CN103436439A - Calibration method for cell counter based on image identification method - Google Patents
Calibration method for cell counter based on image identification method Download PDFInfo
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- CN103436439A CN103436439A CN201310399802XA CN201310399802A CN103436439A CN 103436439 A CN103436439 A CN 103436439A CN 201310399802X A CN201310399802X A CN 201310399802XA CN 201310399802 A CN201310399802 A CN 201310399802A CN 103436439 A CN103436439 A CN 103436439A
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- 238000000034 method Methods 0.000 title abstract description 12
- 210000004027 cell Anatomy 0.000 claims abstract description 178
- 230000004899 motility Effects 0.000 claims abstract description 5
- 238000005516 engineering process Methods 0.000 claims abstract description 4
- 239000010453 quartz Substances 0.000 claims abstract description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000012780 transparent material Substances 0.000 claims abstract description 3
- 238000012567 pattern recognition method Methods 0.000 claims description 14
- 238000001514 detection method Methods 0.000 claims description 6
- 210000000678 band cell Anatomy 0.000 claims description 4
- 230000002123 temporal effect Effects 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 229920003023 plastic Polymers 0.000 claims description 2
- 210000003527 eukaryotic cell Anatomy 0.000 abstract description 4
- 238000005259 measurement Methods 0.000 abstract description 4
- 210000002257 embryonic structure Anatomy 0.000 abstract 1
- 238000011160 research Methods 0.000 abstract 1
- 238000004458 analytical method Methods 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 238000011179 visual inspection Methods 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 238000004164 analytical calibration Methods 0.000 description 1
- 230000002380 cytological effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000016507 interphase Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000003909 pattern recognition Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
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Abstract
The invention discloses a calibration method for a cell counter based on an image identification method. According to the calibration method, a special imitation cell counting plate made of a transparent material is used as a calibration product for a cell counter; the imitation cell counting plate is prepared by a quartz photomask technology and is provided with imitation cells which can be identified by the cell counter, and the imitation cells have the diameters and the motility rates which are similar to those of eukaryotic cells. According to the calibration method for the cell counter based on the image identification method, the problem that the measurement accuracy of the cell counter is indirectly verified through manual counting of an existing cell counting plate method is solved. The imitation cell counting plate can truly reflect information, such as sizes, the concentration and the motility rates, of the eukaryotic cells and can be used for evaluating the precision of measurement of the cell counter under different conditions. The calibration method has an important calibration value and can be widely applied to the field of life science researches on cells, embryos, reproduction and the like and the clinical application.
Description
technical field
The present invention relates to a kind of Biomedical Instruments calibration steps, relate in particular to a kind of cell of the pattern recognition for detection of viable cell quantity gauge calibration steps.
Background technology
Cell counter, as a kind of metering outfit, need to regularly be calibrated to guarantee the precision of measuring.The calibration of cell counter is without standard substance at present, normally by applying unit, utilize homemade viable cell goods monitoring, but, due to condition restriction such as the kind of cell, culture condition, survival rate, suspension concentration Making and banking times, what is more important self-control cell quantity and active uncertainty in time make still can not well use a kind of biological model of viable cell Criterion for calibrating cell counter at present; Cell counter is calibrated at every turn and need to be prepared the accuracy that sample carries out the measurement of indirect verification cell counter with cell counting count board method artificial counting at present, but the precision in the time of can't estimating the different large minicells of cell counter mensuration and Cell viability, also can't estimate error and deviation in the cell counter system, so may there be larger deviation in the accuracy of cell counter both domestic and external and accuracy simultaneously.
Therefore " cell " model of the repeatability of production standard, and the calibration of using it for cell counter just seems particularly important.
Summary of the invention
Can't estimate cell counter measurement different large minicells without repeatably " cell " model of standard for calibration in order to solve current cell counter, precision when different cell concns and Cell viability and the problem that can't estimate error in the cell counter system, the purpose of this invention is to provide a kind of pattern-recognition method cell gauge calibration steps that adopts imitative cell calibration plate to carry out the cell counter calibration, the method tool is estimated the accuracy of cell counter when different cell concns and Cell viability and is estimated the systematic error function of cell counter, easy and simple to handle while having simultaneously, easily go.
