JPH0315144B2 - - Google Patents

Description

Detailed Description of the Invention (Technical Field) The present invention relates to a cell counting method for automatically counting the number of cells in culture.

(Prior Art) In order to identify the type of cells by culturing cells taken from the human or animal body, there is a strong desire to understand changes in the characteristics and number of cells during culture over time. To this end, a method can be considered in which cells in culture are imaged over time via a microscope, enlarged and reproduced on a television monitor, and counted visually.

The contrast between the cells and the culture medium is low, and background mottle image noise (inherent noise such as dirt in the optical system) makes it even more difficult to understand the contours of the cells themselves.

However, since the cells are proliferating, staining methods cannot be used, and the cell lines cannot be clarified by staining methods.

Therefore, considerable skill and patience are required to visually count cell images on a television monitor.

The inventors of the present application have already proposed an image processing method for cell measurement (an invention filed by the same applicant on November 4, 1982) in order to clarify the circulation of cells.

FIG. 1 is a block diagram showing an apparatus for carrying out the method of the invention. This will be briefly explained with reference to this figure.

This image deep processing method for cell measurement relates to a cell measurement method for enlarging and measuring cells attached to one surface of a transparent parallel plate 1a that is in contact with a substantially transparent culture solution 1b.

A cell image is magnified using the microscope 2 from the transparent parallel plate 1a side.

The enlarged image is captured by the television imaging device 3. A frame memory 6 is provided for storing video signals from the television imaging device 3 on a screen-by-screen basis.

A first image at a position where the distance between the objective lens 2a of the microscope 2 and the cell is slightly smaller than the optimal observation position with the naked eye is stored in the first area of the frame memory 6.

A second image at a position slightly larger than the optimum observation position is stored in the second area of the frame memory 6.

The outline of the cell is specified by the image obtained by subtracting the contents of the first area and the second area of the frame memory 6 in corresponding pixel units by the arithmetic unit 7.

The image obtained as described above can be displayed on the television monitor 5.

According to the method, the mottle pattern can be removed and the cell outline can be clearly extracted.

However, even if an image with a clear outline of cells can be obtained by the above-mentioned method, it is not easy to visually confirm the number and characteristics of cells and to quantitatively understand them.

Tracking changes in proliferating cells at regular intervals can require hours of work into the night.

(Objective of the Invention) The object of the present invention is to provide at least a characteristic
In a cell measurement method that measures cells that include a change in shape from one shape to a second shape, measuring the characteristic shape of cells in the process of culture and automatically registering the time point of generation of the number, etc. The purpose of this invention is to provide a cell measurement method that enables the following.

(Structure of the Invention) In order to achieve the above-mentioned object, the cell measurement method according to the present invention includes at least a characteristic change from a first shape to a second shape during repeated cell changes in the process of proliferation including division. In a cell measurement method for measuring cells containing changes, an imaging storage step of imaging a cell proliferation plane using an imaging device and storing the image in a frame memory device, and a calculation device for each cell stored in the frame memory device. A shape discrimination step of discriminating whether the cell has the first shape or the second shape from the outer shape, a cell registration step of storing the discrimination result in a cell registration storage device, and a certain period of time elapsed. Thereafter, a subsequent image storage step is performed in which an imaging device images a cell proliferation plane and the image is stored in a frame memory device, and an arithmetic device determines whether the cell is the first cell based on the outline of each cell stored in the frame memory. a new cell discrimination step of determining whether the cell is a new shape or a second shape and determining whether the cell is a new cell by referring to the contents of a cell registration storage device; and the new cell discrimination step. and a new cell registration step of storing the discrimination results of cells determined to be new in the cell registration storage device.

(Description of Examples) The present invention will be described in more detail below with reference to the drawings and the like.

Cells change in a certain pattern during the process of proliferation.

FIG. 2 is an explanatory diagram showing the process of cell change. This will be explained using mouse L-type cells as an example.

Immediately after the cell divides, at time _t1 , it assumes a special shape as shown in FIG. 2A. Cells with this special shape, that is, neither round nor constricted, are called irregularly shaped cells.

At time _t2 , after a certain period of time has elapsed, the amorphous cells become round cells as shown in FIG. 2B.

At time _t3 , after a certain period of time has elapsed, the round cells assume a constricted shape, which is the shape before division, as shown in FIG. 2C.

At time _t4 , after a certain period of time has elapsed, the constricted cells divide into irregularly shaped cells as shown in FIG. 2A, and each cell continues to change in the pattern described above.

The inventors of the present application focused on the regularity of this proliferation pattern in order to automatically measure the shape of cells, the point of division, etc.

FIG. 3 is a block diagram showing an embodiment of an apparatus for carrying out the method of the present invention.

It is advantageous to use the image processing method for cell measurement proposed above to carry out the method of the present invention because it allows a clear understanding of cell contours.

The culture container 1 is a transparent container, and a culture solution 1b is sealed inside.

