CN109852541A - 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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- CN109852541A CN109852541A CN201811499611.XA CN201811499611A CN109852541A CN 109852541 A CN109852541 A CN 109852541A CN 201811499611 A CN201811499611 A CN 201811499611A CN 109852541 A CN109852541 A CN 109852541A
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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 present invention relates to the technical fields that the microalgae in a kind of pair of ballast water for ship carries out active differentiation, specifically, especially
It is related to the cytoactive detection apparatus and method based on the total gray value of hologram fringe Yu gray scale difference ratio.
Background technique
Ballast water for ship refers to as control ship heel, trim, drinking water, stability or stress and is loaded into the water on ship and suspension
Substance.It can be adapted to rapidly in ballast water for ship containing a large amount of biology, including planktonic organism, microorganism, bacterium, these alien species
Environment expands population, to cause harm to environment.Ocean is raw between the discharge of ballast water for ship causes rationality isolation water body
The main path that object and pathogen are propagated, what ballast water for ship was got worse destroys marine organisms chain.As international carriage produces
The continuous enhancing that industry goes from strength to strength and people realize marine environmental protection, it is external brought by the discharge of ballast water for ship
Marine biological invasion problem causes the extensive concern of various circles of society.Recognize unwanted aquatic organism and pathogen in ballast water for ship
Trans-regional transfer cause very important influence to global ocean ecological environment, Global Environment Facility organizes (GEF)
It has been classified as and has endangered one of four big threats of ocean.For the invasion for preventing adventive in ballast water, International Maritime Organization
The Management of Ships ' Ballast Water management conference held in 2004 has passed through " Management of Ships ' Ballast Water and its sediment monitoring and management
Pact " hereinafter referred to as compressive effect, wherein being 30 for accounting for world merchant shipping tonnage 35% for the clause regulation that pact comes into force
It comes into force after 12 months of state approval.
Occupy very big ratio with microalgae phytoplankton in the water-borne alien species of ballast, microalgae invasion exists
Huge economy and environmental loss are caused in world wide.Endure the harm of red tide algae to the fullest extent all over the world in recent years, red tide algae is big
Part is toxic and be easy to cause the death of birds and fish, greatly endangers to environment and economy-zone, so in ballast water
The Activity determination of frustule just becomes of crucial importance.
The method of algae cell activity detection at present has optical microscopy, Flow cytometry method, dye fluorescence micro-
Mirror counting method, molecule and biochemical method etc..Optical microscopy observes the form of microalgae cell by professional technician come area
Divide frustule, and algae cell activity is judged by the dyeing of frustule.Microscope is expensive, and volume is big, not easy to operate, no
It is suitble to the quick detection of microalgae cell.Flow cytometry needs with fluorochrome label microalgae and is made into suspension, passes through
Fluorescence signal and the scattered signal of frustule are measured to judge that the activity of microalgae cell, this method have high-efficient, accurate
Advantage, but commercial flow cytometer is expensive, bulky, sample treatment is cumbersome, it is complicated for operation.
Summary of the invention
The quick detection for being not suitable for scene according to activity of microalgae detection method in spot ship ballast water set forth above, has
The disadvantages of volume is not big, expensive, easy to operate, activity of microalgae detection method and device are still to be badly in need of solution in ballast water for ship
Certainly the technical issues of, and a kind of cytoactive detection device based on the total gray value of hologram fringe and gray scale difference ratio and side are provided
Method.
