WO2017000700A1 - 一种多荧光通道同步显微成像方法及装置 - Google Patents

一种多荧光通道同步显微成像方法及装置 Download PDF

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WO2017000700A1
WO2017000700A1 PCT/CN2016/082767 CN2016082767W WO2017000700A1 WO 2017000700 A1 WO2017000700 A1 WO 2017000700A1 CN 2016082767 W CN2016082767 W CN 2016082767W WO 2017000700 A1 WO2017000700 A1 WO 2017000700A1
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excitation light
light source
fluorescence
monochromatic
fluorescent excitation
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PCT/CN2016/082767
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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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Publication of WO2017000700A1 publication Critical patent/WO2017000700A1/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 invention relates to the field of fluorescence microscopic imaging technology, and more particularly to a multi-fluorescence channel synchronous microscopic imaging method and device.
  • a conventional fluorescence microscopic imaging apparatus comprising: a monochromatic fluorescence excitation light source 1 and a dichroic phase mirror 2 disposed at an angle of 45 degrees to the illumination direction of the monochromatic fluorescence excitation light source 1
  • the monochromatic fluorescent excitation light source 1 emits excitation light, 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.
  • the existing fluorescence microscopic imaging device can only realize the detection of one fluorescent channel at a time.
  • the detection of multiple fluorescent channels is required, only after one fluorescent channel detection is completed, switching to another fluorescent channel for detection, and then more The detection results of the fluorescent channels were integrated.
  • Many fluorescent dyes today have rapid quenching properties.
  • the quenching effect of fluorescence is difficult to measure due to the difference in the time during which the sample is repeatedly irradiated or irradiated. Therefore, if the multi-fluorescence channel synchronization detection can be realized during the detection of the multi-fluorescence channel, the influence of the fluorescence quenching property due to the step detection can be solved.
  • the present invention provides a multi-fluorescence channel synchronous microscopic imaging method and device, which realizes simultaneous microscopic imaging of multiple fluorescent channels, avoids the influence of fluorescence quenching caused by step detection, and is convenient for experiments; Moreover, the monochromatic fluorescent excitation light entering the objective lens is reduced, so that the acquired fluorescent image is more accurate; in addition, the device has a simple structure and low cost.
  • a multi-fluorescence channel synchronous microscopic imaging method after the sample plate to be tested is placed, includes:
  • a combination of a plurality of monochromatic fluorescent excitation light sources of different colors simultaneously illuminated in a plurality of monochromatic fluorescence excitation light sources is a target light source, wherein each monochromatic fluorescent excitation light source emits a monochromatic fluorescent excitation light a predetermined detection area that is injected into 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 multi-fluorescence channel synchronous microscopic imaging device, comprising:
  • a light source device a sample placement table, an objective lens, a multi-band filter, 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.
  • 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 multi-band filter is disposed on a side of the objective lens facing away from the sample placing table;
  • the image acquisition device is disposed on a side of the multi-band filter 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 excitation light source and the sample placement stage.
  • 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 invention provides a multi-fluorescence channel synchronous microscopic imaging method and device, comprising: simultaneously lighting a combination of a plurality of monochromatic fluorescent excitation light sources of different colors in a plurality of monochromatic fluorescent excitation light sources as a target light source according to an experimental requirement, wherein, the monochromatic fluorescent excitation light emitted by each of the monochromatic fluorescent excitation light sources is obliquely incident into a predetermined detection area of the sample plate to be tested; and the collection side of the sample plate to be tested faces away from the target light source Fluctuating the particles in the predetermined detection area by the illumination of the multi-color fluorescent excitation light emitted by the target light source, and amplifying the preset detection area to a preset multiple; for the preset detection area The fluorescence excited by the particles is subjected to multi-band filter processing; and the fluorescence image of the preset detection area is acquired.
  • the technical solution provided by the present invention is in a plurality of monochromes according to experimental requirements.
  • a plurality of monochromatic fluorescent excitation light sources of different colors are simultaneously illuminated in the fluorescence excitation light source, and the preset detection regions of the sample plate to be tested are synchronously excited by the monochromatic fluorescence excitation light sources of different colors, and are subjected to fluorescence collection and preset detection regions.
  • the fluorescence is subjected to multi-band filtering to remove stray light, and finally a fluorescence image of the preset detection area is obtained.
  • the monochromatic fluorescent excitation light emitted by all the monochromatic fluorescent excitation light sources is obliquely incident on the preset detection area of the sample plate to be tested, thereby greatly reducing the monochromatic fluorescent excitation light entering the objective lens, so that Subsequent acquired fluorescence images are more accurate.
