CN103146800A - Full-automatic cell non-staining image recognizing and counting method - Google Patents
Full-automatic cell non-staining image recognizing and counting method Download PDFInfo
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Abstract
The invention discloses a full-automatic cell non-staining image recognizing and counting method. The full-automatic cell non-staining image recognizing and counting method comprises the following steps of: selecting a full-automatic cell counting meter through an image recognition method, adding a cell suspension to be detected into a counting sheet directly, and inserting the cell suspension to be detected into a counting meter; lowering the receiving capability of a light sensing chip to the light intensity of a red light R and a green light G, intensifying the receiving capability of the light sensing chip to the light intensity of a blue light B, and regulating the aeration time and gain manually so as to change the background of a display to be light blue, and take round hot spots as images of active cells, and take the round blue spots darker than the background color as images of dead cells; and analyzing through cell image recognition software, thereby obtaining accurate large-small cell activity analysis data. According to the full-automatic cell non-staining image recognizing and counting method, dyes are not required to be used for counting cell activities; latent toxicity brought to cells to be detected by trypan blue is prevented; detected cells can continue to live, and the activities of the cells are counted during the culturing process; and meanwhile, according to the full-automatic cell non-staining image recognizing and counting method provided by the invention, non-staining real-time on-line cell counting can be achieved, and the method can be widely applied to the field of life science research.
Description
Technical field
The present invention relates to a kind of detection method, relate in particular to a kind of cell quantity and the active non-dyeing pattern recognition of full-automatic cell method of counting of detecting.
Background technology
Must keep certain concentration just can have when cultivating due to cell active preferably, therefore need repeatedly to carry out cell counting according to practical situation in the recovery of cell, cultivation, frozen process; At present, method for cell count commonly used comprises automated graphics method of identification and electric impedance counting process.
The electric impedance counting process is: to detect resistance change that the cell that suspends in electrolyte solution causes as the basis by counting channel the time, carry out the mensuration of cell counting and volume, this method is called electrical impedance method, also referred to as Coulter principle.
The electrical impedance method shortcoming: 1, the internal diameter due to the aperture pipe in electrical impedance method only has 25 μ m, therefore at present the instrument that adopts electric impedance all can't be counted such as the mankind's ovum diameter reaches 100 μ m greater than the cell of 25 μ m diameter, 2, agglomerate, the impurity agglomerate in diluent and doublet cell or a plurality of cell aggregation of cell debris formation, all can affect Cytometric accuracy, 3, the count signal of cell and undesired signal are exaggerated in amplification process simultaneously, have affected greatly the reliability of result; 4, in order to make instrument can accurately screen cell, appliance requires carries out regular Quality Control, setting threshold, complex operation; 5, detect simultaneously the existence of electric field, also can produce detrimentally affect to the active of cell and cultivation more in the future.
The pattern-recognition method principle is: according to the cell of existing state, cytolemma is complete, and the staining agent trypan blue can not penetrate the cytolemma of viable cell, can only make cytolemma dyeing but the cell interior structure can not be colored.And the cell after death, its cytolemma loses integrity, and trypan blue just can pass the cytolemma of dead cell, makes to be dispersed in intracytoplasmic nucleus after cell cytosol and nuclear fragmentation to be dyed to blueness, thereby is able to survival or the dead state of indirect identification of cell.At microscopically, the viable cell imaging features is: the cell after dyeing, and there is around cytolemma a circle blue, the cell centre zone is bright, is rendered as one bright circular; The image of dead cell is rendered as can distinguish with background color circle or the elliptical spot of the blueness that comes.Amplify by the cell image of optical system with the specific region of known sample volume on cell counting count board, the appliance computer image recognition software grasps by figure, the parameters such as the circularity of analysis image, diameter, color gray scale, can automatically calculate viable cell concentrations in tally (quantity/milliliter), dead cell concentration (quantity/milliliter), total cellular score (containing dead cell and viable cell) value, the data such as viable cell ratio, and can store in instrument or transfer to other computer-readable storage mediums by USB flash disk etc.