The technological means that the present invention uses is as follows:
Cytoactive detection device based on the total gray value of hologram fringe Yu gray scale difference ratio characterized by comprising according to
Light source assembly, light passing aperture member, light propagation component, micro-fluidic chip, the image collection assembly, image processing modules of secondary connection；
The light source assembly includes power supply power supply device, light source fixing structure and fits closely with the light passing aperture member
Light emitting diode, the light beam that the light emitting diode issues becomes spherical wave through the light passing aperture member, and passes through the light
It propagates component to irradiate the micro-fluidic chip and be imaged by image collection assembly, obtains the hologram of microalgae cell；
The micro-fluidic chip includes dimethyl silicone polymer piece and glass slide, and the dimethyl silicone polymer piece is successively recessed
It is carved with detection zone, the detection zone both ends symmetrically connect the first focus channel and the second focus channel, and described first focuses
The other end width in channel is cumulative until wide be connected to first passage one end, and the first passage other end is equipped with inlet opening,
The second focus channel other end width is cumulative until wide be connected to second channel one end, and the second channel other end is set
There is waste liquid hole；
When work, described image acquisition component acquires the hologram that micro-fluidic chip detection zone generates under light beam effect
Sample, the holographic pattern are sent to the image processing modules being connected with described image acquisition component and carry out image analysis, obtain
The speck in bosom and described in the hologram image that is formed with microalgae cell of total gray value for the hologram image that microalgae cell is formed
The ratio of the Crape ring gray value difference of speck outer ring.
Further, the micro-fluidic chip is far smaller than light propagation component to micro- at a distance from described image acquisition component
The distance of fluidic chip.
Further, the light beam that the light emitting diode issues is partially coherent light.
Further, total gray value represents the luminance information of hologram image, the hologram formed by microalgae cell
Total gray value of picture is sentenced with the speck in bosom in hologram image and the ratio of the Crape ring gray value difference of the speck outer ring
The activity of disconnected microalgae.
The present invention also provides the cytoactive detection methods based on the total gray value of hologram fringe Yu gray scale difference ratio, including
Following steps:
Step S1: sample liquid is added dropwise, microalgae cell sample liquid is added in the inlet opening of micro-fluidic chip；Liquid sample is certainly
Pass through the first focus channel flow detection channel along first passage under the action of body tension, surplus liquid sample flows into waste liquid hole
In；
Step S2: starter opens the switch of light emitting diode, by image collection assembly and image processing modules phase
Even；
Step S3: the partially coherent light that light emitting diode issues becomes spherical wave by light passing aperture member, by light propagation
Component, which is radiated on the sample of micro-fluidic chip detection zone, to be formed hologram image and is acquired by image collection assembly；
Step S4: the holographic pattern of image collection assembly acquisition is transmitted in image processing modules through data line；
Step S5: microalgae cell activity is judged according to the microalgae cell drawing feature in holographic pattern.
Further, the microalgae cell drawing feature in the holographic pattern is the total of the hologram image that microalgae cell is formed
The ratio of the Crape ring gray value difference of the speck and speck outer ring in bosom in gray value and hologram image.
Compared with the prior art, the invention has the following advantages that
A kind of cytoactive detection method based on the total gray value of hologram fringe and gray scale difference ratio provided by the invention is suitable
The quick detection for closing scene, the cytoactive detection device based on the total gray value of hologram fringe and gray scale difference ratio have volume
Small, cheap, easy-operating feature has important scientific meaning and realistic price for environmental science.
The present invention can be widely popularized carrying out the fields such as active differentiation to the microalgae in ballast water for ship based on the above reasons.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to do simply to introduce, it should be apparent that, the accompanying drawings in the following description is this hair
Bright some embodiments for those of ordinary skill in the art without any creative labor, can be with
It obtains other drawings based on these drawings.
Fig. 1 is activity of microalgae structure of the detecting device schematic diagram in ballast water for ship；
Fig. 2 is microfluidic chip structure schematic diagram of the present invention；
Fig. 3 is that the present apparatus carries out activity of microalgae detection method flow chart of steps.
In figure: 1, light source assembly；2, light passing aperture member；3, light propagation component；4, micro-fluidic chip；5, Image Acquisition group
Part；6, image processing modules；7, dimethyl silicone polymer piece；8, glass slide；9, first passage；10, the first focus channel；11,
Sense channel；12, the second focus channel；13, second channel；A, inlet opening；B, waste liquid hole.
Specific embodiment
It should be noted that in the absence of conflict, the feature in embodiment and embodiment in the present invention can phase
Mutually combination.The present invention will be described in detail below with reference to the accompanying drawings and embodiments.
