CA1325475C - Method of monitoring cell culture - Google Patents
Method of monitoring cell cultureInfo
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- CA1325475C CA1325475C CA000584597A CA584597A CA1325475C CA 1325475 C CA1325475 C CA 1325475C CA 000584597 A CA000584597 A CA 000584597A CA 584597 A CA584597 A CA 584597A CA 1325475 C CA1325475 C CA 1325475C
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- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
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Abstract
Abstract The present invention is directed to a method of monitoring cell cultures which comprises digitizing an image of the cultured cells inputted to image processing equipment through a TV camera connected to an observing means and measuring the number and cell proliferation of the cultured cells by processing with a spatial filter.
A large number of cells can be treated in a short time and give a precise number of cells.
A large number of cells can be treated in a short time and give a precise number of cells.
Description
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~ Method of Monitoring Cell Culture ,. . _ . _ _ . . .
The present invention relates to a method of monitoring cell cultures. More particularly, it relates to a method of monitoring cell cultures which comprises digi~izing a cultured cell image inputted to image processing equipment and measuring the number of living cells, the number of dead cells and the cell proliferation by processing with a spatial filter.
When cells are cultivated in a vessel, e.g. a microplate, dish or culturing bottle, some of the living cells die if the concentration of the living cells become so high as to reach the so-called "full growth" state. To prevent this, it is necessary to change the culture medium and/or to subculture the cells before the full growth state is reached. ~itherto, a technician determines the timing for changing the cell culture solution or for starting the subculturing by observing the cell proliferation of the j cells in the vessel through a microscope. However, such a conventional method is not quantitative and th~refore not accurate in determining the cell proliferation of cultured cells and the concentration of living cells. In addition, - lt is time-consuming and it is difficult to precisely observe the cell proliferation of a large amount of cultured cells.
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~ Method of Monitoring Cell Culture ,. . _ . _ _ . . .
The present invention relates to a method of monitoring cell cultures. More particularly, it relates to a method of monitoring cell cultures which comprises digi~izing a cultured cell image inputted to image processing equipment and measuring the number of living cells, the number of dead cells and the cell proliferation by processing with a spatial filter.
When cells are cultivated in a vessel, e.g. a microplate, dish or culturing bottle, some of the living cells die if the concentration of the living cells become so high as to reach the so-called "full growth" state. To prevent this, it is necessary to change the culture medium and/or to subculture the cells before the full growth state is reached. ~itherto, a technician determines the timing for changing the cell culture solution or for starting the subculturing by observing the cell proliferation of the j cells in the vessel through a microscope. However, such a conventional method is not quantitative and th~refore not accurate in determining the cell proliferation of cultured cells and the concentration of living cells. In addition, - lt is time-consuming and it is difficult to precisely observe the cell proliferation of a large amount of cultured cells.
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Further, when cells are cloned by a limiting dilution-culture method or when cells are fractionated, both the number of living cells and the number of dead cells should be simultaneously measured. In such cases, even if a blood cell counter is used, visual measurement with the microscope includes large errors and takes a lot of time.
A cell detection method using image processing has been proposed instead of visual observation with a micro-scope. However, it has low detection efficiency as it is done simply by binary digitizing. Although detection of the cells is easy when the background is simple, for example, in the case of an agar medium on which colonies are formed, when the pattern of cells and the background are both comlicated, for example, when the cells in the culturing vessel contain dead cells, dust or other foreign substances, detection is diEficult.
An object of the present invention is to provide a method of monitoring cell cultures so that the number of cells can be precisely measured when the pattern of the celIs and the background are complicated.
Another object of the present 1nvention is to provide a method of monitoring cell cultures which can treat a large number of cells in a short time.
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, _ 3 _ 132~75 In one preferred embodiment of the present invention these and other objects are achieved by a method of monitoring : cell culture comprising the steps of: digitizing an image of . cultured cells which have been input to image processing - 5 equipment through a TV camera connected with observing means;
extracting only living cells by processing with a spatial filter having a round shape, a designated size and positive coefficients in circumferential area wherein said round shape and designated size is determined from an average pattern of cell brightness distribution patterns, and measuring the number of living cells, wherein the cell culture comprises attachment-dependent eukaryotic cells.
