JPS63212862A - Method and apparatus for monitoring cell proliferation state - Google Patents

Abstract

PURPOSE:To rapidly and properly determine the check time of subculture or antibody production, by dividing image data into blocks and selecting Hadamard transformation coefficient calculated from the data of each block to discriminate the presence of cell proliferation. CONSTITUTION:The image from the image pickup apparatus 2 connected to a microscope 1 is digitalized to be stored in a frame memory 4. A block divider 5 divides the obtained image data into the designated number of blocks. An Hadamard converter 7 applies Hadamard transformation to the image data of each block to calculate Hadamard transformation coefficient which is, in turn, stored in an Hadamard transformation coefficient memory 8. A coefficient data selection means 9 selects coefficient data to be used from the memory 8 on the basis of the input from a selection data input means 10. A discrimination analytical means 11 compares said coefficient data with the reference value preset to a reference value memory and setting means to discriminate the presence of the cell proliferation of each block to store the same in a discriminated analytical result memory 14. A proliferation rate calculation means 15 calculates a proliferation rate from the stored presence of the cell proliferation of each block to output the same to an output apparatus 16.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for monitoring the state of cell proliferation and a device for monitoring the same, which allows the state of cells to be measured accurately and in a short period of time.

[Prior Art] When cells are cultured in a cell culture container such as a microplate, if the concentration of living cells becomes too high, a state called full growth occurs, and some cells die.

Therefore, it is necessary to subculture before reaching a full growth state.

Furthermore, when cell fusion is performed, it is necessary to check whether the desired antibody is being produced at an appropriate time after the cells have proliferated. Until now, the time of subculture and the time of antibody production had to be determined by an operator who used a microscope to observe the state of cell proliferation within the culture container and relied on his or her experience.

[Problems to be Solved by the Invention] With conventional methods that rely on experience, the state of proliferation of cultured cells and the concentration of living cells are very unclear and cannot be quantitatively determined. Furthermore, if one were to count the number of living cells under a microscope, it would take an extremely long time and it would be difficult to grasp all the growth states of a large number of cultured cells.

Cell recognition method using image processing instead of microscopic observation,
For example, a method using only binarization (Japanese Unexamined Patent Publication No. 59-78
681) has also been proposed, but the recognition rate was poor. This is true when the background is simple, such as in the case of cell colonies formed on agar plates.
Although it is easy to recognize, when cultured in a culture medium, both the pattern and the background are complex because dead cells, dust, and other foreign substances are included. As a method for improving this, a method using spatial filter processing has also been proposed (Japanese Patent Application Laid-Open No. 60-256154).

However, this method uses a spatial filter, so although it has good accuracy, it has the problem of being time consuming.

[Means for Solving the Problems] The present invention has been made to accurately and quickly measure the proliferation state of cells even when there is a complex pattern or background. The invention involves dividing a digitized cell image into a block of an appropriate number of pixels (4n horizontal pixels x 4n vertical pixels: n≧1) into an image processing device through an imaging device connected to a microscope. It is divided into two-dimensional Hadamard (Hadamard) for each block.
) The second invention provides a method for monitoring the state of cell proliferation by determining the presence or absence of living cells in each block through processing using discriminant analysis after conversion. A microscope, an imaging device connected to the drifting mirror, an A/D converter that converts the signal from the imaging device into a digital signal, a storage device that stores the digitalized image data, and a storage device that stores the image data. an image dividing means for dividing into a specified number; a converting means for performing Hadamard transform on image data of each block obtained by the division; and means for storing Hadamard transform coefficient data after Hadamard transform;
A discriminant analysis means that calculates a discriminant function using a specified part or all of the stored Hadamard transform coefficient data and compares the obtained value with a reference value, and stores the discriminant results of each block. The present invention provides a cell proliferation state monitoring device comprising: a calculation means for calculating the proportion of a set of blocks having the same discrimination result with respect to all images; and an output means for outputting the calculated proportion.

[Effect] The present invention will be explained with reference to the drawings.

FIG. 1 is a flow diagram showing the steps of the method for monitoring the cell proliferation state of the present invention.

Using an imaging device such as a television camera connected to a microscope serving as an observation means, images of cells in the culture container are input to an image processing device.

To import cultured cell images, the diameter of 1 liter of cultured living cells is 1 side of the specified size block.
When the magnification is /2 to 1, that is, the size of the divided block is 4 n × 4 n pixels (n≧1), the size of cultured living cells is 10 to 20 μm in -m, so 1 Pixel size is minimum 5/2n [μm], extra large 1o
/n[μm]. This is because recognition efficiency will be poor if the living cells are too large or too small than the specified block size.