  • the multi-fluorescence channel synchronous microscopic imaging device provided by the present invention does not require a two-color phase mirror, the structure of the device is simpler and cheaper, and the loss of light when passing through the dichroic mirror is avoided, so that the final The acquired fluorescence image is clearer.
  • FIG. 1 is a schematic structural view of a conventional fluorescence microscopic imaging device
  • FIG. 2 is a flowchart of a method for synchronous microscopic imaging of multiple fluorescent channels according to an embodiment of the present application
  • FIG. 3 is a schematic structural diagram of a multi-fluorescence channel synchronous microscopic imaging apparatus according to an embodiment of the present application
  • FIG. 4 is a schematic structural diagram of another multi-fluorescence channel synchronous microscopic imaging apparatus according to an embodiment of the present application.
  • the existing fluorescence microscopic imaging device can only realize the detection of one fluorescent channel at a time.
  • only one fluorescent channel can be switched to another fluorescent channel after the detection of one fluorescent channel is completed. The detection is performed, and then the detection results of the plurality of fluorescent channels are integrated.
  • Many fluorescent dyes today have rapid quenching properties.
  • the quenching effect of fluorescence is difficult to measure due to the difference in the time during which the sample is repeatedly irradiated or irradiated. Therefore, if the multi-fluorescence channel synchronization detection can be realized during the detection of the multi-fluorescence channel, the influence of the fluorescence quenching property due to the step detection can be solved.
  • the embodiments of the present application provide a multi-fluorescence channel synchronous microscopic imaging method and device, which realizes synchronous microscopic imaging of multiple fluorescent channels, avoids the influence of fluorescence quenching caused by step detection, and is convenient for experiments. Moreover, the monochromatic fluorescent excitation light entering the objective lens is reduced, so that the acquired fluorescent image is more accurate; in addition, the device has a simple structure and low cost.
  • FIG. 2 a flowchart of a multi-fluorescence channel synchronous 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 side of the sample plate to be tested is away from the target light source, and the fluorescence of the particles in the preset detection area is excited by the multi-color fluorescent excitation light emitted by the target light source, and the preset detection area is enlarged to a preset multiple;
  • the plurality of monochromatic fluorescent excitation light sources provided by the embodiments of the present application include a monochromatic fluorescent excitation light source with different colors (or bands).
  • the colors of all the monochromatic fluorescent excitation sources provided by the embodiments of the present application are different; or all the monochromatic fluorescent excitation sources are divided into groups, and the colors of the monochromatic fluorescent excitation sources in each group are the same, and between the groups
  • the color is different to increase the brightness of the fluorescent excitation light of each color, and the present application is not specifically limited.
  • a plurality of monochromatic fluorescent excitation light sources of different colors are simultaneously illuminated in a plurality of monochromatic fluorescent excitation light sources according to an experimental requirement, and the preset detection of the sample plate to be tested is performed by a monochromatic fluorescent excitation light source of different colors.
  • the area is synchronously excited and amplified by fluorescence collection and preset detection area After that, the fluorescence is subjected to multi-band filter processing to remove stray light, and finally a fluorescence image of the preset detection area is obtained.
  • the simultaneous microscopic imaging of the multi-fluorescence channel is realized, and the influence of fluorescence quenching caused by the step detection is avoided, which is convenient for the experiment.
  • the monochromatic fluorescence excitation light emitted by all the monochromatic fluorescence excitation sources is obliquely incident on the preset detection area of the sample plate to be tested, which greatly reduces the monochromatic fluorescence excitation light entering the objective lens, so that the subsequent acquired fluorescence image is more accurate.
  • the embodiment of the present application provides a technical solution that does not require a dichroic mirror to perform splitting, thereby making the corresponding device structure simpler and cheaper, and avoiding loss of light when passing through the dichroic mirror, the finally obtained is obtained. Fluorescent images are sharper.
  • the fluorescence color excited by each particle can be analyzed.
  • the wavelength band between the monochromatic fluorescent excitation light sources of different colors in the target light source is non-overlapping, therefore, all the fluorescent bands and any one of the monochromatic fluorescent excitation lights are filtered in the subsequent filtering of the fluorescence excited by the particles.
  • the band is also non-overlapping, avoiding the influence of the monochromatic fluorescence excitation light of the same band on the fluorescence filter, that is, the band transmitted by the multi-band filter and the band of the monochromatic fluorescence excitation source are not intersected. Stacked.