In another embodiment of the invention there is provided a method of monitoring cell culture comprising the steps of: digitizing an image of cultured cells which have been input to image processing equipment through a TV camera connected with observing means; extracting living cells and dead cells by processing with a spatial filter having a round shape, a designated size and positive coefficients in circumferential area wherein said round shape and designated size is determined from an average pattern of cell bri~htness distributed patterns, and measuring the number of living cells and the number of dead cells, wherein the cell culture comprises attachment-dependent eukaryotic cells.
In the drawings which illustrate preferred embodiments of the present invention, Fig. 1 is a flow chart of procedures for measuring the number of living cells;
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; - 3a -Fig. 2 is a flow chart of procedures for measuring the number of living cells and the number of dead cells;
Fig. 3 shows shape and coefficients of a spatial filter having 7 x 7 pixels;
Fig. 4 shows shape and coefficients of an optimized spatial filter having 7 x 7 pixels; and Figs. 5 and 6 respectively show shape and coefficients of spatial filters used in Examples 1 and 2.
-I The method of monitoring cell cultures according to the present invention will be illustrated by reference to the accompanying drawings.
By using a TV camera connected to a microscope as an observing means, an image of the cultured cells in a culturing vessel is input to image processing equipment. According to the procedures shown in Fig. l, the image is processed and the , number of living cells counted.
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Preferably, expansion processing is carried out after the binary digitizing. According to the procedure as shown in Fig. 2, the number of living cells and the number of dead cells are measured.
By displacing the focus from an entirely focused state, the image of the cultured cells is inputted at a focus point where the center area of the cultured living cells is brighter and the circumferential area thereof is darker so that the image has a clear profile. By displacing the focus of the microscope at a distance of ~5 to -~250 micrometers, the image of the living cell has a clear profile. When the displaced distance is less than 5 micrometers, the whole image of the living cell remains dark. When the displaced distance is more than 250 micrometers, the image is blurred.
In this state, when the image of one cultured liv-ing cell is magnified by such magnification that the cellimage inscribes a square of a spatial filter matrix having a designated size, that is, when the spatial filter matrix has the size of n x n pixels (n 25), the image of the cultured cell is inputted at such magnification that one pixel has a size of 10/n x 10/n to 21/n x 21/n (micrometer x micrometer) since one cultured living cell generally has a size of 10 to 21 micrometers. The detection efficiency is poor when the living cell is larger or smaller than the spatial filter matrix of a designated size. A spatial filter matrix having a size not smaller than 5 x 5 pixels is used.
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', - s- ~325~75 When a spatial filter matrix having a size smaller than S
x 5 pixels is used, the filter has poor detection efficiency and cannot be used practically.
When the whole image is filtered through a round shaped spatial filter having negative coefficients in its circumferential area, positive coefficients in its center area and zero coefficients in other intermediate areas, the living cell parts can be emphasized. For example, the shape and coefficients of the spatial filter having 7 x 7 pixels are shown in Fig. 3.
The coefficients and shape of the filter for em-phasizing the living cell parts can be optimized by collect-ing brightness distribution patterns of the images of plural objective living cells and determining an average pattern~
The reason for the above is that since the spatial filtering emphasizes the parts which have better correlation with the shape of the spatial filter (standard pattern), the best correlation is realized when the shape of the spatial filter coincides with the cell brightness pattern. For example, the shape and coefficients of the optimized spatial filter having 7 x 7 pixels are shown in Fig. 4.
In this case, a value Yij of the center pixels is calculated according to the following equation:
1 ~3 +3 Yij A ~ ~ hmn Xi+m,j+n m=-3 n=-3 wherein x is a value of the pixel before filtering, , , ~: : , - 6 - 132547~
y is a value of the pixel after filtering, ; A is a coefficient, and hmn is a coefficient corresponding to each pixel of the filter.
The pixels are binary digitized with a suitable threshold.