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is only
It is only a part of the embodiment of the present invention, instead of all the embodiments.It is real to the description of at least one exemplary embodiment below
It is merely illustrative on border, never as to the present invention and its application or any restrictions used.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
Unless specifically stated otherwise, positioned opposite, the digital table of the component and step that otherwise illustrate in these embodiments
It is not limited the scope of the invention up to formula and numerical value.Simultaneously, it should be clear that for ease of description, each portion shown in attached drawing
The size divided not is to draw according to actual proportionate relationship.Technology known for person of ordinary skill in the relevant, side
Method and equipment may be not discussed in detail, but in the appropriate case, and the technology, method and apparatus should be considered as authorizing explanation
A part of book.In shown here and discussion all examples, appointing should be construed as merely illustratively to occurrence, and
Not by way of limitation.Therefore, the other examples of exemplary embodiment can have different values.It should also be noted that similar label
Similar terms are indicated in following attached drawing with letter, therefore, once it is defined in a certain Xiang Yi attached drawing, then subsequent attached
It does not need that it is further discussed in figure.
In the description of the present invention, it is to be understood that, the noun of locality such as " front, rear, top, and bottom, left and right ", " it is laterally, vertical,
Vertically, orientation or positional relationship indicated by level " and " top, bottom " etc. is normally based on orientation or position shown in the drawings and closes
System, is merely for convenience of description of the present invention and simplification of the description, in the absence of explanation to the contrary, these nouns of locality do not indicate that
It must have a particular orientation or be constructed and operated in a specific orientation with the device or element for implying signified, therefore cannot manage
Solution is limiting the scope of the invention: the noun of locality " inside and outside " refers to inside and outside the profile relative to each component itself.
For ease of description, spatially relative term can be used herein, as " ... on ", " ... top ",
" ... upper surface ", " above " etc., for describing such as a device shown in the figure or feature and other devices or spy
The spatial relation of sign.It should be understood that spatially relative term is intended to comprising the orientation in addition to device described in figure
Except different direction in use or operation.For example, being described as if the device in attached drawing is squeezed " in other devices
It will be positioned as " under other devices or construction after part or construction top " or the device of " on other devices or construction "
Side " or " under its device or construction ".Thus, exemplary term " ... top " may include " ... top " and
" in ... lower section " two kinds of orientation.The device can also be positioned with other different modes and (is rotated by 90 ° or in other orientation), and
And respective explanations are made to the opposite description in space used herein above.
In addition, it should be noted that, limiting components using the words such as " first ", " second ", it is only for be convenient for
Corresponding components are distinguished, do not have Stated otherwise such as, there is no particular meanings for above-mentioned word, therefore should not be understood as to this
The limitation of invention protection scope.
Since the hologram image that the different microalgae cell of activity is formed is different, the holographic pattern feature formed is different, root
According to above-mentioned principle, the present invention relates to cytoactive detection methods and dress based on the total gray value of hologram fringe and gray scale difference ratio
It sets, the present invention is further described with reference to the accompanying drawing.
Embodiment 1
As shown in Figure 1, the present invention provides a kind of cell activity based on hologram fringe total gray value and gray scale difference ratio
Detection device, comprising: sequentially connected light source assembly 1, light passing aperture member 2, light propagation component 3, micro-fluidic chip 4, image are adopted
Collect component 5, image processing modules 6.Light source assembly 1 include power supply power supply device, light source fixing structure and with the light hole
The light emitting diode that component 2 fits closely, the partially coherent light that light emitting diode issues become spherical wave through light passing aperture member 2,
And be irradiated on micro-fluidic chip 4 and be imaged by image collection assembly 5 by light propagation component 3, obtain the hologram of frustule.
Image collection assembly 5 acquires the holographic pattern that 4 detection zone of micro-fluidic chip generates under light beam effect, this holographic pattern is sent
Image analysis is carried out to the image processing modules 6 being connected with image collection assembly 5, obtains the total of the hologram image of microalgae formation
The Crape ring gray value difference of the speck in bosom and the speck outer ring in sum of the grayscale values hologram image.Total gray value represents holography
The luminance information of image, total gray value of hologram image and the speck in bosom in hologram image formed by microalgae and described
The ratio of the Crape ring gray value difference of speck outer ring judges the activity of microalgae.Light emitting diode issued section phase in light source assembly
Noise can be effectively reduced in dry light.Micro-fluidic chip 4 is far smaller than light propagation component 3 to micro- at a distance from image collection assembly 5
The distance of fluidic chip 4, so that the hologram visual field of collected microalgae cell is more much larger than microscopical visual field.
