WO2019169871A1 - Dispositif et méthode de détection d'activité cellulaire utilisant une fibre optique appliquée à une microscopie holographique numérique coaxiale - Google Patents

Dispositif et méthode de détection d'activité cellulaire utilisant une fibre optique appliquée à une microscopie holographique numérique coaxiale Download PDF

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Publication number
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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Prior art keywords
cell
computer
laser light
optical fiber
light source
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PCT/CN2018/110992
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English (en)
Chinese (zh)
Inventor
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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Priority claimed from CN201810187051.8A external-priority patent/CN108570409B/zh
Application filed by 广州博冠光电科技股份有限公司 filed Critical 广州博冠光电科技股份有限公司
Publication of WO2019169871A1 publication Critical patent/WO2019169871A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

Definitions

  • 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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  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Health & Medical Sciences (AREA)
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  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Sustainable Development (AREA)
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  • Computer Vision & Pattern Recognition (AREA)
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  • General Physics & Mathematics (AREA)
  • Theoretical Computer Science (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
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Abstract

L'invention concerne un dispositif et une méthode de détection d'activité cellulaire utilisant une fibre optique appliquée à une microscopie holographique numérique coaxiale, se rapportant au domaine technique de la détection cellulaire. Le dispositif comprend une unité d'émission de source de lumière laser (1), une unité d'acquisition d'image (2), un support (3) et un ordinateur (4). Une surface d'extrémité supérieure du support (3) est pourvue d'une fenêtre de support transparente (5) pour placer un liquide cellulaire. L'unité d'émission de source de lumière laser (1) est disposée à une extrémité inférieure de la fenêtre de support (5). L'unité d'acquisition d'image (2) est disposée à une extrémité supérieure de la fenêtre de support (5). Une extrémité de sortie de l'unité d'acquisition d'image (2) est connectée à une extrémité d'entrée de l'ordinateur (4). Un hologramme de liquide cellulaire est déterminé sur la base du fait qu'une cellule et un milieu environnemental associé ont des indices de réfraction différents pour la lumière, et la lumière traversant la cellule change en phase par rapport à la lumière traversant le milieu environnemental. Une image cellulaire en coupe transversale est reconstruite au moyen d'une inversion à l'aide d'une méthode de transformation de Fourier, et une image en phase réelle de la cellule est obtenue au moyen d'opérations arctangentes. La viabilité cellulaire est déterminée en fonction d'une relation proportionnelle entre la différence de phase cellulaire et la différence d'indice de réfraction, ce qui permet d'obtenir une imagerie quantitative non invasive et sans dommage de cellules biologiques.
PCT/CN2018/110992 2018-03-07 2018-10-19 Dispositif et méthode de détection d'activité cellulaire utilisant une fibre optique appliquée à une microscopie holographique numérique coaxiale WO2019169871A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201820313011.9 2018-03-07
CN201810187051.8A CN108570409B (zh) 2018-03-07 2018-03-07 基于光纤数字同轴全息显微的细胞活性检测装置及方法
CN201810187051.8 2018-03-07
CN201820313011 2018-03-07

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1971253A (zh) * 2006-10-19 2007-05-30 上海大学 数字全息显微测量装置
CN101346673A (zh) * 2005-12-22 2009-01-14 相位全息成像Phi有限公司 用于分析细胞的样本的方法和装置
CN102436063A (zh) * 2011-10-21 2012-05-02 李志扬 一种激光光镊显微镜
CN103063155A (zh) * 2012-12-12 2013-04-24 浙江师范大学 一种数字显微全息相位图的快速去包裹方法
US20130274119A1 (en) * 2010-05-25 2013-10-17 Arryx, Inc. Methods and apparatuses for detection of positional freedom of particles in biological and chemical analyses and applications in immunodiagnostics
CN108570409A (zh) * 2018-03-07 2018-09-25 广州博冠光电科技股份有限公司 基于光纤数字同轴全息显微的细胞活性检测装置及方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101346673A (zh) * 2005-12-22 2009-01-14 相位全息成像Phi有限公司 用于分析细胞的样本的方法和装置
CN1971253A (zh) * 2006-10-19 2007-05-30 上海大学 数字全息显微测量装置
US20130274119A1 (en) * 2010-05-25 2013-10-17 Arryx, Inc. Methods and apparatuses for detection of positional freedom of particles in biological and chemical analyses and applications in immunodiagnostics
CN102436063A (zh) * 2011-10-21 2012-05-02 李志扬 一种激光光镊显微镜
CN103063155A (zh) * 2012-12-12 2013-04-24 浙江师范大学 一种数字显微全息相位图的快速去包裹方法
CN108570409A (zh) * 2018-03-07 2018-09-25 广州博冠光电科技股份有限公司 基于光纤数字同轴全息显微的细胞活性检测装置及方法

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