  • the purple excitation source (band range 385nm ⁇ 10, including the endpoint value), the blue excitation source (band range 480nm ⁇ 10, including the endpoint value), and the green excitation source (band range) are illuminated according to the experimental requirements.
  • 550 nm ⁇ 10, including the endpoint value is the target light source, that is, the bands between the monochromatic fluorescent excitation light sources of different colors in the target light source do not overlap;
  • the target light source emits purple excitation light, blue excitation light, and green excitation light, respectively.
  • the emitted fluorescence is blue (the wavelength band is 460 nm); if the particles in the predetermined detection area are preset Only excited by green excitation light, the emitted fluorescence is red (band is greater than 600nm); if the particles in the preset detection area are only excited by blue excitation light, the emitted fluorescence is green (band 520nm); If the particles in the preset detection area are excited by the blue excitation light and the purple excitation light, the emitted fluorescence is cyan; if the particles in the preset detection area are excited by the purple excitation light and the green excitation light, the emitted fluorescence It is purple; if the particles in the preset detection area are excited by blue excitation light and green excitation light, the emitted fluorescence It is purple; if the particles in the preset detection area are excited by blue excitation light and green excitation light, the emitted fluor
  • the multi-band filter is used to transmit blue light with a wavelength range of 460 nm ⁇ 10 (including the endpoint value), green light of 520 nm ⁇ 10 (including the endpoint value), and not less than A three-band filter of red light in the 600 nm band.
  • the multi-band filter can not only transmit blue fluorescence, green fluorescence and red fluorescence excited by the particles; but also can transmit blue fluorescence, green fluorescence and red fluorescence when the particles are excited by multi-color excitation light.
  • Fluorescence of any combination for example, excited by blue excitation light and purple excitation light, emits cyan fluorescence combined with blue fluorescence and green fluorescence, and multi-band filter transmits blue fluorescence and green fluorescence through cyan fluorescence.
  • the combination Therefore, the wavelength band of the multi-band filter and the monochromatic fluorescence excitation source of different colors in the target light source are set to be non-overlapping, thereby filtering the fluorescence emitted by the particles to remove stray light.
  • multi-fluorescence channel simultaneous microscopic imaging such as two-channel synchronous microscopic imaging and three-channel synchronous microscopic imaging is realized by simultaneously illuminating a plurality of monochromatic fluorescent excitation light sources of different colors.
  • the brightness of the multi-color fluorescent excitation light emitted by the target light source is increased, and the multi-color 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 multi-fluorescence channel synchronous microscopic imaging apparatus provided by an embodiment of the present application, wherein the multi-fluorescence channel synchronous microscopic imaging apparatus includes:
  • a light source device 100 a sample placement table 200, an objective lens 300, a multi-band filter 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 disposed in the axis X, and the monochromatic fluorescent excitation light emitted by each of the monochromatic fluorescent excitation light sources 101 intersects the central axis of the imaging optical path at a preset position of the sample placement table 200, and the control system is in accordance with experimental requirements.
  • the sample placing table 200 is disposed on a monochromatic fluorescent excitation emitted by a plurality of monochromatic fluorescent excitation light sources 101 a light intersection position for placing the sample plate to be tested, and a preset detection area of the sample plate to be tested is set at a preset position of the sample placement table 200;
  • the objective lens 300 is disposed on a side of the sample placing table 200 facing away from the light source device 100 for collecting fluorescence excited by the irradiation of the multi-color fluorescent excitation light emitted by the target light source in the preset detection area, and amplifying the preset detection area to Preset multiple;
  • the multi-band filter 400 is disposed on the side of the objective lens 300 facing away from the sample placing table 200 for performing multi-band filtering on the fluorescence excited by the particles in the preset detection area;
  • the image acquisition device 500 is disposed on the side of the multi-band filter 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 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 An excitation filter between the monochromatic LED fluorescence 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 excitation light source emits full-band white light, and then the excitation filter absorbs light of an unnecessary wavelength band, and the monochromatic fluorescence of the predetermined wavelength band excites the light.
  • 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.
  • a schematic diagram of a multi-fluorescence channel synchronous microscopic imaging device wherein the light source device further comprises:
  • the concentrating module 600 is disposed between the monochromatic fluorescent excitation light source 101 and the sample placing table 200 in the irradiation direction of the monochromatic fluorescence excitation light source 101.
  • 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 this embodiment.
  • the fluorescence microscopic imaging device provided by the embodiment of the present application can fix all the structures thereof through a fixing frame.
