a dichroic mirror to split the monochromatic fluorescence excitation light and the fluorescence, which can make the fluorescence microscopic device simple in structure and low in cost, and avoid the fluorescence in the second pass.
  • the loss of light energy occurs in the hue mirror, which in turn makes the resulting fluorescence image brighter and clearer.
  • the monochromatic fluorescent excitation light is transmitted into the objective lens through the sample plate to be tested, thereby reducing the influence of the monochromatic fluorescent excitation light in subsequent imaging.
  • the acquired fluorescence image is more accurate.
  • the brightness of the monochromatic fluorescent excitation light emitted by the target light source is increased, and the monochromatic fluorescence emitted by the target light source after the target light source is illuminated Before the excitation light is injected into the sample plate to be tested, it also includes:
  • the monochromatic fluorescent excitation light emitted by each of the monochromatic fluorescent excitation sources in the target light source is condensed.
  • FIG. 3 a schematic structural diagram of a fluorescence microscopic imaging apparatus according to an embodiment of the present application, wherein the fluorescence microscopic imaging apparatus includes:
  • a light source device 100 a sample placement table 200, an objective lens 300, an emission filter module 400, and an image acquisition device 500;
  • the light source device 100 includes a plurality of monochromatic fluorescent excitation light sources 101 and a control system (not shown) electrically connected to the plurality of monochromatic fluorescent excitation light sources 101, wherein the plurality of monochromatic fluorescent excitation light sources 101 surround the objective lens 300 and image acquisition
  • the imaging optical path formed by the device 500 is set in the axis X, and each monochromatic fluorescent excitation light
  • the monochromatic fluorescent excitation light emitted by the source 101 intersects the axis X of the imaging optical path at a preset position of the sample placing table 200, and the control system illuminates at least one single color of the same color in the plurality of monochromatic fluorescent excitation light sources 101 according to experimental requirements.
  • the color fluorescent excitation light source is a target light source;
  • the sample placing table 200 is disposed at a position where the monochromatic fluorescent excitation light emitted by the plurality of monochromatic fluorescent excitation light sources 101 meets, and is used for placing the sample plate to be tested, and the preset detection area of the sample plate to be tested is disposed on the sample placing table 200. Preset position
  • the objective lens 300 is disposed on the side of the sample placement table 200 facing away from the light source device 100, and collects fluorescence excited by the illumination of the monochromatic fluorescent excitation light emitted by the target light source in the preset detection area, and enlarges the preset detection area to a preset. multiple;
  • the emission filter module 400 is disposed on the side of the objective lens 300 facing away from the sample placing table 200 for filtering the fluorescence excited by the particles in the preset detection area;
  • the image acquisition device 500 is disposed on the side of the emission filter module 400 facing away from the objective lens 300 for acquiring a fluorescent image of the preset detection area.
  • the image acquisition device may be an eyepiece, a camera, or the like, which is not specifically limited herein.
  • the plurality of monochromatic fluorescent excitation light sources provided by the embodiments of the present application are disposed around the central axis of the imaging optical path formed by the objective lens and the image acquiring device, and the monochromatic fluorescent excitation light emitted by each of the monochromatic fluorescent excitation light sources is The axes of the imaging light path intersect at a preset position on the sample placement stage. That is, the monochromatic fluorescent excitation light emitted by the monochromatic fluorescent excitation source is obliquely incident on the preset position of the sample placing table, that is, the preset fluorescence of the monochromatic fluorescent excitation light emitted by the monochromatic fluorescent excitation source is incident on the sample plate to be tested.
  • the detection area is such that a small amount of fluorescent excitation light is transmitted into the objective lens after passing through the sample plate to be tested, thereby reducing the influence of the monochromatic fluorescence excitation light in subsequent imaging, so that the acquired fluorescence image is more accurate.
  • the multi-fluorescence channel synchronous microscopic imaging device provided by the embodiment of the present application further includes:
  • the bright field light source 102 the full-band white light emitted by the bright field light source 102 faces the sample placement stage 200 and coincides with the axis X of the imaging optical path.
