CN109852541B - Cell activity detection device and method based on total gray value and gray difference ratio of holographic fringes - Google Patents
Cell activity detection device and method based on total gray value and gray difference ratio of holographic fringes Download PDFInfo
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Abstract
The invention provides a cell activity detection device and method based on a ratio of a total gray value to a gray difference of holographic fringes, wherein the device comprises a light source component, a light through hole component, a light propagation component, a microfluidic chip, an image acquisition component and an image processing component which are sequentially connected; when the micro-fluidic chip works, the image acquisition assembly acquires a holographic pattern generated by the detection area of the micro-fluidic chip under the action of a light beam, and the holographic pattern is sent to the image processing assembly connected with the image acquisition assembly for image analysis to obtain the ratio of the total gray value of the holographic image formed by the microalgae cells to the difference value of the central bright spot in the holographic image formed by the microalgae cells and the dark ring gray value of the outer ring of the bright spot. The technical scheme of the invention solves the problems that the existing method for detecting the activity of the microalgae in the ship ballast water is not suitable for quick detection on site, and has large volume, high price, difficult operation and the like.
Description
Technical Field
The invention relates to the technical field of activity judgment of microalgae in ship ballast water, in particular to a cell activity detection device and method based on a holographic stripe total gray value and gray difference ratio.
Background
Ship ballast water refers to water and suspended matter loaded onto a ship to control the roll, pitch, draft, stability or stress of the ship. The ballast water of ships contains a large amount of organisms including plankton, microorganism, bacteria, and these foreign species are rapidly adapted to the environment to expand the population, thereby causing harm to the environment. The discharge of ship ballast water has created a major way to rationally isolate the spread of marine organisms and pathogens between bodies of water, and the ship ballast water increasingly destroys the marine organism chain. With the increasing international shipping industry and the increasing awareness of people on marine environment protection, the problem of foreign marine organism invasion caused by the discharge of ship ballast water has attracted wide attention of all societies. Recognizing that trans-regional transfer of harmful aquatic organisms and pathogens in ship ballast water has had a non-negligible impact on the global marine ecological environment, the Global Environmental Foundation (GEF) has classified it as one of four major threats to the harm of the sea. To prevent the invasion of foreign organisms in ballast water, international ship ballast water management held by international maritime organization in 2004 will pass the international ship ballast water and its deposit control and management convention, hereinafter referred to as the ballast water convention, wherein the terms for the convention to take effect are defined to take effect after 12 months of 30 national approval at 35% of the world commercial ship tonnage.
Microalgae phytoplankton account of a very large proportion of foreign species spread with ballast water, microalgae invasion has caused enormous economic and environmental losses worldwide. In recent years, red tide algae, which is mostly toxic and easily causes death of birds and fishes, is extremely harmful to the environment and economy all over the world, so that the activity detection of algae cells in ballast water becomes extremely important.
The current methods for detecting the cell activity of algae include an optical microscopy method, a flow cytometry counting method, a dye fluorescence microscopy counting method, a molecular and biochemical method and the like. The optical microscopy distinguishes algae cells by observing the morphology of the microalgae cells by a professional technician, and judges the activity of the algae cells by staining the algae cells. The microscope is expensive, large in size, difficult to operate and not suitable for rapid detection of microalgae cells. The flow cytometry method needs to label microalgae with a fluorescent dye and prepare the microalgae into suspension, and the activity of microalgae cells is judged by measuring fluorescent signals and scattering signals of the microalgae cells.
Disclosure of Invention
According to the proposal, the existing method for detecting the activity of the microalgae in the ship ballast water is not suitable for quick detection on site, has the defects of large volume, high price, difficult operation and the like, and the method and the device for detecting the activity of the microalgae in the ship ballast water still have the technical problems to be solved urgently, so that the device and the method for detecting the activity of the cells based on the ratio of the total gray value to the gray difference of the holographic stripes are provided.