  • Embodiments of the present application provide a multi-fluorescence channel synchronous microscopic imaging method and apparatus, including: aiming at simultaneously combining a plurality of monochromatic fluorescent excitation light sources of different colors in a plurality of monochromatic fluorescent excitation light sources according to experimental requirements a light source, wherein the monochromatic fluorescent excitation light emitted by each of the monochromatic fluorescent excitation light sources is obliquely incident on a predetermined detection area of the sample plate to be tested; and the sample plate to be tested faces away from the target light source, Collecting fluorescence of the particles in the preset detection area excited by the multi-color fluorescent excitation light emitted by the target light source, and amplifying the preset detection area to a preset multiple; and the preset detection area The fluorescence excited by the particles is subjected to multi-band filter processing; a fluorescence image of the preset detection area is acquired.
  • the technical solution provided by the embodiments of the present application simultaneously illuminates a plurality of monochromatic fluorescent excitation light sources of different colors in a plurality of monochromatic fluorescence excitation light sources according to experimental requirements, and is excited by monochromatic fluorescence of different colors.
  • the light source is synchronously excited by the preset detection area of the sample plate to be tested, and after the fluorescence collection and the preset detection area are amplified, the multi-band filtering process is performed on the fluorescence to remove the stray light, and finally the fluorescence image of the preset detection area is obtained. .
  • the simultaneous microscopic imaging of the multi-fluorescence channel is realized, and the influence of fluorescence quenching caused by the step detection is avoided, which is convenient for the experiment.
  • the technical solution provided by the embodiment of the present application is a single issued by all the monochromatic fluorescent excitation light sources.
  • the color fluorescence excitation light is obliquely incident on the preset detection area of the sample plate to be tested, which greatly reduces the monochromatic fluorescence excitation light entering the objective lens, so that the subsequently acquired fluorescence image is more accurate.
  • the dichroic phase mirror is not needed, so that the device structure is simpler and cheaper, and the loss of light when passing through the dichroic mirror is avoided. Make the final acquired fluorescence image clearer.

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Abstract

一种多荧光通道同步显微成像方法和装置，通过根据实验要求在多个单色荧光激发光源(101)中同时点亮多个不同颜色的单色荧光激发光源(101)，以通过不同颜色的单色荧光激发光源(101)对待测样品板的预设检测区域进行同步激发，并经过荧光收集和预设检测区域放大后，对荧光进行多波段滤光处理，以将杂散光去除，最终获取预设检测区域的荧光图像。实现了多荧光通道同步显微成像，避免了因分步检测而导致的荧光淬灭性的影响。所有单色荧光激发光源(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，大大减少了进入物镜的单色荧光激发光。由于该方案无需二色相镜，使得装置结构更加简单且成本低廉，避免了光损耗的情况。

Description

一种多荧光通道同步显微成像方法及装置
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。
本申请要求于2015年07月28日提交中国专利局、申请号为201510452279.1、发明名称为“一种多荧光通道同步显微成像方法及装置”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，涉及一种多荧光通道同步显微成像方法及装置。
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：一单色荧光激发光源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一次只能实现一个荧光通道的检测，当需要多个荧光通道的检测时，只能在一个荧光通道检测完成后，切换至另一个荧光通道进行检测，而后对多个荧光通道的检测结果进行整合。现今很多荧光染料具有快速的淬灭性，在进行多个荧光通道分步检测时，由于样品被反复照射或照射的时间有差异，荧光的淬灭性影响很难测定。因此，若在多荧光通道的检测过程中，能够实现多荧光通道同步检测，即能解决因分步检测而导致的荧光淬灭性的影响。