  • the monochromatic fluorescent excitation light source provided by the embodiment of the present application may be a single-color LED (Light Emitting Diode) fluorescent excitation light source.
  • the light source device provided by the embodiment of the present application further includes: being disposed in the illumination direction of the monochromatic LED fluorescent excitation source, and disposed on Monochrome LED An excitation filter between the fluorescent excitation source and the sample placement stage.
  • the light outside the wavelength band of the monochromatic fluorescent excitation light is absorbed by the excitation filter, and only the monochromatic fluorescent excitation light is used, thereby improving the singularity of the monochromatic fluorescent excitation light emitted by the monochromatic LED fluorescent excitation light source.
  • the monochromatic fluorescent excitation light source may further include: a white light excitation light source;
  • the white light excitation source emits full-band white light, and then the excitation filter absorbs light of an unnecessary wavelength band, and the monochromatic fluorescence is excited by a predetermined wavelength band.
  • the white light excitation light source provided by the embodiment of the present application may be a mercury lamp, a xenon lamp, or the like, which is not specifically limited herein.
  • the monochromatic fluorescent excitation light source is preferably a monochromatic LED fluorescent excitation light source.
  • the control system provided by the embodiment of the present application may be a single-chip microcomputer or the like, and the present application does not specifically limit the present application.
  • the emission filter module provided in the embodiment of the present application filters the fluorescence of the particles in the preset detection area after being excited by the monochromatic fluorescence excitation light.
  • the emission filter module is an emission filter turntable, and a plurality of emission filter regions are disposed around the emission filter turntable, and a pass band of each of the emission filter regions is a single color of the particles in the sample plate to be tested.
  • the target light source when the target light source is a blue fluorescent excitation light source, the target light source emits blue fluorescent excitation light, and after irradiating to the preset detection area of the sample to be tested, the particles in the preset detection area are excited to emit green fluorescence, and then doped.
  • the emitted light with green fluorescence and blue stray light passes through the objective lens and enters the emission filter turntable; at this time, the emission filter area corresponding to the green fluorescence is aligned with the objective lens, so that the stray light in the emitted light is Absorbs only through green fluorescence.
  • the emission filter module further includes a transmission region, that is, when the target light source is a bright field light source, the emission filter is activated at this time.
  • the light carousel will pass through the area corresponding to the objective lens.
  • FIG. 4 is a schematic structural diagram of another fluorescent microscopic imaging device according to an embodiment of the present application, wherein the light source device 100 further includes:
  • the concentrating module 600 is disposed in the irradiation direction of the monochromatic fluorescent excitation light source 101 and disposed between the monochromatic fluorescent excitation light source 101 and the sample placing table 200.
  • the concentrating module may be a single concentrating lens or a concentrating lens group composed of a plurality of lenses, which is not specifically limited in itself.
  • the fluorescence microscopic imaging device provided by the embodiment of the present application can fix all the structures thereof through a fixing frame.
  • the embodiment of the present application provides a fluorescence microscopic imaging method and apparatus, comprising: illuminating at least one monochromatic fluorescent excitation light source of the same color as a target light source in a plurality of monochromatic fluorescent excitation light sources according to an experimental requirement, wherein each The monochromatic fluorescent excitation light emitted by the monochromatic fluorescent excitation source is obliquely incident on the predetermined detection area of the sample plate to be tested; and the preset detection is collected on the side of the sample plate to be tested facing away from the target light source
  • the particles in the region are excited by the illumination of the monochromatic fluorescent excitation light emitted by the target light source, and the predetermined detection region is enlarged to a preset multiple; the fluorescence excited by the particles in the predetermined detection region Performing a filtering process; acquiring a fluorescent image of the preset detection area.
  • the technical solution provided by the embodiment of the present invention does not need to provide a dichroic mirror to split the monochromatic fluorescence excitation light and the fluorescence, so that the fluorescence microscopic device has a simple structure and low cost, and the fluorescence is prevented from passing through the dichroic phase.
