WO2019169871A1 - 基于光纤数字同轴全息显微的细胞活性检测装置及方法 - Google Patents

基于光纤数字同轴全息显微的细胞活性检测装置及方法 Download PDF

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WO2019169871A1
WO2019169871A1 PCT/CN2018/110992 CN2018110992W WO2019169871A1 WO 2019169871 A1 WO2019169871 A1 WO 2019169871A1 CN 2018110992 W CN2018110992 W CN 2018110992W WO 2019169871 A1 WO2019169871 A1 WO 2019169871A1
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cell
computer
laser light
optical fiber
light source
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PCT/CN2018/110992
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French (fr)
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陈艳
俞小进
曾德祥
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广州博冠光电科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/70—Determining position or orientation of objects or cameras

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  • the invention relates to the technical field of cell detection, in particular to a cell activity detecting device and method based on optical fiber digital coaxial holographic microscopy.
  • the research on biological cells mainly includes the study of cell size distribution, physical characteristics, distribution of biological substances inside and outside the cell and cell dynamics.
  • most biological cells are colorless and transparent. It is difficult to achieve clear imaging of cells by ordinary optical microscopy.
  • dyeing techniques are used to change the color and brightness of cells, but this will cause damage to cells and affect cell performance. And dynamic behavior.
  • the main object of the present invention is to provide a cell activity detecting device and method based on optical fiber digital coaxial holographic microscopy, aiming at realizing non-invasive and damage-free quantitative imaging of biological cells.
  • the present invention provides a cell activity detecting device based on optical fiber digital coaxial holographic microscopy, the device comprising a laser light source emitting unit, an image collecting unit, a stage and a computer, wherein the upper end of the stage The plane is provided with a transparent load window for placing the cell liquid, the laser light source emitting unit is disposed at the lower end of the load window, the image collecting unit is disposed at the upper end of the load window, and the output end of the image collecting unit is connected to the input end of the computer.
  • the laser light source emitting unit comprises: a laser light source and a single mode fiber, and the laser light source emits laser light, and the laser light is transmitted to the transparent load window of the stage through the single mode fiber.
  • the image acquisition unit adopts an image sensor, and the image sensor collects the cell liquid hologram and sends it to the computer.
  • the laser light is transmitted to the transparent load window of the stage through the single mode fiber, specifically: the laser irradiates the cell liquid of the transparent load window through the single mode fiber to form a cell liquid hologram.
  • the image sensor collects the cell liquid hologram and sends it to the computer, specifically: the image sensor collects the hologram, records the light intensity of the hologram and inputs it into the computer.
  • the inversion cell cross-sectional image is reconstructed by Fourier transform, and the true phase map of the cell is obtained by using an arctangent operation, and the cell activity is determined according to the proportional relationship between the cell phase difference and the refractive index difference.
  • a detection method using a cell activity detecting device based on optical fiber digital coaxial holographic microscopy includes the following steps:
  • a laser light source is activated, and the laser irradiates the cell liquid of the transparent load window through the single mode fiber to obtain a cell liquid hologram;
  • the image sensor collects the cell liquid hologram and sends it to the computer;
  • the computer reconstructs the inversion cell cross-sectional image by Fourier transform method
  • the computer uses the arctangent operation to obtain the true phase map of the cell
  • the computer determines cell viability based on the proportional relationship between cell phase difference and refractive index difference.
  • the computer uses an arctangent operation to obtain a true phase map of the cell, specifically: performing an arctangent operation on the cell light wave, obtaining a wrap phase, and obtaining a true phase map of the cell by using the unwrapped phase.
  • the computer determines the cell activity according to a proportional relationship between the cell phase difference and the refractive index difference, specifically: the lower the cell refractive index, the lower the cell activity.
  • the invention provides a cell activity detecting device and a method based on optical fiber digital coaxial holographic microscopy.
  • the cell and its environmental medium have different refractive indices for light, and the light passing through the cell is in phase with respect to the light passing through the environmental medium.
  • the change will be made, the cell liquid hologram is determined, the inversion cell cross-section image is reconstructed by Fourier transform method, and the true phase map of the cell is obtained by the inverse tangent operation; the cell activity is determined according to the proportional relationship between the cell phase difference and the refractive index difference. This enables non-invasive, non-invasive quantitative imaging of biological cells.
  • FIG. 1 is a schematic structural view of a cell activity detecting device based on optical fiber digital coaxial holographic microscopy according to an embodiment of the present invention
  • FIG. 2 is a flow chart of a cell activity detecting method based on optical fiber digital coaxial holographic microscopy in an embodiment of the present invention