Technical scheme of the present invention realizes in the following manner:
A kind of pattern-recognition method cell counter calibration steps, use the calibration object of the imitative cell counting count board of special-purpose transparent material as cell counter in calibration steps, its imitative cell counting count board is the imitative cell counting count board that adopts the preparation of quartz photomask technology, has the imitative cell similar with motility rate to eukaryotic diameter that can make cell counter identification.
Imitative cell counting count board is glass or transparent plastics material, and two same detection zones are arranged on it, and each surveyed area area is at least 3mm * 3mm; On imitative cell counting count board, surveyed area has two classes circles: a class is the imitative viable cell of transparent ring representative; Another kind of is the imitative dead cell of opaque filled circles representative.
The surveyed area of imitative cell counting count board is divided into 9 large zones by 1mm * 1mm, and the line width between large zone is 9um, and the line degree of depth is 4um.
Be divided in 16 zone by 0.25mm * 0.25mm in each large zone, the line width between middle zone is 6um, and the line degree of depth is 4um, in zone, 8 band cell area of the pattern and 8 white spaces, band cell pattern area and white space alternative arrangements is arranged in 16.
Each zone with the cell pattern is divided into 4 zonules by 0.125mm * 0.125mm, and between zone, live width is 4um, and the line degree of depth is 4um.
Each zone with the cell pattern is by from left to right, principle of temporal sequence mark Chinese character " "～" ten " sequence number marks respectively work or the dead imitative cell of the imitative cell transversely arranged work of ten row or dead and ten row longitudinal arrangements from top to bottom.
Each zonule centre compartment 25um * 25um, from small to large, order from left to right, principle is arranged from top to bottom for the two class circles that the imitative cell of two classes is 5um, 10um, 15um, 20um, 25um by diameter.
4 zonule cell patterns are divided into two imitative viable cell area of the pattern and two imitative dead cell area of the pattern, two imitative viable cell area of the pattern centrosymmetry, two imitative dead cell area of the pattern centrosymmetry, it is that the imitative viable cell of 25um, 3 diameters are that the imitative viable cell of 20um, 5 diameters are that the imitative viable cell of 15um, 7 diameters are that the imitative viable cell of 10um, 9 5um diameters are imitative viable cell that each imitative viable cell area of the pattern has 1 diameter; The imitative dead cell quantity of each imitative dead cell area of the pattern is identical with diameter with the imitative viable cell area of the pattern of diameter and each viable cell quantity.
Pattern-recognition method cell counter structure comprises light source, count slice, focusing object lens, sensitive chip, data line, controller, image knowledge software, indicating meter.
Cell counter and imitative cell counting count board precision are evaluated as:
Withinday precision: same imitative cell counting count board tie-in 5 times, calculate RSD(relative standard variance), RSD should reach 5%---and 10% for qualified;
Day to day precision: same imitative cell counting count board is not respectively surveyed 3 times on the same day, calculates RSD, and RSD be take interior as qualified at 5-15%;
Withinrun precision: be the precision of measuring with once, usually adopt each 7-10 part of same sample of high, medium and low three kinds of concentration, the analytical procedure operation that the sample of every kind of concentration is drafted, once after the start, measure one by one, calculate the RSD value of every kind of concentration sample, and gained RSD should be 5%---10% is qualified;
Betweenrun precision: be the precision that homogeneous is not measured, usually adopt the same sample of high, medium and low three kinds of concentration, every kind of concentration preparation 7-10 part, by drafted analytical procedure operation, take out a mensuration every day, calculate the RSD value of every kind of concentration sample, RSD should be controlled in 5-15%.