As shown in Fig. 2, micro-fluidic chip 4 includes dimethyl silicone polymer piece 7 and glass slide 8, dimethyl silicone polymer piece)
Successively intaglio has detection zone 11, and 11 both ends of detection zone symmetrically connect the first focus channel 10 and the second focus channel 12, the
The other end width of one focus channel 10 is cumulative until wide be connected to first passage 9, the first passage other end be equipped with into
Fluid apertures, 12 other end width of the second focus channel is cumulative until wide be connected to 1 one end of second channel, 13 other end of second channel
Equipped with waste liquid hole；
Microalgae cell sample when in use, is placed on micro-fluidic chip first, micro-fluidic chip is set by apparatus of the present invention
In objective table, the partially coherent light that light emitting diode issues is radiated at and is placed on objective table by light hole and light propagation component
Sample on the diffraction image that is formed and acquired by image collection assembly.The device selects light emitting diode as light source, light source
The light that light emitting diode issues in component is partially coherent light, can effectively inhibit relevant speckle noise and interference.Light source group
The partially coherent light that LED light source in part issues is dissipated by diverging to a branch of spherical wave after the light hole in light passing aperture member
The spherical wave of formation travels to sample surface by suitable propagation distance.The device is using the branch light transmission across sample as reference
Light, without in addition introducing reference light.Sample is very close at a distance from charge coupled cell in image collection assembly (cmos), distance
At several millimeters or so, partially coherent light is radiated at the holographic pattern formed on sample and is acquired by image collection assembly, finally will
Collected hologram image is handled by image processing modules.
The hologram image that the different microalgae cell of activity is formed is different, distinguishes micro- by judging some features of hologram
The active situation of frustule.
The speck in bosom and the Crape ring gray value of speck outer ring are variant in the hologram that microalgae cell is formed, by dividing
Analyse the dark of total gray value of hologram image and the speck in bosom in hologram image that microalgae cell is formed and the speck outer ring
The ratio of ring gray value difference judges the activity of microalgae.
Embodiment 2
On the basis of embodiment 1, as shown in figure 3, the present invention also provides the activity of microalgae detections based on above-mentioned apparatus
Method, it is characterised in that: it includes the following steps:
Step S1: sample liquid is added dropwise, microalgae cell sample liquid is added in the inlet opening of micro-fluidic chip；Liquid sample is certainly
Pass through the first focus channel flow detection channel along first passage under the action of body tension, surplus liquid sample flows into waste liquid hole
In；
Step S2: starter opens the switch of light emitting diode, by image collection assembly and image processing modules phase
Even；
Step S3: the partially coherent light that light emitting diode issues becomes spherical wave by light passing aperture member, by light propagation
Component, which is radiated on the sample of micro-fluidic chip detection zone, to be formed hologram image and is acquired by image collection assembly；
Step S4: the holographic pattern of image collection assembly acquisition is transmitted in image processing modules through data line；
Step S5: microalgae cell activity is judged according to the microalgae cell drawing feature in holographic pattern；In holographic pattern
Microalgae cell drawing feature be the total gray value for the hologram image that microalgae cell is formed and the speck in bosom in hologram image and
The ratio of the Crape ring gray value difference of the speck outer ring.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (6)
1. the cytoactive detection device based on the total gray value of hologram fringe Yu gray scale difference ratio characterized by comprising successively
The light source assembly (1) of connection, light passing aperture member (2), light propagation component (3), micro-fluidic chip (4), image collection assembly (5),
Image processing modules (6)；
The light source assembly (1) includes power supply power supply device, light source fixing structure and closely pastes with the light passing aperture member (2)
The light emitting diode of conjunction, the light beam that the light emitting diode issues becomes spherical wave through the light passing aperture member (2), and passes through institute
It states light propagation component (3) to irradiate the micro-fluidic chip (4) and be imaged by image collection assembly (5), obtains the complete of microalgae cell
Breath figure；
The micro-fluidic chip (4) includes dimethyl silicone polymer piece (7) and glass slide (8), the dimethyl silicone polymer piece
(7) successively intaglio has detection zone (11), and the detection zone both ends symmetrically connect the first focus channel (10) and second and focus
Channel (12), the other end width of first focus channel (10) is cumulative until wide be connected to first passage (9) one end, institute
The first passage other end is stated equipped with inlet opening, the second focus channel (12) other end width is cumulative until wide be connected to the
Two channels (13) one end, second channel (13) other end are equipped with waste liquid hole；
When work, described image acquisition component (5) acquires the holography that micro-fluidic chip (4) detection zone generates under light beam effect
Pattern, the holographic pattern are sent to the image processing modules (6) being connected with described image acquisition component (5) and carry out image point
Analysis obtains the speck in bosom in total gray value of the hologram image of microalgae cell formation and the hologram image of microalgae cell formation
With the ratio of the Crape ring gray value difference of the speck outer ring.