  • FIG. 3 is a cross-sectional view of reconstructing an inversion cell by Fourier transform in an embodiment of the present invention
  • Figure 5 is a graph showing the true phase difference of a cell as a function of distance in an embodiment of the present invention.
  • FIG. 6 is a cell phase diagram of a cell liquid after standing for a certain period of time in an embodiment of the present invention
  • 1-laser light source emitting unit 2-image acquisition unit, 3-stage, 4-computer, 5-transparent load window, 101-laser source, 102-single mode fiber;
  • the directional indication is only used to explain in a certain posture (as shown in the drawing)
  • the relative positional relationship between the components, the motion situation, and the like if the specific posture changes, the directional indication also changes accordingly.
  • first”, “second”, etc. in the embodiments of the present invention, the description of the "first”, “second”, etc. is used for the purpose of description only, and is not to be construed as an Its relative importance or implicit indication of the number of technical features indicated.
  • features defining “first” or “second” may include at least one of the features, either explicitly or implicitly.
  • the technical solutions between the various embodiments may be combined with each other, but must be based on the realization of those skilled in the art, and when the combination of the technical solutions is contradictory or impossible to implement, it should be considered that the combination of the technical solutions does not exist. It is also within the scope of protection required by the present invention.
  • the invention provides a cell activity detecting device based on optical fiber digital coaxial holographic microscopy
  • the apparatus includes a laser light source emitting unit 1, an image capturing unit 2, a stage 3, and a computer 4, wherein the upper end plane of the stage 3 is provided for The transparent load window 5 for placing the cell liquid, the laser light source emitting unit 1 is disposed at the lower end of the load window 5, the image collecting unit 2 is disposed at the upper end of the load window 5, and the output end of the image collecting unit 2 is connected to the input end of the computer 4.
  • the laser light source emitting unit 1 includes a laser light source 101 and a single mode fiber 102.
  • the laser light source 101 emits laser light, and the laser light is transmitted through the single mode fiber 102 to the load.
  • the transparent load window 5 of the object table 3 is specifically such that the laser irradiates the cell liquid of the transparent load window 5 through the single mode fiber 102 to form a cell liquid hologram.
  • the image acquisition unit 2 adopts an image sensor, and the image sensor collects the cell liquid hologram and sends it to the computer 4, specifically: the image sensor collects the hologram, and records the light intensity of the hologram. And enter it into your computer.
  • the computer 4 reconstructs an inversion cell cross-sectional image by Fourier transform, and obtains a true phase map of the cell by using an arctangent operation, according to a ratio between a cell phase difference and a refractive index difference. The relationship determines cell viability.
  • the laser light source (monochrome laser wavelength is 405 nm) is activated, and the laser light is transmitted to the stage via a single mode fiber, and the output spot is 1 ⁇ m, and the numerical aperture is 0.13, which can be approximated as a point source.
  • the point source emits a spherical wave, and irradiates a certain concentration of cell liquid placed on the stage. Since the refractive index of the cell is different from that of the surrounding medium, the light passing through the cell changes in phase with respect to the light passing through the surrounding medium. The phase difference is formed such that the changed cell light wave is superimposed with the original reference wave that has not changed to form a hologram; the intensity of the hologram is calculated by the following formula:
  • O(x, y) is sample light
  • R(x, y) is reference light
  • * is a conjugate symbol
  • the image sensor collects the cell liquid hologram and sends it to the computer;
  • the computer reconstructs the inverted cross-sectional image of the cell by using a Fourier transform method
  • the image sensor records the light intensity of the hologram and inputs it into the computer, and the computer reconstructs the inverted cross-sectional image of the cell by Fourier transform, as shown in FIG. 3;
  • the computer uses the arctangent operation to obtain the true phase map of the cell; specifically: performing an arctangent operation on the cell light wave, obtaining a wrap phase, and obtaining a true phase map of the cell by using the unwrapped phase;
  • Im is the imaginary part of the function
  • Re is the real part of the function
  • FIG. 5 is a graph of phase difference as a function of distance
  • the computer determines the cell activity according to a proportional relationship between the cell phase difference and the refractive index difference.
  • phase difference is proportional to the refractive index difference between the cell and the surrounding environment, as follows:
  • ⁇ OPL is the optical path difference
  • n obj (x, y) is the refractive index of the cell
  • n m is the refractive index of the medium surrounding the cell
  • h obj (x, y) is the cell thickness
  • the cell phase diagram was observed again, and the phase difference was changed (as shown in Fig. 6), which was proportional to the phase difference and the refractive index difference (the difference between the cell refractive index and the environmental refractive index). It can be seen that the refractive index of the cells is lowered and the cells are inactivated.