Principle of the present invention is: according to the principle that manually can prepare imitative cell counting count board that physics and optical characteristics and eukaryotic cell are close and indirectly reflect cell counter precision.The feature of imitative cell counting count board is: the bright-dark degree of imitative cell pattern diameter, imitative cell pattern and the diameter of eukaryotic cells and life or death state are similar.Imitative cell pattern density interphase, the actual concentrations situation of Reality simulation cell.
Positively effect of the present invention is:
The various parameters of " cell " model of the repeatability of imitative cell counting count board are known controlled, and can directly on cell counter, use, typical curve when drawing different cell concns and Cell viability and calculate the coefficient of variation, in order to estimate the precision of cell counter; Solved and with cell counting count board method artificial counting, carried out the helpless of accuracy that the indirect verification cell counter measures at present.Imitative cell counting count board can truly reflect the information such as eukaryotic size, concentration and motility rate, can estimate the precision that cell counter is measured under different condition.There is important calibration and be worth, can be widely used in life science and the clinical application fields such as cell, embryo, reproduction.
figure of description:
Fig. 1 is imitative cell counting count board surface structure schematic diagram.
Fig. 2 is the enlarged diagram of imitative each surveyed area of cell counting count board.
Fig. 3 is regional enlarged diagram in imitative cell counting strip cytological map case.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described:
Embodiment mono-
As seen from Figure 1: the present invention selects the calibration object of imitative cell counting count board 1 as cell counter, imitative cell counting count board 1 is transparent, have two same detection zones 2 to calibrate detection so that two ends all can be inserted in counter, each surveyed area area is at least 3mm * 3mm; Be provided with two class circles on imitative cell counting count board in surveyed area, a class is transparent ring 5, and the groove of annulus is opaque, loop line middle portion and inner circle central, transparent, the imitative viable cell of representative; Another kind of is opaque filled circles 6, the imitative dead cell of representative;
As seen from Figure 2: in order to facilitate visual inspection, the surveyed area of imitative cell counting count board is divided into 9 large zones by 1mm * 1mm, and the live width between large zone is 9um, and the line degree of depth is 4um;
By Fig. 2, be it can also be seen that: in order to facilitate visual inspection, in each large zone, by 0.25mm * 0.25mm, be divided in 16 zone, between middle zone, live width is 6um, and the line degree of depth is 4um, in zone, 8 band cell area of the pattern 3 and 8 white spaces 4 is arranged in 16;
As seen from Figure 3: in order to facilitate visual inspection and number number, cause image recognition software erroneous judgement for keeping away, each middle zone with the cell pattern is by from left to right, " one " to " ten " sequence number of principle of temporal sequence mark Chinese character marks respectively work or the dead imitative cell of the imitative cell transversely arranged work of ten row or dead and ten row longitudinal arrangements from top to bottom simultaneously.
By Fig. 3, be it can also be seen that: each is divided into 4 zonules with zone in the cell pattern by 0.125mm * 0.125mm, and between zone, live width is 4um, and the line degree of depth is 4um;
By Fig. 3, be it can also be seen that: with the zonule of cell pattern, with centre compartment 25um * 25um, from small to large, order from left to right, principle is arranged from top to bottom for the two classes circles that the imitative cell of two classes is 5um, 10um, 15um, 20um, 25um by diameter.
By Fig. 3, be it can also be seen that: 4 zonule cell patterns are divided into two imitative viable cell area of the pattern and two imitative dead cell area of the pattern, two imitative viable cell area of the pattern centrosymmetry, two imitative dead cell area of the pattern centrosymmetry, it is that the imitative viable cell of 25um, 3 diameters are that the imitative viable cell of 20um, 5 diameters are that the imitative viable cell of 15um, 7 diameters are that the imitative viable cell of 10um, 9 5um diameters are imitative viable cell that each imitative viable cell area of the pattern has 1 diameter; The imitative dead cell quantity of each imitative dead cell area of the pattern is identical with diameter with the imitative viable cell area of the pattern of diameter and each viable cell quantity.
During detection:
The cell counter structure adopted comprises light source, count slice, focusing object lens, sensitive chip, data line, controller, image knowledge software, indicating meter.