The technical means adopted by the invention are as follows:
cell activity detection device based on total grey scale value of holographic stripe and grey scale difference ratio, its characterized in that includes: the light source assembly, the light through hole assembly, the light propagation assembly, the micro-fluidic chip, the image acquisition assembly and the image processing assembly are connected in sequence;
the light source assembly comprises a power supply device, a light source fixing structure and a light emitting diode tightly attached to the light through hole assembly, light beams emitted by the light emitting diode are changed into spherical waves through the light through hole assembly, and the spherical waves irradiate the microfluidic chip through the light transmission assembly and are imaged by the image acquisition assembly to obtain a hologram of the microalgae cells;
the microfluidic chip comprises a polydimethylsiloxane sheet and a glass slide, wherein a detection area is sequentially concavely etched on the polydimethylsiloxane sheet, two ends of the detection area are symmetrically connected with a first focusing channel and a second focusing channel, the width of the other end of the first focusing channel is gradually increased until the other end of the first focusing channel is connected to one end of the first channel in an equal-width mode, a liquid inlet hole is formed in the other end of the first channel, the width of the other end of the second focusing channel is gradually increased until the other end of the second focusing channel is connected to one end of the second channel in an equal-width mode, and a waste liquid hole is formed in the other end of the second channel;
when the micro-fluidic chip works, the image acquisition assembly acquires a holographic pattern generated by the detection area of the micro-fluidic chip under the action of a light beam, and the holographic pattern is sent to the image processing assembly connected with the image acquisition assembly for image analysis to obtain the ratio of the total gray value of the holographic image formed by the microalgae cells to the difference value of the central bright spot in the holographic image formed by the microalgae cells and the dark ring gray value of the outer ring of the bright spot.
Further, the distance between the microfluidic chip and the image acquisition assembly is far smaller than the distance between the light transmission assembly and the microfluidic chip.
Further, the light beam emitted by the light emitting diode is partially coherent light.
Further, the total gray value represents brightness information of the holographic image, and the activity of the microalgae is judged according to the ratio of the total gray value of the holographic image formed by the microalgae cells to the difference value of the gray value of the bright spot at the center in the holographic image and the gray value of the dark ring at the outer ring of the bright spot.
The invention also provides a cell activity detection method based on the ratio of the total gray value to the gray difference of the holographic fringes, which comprises the following steps:
step S1: dropwise adding the sample solution, and adding the microalgae cell sample solution into a liquid inlet hole of the microfluidic chip; the liquid sample flows to the detection channel along the first channel through the first focusing channel under the action of self tension, and redundant liquid sample flows into the waste liquid hole;
step S2: the starting device is used for turning on a switch of the light-emitting diode and connecting the image acquisition assembly with the image processing assembly;
step S3: part of coherent light emitted by the light emitting diode is changed into spherical waves through the light through hole assembly, and the spherical waves are irradiated on a sample in the detection area of the microfluidic chip through the light transmission assembly to form a holographic image and are collected by the image collection assembly;
step S4: the holographic pattern collected by the image collecting assembly is transmitted to the image processing assembly through a data line;
step S5: and judging the activity of the microalgae cells according to the pattern characteristics of the microalgae cells in the holographic pattern.
Further, the microalgae cell pattern in the holographic pattern is characterized by a ratio of a total gray value of the holographic image formed by the microalgae cells to a difference value of a gray value of a central bright spot in the holographic image and a dark ring of an outer ring of the bright spot.
Compared with the prior art, the invention has the following advantages:
the cell activity detection method based on the ratio of the total gray value to the gray difference of the holographic fringes is suitable for rapid detection on site, and the cell activity detection device based on the ratio of the total gray value to the gray difference of the holographic fringes has the characteristics of small volume, low price and easiness in operation, and has important scientific significance and practical value in the field of environmental science.
For the above reasons, the present invention can be widely applied to the field of determination of the activity of microalgae in ship ballast water.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a device for detecting microalgae activity in ship ballast water;
FIG. 2 is a schematic structural diagram of a microfluidic chip according to the present invention;
FIG. 3 is a flow chart of the steps of the method for detecting the activity of microalgae by the present device.
