CN112540039A - Method for directly calculating number of adherent living cells - Google Patents
Method for directly calculating number of adherent living cells Download PDFInfo
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- CN112540039A CN112540039A CN202011613070.6A CN202011613070A CN112540039A CN 112540039 A CN112540039 A CN 112540039A CN 202011613070 A CN202011613070 A CN 202011613070A CN 112540039 A CN112540039 A CN 112540039A
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- 238000000034 method Methods 0.000 title claims abstract description 20
- 230000001464 adherent effect Effects 0.000 title claims abstract description 12
- 230000000007 visual effect Effects 0.000 claims abstract description 6
- 238000012545 processing Methods 0.000 claims abstract description 5
- 238000004113 cell culture Methods 0.000 claims description 4
- 238000005070 sampling Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 64
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- 230000000052 comparative effect Effects 0.000 description 2
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- 238000002474 experimental method Methods 0.000 description 2
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- 238000012258 culturing Methods 0.000 description 1
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- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
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- 210000003954 umbilical cord Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
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- G—PHYSICS
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- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
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- G06T7/0002—Inspection of images, e.g. flaw detection
- G06T7/0012—Biomedical image inspection
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- G06T7/187—Segmentation; Edge detection involving region growing; involving region merging; involving connected component labelling
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- G—PHYSICS
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- G06T7/194—Segmentation; Edge detection involving foreground-background segmentation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
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- G01N2015/1024—
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Abstract
The invention discloses a method for directly calculating the number of adherent living cells, which comprises the following steps: a. taking a picture of cells under a microscope; b. performing programmed processing on the picture, and reading the number of cells; c. calculating the number of cells in the visual field; d. and (4) calculating the total number of the cells adhered to the wall in the culture container. The invention has the advantages that the number of cells can be obtained before the cells are not digested, the efficiency is improved, the time is saved, the consumption of reagent consumables is reduced, and the cost is reduced.
Description
Technical Field
The invention belongs to the technical field of cell counting, and particularly relates to a method for directly calculating the number of adherent living cells.
Background
In a large number of cell biology research experiments, the number of cells is required to be detected, and the number of cells is also a necessary parameter in many experimental projects. The existing counting means mainly include a manual counting method using a cell counting plate, an automatic counting instrument based on an image analysis technology, and an automatic counting instrument using a resistance method (coulter principle).
The main disadvantages of the existing counting means are:
1. the count is not accurate enough: because the depth of the cell counting plate is several times of the cell size, the cell sample can be layered and suspended in the cell counting plate after being injected, so that the observed cell morphology can be different, and the inaccurate counting result and the wrong judgment on the cell activity can be caused; the sample injected into the cell counting plate according to the rule is usually 10 mu L, but the sample amount in the observation area of the microscope is only a small part and is less than 1 mu L, so whether the cell sample is uniformly distributed in the counting cell or not can cause great influence on the result;
2. the use efficiency is low: the cell counting plate is complicated in use process, and needs to be calibrated regularly to ensure the precision of measurement; and only one sample can be measured on the cell counting plate at one time, which is inconvenient; generally, one cell count takes 20 minutes on average, and the efficiency of cell counting is low.
3. Although the automated instrument based on the image analysis technology avoids the difficulty of visual observation, the following defects still exist: (1) the disposable counting sheet consumables are introduced, so that the detection cost of a user is increased; (2) the counting sheet is similar to a cell counting plate in structure, so that the problems of inaccurate results and misjudgment of activity caused by layered suspension of cells on the counting plate also exist; (3) like manual counting, most of the instruments based on the image method have the problem of large deviation of results caused by small amount of detected samples.
4. The traditional coulter counter has low integral integration level and is not concise enough in operation, and in addition, the traditional coulter counter has no function of judging the viability of cell samples, and cells dead due to various reasons always exist in cell groups, so that the counting is not accurate enough.
Disclosure of Invention
The present invention is directed to solving the problems of the background art and providing a method that can be used to directly count the number of adherent living cells.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method useful for directly calculating the number of adherent living cells, comprising the steps of:
a. taking a picture of cells under a microscope;
b. performing programmed image processing on the picture, and reading the number of cells;
c. calculating the number of cells in the visual field;
d. and (4) calculating the total number of the cells adhered to the wall in the culture container.
Preferably, in step a, images are taken at four corners and the center of the cell culture dish or the cell culture bottle according to the principle of statistical five-point sampling.
Preferably, step b specifically comprises the steps of:
1) reading a cell scanning image under a microscope;
2) acquiring single-channel data of an image; the foreground and the background of the image can be better distinguished through the step;
3) performing binarization negation operation on the image obtained in the step 2) to obtain a binarized image;
4) performing Gaussian blur operation on the binary image obtained in the step 3);
5) searching a connected domain of the image obtained in the step 4);
6) screening out the cell regions which meet the requirements in all the regions obtained in the step 5) according to the set threshold value, and obtaining the number of the cells in the cell scanning image under the microscope.
Preferably, step c comprises: and (4) the cell number obtained after all the cell pictures sampled at the five points are subjected to cell program reading, and the average value is used for representing the number n of the cells in the visual field area range in the culture container.
Preferably, step d comprises: the total area of the culture vessel was calculated to be a times the area of the field of view taken, and the total number of cells in the whole culture vessel was found to be: a n.
The invention has the advantages that the number of cells can be obtained before the cells are not digested, the efficiency is improved, the time is saved, the consumption of reagent consumables is reduced, and the cost is reduced.
Drawings
FIG. 1 is a flow chart of a method of the present invention that can be used to directly count the number of adherent living cells.