After digitizing the image captured by the image processing device,
Divide into blocks. The size of the block to be divided is 4.
A number of n×4n pixels (n≧1) is used. This is 2
This is because the order of the Hadamard matrix used to perform the dimensional Hadamard transformation must be a multiple of four.

Next, a two-dimensional Hadamard transform is applied to each block, using a Hadamard matrix of the same order as the size of the block. The natural Hadamard matrix is expressed by the formula (% Formula %): The matrix C4n of Hadamard transform coefficients is given by formula (2) where X4n is the matrix of luminance values in the divided blocks.

C4-=H4-X4nH411 (2) where C4,
:4nX4n Hadamard transform coefficient matrix, X4. :4nX
Matrix of luminance values of a block of 4n pixel size, H411:
4n x 4n Hadamard matrix, n: an integer of n≧1.

In particular, the natural Hadamard matrix in the case of 4n=2 (k is an integer of ≧2) is given by:

The three values of the Hadamard-transformed coefficient matrix C4fl represent the amplitude of each sequence component represented by the Hadamard matrix. Using all or some of the values of C4°, it is determined whether or not living cells exist within the divided blocks.

Two-group discrimination using Mahalanobis general distance can be used to determine the presence or absence of living cells within a block.

Let the number of elements of the feature parameter PETAR, which is characterized by all or a part of the elements of the coefficient matrix C4++ after Hadamard transformation, be p.

Furthermore, when the number of groups j = 2, the presence or absence of living cells in a block is determined by the discriminant function Z )/2(3
) can be determined by the sign of

Here, χ: feature parameter vector, χJ: average value vector within each group, R: variance/covariance matrix, and t and -1 on the right shoulder are transposed matrices, respectively.

Indicates that it is an inverse matrix.

In advance, E + + Fang 2 and R (
Once χl−χ2) is determined, the presence or absence of living cells can be determined by substituting the Hadamard transform coefficient of each block into this equation.

Furthermore, the degree of proliferation of cultured cells can be expressed as a ratio of the number of blocks determined to have living cells to the total number of blocks.

FIG. 2 is a block diagram of a cell proliferation state monitoring device according to the second invention. (1) is a microscope, and (2) is an imaging device such as a television camera connected to the microscope (1). The image signal from the imaging device (2) is sent to an A/D converter (
3) and stored in the frame memory (4).
).

On the other hand, (5) is a block divider which divides the image data stored in the frame memory into block number input means (6).
Divide into the input number of blocks.

The image data in one block is processed by Hadamard transform (7
) to calculate Hadamard transform coefficients. The calculated Hadamard transform coefficients are stored in the Hadamard transform coefficient memory (8). Similarly, Hadamard transform coefficients are calculated and stored for the image data in each of the other blocks. (9) is a coefficient data selection means for selecting Hadamard transform coefficient data to be used for discriminant analysis, and selection is made based on the transform coefficient to be selected inputted from the selection data input means (1o).

The data of the selected coefficients is compared by the discriminant analysis means (11) with the reference value input in advance from the reference value input means (12) and set in the reference value storage setting device (13), and The presence or absence of cell proliferation is determined.

The presence or absence of cell proliferation in each block is stored in a discriminant analysis result memory (14), and the proliferation rate calculation means (15) calculates the proliferation rate from the stored presence or absence of cell proliferation in each block.

(16) is an output device for displaying the proliferation rate, such as a printer, a display device, etc.

[Example] An 'X4R mirror image in which colonies of cultured live cells were present among dead cells, foreign matter, etc. was input to an image processing device via a television camera and an 8-bit A/D converter. One image was composed of 512×480 pixels, and the magnification of the input system was adjusted so that the diameter of the cell was about 4 pixels.

The image was divided into blocks such that one block had 8 pixels x 8 pixels, and a two-dimensional Hadamard transform was applied to each block using the 8 x 8 Hadamard matrix shown in equation (1).

Among the Hadamard transform coefficients obtained, eight Hadamard transform coefficients corresponding to the sequence components shown in FIG. 2 were used as parameters for discriminant analysis.

From the image ■, extract 100 blocks each with cultured cells present and 100 blocks without cultured cells, and from these, the formula (
3) R, tusk t, and te2 were calculated. Here, (at + a2-aa) = (R(rt H2)
The coupling coefficient al expressed as 1 is shown in Table 1.