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Abstract

一种基于光纤数字同轴全息显微的细胞活性检测装置及方法，涉及细胞检测技术领域，该装置包括激光光源发射单元（1）、图像采集单元（2）、载物台（3）和计算机（4），其中，所述载物台（3）上端平面设置有用于放置细胞液的透明载物窗口（5），激光光源发射单元（1）设置于载物窗口（5）下端，图像采集单元（2）设置于载物窗口（5）上端，图像采集单元（2）的输出端连接计算机（4）的输入端；根据细胞及其环境介质对光具有不同折射率，通过细胞的光相对于通过环境介质的光在相位上会发生变化，确定细胞液全息图，通过傅里叶变换法重建反演细胞截面图像，利用反正切运算得到细胞真实相位图；根据细胞相位差与折射率差之间的比例关系确定细胞活性；实现对生物细胞的非侵入、无损伤的定量成像。

Description

基于光纤数字同轴全息显微的细胞活性检测装置及方法 技术领域
本发明涉及细胞检测技术领域，尤其涉及一种基于光纤数字同轴全息显微的细胞活性检测装置及方法。
背景技术
现阶段对生物细胞的研究主要包括对细胞的大小分布、形体特征、细胞内外生物物质分布以及细胞动力学行为的研究。但大部分生物细胞本身无色透明，采用普通光学显微技术难以实现细胞的清晰成像，为克服这一困难，常用染色技术改变细胞的颜色和亮度，但这会对细胞造成损伤，影响细胞性能及动力学行为。
发明内容
本发明的主要目的在于提供一种基于光纤数字同轴全息显微的细胞活性检测装置及方法，旨在实现对生物细胞的非侵入、无损伤的定量成像。
为了实现上述目的，本发明提出一种基于光纤数字同轴全息显微的细胞活性检测装置，该装置包括激光光源发射单元、图像采集单元、载物台和计算机，其中，所述载物台上端平面设置有用于放置细胞液的透明载物窗口，激光光源发射单元设置于载物窗口下端，图像采集单元设置于载物窗口上端，图像采集单元的输出端连接计算机的输入端。
优选地，所述的激光光源发射单元，包括：激光光源和单模光纤，激光光源发射激光，激光通过单模光纤传输至载物台的透明载物窗口。
优选地，所述的图像采集单元采用图像传感器，图像传感器采集细胞液全息图发送至计算机中。
优选地，所述的激光通过单模光纤传输至载物台的透明载物窗口，具体为：激光通过单模光纤照射透明载物窗口的细胞液，形成细胞液全息图。
优选地，所述的图像传感器采集细胞液全息图发送至计算机中，具体为：图像传感器采集全息图，将全息图的光强进行记录并输入计算机中。
优选地，所述的计算机，通过傅里叶变换法重建反演细胞截面图像，利用反正切运算得到细胞真实相位图，根据细胞相位差与折射率差之间的比例关系确定细胞活性。
采用基于光纤数字同轴全息显微的细胞活性检测装置进行的检测方法，包括如下步骤：
启动激光光源，激光通过单模光纤照射透明载物窗口的细胞液，获得细胞液全息图；
图像传感器采集细胞液全息图发送至计算机中；
计算机通过傅里叶变换法重建反演细胞截面图像；
计算机利用反正切运算得到细胞真实相位图；
计算机根据细胞相位差与折射率差之间的比例关系确定细胞活性。
优选地，所述的计算机利用反正切运算得到细胞真实相位图，具体为：对细胞光波进行反正切运算，获得包裹相位，利用解包裹相位得到细胞真实相位图。
优选地，所述的计算机根据细胞相位差与折射率差之间的比例关系确定细胞活性，具体为：细胞折射率越低，细胞活性越低。
本发明提出一种基于光纤数字同轴全息显微的细胞活性检测装置及方法，本发明根据细胞及其环境介质对光具有不同的折射率，通过细胞的光相对于通过环境介质的光在相位上会发生变化，确定细胞液全息图，通过傅里叶变换法重建反演细胞截面图像，利用反正切运算得到细胞真实相位图；根据细胞相位差与折射率差之间的比例关系确定细胞活性；这样实现对生物细胞的非侵入、无损伤的定量成像。
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一些实施例，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，还可以根据这些附图示出的结构获得其他的附图。
图1为本发明一种实施例中基于光纤数字同轴全息显微的细胞活性检测装置结构示意图；
图2为本发明一种实施例中基于光纤数字同轴全息显微的细胞活性检测方法流程图；
图3为本发明一种实施例中通过傅里叶变换法重建反演细胞截面图像；
图4为本发明一种实施例中细胞真实图像；
图5为本发明一种实施例中细胞真实相位差随距离变化的曲线图；
图6为本发明一种实施例中细胞液静置一定时间后细胞相位图；
标号说明：
1-激光光源发射单元，2-图像采集单元，3-载物台，4-计算机，5-透明载物窗口，101-激光光源，102-单模光纤；
本发明目的的实现、功能特点及优点将结合实施例，参照附图做进一步说明。