Its operation steps is as follows:
1, will imitate cell counting count board and put into ultrasonic cleaner and repeatedly clean 5 times, use the ultrapure water of fresh preparation instead, the ultrapure water specific conductivity: 18.2M Ω at every turn;
2, in super clean bench, use dehydrated alcohol or clear water to clean imitative cell counting count board, upper and lower surface respectively cleans five times;
3, use rubber suction bulb to dry up, stand-by;
4, open the cell counter power supply, instrument self checking finishes, and inserts stand-by imitative cell counting count board;
5, by focusing knob, that Image Adjusting is extremely clear;
6, press counting key counting (press in a few days/day to day precision and batch intra/inter-precision requirement are measured the corresponding number of times of different calibration plates);
7, result shows and stores;
8, cell counter and imitative cell counting count board precision are estimated:
Withinday precision: same imitative cell counting count board tie-in five times, calculate relative standard's variance (RSD), RSD should reach 5%---and 10%, counter is qualified, otherwise is defective;
Day to day precision: same imitative cell counting count board is not respectively surveyed three times on the same day, calculates RSD, and RSD is controlled at 5-15% and take the inside counting device as qualified product.
Withinrun precision: be the precision of measuring with once, usually adopt each 7-10 part of same sample of high, medium and low three kinds of concentration, the analytical procedure operation that the sample of every kind of concentration is drafted, once, after the start, measure one by one.Calculate the RSD value of every kind of concentration sample, and gained RSD should strive for 5%---10% take the inside counting device as qualified.
Betweenrun precision: be the precision that homogeneous is not measured.Usually adopt the same sample of high, medium and low three kinds of concentration, every kind of concentration preparation 7-10 part, by drafted analytical procedure operation, take out a mensuration every day, calculate the RSD value of every kind of concentration sample, RSD should be controlled in 5-15%, and counter is qualified product.
Claims (9)
1. a pattern-recognition method cell counter calibration steps, use the calibration object of the imitative cell counting count board (1) of special-purpose transparent material as cell counter in calibration steps, it is characterized in that: imitative cell counting count board (1) is the imitative cell counting count board that adopts the preparation of quartz photomask technology, has the imitative cell similar with motility rate to eukaryotic diameter that can make cell counter identification.
2. pattern-recognition method cell counter calibration steps according to claim 1, it is characterized in that: imitative cell counting count board is glass or transparent plastics material, and two same detection zones (2) are arranged on it, each surveyed area area is at least 3mm * 3mm; On imitative cell counting count board, surveyed area has two classes circles: a class is the imitative viable cell of transparent ring (5) representative; Another kind of is the imitative dead cell of opaque filled circles (6) representative.
3. pattern-recognition method cell counter calibration steps according to claim 2 is characterized in that: the surveyed area of imitative cell counting count board is divided into 9 large zones by 1mm * 1mm, and the line width between zone is 9um greatly, and the line degree of depth is 4um.
4. pattern-recognition method cell counter calibration steps according to claim 3, it is characterized in that: in each large zone, by 0.25mm * 0.25mm, be divided into zone in 16, line width between middle zone is 6um, the line degree of depth is 4um, 8 band cell area of the pattern (3) and 8 white spaces (4) is arranged in zone in 16, be with cell area of the pattern and white space alternative arrangement.
5. pattern-recognition method cell counter calibration steps according to claim 4, it is characterized in that: each zone with the cell pattern is divided into 4 zonules by 0.125mm * 0.125mm, and between zone, live width is 4um, and the line degree of depth is 4um.
6. pattern-recognition method cell counter calibration steps according to claim 5 is characterized in that: each zone with the cell pattern is by from left to right, principle of temporal sequence mark Chinese character " "～" ten " sequence number marks respectively work or the dead imitative cell of the imitative cell transversely arranged work of ten row or dead and ten row longitudinal arrangements from top to bottom.