Further, Table 2 shows the results of identification performed using this coupling coefficient a for three types of images 1 to 2.

As a result, a classification rate of about 80% was obtained not only for the image for which the coupling coefficient al of the discriminant function was determined but also for other cell images.

Here, the identification rate was determined as follows: identification rate [%] = (number of blocks in which the presence or absence of living cells was correctly determined)/(total number of blocks in one stroke) x 100.

Table 1 Coupling coefficient at Table 2 Identification results (distribution of number of blocks) [Effects of the invention] Through a combination of dividing an image containing living cells into blocks of appropriate size and Hadamard transformation that converts the luminance distribution of each block into a sequence component. Since the size of sequence components representing the characteristics of living cells can be easily measured, blocks containing living cells can be identified from among them even in images with complex patterns and backgrounds. be able to.

Moreover, (a) Since the Hadamard matrix is composed of only "1" and "-1", the Hadamard transform can be calculated only by addition and subtraction. (b) Hadamard transformation need only be performed for the number of divided blocks, and there is no need for processing for each pixel that is required for filtering or the like. (c) The presence or absence of living cells within a block can be determined without using all of the Hadamard transform coefficients. (d) Once the coupling coefficients for discriminant analysis are determined, they do not need to be determined for each image.

For the above reasons, processing can be performed at high speed and the growth state of a large number of cultured cells can be monitored in a short period of time.

Therefore, the timing of subculture and the timing of checking antibody production can be quickly and appropriately determined, and can be carried out simultaneously.

[Brief explanation of the drawing]

Fig. 1 is a processing flow diagram of a method for monitoring cell proliferation status using Hadamard transformation and decision analysis, Fig. 2 is a block diagram of the cell proliferation status monitoring device of the present invention, and Fig. 3 is a flow diagram of each block in the embodiment. FIG. 11 is a diagram showing Siegenshi 11m juice corresponding to the Hadamard transformation coefficients used to determine the presence or absence of viable cells. (1)...Microscope, (2)...Imaging device, (3)...
・・A/D converter, (4) ・・Frame memory, (
5)...Block divider, <7)...Hadamard transformer, (8)...Hadamard transform coefficient memory, (9
)...Coefficient data selection means, (11)...Discriminant analysis means, (13)...Reference value storage setting device, (14)...
・Discriminant analysis result memory, (15) ・Proliferation rate calculation means, (16) ・Output device San +: 11 1 6's use t mi to na hozu. Hz Hadamard i- & ty+ u +
Sui J[.

Claims (6)

[Claims] (1) The state of cultured cells is taken with an imaging device connected to a microscope, the image is digitized with an A/D converter, and then the image is divided into an appropriate number of pixels (horizontal 4n x vertical 4n: n≧
1 integer), and each block consists of 2
1. A method for monitoring cell proliferation status, comprising performing dimensional Hadamard transformation and then using discriminant analysis to determine the presence or absence of living cells in each block. (2) The method for monitoring the state of cell proliferation according to claim (1), in which the discriminant analysis uses all or part of the (4n) Hadamard transform coefficients obtained as a result of Hadamard transform. (3) The cell proliferation state monitoring method according to item (1), wherein the discriminant analysis is two-group discrimination using Mahalanobis general distance. (4) A microscope, an imaging device connected to the microscope, an A/D converter that converts the signal from the imaging device into a digital signal, a storage device that stores the digitized image data, and a storage device that stores the digitized image data. an image dividing means for dividing into a specified number; a converting means for performing Hadamard transform on image data of each block obtained by the division; means for storing Hadamard transform coefficient data after Hadamard transform; and stored Hadamard transform coefficient data. A discriminant analysis means that calculates a discriminant function using a specified part or all of the blocks and compares the obtained value with a reference value, a means that stores the discrimination results of each block, and a discriminant analysis means that calculates a discriminant function using a specified part or all of the blocks, A cell proliferation state monitoring device comprising calculation means for calculating the proportion of a set of blocks as a result of discrimination, and output means for outputting the calculated proportion. (5) The cell proliferation state monitoring device according to claim (4), wherein the discriminant analysis means uses all or part of (4n) numbers obtained as a result of Hadamard transformation. (6) Discriminant analysis means uses Mahalanobis generalized distance 2
The cell proliferation state monitoring device according to item (4), which is a means for group discrimination.