  • the loss of light energy occurs in the mirror, which makes the final acquired fluorescence image brighter and clearer.
  • the monochromatic fluorescent excitation light is transmitted into the objective lens through the sample plate to be tested, thereby reducing the influence of the monochromatic fluorescent excitation light in subsequent imaging.
  • the acquired fluorescence image is more accurate.

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Abstract

一种荧光显微成像方法和装置，包括：根据实验要求在多个单色荧光激发光源(101)中点亮至少一个相同颜色的单色荧光激发光源(101)为目标光源，其中，每个单色荧光激发光源(101)所发出的单色荧光激发光均倾斜射入待测样品板的预设检测区域；在待测样品板背离目标光源一侧，收集预设检测区域内的颗粒受目标光源发出的单色荧光激发光的照射而激发的荧光，并将预设检测区域放大至预设倍数；对预设检测区域内的颗粒激发的荧光进行滤光处理；获取预设检测区域的荧光图像。无需设置二色相镜，使得荧光显微装置结构简单且成本低，避免了光能损耗的情况。由于单色荧光激发光倾斜照射至待测样品板，使得单色荧光激发光透过待测样品板后少量射入物镜中。

Description

一种荧光显微成像方法和装置
本申请要求于2015年07月01日提交中国专利局、申请号为201510376530.0、发明名称为“集成式荧光激发光源装置”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
本申请还要求于2015年07月28日提交中国专利局、申请号为201510452593.X、发明名称为“一种荧光显微成像方法和装置”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及荧光显微成像技术领域，更为具体的说，涉及一种荧光显微成像方法和装置。
背景技术
参考图1所示，为现有的一种荧光显微成像装置，其包括：一荧光激发光源1、与单色荧光激发光源1的照射方向呈45度角设置的二色相镜2、物镜3、样品放置台4、发射滤光片5和相机6。其中，单色荧光激发光源1发出激发光，经过二色相镜2反射至物镜3后，照射至样品放置台4的待测样品板上，而待测样品板内颗粒受激发发出荧光，通过二色相镜2和发射滤光片5后，进入相机6成像。现有的荧光显微成像装置需要二色相镜分光，使得其结构复杂，且成本高昂。
发明内容
有鉴于此，本发明提供了一种荧光显微成像方法和装置，无需二色相镜分光，使得装置不仅结构简单且成本低；而且减少了进入物镜的单色荧光激发光，使得获取的荧光图像更加精确。
为实现上述目的，本发明提供的技术方案如下：
一种荧光显微成像方法，其特征在于，将待测样品板放置完毕后，包括：
根据实验要求在多个单色荧光激发光源中点亮至少一个相同颜色的单色 荧光激发光源为目标光源，其中，每个单色荧光激发光源所发出的单色荧光激发光均倾斜射入所述待测样品板的预设检测区域；
在所述待测样品板背离所述目标光源一侧，收集所述预设检测区域内的颗粒受所述目标光源发出的单色荧光激发光的照射而激发的荧光，并将所述预设检测区域放大至预设倍数；
对所述预设检测区域内的颗粒激发的荧光进行滤光处理；
获取所述预设检测区域的荧光图像。