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的一部分实施例，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作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。
需要说明，若本发明实施例中有涉及方向性指示(诸如上、下、左、右、前、后……)，则该方向性指示仅用于解释在某一特定姿态(如附图所示)下各部件之间的相对位置关系、运动情况等，如果该特定姿态发生改变时，则该方向性指示也相应地随之改变。
另外，若本发明实施例中有涉及“第一”、“第二”等的描述，则该“第一”、“第二”等的描述仅用于描述目的，而不能理解为指示或暗示其相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。另外，各个实施例之间的技术方案可以相互结合，但是必须是以本领域普通技术人员能够实现为基础，当技术方案的结合出现相互矛盾或无法实现时应当认为这种技术方案的结合不存在，也不在本发明要求的保护范围之内。
本发明提供一种基于光纤数字同轴全息显微的细胞活性检测装置；
本发明一种优选实施例中，如图1所示，该装置包括激光光源发射单元1、图像采集单元2、载物台3和计算机4，其中，所述载物台3上端平面设置有用于放置细胞液的透明载物窗口5，激光光源发射单元1设置于载物窗口5下端，图像采集单元2设置于载物窗口5上端，图像采集单元2的输出端连接计算机4的输入端。
本发明一种优选实施例中，如图1所示，所述的激光光源发射单元1，包括：激光光源101和单模光纤102，激光光源101发射激光，激光通过单模光纤102传输至载物台3的透明载物窗口5，具体为：激光通过单模光纤102照射透明载物窗口5的细胞液，形成细胞液全息图。
本发明一种优选实施例中，所述的图像采集单元2采用图像传感器，图像传感器采集细胞液全息图发送至计算机4中，具体为：图像传感器采集全息图，将全息图的光强进行记录并输入计算机中。
本发明一种优选实施例中，所述的计算机4，通过傅里叶变换法重建反演细胞截面图像，利用反正切运算得到细胞真实相位图，根据细胞相位差与折射率差之间的比例关系确定细胞活性。
采用基于光纤数字同轴全息显微的细胞活性检测装置进行的检测方法；
本发明一种优选实施例中，如图2所示，包括如下步骤：
S10、启动激光光源，激光通过单模光纤照射透明载物窗口的细胞液，获得细胞液全息图；
本发明实施例中，启动激光光源(单色激光波长为405nm)，激光经单模光纤传输至载物台，其输出光斑为1μm，数值孔径为0.13，可近似等效为点光源。其次点光源发出球面波，照射放置在载物台上一定浓度的细胞液，由于细胞与其周围环境介质的折射率不同，所以通过细胞的光相对于通过周围环境介质的光在相位上发生变化，构成了相位差，使得发生变化的细胞光波与原来未发生变化的参考波干涉叠加形成全息图；全息图的光强大小由下式计算可得：
I(x,y)＝|O(x,y)+R(x,y)| 2＝
O(x,y)O(x,y) *+R(x,y)R(x,y) *+O(x,y)R(x,y) *+R(x,y)O(x,y) *  (1)
其中，O(x,y)为样品光，R(x,y)为参考光，*为共轭符号；
S20、图像传感器采集细胞液全息图发送至计算机中；
S30、计算机通过傅里叶变换法重建反演细胞截面图像；
图像传感器(CCD)将全息图的光强记录下来并输入计算机，计算机通过傅里叶变换法重建反演细胞截面图像，如图3所示；
S40、计算机利用反正切运算得到细胞真实相位图；具体为：对细胞光波进行反正切运算，获得包裹相位，利用解包裹相位得到细胞真实相位图；
对细胞光波(样品光)O(x,y)进行反正切运算，获得包裹相位
Figure PCTCN2018110992-appb-000001
如下式：
Figure PCTCN2018110992-appb-000002
其中，Im为取函数虚部，Re为取函数实部；
再利用解包裹相位得到细胞真实相位图。如图4所示，图5为相位差随距离变化的曲线图；
S50、计算机根据细胞相位差与折射率差之间的比例关系确定细胞活性。
又因为相位差与细胞和周围环境介质的折射率差成正比，如下式：
Figure PCTCN2018110992-appb-000003
其中，
Figure PCTCN2018110992-appb-000004
为细胞相位差，ΔOPL为光程差，n obj(x,y)为细胞折射率，n m为细胞周围环境介质折射率，h obj(x,y)为细胞厚度；
将细胞液静置一定时间后，再次观察细胞相位图，可知相位差发生了变化(如图6所示)，由相位差与折射率差(细胞折射率与环境折射率的差值)成正比可知，细胞折射率降低，细胞失去活性。
以上所述仅为本发明的优选实施例，并非因此限制本发明的专利范围，凡是在本发明的发明构思下，利用本发明说明书及附图内容所作的等效结构变换，或直接/间接运用在其他相 关的技术领域均包括在本发明的专利保护范围内。