In the figure: 1. a light source assembly; 2. a light through hole assembly; 3. a light propagating assembly; 4. a microfluidic chip; 5. an image acquisition component; 6. an image processing component; 7. a polydimethylsiloxane sheet; 8. a glass slide; 9. a first channel; 10. a first focusing channel; 11. a detection channel; 12. a second focusing channel; 13. a second channel; A. a liquid inlet hole; B. a waste liquid hole.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise. Meanwhile, it should be understood that the sizes of the respective portions shown in the drawings are not drawn in an actual proportional relationship for the convenience of description. Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate. Any specific values in all examples shown and discussed herein are to be construed as exemplary only and not as limiting. Thus, other examples of the exemplary embodiments may have different values. It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, further discussion thereof is not required in subsequent figures.
In the description of the present invention, it is to be understood that the orientation or positional relationship indicated by the directional terms such as "front, rear, upper, lower, left, right", "lateral, vertical, horizontal" and "top, bottom", etc., are generally based on the orientation or positional relationship shown in the drawings, and are used for convenience of description and simplicity of description only, and in the absence of any contrary indication, these directional terms are not intended to indicate and imply that the device or element so referred to must have a particular orientation or be constructed and operated in a particular orientation, and therefore should not be considered as limiting the scope of the present invention: the terms "inner and outer" refer to the inner and outer relative to the profile of the respective component itself.
Spatially relative terms, such as "above … …," "above … …," "above … … surface," "above," and the like, may be used herein for ease of description to describe one device or feature's spatial relationship to another device or feature as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is turned over, devices described as "above" or "on" other devices or configurations would then be oriented "below" or "under" the other devices or configurations. Thus, the exemplary term "above … …" can include both an orientation of "above … …" and "below … …". The device may be otherwise variously oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
It should be noted that the terms "first", "second", and the like are used to define the components, and are only used for convenience of distinguishing the corresponding components, and the terms have no special meanings unless otherwise stated, and therefore, the scope of the present invention should not be construed as being limited.
The invention relates to a cell activity detection method and a cell activity detection device based on the ratio of the total gray value to the gray difference of holographic fringes according to the principle that the holographic patterns formed by microalgae cells with different activities have different characteristics, and is further described with reference to the accompanying drawings.
Example 1
As shown in fig. 1, the present invention provides a cell activity detecting device based on the ratio of the total gray-level value to the gray-level difference of the holographic fringes, comprising: the device comprises a light source component 1, a light through hole component 2, a light transmission component 3, a micro-fluidic chip 4, an image acquisition component 5 and an image processing component 6 which are connected in sequence. The light source assembly 1 comprises a power supply device, a light source fixing structure and a light emitting diode tightly attached to the light through hole assembly 2, partial coherent light emitted by the light emitting diode is changed into spherical waves through the light through hole assembly 2, and the spherical waves irradiate the microfluidic chip 4 through the light transmission assembly 3 and are imaged by the image acquisition assembly 5 to obtain the hologram of the algae cells. The image acquisition component 5 acquires a holographic pattern generated by the detection area of the microfluidic chip 4 under the action of light beams, and the holographic pattern is sent to the image processing component 6 connected with the image acquisition component 5 for image analysis to obtain the total gray value of the holographic image formed by the microalgae, and the difference value between the central bright spot in the holographic image and the dark ring gray value of the outer ring of the bright spot. The total gray value represents the brightness information of the holographic image, and the activity of the microalgae is judged according to the ratio of the total gray value of the holographic image formed by the microalgae to the difference value of the gray value of the bright spot at the center in the holographic image and the gray value of the dark ring at the outer ring of the bright spot. The light emitting diodes in the light source component emit partially coherent light, so that noise can be effectively reduced. The distance between the microfluidic chip 4 and the image acquisition component 5 is far less than the distance between the light propagation component 3 and the microfluidic chip 4, so that the field of view of the acquired hologram of the microalgae cell is much larger than that of the microscope.