发明内容
有鉴于此，本发明提供了一种多荧光通道同步显微成像方法和装置，实现了多荧光通道同步显微成像，避免了因分步检测而导致的荧光淬灭性的影响，便于实验；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；此外，该装置结构简单且成本低。
为实现上述目的，本发明提供的技术方案如下：
一种多荧光通道同步显微成像方法，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同时点亮多个不同颜色的单色荧光激发光源的组合为目标光源，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多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多波段滤光处理；
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多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多荧光通道同步显微成像装置，包括：
光源装置、样品放置台、物镜、多波段滤光片和图像获取装置；
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***，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***根据实验要求在多个单色荧光激发光源中同时点亮多个不同颜色的单色荧光激发光源的组合为目标光源；
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；
所述多波段滤光片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多波段滤光片背离所述物镜一侧。
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、且设置于所述白色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：
本发明提供了一种多荧光通道同步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同时点亮多个不同颜色的单色荧光激发光源的组合为目标光源，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多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多波段滤光处理；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，通过根据实验要求在多个单色 荧光激发光源中同时点亮多个不同颜色的单色荧光激发光源，以通过不同颜色的单色荧光激发光源对待测样品板的预设检测区域进行同步激发，并经过荧光收集和预设检测区域放大后，对荧光进行多波段滤光处理，以将杂散光去除，最终获取预设检测区域的荧光图像。由此实现了多荧光通道同步显微成像，避免了因分步检测而导致的荧光淬灭性的影响，便于实验。
另外，本发明提供的技术方案，所有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，大大减少了进入物镜的单色荧光激发光，使得后续获取的荧光图像更加精确。此外，由于本发明提供的多荧光通道同步显微成像装置中，无需二色相镜，使得该装置结构更加简单且成本低廉，并且避免了光在透过二色相镜时出现损耗的情况，使得最终获取的荧光图像更加清晰。
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多荧光通道同步显微成像方法的流程图；
图3为本申请实施例提供的一种多荧光通道同步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多荧光通道同步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一次只能实现一个荧光通道的检测，当需要多个荧光通道的检测时，只能在一个荧光通道检测完成后，切换至另一个荧光通道进行检测，而后对多个荧光通道的检测结果进行整合。现今很多荧光染料具有快速的淬灭性，在进行多个荧光通道分步检测时，由于样品被反复照射或照射的时间有差异，荧光的淬灭性影响很难测定。因此，若在多荧光通道的检测过程中，能够实现多荧光通道同步检测，即能解决因分步检测而导致的荧光淬灭性的影响。
基于此，本申请实施例提供了一种多荧光通道同步显微成像方法和装置，实现了多荧光通道同步显微成像，避免了因分步检测而导致的荧光淬灭性的影响，便于实验；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；此外，该装置结构简单且成本低。
具体的，参考图2所示，为本申请实施例提供的一种多荧光通道同步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同时点亮多个不同颜色的单色荧光激发光源的组合为目标光源，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多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多波段滤光处理；
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，包括有不同颜色(或波段)的单色荧光激发光源。其中，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，通过根据实验要求在多个单色荧光激发光源中同时点亮多个不同颜色的单色荧光激发光源，以通过不同颜色的单色荧光激发光源对待测样品板的预设检测区域进行同步激发，并经过荧光收集和预设检测区域放大 后，对荧光进行多波段滤光处理，以将杂散光去除，最终获取预设检测区域的荧光图像。由此实现了多荧光通道同步显微成像，避免了因分步检测而导致的荧光淬灭性的影响，便于实验。
另外，所有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，大大减少了进入物镜的单色荧光激发光，使得后续获取的荧光图像更加精确。此外，由于本申请实施例提供技术方案中无需二色相镜进行分光，进而使得相应的装置结构更加简单且成本低廉，并且避免了光在透过二色相镜时出现损耗的情况，使得最终获取的荧光图像更加清晰。
需要说明的是，对于本申请实施例提供技术方案最终获取的荧光图像的分析，可以通过对每个颗粒激发的荧光颜色进行分析。其中，目标光源中不同颜色的单色荧光激发光源之间的波段是无交叠的，因此，在后续对颗粒激发的荧光进行滤光时，所有荧光的波段与任意一单色荧光激发光的波段同样是无交叠的，避免相同波段的单色荧光激发光对荧光滤光时造成影响，亦即，采用的多波段滤光片透过的波段与单色荧光激发光源的波段是无交叠的。
举例说明，假设根据实验要求同时点亮紫色激发光源(波段范围为385nm±10，包括端点值)、蓝色激发光源(波段范围为480nm±10，包括端点值)和绿色激发光源(波段范围为550nm±10，包括端点值)为目标光源时，即，目标光源中不同颜色的单色荧光激发光源之间的波段无交叠；
目标光源相应发出紫色激发光、蓝色激发光和绿色激发光。其中，当目标光源照射至预设检测区域时，若预设检测区域内的颗粒仅受紫色激发光的激发，则发出的荧光为蓝色(波段为460nm)；若预设检测区域内的颗粒仅受绿色激发光的激发，则发出的荧光为红色(波段为大于600nm)；若预设检测区域内的颗粒仅受蓝色激发光的激发，则发出的荧光为绿色(波段为520nm)；若预设检测区域内的颗粒受蓝色激发光和紫色激发光的激发，则发出的荧光为青色；若预设检测区域内的颗粒受紫色激发光和绿色激发光的激发，则发出的荧光为紫色；若预设检测区域内的颗粒受蓝色激发光和绿色激发光的激发，则发出的荧光为黄色；若预设检测区域内的颗粒受紫色激发光、绿色激发光和蓝色激发光的激发，则发出的荧光为白色。由于颗粒受不同颜色的荧光激发光的激发，而发出的颜色不同，因此可以通过此原理对获取的荧光图像进行分析。