Cells to be measured are implanted on the upper surface of the transparent parallel plate 1a on the bottom of the container 1, and allowed to divide and proliferate. This container 1 is illuminated from above, magnified by an objective lens 2a of an inverted microscope 2, and imaged by a television imaging device 3, which is taken out as a video signal.

This video signal is transmitted to a frame memory device 1 consisting of a plurality of frame memories having addresses corresponding to pixels.
6 is stored in a predetermined frame memory, and processing is performed to clarify the outline of the cells by the procedure described above.

The arithmetic and control device 17 is connected to the frame memory device 1.
6 performs the image processing at regular intervals, and performs the processing shown in FIG. 4 on the contents of the frame memory in which the image processing results are stored, and registers the processing results in the cell registration storage device 18, as needed. Update its contents.

In FIG. 3, 14 is a television signal processing device, and 15 is a monitor, which are mainly used during the image processing.

Next, the measurement method will be explained mainly with reference to FIG.

(Step 100) Adjust and set the front and rear focal positions, input the imaging interval and number of times, etc., and start the device.

(Step 101) An image is taken and stored in the first and second frame memories of the frame memory device 16, and the result of the image processing to clarify the contour described above is stored in the third frame memory.

Note that the processing results for the image obtained in the next imaging are stored in the fourth frame memory. The third frame memory or the fourth frame memory is used alternately so that the memory of the previous image capture is always retained.

Subsequent processing for determination is performed for each cell of this frame memory.

(Step 102) Determine whether an arbitrary cell is a round cell.

Round cells are determined by the maximum length of the cell in the X direction.
This is done based on Xm, the maximum length in the Y direction Ym, the length L of the ring, and the area S.

The algorithm for determining round cells will be explained with reference to FIG.

When the following three conditions are satisfied, this is determined to be a round cell.

|Xm−Ym|<δd |S−(π/16)(Xm+Ym) ² |<δs |L−π(Xm+Ym)/2|<δl However, δd, δs, and δl are selected depending on the cell type, expansion rate, etc. is a constant.

(Step 103) When it is determined that the cell is round according to the above determination,
It is determined whether the cell is a new cell.

The determination as to whether the cell is a new cell is made by comparing the center position of the cell already registered in the memory 18 with the center position of the cell.

If the position matches or is very close to the center position of a previously registered round cell, the cell is not a new round cell.

In the case of processing the results of the first image capture and storage, since there are no cells that have already been registered, it is determined that the cells are new round cells.

(Step 104) The center position of the cell determined to be a new round cell is registered in the cell registration storage device 18.

At this time, the time when the cell was imaged is also registered.

(Step 105) For cells that were determined not to be round cells in step 103 described above, a determination is made as to whether or not they are waist-shaped cells.

An algorithm for determining whether a cell is a constriction-shaped cell or not will be explained with reference to FIG.

The determination of waist-shaped cells is based on the position of the cell's center of gravity p.
, the maximum length Lmax passing through the center of gravity, the minimum length Lmin passing through the center of gravity, and the areas S ₁ , S ₂ , S ₃ and S ₄ divided by Lmax and Lmin.

When the following five conditions are satisfied, this is determined to be a constriction-shaped cell.

Lmax−Lmin＞δL |S ₁ −S ₂ |＜δs ₁ |S ₂ −S ₃ |＜δs ₂ |S ₃ −S ₄ |＜δs ₃ |S ₄ −S ₁ |＜δs _4However , δL, δs ₁ ~δs ₄ This is a constant selected depending on the type of cell, magnification, etc.

The formula Lmax−Lmin>ΔL is used to confirm that the cells are not round cells.

This is because constriction-shaped cells have a certain degree of symmetry. The following equations are equations that confirm the symmetry of the cell.

(Step 106) When it is determined that the cell is a constriction-shaped cell, it is determined whether or not the cell is a new cell.

The determination as to whether the cell is a new constriction-shaped cell is made by comparing the position of the center of gravity of the constriction-shaped cell already registered in the memory 18 with the position of the center of gravity of the cell. If the position of the center of gravity of the previously registered constriction-shaped cell matches or is very close to that of the constriction-shaped cell, the cell is not a new constriction-shaped cell.

In the case of processing the results of the first image capture and storage, since there are no cells that have already been registered, the cells are determined to be new constriction-shaped cells.

(Step 107) The position of the center of gravity of the cell determined to be a new constriction-shaped cell is registered in the cell registration storage device 18.

At this time, the time when the cell was imaged is also registered.

For cells that are not round or constricted, the centroid position may be registered as amorphous.

(Step 108) Frame memory (1st and even numbered) or frame memory (2nd and even numbered)
The above-mentioned process is performed until the above-mentioned determination and registration are completed for all cells stored in the memory (step 102).
The steps from to (step 107) are repeated.

(Step 109) It is determined whether there is a request to read the contents of the cell registration storage device 18.

(Step 110) The contents of memory 18 are output to output device 19.

This allows us to determine the shape of the cells and the time point of their development.