The pixel ir. which the living cell is presen~ is digitized to be "1" and the pixel in which no living cell is present is digitized to be "O". The optimized binary digitized threshold is determined so that in one image, a visually counted number or the actual number of the living cel.ls coincides with the number of masses of the pixels which are digitized as "1" and considered to have the living . cell, and an average value of those in plural images can . be used as a fixed binary digitized threshold. The reason for this is that the correlation of the shape is observed ~; irrespective of background brightness. The number of the ~: living cells is calculated from the number of pixels i considered to have the living cell (the number of the ;~; pixels which are digitlzed as "1"). Since the number of pixels which one living cell occupies is constant, the number of the living cells is calculated according to the following equation:
The number of living cells =
The number of pixels which are ~: considered to have the living cells , The~ number of pixels which~~o-ne~~~-~
living pixel occupies ' '~
. . .
;~ , . : . .
.'` . :, ' - 7 - ~32~75 It is possible to decrease the errors resulting when the number of pixels is converted to the number of the cells, by subjecting the binary digitized image to a 4-neighbour expansion processing, 8-neighbour expansion processing or combination thereof one or more times before calculating the number of pixels from the binary digitized image. By the expansion processing, the images which are damaged by binary digitizing can be restored, and the number of pixels which one cell occupies is increased so that the influence of one pixel is decreased.
The image emphasized by spatial filtering is binary digitized again with a threshold different from, usually lower than, the threshold which is used to deter-mine the number of living cells and then inverted. Tbe pixel in which the dead cell is present is digitized to be "1" and the pixel in which no dead cell is present is digitized to be 1~0l~. From the number of pixels which are considered to have the dead cell (the number of pixels which are digitized as "1"), the number of dead cells is calculated. Since the num~er of pixels which one dead cell occupies is constant, the number of dead cells is calculated according to the following equation:
The number of dead cells=
The number of pixels which are considered to have the dead cells , The num~er of plxels whlcn one dead cell occupies .~ , , ; ~ ,. .. .
. , . , :
.
- 8 - ~32~75 The circumferential area (profile~ of the living cell may also be contained in the pixel of the dead cell depending on the absolute value of the threshold. This is removed by a procedure wherein the spatially filtered image is binary digitized with the threshold for determining the number of dead cells and then inverted r and a binary digi-tized image which is binary digitized with the threshold for determining the number of living cells or a binary digitized image which is subjected to an expansion pro-cessing for an appropriate number of times is deducted from said inverted image on the display. The expansion pro-cessing is preferably carried out several times. When the expansion processing is carried out too often, the neces-sary image is erased. In the same manner as for the living cells, the error caused when the number of cells is calcu-lated from the number of pixels is reduced by subjecting the binary digitized image from which the noise of the circumferential area of the living cell is removed to a 4-neighbour expansion processing, 8-neighbour expansion processing or combination thereof one or more times.
Now, the present invention is explained by the following examples.
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Example 1 A complicated image in which colonies of cultured living cells were present together with dead cells, foreign substances and the like was inputted to image processing equipment PIAS-l* (available from PC Systems, Japan~
through a microscope connected to a CCD camera by dis-placing the focus a distance of 30 micrometers from the entirely focused state.
The magnification was such that the profile of cultured living cell inscribed a spatial filter having 7 x 7 pixels, that is, one pixel had a size of 1.8 micrometers x 1.8 micrometers.
When the image was digitized with the image pro-i cessing equipment PIAS-l and filtered with a spatial filter having shape and coefficients shown in Fig. 5, detection rate was 67 % and 70 % with an erroneous detection rate of 10 % and 20 %, respectively. The detection rate and the , erroneous detection rate were determined according to the following equations:
Detection rate =
The number of actually present living cells which are detected by the image processing :
The number of living cells actually present in the original image Erroneous detection rate =
` The number of living cells considered to be present by the image processing but not actually present in an original image The number of all living cells which are consi-dered to be present by the image processing *Ir~de Mark '1.
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Example 2 A complicated image in which colonies of cultured living cells were present with dead cells, foreign sub-stances and the like was inputted to image processing equipment PIP-4000* (available from ADS Limited, Japan) through a microscope connected to a CCD camera by displacing the focus a distance of 30 micrometers from the entirely focused state.