与此同时，采用多波段滤光片透过波段为能够透过波段范围为460nm±10的波段(包括端点值)的蓝色光、520nm±10的波段(包括端点值)的绿色光和不小于600nm的波段的红色光的三波段滤光片。进而，多波段滤光片不仅能够透过颗粒激发出的蓝色荧光、绿色荧光和红色荧光；而且当颗粒受多色激发光激发时，同样能够透过蓝色荧光、绿色荧光和红色荧光中任意组合的荧光，例如，受蓝色激发光和紫色激发光激发，则发出蓝色荧光和绿色荧光组合的青色荧光，多波段滤光片透过青色荧光即为透过蓝色荧光和绿色荧光的组合。因此，将多波段滤光片的透过波段与目标光源中不同颜色的单色荧光激发光源的波段设置为无交叠，以此对颗粒发出的荧光进行滤光处理，以清除杂散光。
基于此，通过同时点亮多个不同颜色的单色荧光激发光源，以实现二通道同步显微成像、三通道同步显微成像等多荧光通道同步显微成像。
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多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多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多荧光通道同步显微成像装置的结构示意图，其中，多荧光通道同步显微成像装置包括：
光源装置100、样品放置台200、物镜300、多波段滤光片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***(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相交于样品放置台200的预设位置，控制***根据实验要求在多个单色荧光激发光源101中同时点亮多个不同颜色的单色荧光激发光源的组合为目标光源；
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，用于收集预设检测区域内的颗粒受目标光源发出的多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；
多波段滤光片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多波段滤光处理；
图像获取装置500设置于多波段滤光片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。
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、且设置于白色激发光源与样品放置台之间的激发滤光片。其中，白色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***可以为单片机等，对此本申请不作具体限制。
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，即，提高目标光源所发出的多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多荧光通道同步显微成像装置的结构示意图，其中，光源装置还包括：
设置于单色荧光激发光源101的照射方向上、且设置于单色荧光激发光源101与样品放置台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，对此本申请实施例不作具体限制。
为了将光源装置、样品放置台、物镜、多波段滤光片、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多荧光通道同步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同时点亮多个不同颜色的单色荧光激发光源的组合为目标光源，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多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多波段滤光处理；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，通过根据实验要求在多个单色荧光激发光源中同时点亮多个不同颜色的单色荧光激发光源，以通过不同颜色的单色荧光激发光源对待测样品板的预设检测区域进行同步激发，并经过荧光收集和预设检测区域放大后，对荧光进行多波段滤光处理，以将杂散光去除，最终获取预设检测区域的荧光图像。由此实现了多荧光通道同步显微成像，避免了因分步检测而导致的荧光淬灭性的影响，便于实验。
另外，本申请实施例提供的技术方案，所有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，大大减少了进入物镜的单色荧光激发光，使得后续获取的荧光图像更加精确。此外，由于本申请实施例提供的多荧光通道同步显微成像装置中，无需二色相镜，使得该装置结构更加简单且成本低廉，并且避免了光在透过二色相镜时出现损耗的情况，使得最终获取的荧光图像更加清晰。
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 (10)

  1. 一种多荧光通道同步显微成像方法，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同时点亮多个不同颜色的单色荧光激发光源的组合为目标光源，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多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多波段滤光处理；
    获取所述预设检测区域的荧光图像。
  2. 根据权利要求1所述的多荧光通道同步显微成像方法，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多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. 一种多荧光通道同步显微成像装置，其特征在于，包括：
    光源装置、样品放置台、物镜、多波段滤光片和图像获取装置；
    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***，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***根据实验要求在多个单色荧光激发光源中同时点亮多个不同颜色的单色荧光激发光源的组合为目标光源；
    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；
    所述多波段滤光片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多波段滤光片背离所述物镜一侧。
  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所述的多荧光通道同步显微成像装置，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：
    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多荧光通道同步显微成像装置，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、且设置于所述白色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：
    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。
  9. 根据权利要求8所述的多荧光通道同步显微成像装置，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。
  10. 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，所述图像获取装置为目镜或相机。
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