Claims (9)

  1. 一种基于光纤数字同轴全息显微的细胞活性检测装置，其特征在于，该装置包括激光光源发射单元、图像采集单元、载物台和计算机，其中，所述载物台上端平面设置有用于放置细胞液的透明载物窗口，激光光源发射单元设置于载物窗口下端，图像采集单元设置于载物窗口上端，图像采集单元的输出端连接计算机的输入端。
  2. 根据权利要求1所述的基于光纤数字同轴全息显微的细胞活性检测装置，其特征在于，所述的激光光源发射单元，包括：激光光源和单模光纤，激光光源发射激光，激光通过单模光纤传输至载物台的透明载物窗口。
  3. 根据权利要求1所述的基于光纤数字同轴全息显微的细胞活性检测装置，其特征在于，所述的图像采集单元采用图像传感器，图像传感器采集细胞液全息图发送至计算机中。
  4. 根据权利要求2所述的基于光纤数字同轴全息显微的细胞活性检测装置，其特征在于，所述的激光通过单模光纤传输至载物台的透明载物窗口，具体为：激光通过单模光纤照射透明载物窗口的细胞液，形成细胞液全息图。
  5. 根据权利要求3所述的基于光纤数字同轴全息显微的细胞活性检测装置，其特征在于，所述的图像传感器采集细胞液全息图发送至计算机中，具体为：图像传感器采集全息图，将全息图的光强进行记录并输入计算机中。
  6. 根据权利要求5所述的基于光纤数字同轴全息显微的细胞活性检测装置，其特征在于，所述的计算机，通过傅里叶变换法重建反演细胞截面图像，利用反正切运算得到细胞真实相位图，根据细胞相位差与折射率差之间的比例关系确定细胞活性。
  7. 采用权利要求1所述的基于光纤数字同轴全息显微的细胞活性检测装置进行的检测方法，其特征在于，包括如下步骤：
    启动激光光源，激光通过单模光纤照射透明载物窗口的细胞液，获得细胞液全息图；
    图像传感器采集细胞液全息图发送至计算机中；
    计算机通过傅里叶变换法重建反演细胞截面图像；
    计算机利用反正切运算得到细胞真实相位图；
    计算机根据细胞相位差与折射率差之间的比例关系确定细胞活性。
  8. 根据权利要求7所述的检测方法，其特征在于，所述的计算机利用反正切运算得到细胞真实相位图，具体为：对细胞光波进行反正切运算，获得包裹相位，利用解包裹相位得到细胞真实相位图。
  9. 根据权利要求7所述的检测方法，其特征在于，所述的计算机根据细胞相位差与折射率差之间的比例关系确定细胞活性，具体为：细胞折射率越低，细胞活性越低。
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