优选的，在点亮所述目标光源后，且在所述目标光源所发出的单色荧光激发光未射入所述待测样品板之前，还包括：
对所述目标光源中每个单色荧光激发光源所发出的单色荧光激发光进行聚光处理。
相应的，本发明还提供了一种荧光显微成像装置，包括：
光源装置、样品放置台、物镜、发射滤光模组和图像获取装置；
所述光源装置包括：多个单色荧光激发光源和与所述多个单色荧光激发光源电连接的控制系统，其中，所述多个单色荧光激发光源环绕所述物镜和图像获取装置所构成的成像光路中轴设置，且每个单色荧光激发光源所发出的单色荧光激发光与所述成像光路中轴相交于所述样品放置台的预设位置，所述控制系统根据实验要求在所述多个单色荧光激发光源中点亮至少一个相同颜色的单色荧光激发光源为目标光源；
所述样品放置台设置于所述多个单色荧光激发光源所发出的单色荧光激发光交汇位置，用于放置待测样品板，且所述待测样品板的预设检测区域设置于所述样品放置台的预设位置；
所述物镜设置于所述样品放置台背离所述光源装置一侧；
所述发射滤光模组设置于所述物镜背离所述样品放置台一侧；
所述图像获取装置设置于所述发射滤光模组背离所述物镜一侧。
优选的，所述光源装置还包括：
明场光源，所述明场光源所发出的全波段白光朝向所述样品放置台、且与所述成像光路中轴重合。
优选的，所述单色荧光激发光源为单色LED荧光激发光源。
优选的，所述光源装置还包括：
设置于所述单色LED荧光激发光源的照射方向上、且设置于所述单色LED荧光激发光源与所述样品放置台之间的激发滤光片。
优选的，所述单色荧光激发光源包括：
白光激发光源；
以及，设置于所述白光激发光源的照射方向上、且设置于所述白光激发光源与所述样品放置台之间的激发滤光片。
优选的，所述发射滤光模组为发射滤光转盘，且所述发射滤光转盘四周设置有多个发射滤光区域，每一发射滤光区域的通过波段为所述待测样品板内颗粒受一颜色的单色荧光激发光源的激发而发出的荧光的波段。
优选的，所述光源装置还包括：
设置于所述单色荧光激发光源的照射方向上、且设置于所述单色荧光激发光源与所述样品放置台之间的聚光模组。
优选的，所述聚光模组为聚光透镜或多个镜片组成的聚光镜片组。
优选的，所述图像获取装置为目镜或相机。
相较于现有技术，本发明提供的技术方案至少具有以下优点：
本发明提供了一种荧光显微成像方法和装置，包括：根据实验要求在多个单色荧光激发光源中点亮至少一个相同颜色的单色荧光激发光源为目标光源，其中，每个单色荧光激发光源所发出的单色荧光激发光均倾斜射入所述待测样品板的预设检测区域；在所述待测样品板背离所述目标光源一侧，收集所述预设检测区域内的颗粒受所述目标光源发出的单色荧光激发光的照射而激发的荧光，并将所述预设检测区域放大至预设倍数；对所述预设检测区域内的颗粒激发的荧光进行滤光处理；获取所述预设检测区域的荧光图像。
由上述内容可知，本发明提供的技术方案，无需设置二色相镜对单色荧光激发光和荧光进行分光，使得荧光显微装置结构简单且成本低，而且避免了荧光在透过二色相镜时出现光能损耗的情况，进而使得最终获取的荧光图像更加明亮清晰。另外，由于单色荧光激发光源的倾斜照射至待测样品板，使得单色荧光激发光透过待测样品板后少量射入物镜中，减少了后续成像时单色荧光激发光的影响，使得获取的荧光图像更加精确。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为现有的一种荧光显微成像装置的结构示意图；
图2为本申请实施例提供的一种荧光显微成像方法的流程图；
图3为本申请实施例提供的一种荧光显微成像装置的结构示意图；
图4为本申请实施例提供的另一种荧光显微成像装置的结构示意图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
正如背景技术所述，现有的荧光显微成像装置需要二色相镜分光，使得其结构复杂，且成本高昂。