` The magnification was such that a profile of ! cultured living cell inscribed a spatial filter having 7 x 7 pixels, that is, one pixel had a size of 2.4 micrometers x 2.4 micrometers.
The image was digitized with the image processing equipment PIP-4000 and filtered with the spatial filter having shape and coefficients shown in Fig. 6. When the ~; binary digitizing threshold which is based on the visually counted number of living cells was used, the detection rate was 73% and 81% with the erroneous detection rate of 10~
and 20%, respectively. When the same image was subjected to the binary digitizing alone, the detection rate was 57%
and 60% with the erroneous detection rate of 10% and 20%, respectively.
! The image in which living cells and dead cells were randomly present was inputted in the same manner as above, and the number of living cells and the number of ! dead cells were measured. As for the living cells, the .
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L3~5475 visually counted number was 47 and the number measured by the image processing was 50, an error of 6.4%. As for the dead cells, the visually counted number was 453 and the number measured by the image processing was 453, an error of 0~. Further, the error of the measured number of total cells (the number of living cells and the number of the dead cells) had a standard deviation of +10.2~ in one sigma for 39 samples. The error was determined according to the following equation:
Error =
; (The number measured by image processing) -(The visually counted number) The visually counted number Example 3 The procedure as in Example 2 was repeated except that the binary digitizing threshold based on the actual number of living cells was used instead of the binary digitizing threshold based on the visually counted number of ~' living cells. When the image was filcered with the spatial filter having the same shape and coefficients as used in Example 2, the detection rate was 73 % and 82 ~ in the erroneous detection rate of 10 % and 20 %, respectively.
When the binary digitized image was subjected to 8-neighbor expansion processing once, the error caused by `~ converting the number of pixels to the number of , living cells was 8 ~. It was 17 % when the image had not been subjected to the expansion processing.
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Further, when cells are cloned by a limiting dilution-culture method or when cells are fractionated, both the number of living cells and the number of dead cells should be simultaneously measured. In such cases, even if a blood cell counter is used, visual measurement with the microscope includes large errors and takes a lot of time.
A cell detection method using image processing has been proposed instead of visual observation with a micro-scope. However, it has low detection efficiency as it is done simply by binary digitizing. Although detection of the cells is easy when the background is simple, for example, in the case of an agar medium on which colonies are formed, when the pattern of cells and the background are both comlicated, for example, when the cells in the culturing vessel contain dead cells, dust or other foreign substances, detection is diEficult.
An object of the present invention is to provide a method of monitoring cell cultures so that the number of cells can be precisely measured when the pattern of the celIs and the background are complicated.
Another object of the present 1nvention is to provide a method of monitoring cell cultures which can treat a large number of cells in a short time.
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, _ 3 _ 132~75 In one preferred embodiment of the present invention these and other objects are achieved by a method of monitoring : cell culture comprising the steps of: digitizing an image of . cultured cells which have been input to image processing - 5 equipment through a TV camera connected with observing means;
extracting only living cells by processing with a spatial filter having a round shape, a designated size and positive coefficients in circumferential area wherein said round shape and designated size is determined from an average pattern of cell brightness distribution patterns, and measuring the number of living cells, wherein the cell culture comprises attachment-dependent eukaryotic cells.
In another embodiment of the invention there is provided a method of monitoring cell culture comprising the steps of: digitizing an image of cultured cells which have been input to image processing equipment through a TV camera connected with observing means; extracting living cells and dead cells by processing with a spatial filter having a round shape, a designated size and positive coefficients in circumferential area wherein said round shape and designated size is determined from an average pattern of cell bri~htness distributed patterns, and measuring the number of living cells and the number of dead cells, wherein the cell culture comprises attachment-dependent eukaryotic cells.
In the drawings which illustrate preferred embodiments of the present invention, Fig. 1 is a flow chart of procedures for measuring the number of living cells;
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; - 3a -Fig. 2 is a flow chart of procedures for measuring the number of living cells and the number of dead cells;
Fig. 3 shows shape and coefficients of a spatial filter having 7 x 7 pixels;
Fig. 4 shows shape and coefficients of an optimized spatial filter having 7 x 7 pixels; and Figs. 5 and 6 respectively show shape and coefficients of spatial filters used in Examples 1 and 2.