As shown in fig. 2, the microfluidic chip 4 includes a polydimethylsiloxane sheet 7 and a glass slide 8, the polydimethylsiloxane sheet) is sequentially concavely etched with a detection region 11, two ends of the detection region 11 are symmetrically connected with a first focusing channel 10 and a second focusing channel 12, the width of the other end of the first focusing channel 10 is gradually increased until the other end is connected with a first channel 9 in an equal width manner, the other end of the first channel is provided with a liquid inlet hole, the width of the other end of the second focusing channel 12 is gradually increased until the other end is connected with one end of a second channel 1 in an equal width manner, and the other end of the second channel 13 is provided with a waste liquid hole;
when the device is used, firstly, a microalgae cell sample is placed on the microfluidic chip, the microfluidic chip is placed on the objective table, and partial coherent light emitted by the light emitting diode irradiates a diffraction image formed on the sample placed on the objective table through the light through hole and the light transmission assembly and is collected by the image collection assembly. The device chooses emitting diode as the light source for use, and the light that emitting diode sent in the light source subassembly is partial coherent light, can effectual suppression coherent speckle noise and interference. Partial coherent light emitted by an LED light source in the light source component is dispersed into a beam of spherical wave after passing through a light passing hole in the light passing hole component, and the spherical wave formed by dispersion is transmitted to a sample surface through a proper transmission distance. The device utilizes the branch light penetrating through the sample as the reference light without introducing the reference light additionally. The distance between the sample and a charge coupled device (cmos) in the image acquisition assembly is very close, the distance is about a few millimeters, a holographic pattern formed by partially coherent light irradiating on the sample is acquired by the image acquisition assembly, and finally the acquired holographic image is processed by the image processing assembly.
The holographic images formed by the microalgae cells with different activities are different, and the activity condition of the microalgae cells is distinguished by judging some characteristics of the holographic images.
The method comprises the steps of obtaining a hologram formed by microalgae cells, wherein gray values of a central bright spot and a dark ring on the outer ring of the bright spot in the hologram formed by the microalgae cells are different, and judging the activity of microalgae by analyzing the ratio of the total gray value of the hologram formed by the microalgae cells to the difference value of the gray values of the central bright spot and the dark ring on the outer ring of the bright spot in the hologram.
Example 2
On the basis of the embodiment 1, as shown in fig. 3, the invention also provides a microalgae activity detection method based on the device, which is characterized in that: which comprises the following steps:
step S1: dropwise adding the sample solution, and adding the microalgae cell sample solution into a liquid inlet hole of the microfluidic chip; the liquid sample flows to the detection channel along the first channel through the first focusing channel under the action of self tension, and redundant liquid sample flows into the waste liquid hole;
step S2: the starting device is used for turning on a switch of the light-emitting diode and connecting the image acquisition assembly with the image processing assembly;
step S3: part of coherent light emitted by the light emitting diode is changed into spherical waves through the light through hole assembly, and the spherical waves are irradiated on a sample in the detection area of the microfluidic chip through the light transmission assembly to form a holographic image and are collected by the image collection assembly;
step S4: the holographic pattern collected by the image collecting assembly is transmitted to the image processing assembly through a data line;
step S5: judging the activity of microalgae cells according to the pattern characteristics of the microalgae cells in the holographic pattern; the microalgae cell pattern in the holographic pattern is characterized by the ratio of the total gray value of the holographic image formed by the microalgae cells to the difference value of the gray value of the bright spot at the center in the holographic image and the gray value of the dark ring at the outer ring of the bright spot.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (2)
1. Cell activity detection device based on total grey scale value of holographic stripe and grey scale difference ratio, its characterized in that includes: the device comprises a light source component (1), a light through hole component (2), a light transmission component (3), a micro-fluidic chip (4), an image acquisition component (5) and an image processing component (6) which are connected in sequence; the distance between the microfluidic chip (4) and the image acquisition assembly (5) is far smaller than the distance between the light transmission assembly (3) and the microfluidic chip (4);