基于此，本申请实施例提供了一种荧光显微成像方法和装置，无需二色相镜分光，使得装置不仅结构简单且成本低；而且减少了进入物镜的单色荧光激发光，使得获取的荧光图像更加精确。
具体的，参考图2所示，为本申请实施例提供的一种荧光显微成像方法的流程图，其中，将待测样品板放置完毕后，包括：
S1、根据实验要求在多个单色荧光激发光源中点亮至少一个相同颜色的单色荧光激发光源为目标光源，其中，每个单色荧光激发光源所发出的单色荧光激发光均倾斜射入待测样品板的预设检测区域；
S2、在待测样品板背离目标光源一侧，收集预设检测区域内的颗粒受目标光源发出的单色荧光激发光的照射而激发的荧光，并将预设检测区域放大至预设倍数；
S3、对预设检测区域内的颗粒激发的荧光进行滤光处理；
S4、获取预设检测区域的荧光图像。
其中，本申请实施例提供的多个单色荧光激发光源中，可以包括有不同颜色(或波段)的单色荧光激发光源，或者包括有全部相同颜色(或波段)的单色荧光激发光源。当多个单色荧光激发光源为不同颜色的单色荧光激发光源时，分为多种情况，即，本申请实施例提供的所有单色荧光激发光源的颜色均不相同；或者，所有单色荧光激发光源分为多组，每组中单色荧光激发光源的颜色相同，且各组之间的颜色不同，以此通过点亮多个相同颜色的单色荧光激发光源，提高每个颜色的荧光激发光的亮度，对此本申请不作具体限制。
由上述内容可知，本申请实施例提供的技术方案，无需设置二色相镜对单色荧光激发光和荧光进行分光，可以使得荧光显微装置结构简单且成本低，而且避免了荧光在透过二色相镜时出现光能损耗的情况，进而使得最终获取的荧光图像更加明亮清晰。另外，由于单色荧光激发光源的倾斜照射至待测样品板，使得单色荧光激发光透过待测样品板后少量射入物镜中，减少了后续成像时单色荧光激发光的影响，使得获取的荧光图像更加精确。
进一步的，为了提高单色荧光激发光源发出的荧光激发光的亮度，以提高目标光源所发出的单色荧光激发光的亮度，在点亮目标光源后，且在目标光源所发出的单色荧光激发光未射入待测样品板之前，还包括：
对目标光源中每个单色荧光激发光源所发出的单色荧光激发光进行聚光处理。
相应的，参考图3所述，为本申请实施例提供的一种荧光显微成像装置的结构示意图，其中，荧光显微成像装置包括：
光源装置100、样品放置台200、物镜300、发射滤光模组400和图像获取装置500；
光源装置100包括：多个单色荧光激发光源101和与多个单色荧光激发光源101电连接的控制系统(未画出)，其中，多个单色荧光激发光源101环绕物镜300和图像获取装置500所构成的成像光路中轴X设置，且每个单色荧光激发光 源101所发出的单色荧光激发光与成像光路中轴X相交于样品放置台200的预设位置，控制系统根据实验要求在多个单色荧光激发光源101中点亮至少一个相同颜色的单色荧光激发光源为目标光源；
样品放置台200设置于多个单色荧光激发光源101所发出的单色荧光激发光交汇位置，用于放置待测样品板，且待测样品板的预设检测区域设置于样品放置台200的预设位置；
物镜300设置于样品放置台200背离光源装置100一侧，收集预设检测区域内的颗粒受目标光源发出的单色荧光激发光的照射而激发的荧光，并将预设检测区域放大至预设倍数；
发射滤光模组400设置于物镜300背离样品放置台200一侧，用于对预设检测区域内的颗粒激发的荧光进行滤光处理；
图像获取装置500设置于发射滤光模组400背离物镜300一侧，用于获取预设检测区域的荧光图像，其中，图像获取装置可以为目镜、相机等，对此本申请不作具体限制。
由上述内容可知，本申请实施例提供的多个单色荧光激发光源环绕物镜和图像获取装置所构成的成像光路中轴设置，且每个单色荧光激发光源所发出的单色荧光激发光与成像光路中轴相交于样品放置台的预设位置。即，单色荧光激发光源发出的单色荧光激发光倾斜射入样品放置台的预设位置，亦即，单色荧光激发光源发出的单色荧光激发光倾斜射入待测样品板的预设检测区域，使得单色荧光激发光透过待测样品板后少量射入物镜中，减少了后续成像时单色荧光激发光的影响，使得获取的荧光图像更加精确。