-I The method of monitoring cell cultures according to the present invention will be illustrated by reference to the accompanying drawings.
By using a TV camera connected to a microscope as an observing means, an image of the cultured cells in a culturing vessel is input to image processing equipment. According to the procedures shown in Fig. l, the image is processed and the , number of living cells counted.
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Preferably, expansion processing is carried out after the binary digitizing. According to the procedure as shown in Fig. 2, the number of living cells and the number of dead cells are measured.
By displacing the focus from an entirely focused state, the image of the cultured cells is inputted at a focus point where the center area of the cultured living cells is brighter and the circumferential area thereof is darker so that the image has a clear profile. By displacing the focus of the microscope at a distance of ~5 to -~250 micrometers, the image of the living cell has a clear profile. When the displaced distance is less than 5 micrometers, the whole image of the living cell remains dark. When the displaced distance is more than 250 micrometers, the image is blurred.
In this state, when the image of one cultured liv-ing cell is magnified by such magnification that the cellimage inscribes a square of a spatial filter matrix having a designated size, that is, when the spatial filter matrix has the size of n x n pixels (n 25), the image of the cultured cell is inputted at such magnification that one pixel has a size of 10/n x 10/n to 21/n x 21/n (micrometer x micrometer) since one cultured living cell generally has a size of 10 to 21 micrometers. The detection efficiency is poor when the living cell is larger or smaller than the spatial filter matrix of a designated size. A spatial filter matrix having a size not smaller than 5 x 5 pixels is used.
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', - s- ~325~75 When a spatial filter matrix having a size smaller than S
x 5 pixels is used, the filter has poor detection efficiency and cannot be used practically.
When the whole image is filtered through a round shaped spatial filter having negative coefficients in its circumferential area, positive coefficients in its center area and zero coefficients in other intermediate areas, the living cell parts can be emphasized. For example, the shape and coefficients of the spatial filter having 7 x 7 pixels are shown in Fig. 3.
The coefficients and shape of the filter for em-phasizing the living cell parts can be optimized by collect-ing brightness distribution patterns of the images of plural objective living cells and determining an average pattern~
The reason for the above is that since the spatial filtering emphasizes the parts which have better correlation with the shape of the spatial filter (standard pattern), the best correlation is realized when the shape of the spatial filter coincides with the cell brightness pattern. For example, the shape and coefficients of the optimized spatial filter having 7 x 7 pixels are shown in Fig. 4.
In this case, a value Yij of the center pixels is calculated according to the following equation:
1 ~3 +3 Yij A ~ ~ hmn Xi+m,j+n m=-3 n=-3 wherein x is a value of the pixel before filtering, , , ~: : , - 6 - 132547~
y is a value of the pixel after filtering, ; A is a coefficient, and hmn is a coefficient corresponding to each pixel of the filter.
The pixels are binary digitized with a suitable threshold.
The pixel ir. which the living cell is presen~ is digitized to be "1" and the pixel in which no living cell is present is digitized to be "O". The optimized binary digitized threshold is determined so that in one image, a visually counted number or the actual number of the living cel.ls coincides with the number of masses of the pixels which are digitized as "1" and considered to have the living . cell, and an average value of those in plural images can . be used as a fixed binary digitized threshold. The reason for this is that the correlation of the shape is observed ~; irrespective of background brightness. The number of the ~: living cells is calculated from the number of pixels i considered to have the living cell (the number of the ;~; pixels which are digitlzed as "1"). Since the number of pixels which one living cell occupies is constant, the number of the living cells is calculated according to the following equation:
The number of living cells =
The number of pixels which are ~: considered to have the living cells , The~ number of pixels which~~o-ne~~~-~
living pixel occupies ' '~
. . .
;~ , . : . .