But the shortcoming of pattern-recognition method: 1, in these class methods, dyestuff commonly used is the core dyestuff, and viable cell is had certain toxicity; 2, can only realize the off-line counting, cell must break away from culture system can be counted, and can not realize that the real-time online counting does not namely break away from culture system; 3, it is out of use needing the cell of follow-up cultivation propagation for embryonic cell.4, can't judge activity and the quantity with endocytosis cell, and the cell that is in the apoptosis process middle and later periods, this moment, cell entered irreversible dead program, but still refused to dye trypan blue.Therefore there is larger limitation in traditional cell cell counting of carrying out again pattern recognition after dyed.
Summary of the invention
that carries out in order to solve traditional cell that the cell counting of pattern recognition exists after dyed produces toxicity to viable cell again, can't on-line counting, can't carry out follow-up cultivation propagation and can't judge activity with endocytosis cell and the problem of quantity the embryonic cell after dyeing, the purpose of this invention is to provide the non-dyeing pattern recognition of a kind of full-automatic cell method of counting, the method is avoided the genotoxic potential that uses trypan blue to bring, can not only carry out the activity counting to all size cell, and can be to using cell such as the sexual cell of Trypan Blue, embryonic cell carries out the activity counting, simultaneously also can easily realize accurate on-line counting.
Technical scheme of the present invention realizes in the following manner:
The non-dyeing pattern recognition of a kind of full-automatic cell method of counting, the method is selected pattern-recognition method full-automatic cell calculating instrument, and this calculating instrument structure comprises pointolite, convex lens, count slice, focusing object lens, spectral filter, sensitive chip, data line, controller, image knowledge software, indicating meter;
Operation steps is as follows:
Get cell suspension to be measured with pipettor, directly add in count slice and insert calculating instrument;
Open the parameter setting of sensitive chip;
First do white balance, record all parameter values after white balance, with these parameter values as basic value;
Reduce ruddiness R gain, green glow G gain, namely reduce sensitive chip to the receiving capability of ruddiness R, green glow G light intensity, instrument transferred to optimum value and was made as default value this moment;
Increase the gain of blue light B, namely strengthened the receiving capability of sensitive chip to blue light B light intensity, the increase amplitude is 1 to 2 times of basic value, and calculating instrument transferred to optimum value and was made as default value this moment;
This moment, display background was light blue, the light planoconvex lens that pointolite sends, count slice, focusing object lens, colour filter, sensitive chip convert optical signal to electrical signal, and be transferred to controller through data line, the final background that shows on indicating meter is light blue, and instrument transferred to optimum value and was made as default value this moment;
Spectral filter in this moment calculating instrument light path has stoped the interference of ruddiness R, green glow G light, only allows blue light B light to pass through, and the blue light B light intensity of at this moment viable cell reflection is greater than the blue light B light intensity of dead cell reflection;
Turn off automatic exposure function, manual regulation time shutter and exposure gain, obviously distinguish with can checkmate cell image and background and be as the criterion, instrument transferred to optimum value and was made as default value this moment, concrete control method: the blue light B of viable cell reflection is large because of intensity, at first imaging on sensitive chip, indicating meter can be seen the circular locus coeruleus darker than background color gradually; Continuing increases the time shutter, because the time shutter is long, the overexposure phenomenon occurs, and the dark circular locus coeruleus of ratio background color that first occurs becomes circular speck, has simultaneously the dark circular locus coeruleus of new ratio background color and occurs; At this moment, the background of indicating meter is light blue, and circular speck is the image of viable cell, and is exactly the image of dead cell than the circular locus coeruleus of background blue color depth, by the cell image recognition software analysis, obtains accurately to all size cytoactive analytical data.
Described all parameter values that record after white balance: comprise ruddiness R gain, green glow G gain, blue light B gain, gamma value, contrast gradient, auto color gain, automatic exposure value.
Described reduction sensitive chip is during to the receiving capability of ruddiness R, green glow G light intensity, its value be reduced to basic value 1/3 to 2/3 between.
Principle of the present invention is: little to the refractive index of light according to viable cell, luminous energy absorbs few, and the light intensity of reflection is large; Dead cell is large to the refractive index of light, and luminous energy absorbs many, the principle that the light intensity of reflection is little.Under same white light source irradiation, the light intensity of dead cell and viable cell reflection is different, and the light intensity of viable cell reflection is greater than dead cell.