进一步的，参考图3所示，本申请实施例提供的多荧光通道同步显微成像装置，其光源装置100还包括：
明场光源102，明场光源102所发出的全波段白光朝向样品放置台200、且与成像光路中轴X重合。
具体的，本申请实施例提供的单色荧光激发光源可以为单色LED(Light Emitting Diode，发光二极管)荧光激发光源。进一步的，为了提高单色LED荧光激发光源所发出的单色荧光激发光的单一性，本申请实施例提供的光源装置还包括：设置于单色LED荧光激发光源的照射方向上、且设置于单色LED 荧光激发光源与样品放置台之间的激发滤光片。其中，通过激发滤光片将除单色荧光激发光的波段外的光吸收，仅仅通过单色荧光激发光，进而提高单色LED荧光激发光源所发出的单色荧光激发光的单一性。
或者，
在本申请实施例中，单色荧光激发光源还可以包括：白光激发光源；
以及，设置于白光激发光源的照射方向上、且设置于白光激发光源与样品放置台之间的激发滤光片。其中，白光激发光源发出全波段的白光，而后通过激发滤光片吸收不需要的波段的光，而通过预设波段的单色荧光激发光。需要说明的是，本申请实施例提供的白光激发光源可以为汞灯、氙灯等，对此本申请不作具体限制。
需要说明的是，由于LED具有响应时间短、耗能低、成本低、使用寿命长、体积小等诸多优点，因此，在本申请实施例中单色荧光激发光源优选为单色LED荧光激发光源。此外，本申请实施例提供的控制系统可以为单片机等，对此本申请不作具体限制。
本申请实施例提供的发射滤光模组，对预设检测区域内颗粒受单色荧光激发光激发后的荧光进行滤光处理。其中，发射滤光模组为发射滤光转盘，且发射滤光转盘四周设置有多个发射滤光区域，每一发射滤光区域的通过波段为待测样品板内颗粒受一颜色的单色荧光激发光源的激发而发出的荧光的波段。
举例说明，当目标光源为蓝色荧光激发光源时，目标光源发出蓝色荧光激发光，照射至待测样品的预设检测区域后，激发预设检测区域内的颗粒发出绿色荧光，而后掺杂有绿色荧光和蓝色杂散光的发射光通过物镜后，进入发射滤光转盘；此时，发射滤光转盘中对应透过绿色荧光的发射滤光区域对准物镜，使得发射光中杂散光被吸收，而只通过绿色荧光。
此外，当光源装置还包括有明场光源时，由于明场光源发出全波段白光，因此，发射滤光模组还包括有透过区，即，当目标光源为明场光源时，此时发射滤光转盘将透过区域对应物镜。
进一步的，为了提高单色荧光激发光源发出的单色荧光激发光的亮度，参 考图4所示，为本申请实施例提供的另一种荧光显微成像装置的结构示意图，其中，光源装置100还包括：
设置于所述单色荧光激发光源101的照射方向上、且设置于所述单色荧光激发光源101与所述样品放置台200之间的聚光模组600。其中，聚光模组可以为单个的聚光透镜，或者为多个镜片组成的聚光镜片组，对此本身不作具体限制。
为了将光源装置、样品放置台、物镜、发射滤光模组、图像获取装置等结构固定，本申请实施例提供的荧光显微成像装置可以通过一固定架将其所有结构固定。
本申请实施例提供了一种荧光显微成像方法和装置，包括：根据实验要求在多个单色荧光激发光源中点亮至少一个相同颜色的单色荧光激发光源为目标光源，其中，每个单色荧光激发光源所发出的单色荧光激发光均倾斜射入所述待测样品板的预设检测区域；在所述待测样品板背离所述目标光源一侧，收集所述预设检测区域内的颗粒受所述目标光源发出的单色荧光激发光的照射而激发的荧光，并将所述预设检测区域放大至预设倍数；对所述预设检测区域内的颗粒激发的荧光进行滤光处理；获取所述预设检测区域的荧光图像。
由上述内容可知，本申请实施例提供的技术方案，无需设置二色相镜对单色荧光激发光和荧光进行分光，使得荧光显微装置结构简单且成本低，而且避免了荧光在透过二色相镜时出现光能损耗的情况，进而使得最终获取的荧光图像更加明亮清晰。另外，由于单色荧光激发光源的倾斜照射至待测样品板，使得单色荧光激发光透过待测样品板后少量射入物镜中，减少了后续成像时单色荧光激发光的影响，使得获取的荧光图像更加精确。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (11)