7. pattern-recognition method cell counter calibration steps according to claim 6, it is characterized in that: each zonule centre compartment 25um * 25um, from small to large, order from left to right, principle is arranged from top to bottom for the two class circles that the imitative cell of two classes is 5um, 10um, 15um, 20um, 25um by diameter.
8. pattern-recognition method cell counter calibration steps according to claim 7, it is characterized in that: 4 zonule cell patterns are divided into two imitative viable cell area of the pattern and two imitative dead cell area of the pattern, two imitative viable cell area of the pattern centrosymmetry, two imitative dead cell area of the pattern centrosymmetry, it is that the imitative viable cell of 25um, 3 diameters are that the imitative viable cell of 20um, 5 diameters are that the imitative viable cell of 15um, 7 diameters are that the imitative viable cell of 10um, 9 5um diameters are imitative viable cell that each imitative viable cell area of the pattern has 1 diameter; The imitative dead cell quantity of each imitative dead cell area of the pattern is identical with diameter with the imitative viable cell area of the pattern of diameter and each viable cell quantity.
9. according to claim 1---8 arbitrary described pattern-recognition method cell counter calibration stepss is characterized in that: pattern-recognition method cell counter structure comprises light source, count slice, focusing object lens, sensitive chip, data line, controller, image knowledge software, indicating meter.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109269964A (en) * | 2018-10-15 | 2019-01-25 | 深圳博大博聚科技有限公司 | A kind of artificial counting and calibration equipment based on image recognition method cell counter |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106056118B (en) * | 2016-06-12 | 2018-08-24 | 合肥工业大学 | A kind of identification method of counting for cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2472233Y (en) * | 2001-03-09 | 2002-01-16 | 彭钧 | Cell counter |
| CN1793919A (en) * | 2005-12-09 | 2006-06-28 | 天津理工大学 | Automatic counting method of leucocyte number in blood microimage |
| CN101236195A (en) * | 2007-02-01 | 2008-08-06 | 希森美康株式会社 | Blood cell analyzer, body fluid analysis method, and control system therefor |
| CN202093554U (en) * | 2011-06-27 | 2011-12-28 | 深圳市博锐德生物科技有限公司 | Disposable cell counting plate |
| CN103146800A (en) * | 2013-03-27 | 2013-06-12 | 朱耀辉 | Full-automatic cell non-staining image recognizing and counting method |
-
2013
- 2013-09-06 CN CN201310399802.XA patent/CN103436439B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2472233Y (en) * | 2001-03-09 | 2002-01-16 | 彭钧 | Cell counter |
| CN1793919A (en) * | 2005-12-09 | 2006-06-28 | 天津理工大学 | Automatic counting method of leucocyte number in blood microimage |
| CN101236195A (en) * | 2007-02-01 | 2008-08-06 | 希森美康株式会社 | Blood cell analyzer, body fluid analysis method, and control system therefor |
| CN202093554U (en) * | 2011-06-27 | 2011-12-28 | 深圳市博锐德生物科技有限公司 | Disposable cell counting plate |
| CN103146800A (en) * | 2013-03-27 | 2013-06-12 | 朱耀辉 | Full-automatic cell non-staining image recognizing and counting method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109269964A (en) * | 2018-10-15 | 2019-01-25 | 深圳博大博聚科技有限公司 | A kind of artificial counting and calibration equipment based on image recognition method cell counter |
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Application publication date: 20131211 Assignee: Shenzhen Boda Technology Co., Ltd. Assignor: Zhao Guang|Zhu Yaohui|Yu Haibin|Li Jing|Yang Hong Contract record no.: 2015440020033 Denomination of invention: Calibration device for cell counter based on image identification method Granted publication date: 20141217 License type: Exclusive License Record date: 20150206 |
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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: 518000 Guangdong province Shenzhen city Longhua District Office of Longhua Qing Xiang Lu Qing Hu Bao Industrial Park Science and Technology Park 9 C block 16 building 1601 room Patentee before: Shenzhen Boda Boju Technology Co., Ltd. |
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