Positively effect of the present invention is:
Need not to use dyestuff to carry out the activity counting to cell, avoid the genotoxic potential that uses Trypan Blue to bring to cell to be detected, cell after detection can be survived, especially to such as embryonic cell, the more precious cell of stem cell, carry out the activity counting in culturing process, have important practical value.This invention simultaneously can realize the cell counting of non-dyeing real-time online, can be widely used in the life science such as cell, embryo, reproduction field.
Figure of description:
Fig. 1 is the pattern-recognition method full-automatic cell calculating instrument structural representation that the present invention adopts;
Fig. 2 is that the parameter of sensitive chip arranges table;
Dead cell and the viable cell picture of Fig. 3 for adopting the inventive method to take.
Embodiment
The invention will be further described below in conjunction with drawings and Examples:
Embodiment one
As seen from Figure 1: the present invention selects pattern-recognition method full-automatic cell calculating instrument, and this calculating instrument structure comprises pointolite 1, convex lens 2, count slice 3, focusing object lens 4, spectral filter 5, sensitive chip 6, data line 7, controller 8, image knowledge software 9, indicating meter 10; Its operation steps is as follows:
At first get cell suspension 10ul to be measured with pipettor, the cell suspension of non-dyeing is directly added in 3 count slices and inserts calculating instrument;
As seen from Figure 2: the parameter setting of opening sensitive chip 6 has transferred to optimum value and has been made as default value;
First do white balance, record all parameter values after white balance: comprise ruddiness R gain, green glow G gain, blue light B gain, gamma value, contrast gradient, auto color gain, automatic exposure value, with these parameter values as basic value;
Reduce the gain of ruddiness R, green glow G, make ruddiness R, green glow G reduce to basic value 1/3 to 2/3 between, namely reduced sensitive chip 6) to the receiving capability of ruddiness R, green glow G light intensity, calculating instrument transferred to optimum value and was made as default value this moment;
Increase the gain of blue light B, be increased to 1 to 2 times of basic value, namely strengthened the receiving capability of 6 pairs of blue light B light intensities of sensitive chip, calculating instrument transferred to optimum value and was made as default value this moment;
This moment, indicating meter 10 backgrounds were light blue: the light planoconvex lens 2 that pointolite 1 sends, count slice 3, focusing object lens 4, colour filter 5, sensitive chip 6 convert optical signal to electrical signal, and be transferred to controller 8 through data line 7, the final background that shows on indicating meter 10 is light blue, and instrument transferred to optimum value and was made as default value this moment;
Spectral filter 5 in the instrument light path that counts this moment has stoped the interference of ruddiness R, green glow G, only allows blue light B to pass through, and the blue light B intensity of at this moment viable cell reflection is greater than the blue light B intensity of dead cell reflection;
Turn off automatic exposure function, manual regulation time shutter and exposure gain, the sensitive chip 6 of different sorts and manufacturer production needs the scope of adjusting different, with can checkmate cell image and background obviously distinguish be as the criterion this moment calculating instrument transferred to optimum value and be made as default value, concrete control method: the blue light B of viable cell reflection is large because of intensity, at first imaging on sensitive chip 6, indicating meter 10 can be seen the circular locus coeruleus darker than background color gradually; Continue to increase the time shutter, the dark circular locus coeruleus of ratio background color that first occurs becomes circular speck, because the time shutter is long, the overexposure phenomenon appears, and have simultaneously the dark circular locus coeruleus of new ratio background color and occur, as seen from Figure 3: at this moment, the background of indicating meter 10 is light blue, and circular speck is the image of viable cell, and is exactly the image of dead cell than the circular locus coeruleus of background blue color depth, analyze by cell image recognition software 9, draw dead cell concentration: 1 * 10
5, viable cell concentrations: 2 * 10
6
Simultaneous test
Use pipettor obtained cell suspension 50ul respectively, add A pipe and B pipe; The B pipe is added the rear mixing of 50ul trypan blue (0.4%);
Select with embodiment in pattern-recognition method full-automatic cell calculating instrument, this pattern-recognition method full-automatic cell calculating instrument is provided with two function digits, i.e. " non-dyeing " function counting " 1 " position, " dyeing " function counting " 2 ";
Get from the B pipe count slice that 10ul adds pattern-recognition method full-automatic cell calculating instrument, use " dyeing " function to count " 2 " position, by traditional pattern-recognition method step operation.