  1. 一种荧光显微成像方法，其特征在于，将待测样品板放置完毕后，包括：
    根据实验要求在多个单色荧光激发光源中点亮至少一个相同颜色的单色荧光激发光源为目标光源，其中，每个单色荧光激发光源所发出的单色荧光激发光均倾斜射入所述待测样品板的预设检测区域；
    在所述待测样品板背离所述目标光源一侧，收集所述预设检测区域内的颗粒受所述目标光源发出的单色荧光激发光的照射而激发的荧光，并将所述预设检测区域放大至预设倍数；
    对所述预设检测区域内的颗粒激发的荧光进行滤光处理；
    获取所述预设检测区域的荧光图像。
  2. 根据权利要求1所述的荧光显微成像方法，其特征在于，在点亮所述目标光源后，且在所述目标光源所发出的单色荧光激发光未射入所述待测样品板之前，还包括：
    对所述目标光源中每个单色荧光激发光源所发出的单色荧光激发光进行聚光处理。
  3. 一种荧光显微成像装置，其特征在于，包括：
    光源装置、样品放置台、物镜、发射滤光模组和图像获取装置；
    所述光源装置包括：多个单色荧光激发光源和与所述多个单色荧光激发光源电连接的控制系统，其中，所述多个单色荧光激发光源环绕所述物镜和图像获取装置所构成的成像光路中轴设置，且每个单色荧光激发光源所发出的单色荧光激发光与所述成像光路中轴相交于所述样品放置台的预设位置，所述控制系统根据实验要求在所述多个单色荧光激发光源中点亮至少一个相同颜色的单色荧光激发光源为目标光源；
    所述样品放置台设置于所述多个单色荧光激发光源所发出的单色荧光激发光交汇位置，用于放置待测样品板，且所述待测样品板的预设检测区域设置于所述样品放置台的预设位置；
    所述物镜设置于所述样品放置台背离所述光源装置一侧；
    所述发射滤光模组设置于所述物镜背离所述样品放置台一侧；
    所述图像获取装置设置于所述发射滤光模组背离所述物镜一侧。
  4. 根据权利要求3所述的多荧光通道同步显微成像装置，其特征在于，所述光源装置还包括：
    明场光源，所述明场光源所发出的全波段白光朝向所述样品放置台、且与所述成像光路中轴重合。
  5. 根据权利要求3所述的荧光显微成像装置，其特征在于，所述单色荧光激发光源为单色LED荧光激发光源。
  6. 根据权利要求5所述的荧光显微成像装置，其特征在于，所述光源装置还包括：
    设置于所述单色LED荧光激发光源的照射方向上、且设置于所述单色LED荧光激发光源与所述样品放置台之间的激发滤光片。
  7. 根据权利要求3所述的荧光显微成像装置，其特征在于，所述单色荧光激发光源包括：
    白光激发光源；
    以及，设置于所述白光激发光源的照射方向上、且设置于所述白光激发光源与所述样品放置台之间的激发滤光片。
  8. 根据权利要求3所述的荧光显微成像装置，其特征在于，所述发射滤光模组为发射滤光转盘，且所述发射滤光转盘四周设置有多个发射滤光区域，每一发射滤光区域的通过波段为所述待测样品板内颗粒受一颜色的单色荧光激发光源的激发而发出的荧光的波段。
  9. 根据权利要求3所述的荧光显微成像装置，其特征在于，所述光源装置还包括：
    设置于所述单色荧光激发光源的照射方向上、且设置于所述单色荧光激发光源与所述样品放置台之间的聚光模组。
  10. 根据权利要求9所述的荧光显微成像装置，其特征在于，所述聚光模组为聚光透镜或多个镜片组成的聚光镜片组。
  11. 根据权利要求3所述的荧光显微成像装置，其特征在于，所述图像获取装置为目镜或相机。
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