(Step 112) Pauses until a predetermined period of time has elapsed since the previous imaging storage step 101, after all determinations and registrations have been completed and after the requested contents of the cell registration storage device 18 have been read out. After the predetermined time has elapsed, the next imaging storage step 101 is started.

Then, the determination and registration steps and the reading step are repeated in response to a request.

In this way, by performing multiple imaging storages, determinations, and registration of determination results, all data on the process of proliferation of cell groups is stored in the cell registration storage device 1.
It is accumulated in 8.

A summary of the above flow is summarized in Figure 7.

FIG. 7 corresponds to the flow of FIG. 2.

(Description of Effects of the Invention) As explained above, the cell measurement method according to the present invention can detect characteristic first shapes and second shapes that are included in repeated cell changes in the process of proliferation including division.
Since the cell shape is focused on and the occurrence of a change in shape is repeatedly determined and recorded, changes in the cell can be automatically tracked.

A record of the division process of each cell can then be obtained from the contents of the cell registration storage device. In addition, the number of cells of each shape at each time point can be easily obtained by calculation and aggregation. It has now become possible to automatically track and record changes in large numbers of cells over long periods of time, which was previously considered impossible.

[Brief explanation of the drawing]

FIG. 1 is a block diagram showing the configuration of a conventional cell measuring device. FIG. 2 is an explanatory diagram showing the process of cell change. FIG. 3 is a block diagram showing an embodiment of an apparatus for carrying out the method of the present invention. Fourth
The figure is a flowchart showing an example of a program for implementing the method. FIG. 5 is a schematic diagram for explaining the algorithm of the program for determining the round shape of cells. FIG. 6 is a schematic diagram for explaining the algorithm of the program for determining the constriction shape of cells. FIG. 7 is a flowchart showing an extracted summary of the program shown in FIG. 1...Cell culture container, 1a...Parallel plate, 1b
...Culture solution, 2...Microscope, 3...Television imaging device, 14...Television signal processing device,
15...Television monitor, 16...Frame memory device, 17...Arithmetic control device, 18...Storage device for cell registration, 19...Output device.

Claims (1)

[Scope of Claims] 1. A cell measurement method for measuring cells that include at least a characteristic change from a first shape to a second shape during repeated changes in cells during the process of proliferation including division, comprising: an imaging storage step in which a device takes an image of a cell proliferation plane and stores the image in a frame memory device; and an arithmetic device determines that the cell has the first shape based on the outline of each cell stored in the frame memory device; a shape discrimination step of discriminating whether it is a second shape or a second shape; a cell registration step of storing the discrimination result in a cell registration storage device; and after a certain period of time, an imaging device images the cell growth plane and frames A subsequent imaging storage step of storing the image in the memory device and a calculation device calculate the outline of each cell stored in the frame memory.
New cell discrimination that determines whether the cell has the first shape or the second shape and determines whether the cell is a new cell by referring to the contents of the cell registration storage device. and a new cell registration step of storing the determination result of the cells determined as new in the new cell determination step in the cell registration storage device. 2. The cell counting method according to claim 1, wherein the imaging device is a television camera. 3. The cell measuring method according to claim 1, wherein the determination result stored in the cell registration storage device in the cell registration step is the shape and the position of the center of gravity of the cell. 4. The cell measurement method according to claim 1, wherein the determination result stored in the cell registration storage device in the new cell registration step relates to the shape and center of gravity of the cell, and the time at which the cell was imaged. . 5 The first shape of the cell is round, the second shape is constricted, the cell changes from a round cell to a constricted cell, and then divides into an irregular shape, and the divided cell waits for a certain period of time to pass. The method for measuring cells according to claim 1, wherein the cells later change into round cells. 6 The round cell is determined based on the maximum length Xm in the X direction and the maximum length Ym in the Y direction of the cell, and if the difference between Xm and Ym is less than a certain value, it is determined as a round cell. A method for measuring cells according to claim 5. 7 The determination of the round cell is performed based on the maximum length Xm in the X direction, the maximum length Ym in the Y direction, and the area S of the cell, and the calculated value estimated from Xm and Ym and the area S 6. The cell measuring method according to claim 5, wherein the cell is determined to be a round cell when the difference between the two cells is less than a certain value. 8 The constriction-shaped cell is determined based on the position p of the cell's center of gravity, the maximum length Lmax passing through the center of gravity, the minimum length Lmin passing through the center of gravity, and the area divided by the line indicating the maximum length or minimum length. carried out,
Claim 5, wherein the cell is determined to be a constriction-shaped cell when the difference between the maximum length Lmax and the minimum length Lmin is a certain value or more, and considerable symmetry is confirmed by comparison of the divided areas. Cell measurement method. 9 In the new cell discrimination step, the determination that the cell is a new round cell is made when there is no previous round cell registered whose center coincides with or is close to the center of the cell. The cell counting method described in Section 5. 10 In the new cell discrimination step, the determination that the cell is a new waist-shaped cell is made when there is no previous waist-shaped cell registered whose center of gravity is coincident with or close to the center of gravity of the cell. The cell counting method described in Section 5.