Get from the A pipe count slice that 10ul adds pattern-recognition method full-automatic cell calculating instrument, use " non-dyeing " function to count " 1 " position, press embodiment one operation steps operation.
Result shows that two kinds of method count results errors are within 5%.
Claims (5)
1. the non-dyeing pattern recognition of full-automatic cell method of counting, the method is selected pattern-recognition method full-automatic cell calculating instrument, and this calculating instrument structure comprises pointolite (1), convex lens (2), count slice (3), focusing object lens (4), spectral filter (5), sensitive chip (6), data line (7), controller (8), image recognition software (9), indicating meter (10); It is characterized in that operation steps is as follows:
Get cell suspension to be measured with pipettor, directly add in count slice (3) and insert calculating instrument;
Open the parameter setting of sensitive chip (6);
Reduce ruddiness R gain, green glow G gain, namely reduce sensitive chip (6) to the receiving capability of R, G light intensity, instrument transferred to optimum value and was made as default value this moment;
Increase the gain of blue light B, namely strengthened the receiving capability of sensitive chip (6) to the B light intensity;
This moment, indicating meter (10) background was light blue, the light planoconvex lens (2) that pointolite (1) sends, count slice (3), focusing object lens (4), colour filter (5), sensitive chip (6) convert optical signal to electrical signal, and be transferred to controller (8) through data line (7), be finally light blue in the upper background that shows of indicating meter (10), instrument transferred to optimum value and was made as default value this moment;
Spectral filter (5) in this moment calculating instrument light path has stoped the interference of R, G light, only allows B light to pass through, and the B light intensity of at this moment viable cell reflection is greater than the B light intensity of dead cell reflection;
Turn off automatic exposure function, manual regulation time shutter and exposure gain, obviously distinguish with can checkmate cell image and background and be as the criterion, instrument transferred to optimum value and was made as default value this moment, concrete control method: the blue light B of viable cell reflection is large because of intensity, at first in the upper imaging of sensitive chip (6), indicating meter (10) can be seen the circular locus coeruleus darker than background color gradually; Continuing increases the time shutter, because the time shutter is long, the overexposure phenomenon occurs, and the dark circular locus coeruleus of ratio background color that first occurs becomes circular speck, has simultaneously the dark circular locus coeruleus of new ratio background color and occurs; At this moment, the background of indicating meter (10) is light blue, and circular speck is the image of viable cell, and is exactly the image of dead cell than the circular locus coeruleus of background blue color depth, by cell image recognition software analysis (9), obtain accurately to all size cytoactive analytical data.
2. the non-dyeing pattern recognition of full-automatic cell according to claim 1 method of counting is characterized in that: sensitive chip (6) is 1 to 2 times of basic value to the receiving capability increase amplitude of B light intensity.
3. the non-dyeing pattern recognition of full-automatic cell according to claim 2 method of counting, it is characterized in that: the parameter of opening sensitive chip (6) arranges the rear white balance of first doing, and records all parameter values after white balance, with these parameter values as basic value.
4. the non-dyeing pattern recognition of full-automatic cell according to claim 3 method of counting is characterized in that: described all parameter values that record after white balance: comprise ruddiness R gain, green glow G gain, blue light B gain, gamma value, contrast gradient, auto color gain, automatic exposure value.