the light source assembly (1) comprises a power supply device, a light source fixing structure and a light emitting diode tightly attached to the light through hole assembly (2), wherein part of coherent light emitted by the light emitting diode is changed into spherical waves through the light through hole assembly (2), and the spherical waves irradiate the microfluidic chip (4) through the light propagation assembly (3) and are imaged by the image acquisition assembly (5) to obtain a hologram of microalgae cells; the distance between the microfluidic chip (4) and the image acquisition component (5) is smaller than the distance between the light transmission component (3) and the microfluidic chip (4), so that the field of view of the acquired hologram of the microalgae cell is larger than that of the microscope;
the microfluidic chip (4) comprises a polydimethylsiloxane sheet (7) and a glass slide (8), wherein a detection region (11) is sequentially concavely etched on the polydimethylsiloxane sheet (7), two ends of the detection region are symmetrically connected with a first focusing channel (10) and a second focusing channel (12), the width of the other end of the first focusing channel (10) is gradually increased until the other end is connected with one end of a first channel (9) in an equal-width mode, a liquid inlet hole is formed in the other end of the first channel, the width of the other end of the second focusing channel (12) is gradually increased until the other end is connected with one end of a second channel (13) in an equal-width mode, and a waste liquid hole is formed in the other end of the second channel (13);
when the micro-fluidic chip works, the image acquisition component (5) acquires a holographic pattern generated by a detection area of the micro-fluidic chip (4) under the action of a light beam, and the holographic pattern is sent to the image processing component (6) connected with the image acquisition component (5) for image analysis to obtain the ratio of the total gray value of the holographic image formed by the microalgae cells to the difference value of the bright spot at the center in the holographic image formed by the microalgae cells and the dark ring gray value at the outer ring of the bright spot;
the total gray value represents the brightness information of the holographic image, and the activity of the microalgae is judged according to the ratio of the total gray value of the holographic image formed by the microalgae cells to the difference value of the gray value of the bright spot at the center in the holographic image and the gray value of the dark ring at the outer ring of the bright spot.
2. The method for detecting cell activity based on the ratio of the total gray scale value to the gray scale difference of the holographic fringes, which is realized based on the device for detecting cell activity based on the ratio of the total gray scale value to the gray scale difference of the holographic fringes in claim 1, is characterized by comprising the following steps:
step S1: dropwise adding the sample solution, and adding the microalgae cell sample solution into a liquid inlet hole of the microfluidic chip; the liquid sample flows to the detection channel along the first channel through the first focusing channel under the action of self tension, and redundant liquid sample flows into the waste liquid hole;
step S2: the starting device is used for turning on a switch of the light-emitting diode and connecting the image acquisition assembly with the image processing assembly;
step S3: part of coherent light emitted by the light emitting diode is changed into spherical waves through the light through hole assembly, and the spherical waves are irradiated on a sample in the detection area of the microfluidic chip through the light transmission assembly to form a holographic image and are collected by the image collection assembly;
step S4: the holographic pattern collected by the image collecting assembly is transmitted to the image processing assembly through a data line;
step S5: and judging the activity of the microalgae cells according to the pattern characteristics of the microalgae cells in the holographic pattern, wherein the pattern characteristics of the microalgae cells in the holographic pattern are the ratio of the total gray value of the holographic image formed by the microalgae cells to the difference value of the bright spot at the center in the holographic image and the dark ring gray value at the outer ring of the bright spot.
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| CN110487221B (en) * | 2019-08-20 | 2021-05-25 | 大连海事大学 | Micro-plastic detection device and method for area-to-size ratio of central bright spot of holographic stripe |
| CN110487207B (en) * | 2019-08-20 | 2021-05-25 | 大连海事大学 | Micro-plastic detection device and method based on three-dimensional outline chain code and bright spot perimeter ratio |
| CN114015741B (en) * | 2021-11-08 | 2024-01-30 | 中山大学 | Non-invasive cell activity analysis method |
| CN114136935A (en) * | 2021-11-10 | 2022-03-04 | 山东交通学院 | Integrated ship ballast water biological activity detector |
| CN118033071B (en) * | 2024-01-24 | 2024-09-13 | 广东海洋大学 | Aquaculture water quality monitoring system based on artificial intelligence |
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