FIG. 2 is a flow chart of the present invention for reading cell numbers by performing image programming on pictures.
FIG. 3 is a diagram showing five fields and the number of cells read under a microscope 10 Xobjective lens according to the present invention, wherein (a) - (b) correspond to the five fields, respectively.
FIG. 4 shows the number data of cells calculated by a conventional counter.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. 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.
In addition, unless otherwise specified, each step in the examples can be realized by using materials and conventional technical means in the field.
Examples
In this example, the number of cells was counted by culturing P3 instead of umbilical cord, and the experimental procedure included the following steps:
1) and taking out the T175 cell culture flask.
2) Observing the cells under an Olympus CKX53 microscope at 10 Xobjective; and photographing at four corners and the middle position according to a five-point sampling method.
In this step, observation can be performed under objective lenses of other multiples, which is not limited by the present invention.
3) Saving the shot cell picture in a computer, running a program in the computer to read the shot cell picture, and processing the picture by using the program, wherein the program processing flow is shown in figure 2 and comprises the following steps:
3-1) reading a cell scanning image under a microscope;
in the step, a general image processing library OPENCV is used for reading the image, and no special requirement exists; the picture format is not limited, and jpg, png, tif, etc. can be used.
3-2) acquiring single-channel data of the image;
3-3) carrying out binarization negation operation on the image obtained in the step 3-2) to obtain a binarized image;
3-4) carrying out Gaussian blur operation on the binary image obtained in the step 3-3);
3-5) searching a connected domain of the image obtained in the step 3-4);
3-6) screening out cell areas which meet the requirements in all the areas obtained in the step 3-5) according to the set threshold value, and obtaining the number of cells in the cell scanning image under the microscope.
Based on the above provided program processing flow, those skilled in the art can implement the step by conventional technical means according to actual needs, and is not limited specifically herein.
4) The average number of cells in five fields was calculated (see FIG. 3)
n=（2397+2391+2418+2403+2352）/5=2392.2。
5) The actual field diameter under the 10 Xobjective lens of the Olympus CKX53 microscope is 1.9mm, the corresponding field area S = pi (0.19/2) ^2, and the whole bottle area is the multiple a of the field area a ≈ 6172.2.
6) The total number of cells = a × n ≈ 1.48 × 10^7 is calculated by the present invention.
Comparative example
The number of cells was calculated to be 1.17X 10^7 using a conventional counter (brand: countstar, model: IC 1000), as shown in FIG. 4.
Countstar counting procedure:
1) 20ul of cell suspension was taken and mixed well with 20ul of trypan blue solution.
2) The cells were counted in a counter using corresponding software on a computer.
3) And multiplying the obtained cell number by the cell suspension volume to obtain the total cell number.
The new method adopted at this time directly calculates the number of the adherent living cells to be 1.48 x 10^7, the number of the cells obtained in the comparative example to be 1.17 x 10^7, and the result is accurate and reasonable in view of the fact that the used cells are adherent stem cells and the loss of the cells is inevitable in the process of obtaining the cell suspension after the cells are digested.
Claims (5)
1. A method useful for directly calculating the number of adherent living cells, comprising the steps of:
taking a picture of cells under a microscope;
performing programmed image processing on the picture, and reading the number of cells;
calculating the number of cells in the visual field;
and (4) calculating the total number of the cells adhered to the wall in the culture container.
2. The method of claim 1, wherein in step a, images are taken at four corners and the center of the cell culture dish or bottle according to the principle of statistical five-point sampling.
3. The method for directly calculating the number of adherent living cells according to claim 1, wherein step b comprises the following steps:
reading a cell scanning image under a microscope;
acquiring single-channel data of an image;
performing binarization negation operation on the image obtained in the step 2) to obtain a binarized image;
performing Gaussian blur operation on the binary image obtained in the step 3);
searching a connected domain of the image obtained in the step 4);
screening out the cell regions which meet the requirements in all the regions obtained in the step 5) according to the set threshold value, and obtaining the number of the cells in the cell scanning image under the microscope.
4. A method for directly calculating the number of adherent living cells according to any one of claims 1 to 3, wherein step c comprises: and (4) the cell number obtained after all the cell pictures sampled at the five points are subjected to cell program reading, and the average value is used for representing the number n of the cells in the visual field area range in the culture container.
5. The method of claim 4, wherein step d comprises: the total area of the culture vessel was calculated to be a times the area of the field of view taken, and the total number of cells in the whole culture vessel was found to be: a n.
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| CN202011613070.6A CN112540039A (en) | 2020-12-31 | 2020-12-31 | Method for directly calculating number of adherent living cells |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113376079A (en) * | 2021-05-31 | 2021-09-10 | 上海碧博生物医药科技有限公司 | Method for analyzing transfection efficiency using CountStar Rigel System in combination with cell staining |
| CN114276916A (en) * | 2022-01-26 | 2022-04-05 | 辽宁中添干细胞与再生医学创新研究院有限公司 | Non-digestion counting method and device for adherent cells |
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| CN111192273A (en) * | 2019-12-27 | 2020-05-22 | 西北工业大学 | Digital shot blasting coverage rate measuring method based on computer vision technology |
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2020
- 2020-12-31 CN CN202011613070.6A patent/CN112540039A/en active Pending
Patent Citations (6)
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| CN101877074A (en) * | 2009-11-23 | 2010-11-03 | 常州达奇信息科技有限公司 | Tubercle bacillus target recognizing and counting algorithm based on diverse characteristics |
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Cited By (3)
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