5. the non-dyeing pattern recognition of full-automatic cell according to claim 4 method of counting is characterized in that: described reduction sensitive chip (6) is during to the receiving capability of ruddiness R, green glow G light intensity, its value be reduced to basic value 1/3 to 2/3 between.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103436439A (en) * | 2013-09-06 | 2013-12-11 | 朱耀辉 | Calibration method for cell counter based on image identification method |
| CN105572017A (en) * | 2015-12-21 | 2016-05-11 | 深圳联开生物医疗科技有限公司 | Smart gain adjustment system and blood cell analyzer |
| CN108088781A (en) * | 2016-11-23 | 2018-05-29 | 上海迈泰君奥生物技术有限公司 | A kind of reagent for cell counter combines |
| CN109100286A (en) * | 2018-10-31 | 2018-12-28 | 江苏卓微生物科技有限公司 | cell counter |
| CN109825427A (en) * | 2019-02-25 | 2019-05-31 | 广州牛顿光学研究院有限公司 | A kind of full-automatic cell activity analysis system |
| CN113781378A (en) * | 2020-05-20 | 2021-12-10 | 广州博大博聚科技有限公司 | Absolute value counting method for obtaining quantity of micro cells in cell suspension |
| CN114004851A (en) * | 2021-11-26 | 2022-02-01 | 广州市艾贝泰生物科技有限公司 | Cell image segmentation method and device and cell counting method |
| CN114067315A (en) * | 2021-10-23 | 2022-02-18 | 广州市艾贝泰生物科技有限公司 | Cell counting method, cell counting device, computer device, and storage medium |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436439B (en) * | 2013-09-06 | 2014-12-17 | 朱耀辉 | Calibration device for cell counter based on image identification method |
| CN103436439A (en) * | 2013-09-06 | 2013-12-11 | 朱耀辉 | Calibration method for cell counter based on image identification method |
| CN105572017A (en) * | 2015-12-21 | 2016-05-11 | 深圳联开生物医疗科技有限公司 | Smart gain adjustment system and blood cell analyzer |
| CN105572017B (en) * | 2015-12-21 | 2018-07-06 | 深圳联开生物医疗科技有限公司 | A kind of Intelligent Gain regulating system and cellanalyzer |
| CN108088781B (en) * | 2016-11-23 | 2020-07-21 | 上海迈泰君奥生物技术有限公司 | Reagent combination for cell counter |
| CN108088781A (en) * | 2016-11-23 | 2018-05-29 | 上海迈泰君奥生物技术有限公司 | A kind of reagent for cell counter combines |
| CN109100286A (en) * | 2018-10-31 | 2018-12-28 | 江苏卓微生物科技有限公司 | cell counter |
| CN109825427A (en) * | 2019-02-25 | 2019-05-31 | 广州牛顿光学研究院有限公司 | A kind of full-automatic cell activity analysis system |
| CN113781378A (en) * | 2020-05-20 | 2021-12-10 | 广州博大博聚科技有限公司 | Absolute value counting method for obtaining quantity of micro cells in cell suspension |
| CN114067315A (en) * | 2021-10-23 | 2022-02-18 | 广州市艾贝泰生物科技有限公司 | Cell counting method, cell counting device, computer device, and storage medium |
| CN114067315B (en) * | 2021-10-23 | 2022-11-29 | 广州市艾贝泰生物科技有限公司 | Cell counting method, cell counting device, computer device, and storage medium |
| CN114004851A (en) * | 2021-11-26 | 2022-02-01 | 广州市艾贝泰生物科技有限公司 | Cell image segmentation method and device and cell counting method |
| CN114004851B (en) * | 2021-11-26 | 2022-11-29 | 广州市艾贝泰生物科技有限公司 | Cell image segmentation method and device and cell counting method |
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Application publication date: 20130612 Assignee: Shenzhen Boda Technology Co., Ltd. Assignor: Zhu Yaohui|Yu Haibin|Zhao Guang Contract record no.: 2014440020354 Denomination of invention: Full-automatic cell non-staining image recognizing and counting method Granted publication date: 20140813 License type: Exclusive License Record date: 20141031 |
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Effective date of registration: 20200605 Address after: 510000 Room 401, building 2, self compiled, No.188 Kaiyuan Avenue, Huangpu District, Guangzhou City, Guangdong Province Patentee after: Guangzhou Boda Boju Technology Co.,Ltd. Address before: 509, room 450008, Henan Star building, 23 East agricultural road, Henan, Zhengzhou Co-patentee before: Yu Haibin Patentee before: Zhu Yaohui Co-patentee before: Zhao Guang |