2. the cytoactive detection device according to claim 1 based on the total gray value of hologram fringe Yu gray scale difference ratio,
It is characterized in that, the micro-fluidic chip (4) is far smaller than light propagation component (3) at a distance from described image acquisition component (5)
To the distance of micro-fluidic chip (4).
3. the cytoactive detection device according to claim 1 based on the total gray value of hologram fringe Yu gray scale difference ratio,
It is characterized in that, the light beam that the light emitting diode issues is partially coherent light.
4. the cytoactive detection device according to claim 1 based on the total gray value of hologram fringe Yu gray scale difference ratio,
It is characterized in that, total gray value represents the luminance information of hologram image, the hologram image that is formed by microalgae cell it is total
Gray value judges microalgae with the speck in bosom in hologram image and the ratio of the Crape ring gray value difference of the speck outer ring
Activity.
5. the cytoactive detection method based on the total gray value of hologram fringe Yu gray scale difference ratio, which is characterized in that including as follows
Step:
Step S1: sample liquid is added dropwise, microalgae cell sample liquid is added in the inlet opening of micro-fluidic chip；Liquid sample is opened at itself
Pass through the first focus channel flow detection channel along first passage under the action of power, surplus liquid sample flows into waste liquid hole；
Step S2: starter opens the switch of light emitting diode, and image collection assembly is connected with image processing modules；
Step S3: the partially coherent light that light emitting diode issues becomes spherical wave by light passing aperture member, by light propagation component
It is radiated on the sample of micro-fluidic chip detection zone and forms hologram image and acquired by image collection assembly；
Step S4: the holographic pattern of image collection assembly acquisition is transmitted in image processing modules through data line；
Step S5: microalgae cell activity is judged according to the microalgae cell drawing feature in holographic pattern.
6. the cytoactive detection method according to claim 5 based on the total gray value of hologram fringe Yu gray scale difference ratio,
It is characterized in that, the microalgae cell drawing feature in the holographic pattern is the total gray value for the hologram image that microalgae cell is formed
With the ratio of the Crape ring gray value difference of the speck and speck outer ring in bosom in hologram image.
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Cited By (5)
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| CN110487221A (en) * | 2019-08-20 | 2019-11-22 | 大连海事大学 | Hologram fringe central bright spot area and the micro- plastics detection device of dimension ratio and method |
| CN110487207A (en) * | 2019-08-20 | 2019-11-22 | 大连海事大学 | Micro- plastics detection device and method based on three-D profile chain code Yu speck perimeter ratio |
| CN114015741A (en) * | 2021-11-08 | 2022-02-08 | 中山大学 | Non-invasive microbial activity analysis method and system |
| CN114136935A (en) * | 2021-11-10 | 2022-03-04 | 山东交通学院 | An integrated marine ballast water biological activity detector |
| CN118033071A (en) * | 2024-01-24 | 2024-05-14 | 广东海洋大学 | Aquaculture water quality monitoring system based on artificial intelligence |
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