.'` . :, ' - 7 - ~32~75 It is possible to decrease the errors resulting when the number of pixels is converted to the number of the cells, by subjecting the binary digitized image to a 4-neighbour expansion processing, 8-neighbour expansion processing or combination thereof one or more times before calculating the number of pixels from the binary digitized image. By the expansion processing, the images which are damaged by binary digitizing can be restored, and the number of pixels which one cell occupies is increased so that the influence of one pixel is decreased.
The image emphasized by spatial filtering is binary digitized again with a threshold different from, usually lower than, the threshold which is used to deter-mine the number of living cells and then inverted. Tbe pixel in which the dead cell is present is digitized to be "1" and the pixel in which no dead cell is present is digitized to be 1~0l~. From the number of pixels which are considered to have the dead cell (the number of pixels which are digitized as "1"), the number of dead cells is calculated. Since the num~er of pixels which one dead cell occupies is constant, the number of dead cells is calculated according to the following equation:
The number of dead cells=
The number of pixels which are considered to have the dead cells , The num~er of plxels whlcn one dead cell occupies .~ , , ; ~ ,. .. .
. , . , :
.
- 8 - ~32~75 The circumferential area (profile~ of the living cell may also be contained in the pixel of the dead cell depending on the absolute value of the threshold. This is removed by a procedure wherein the spatially filtered image is binary digitized with the threshold for determining the number of dead cells and then inverted r and a binary digi-tized image which is binary digitized with the threshold for determining the number of living cells or a binary digitized image which is subjected to an expansion pro-cessing for an appropriate number of times is deducted from said inverted image on the display. The expansion pro-cessing is preferably carried out several times. When the expansion processing is carried out too often, the neces-sary image is erased. In the same manner as for the living cells, the error caused when the number of cells is calcu-lated from the number of pixels is reduced by subjecting the binary digitized image from which the noise of the circumferential area of the living cell is removed to a 4-neighbour expansion processing, 8-neighbour expansion processing or combination thereof one or more times.
Now, the present invention is explained by the following examples.
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~ ::., , -, "
., , . ~ , ~ :: . , :,. : ~ ' ' : ' I
- 9 - 1~ 2 ~ 47 ~
Example 1 A complicated image in which colonies of cultured living cells were present together with dead cells, foreign substances and the like was inputted to image processing equipment PIAS-l* (available from PC Systems, Japan~
through a microscope connected to a CCD camera by dis-placing the focus a distance of 30 micrometers from the entirely focused state.
The magnification was such that the profile of cultured living cell inscribed a spatial filter having 7 x 7 pixels, that is, one pixel had a size of 1.8 micrometers x 1.8 micrometers.
When the image was digitized with the image pro-i cessing equipment PIAS-l and filtered with a spatial filter having shape and coefficients shown in Fig. 5, detection rate was 67 % and 70 % with an erroneous detection rate of 10 % and 20 %, respectively. The detection rate and the , erroneous detection rate were determined according to the following equations:
Detection rate =
The number of actually present living cells which are detected by the image processing :
The number of living cells actually present in the original image Erroneous detection rate =
` The number of living cells considered to be present by the image processing but not actually present in an original image The number of all living cells which are consi-dered to be present by the image processing *Ir~de Mark '1.
:
," ~ .
~' ' ;. ~ ` ' ' ' , .' ' ' ":
. ' ' ' ~ . ' ' ,,, ~.' ' " ``,'' "'' ` '' ' - lo ~2~7~
Example 2 A complicated image in which colonies of cultured living cells were present with dead cells, foreign sub-stances and the like was inputted to image processing equipment PIP-4000* (available from ADS Limited, Japan) through a microscope connected to a CCD camera by displacing the focus a distance of 30 micrometers from the entirely focused state.
` The magnification was such that a profile of ! cultured living cell inscribed a spatial filter having 7 x 7 pixels, that is, one pixel had a size of 2.4 micrometers x 2.4 micrometers.
The image was digitized with the image processing equipment PIP-4000 and filtered with the spatial filter having shape and coefficients shown in Fig. 6. When the ~; binary digitizing threshold which is based on the visually counted number of living cells was used, the detection rate was 73% and 81% with the erroneous detection rate of 10~
and 20%, respectively. When the same image was subjected to the binary digitizing alone, the detection rate was 57%
and 60% with the erroneous detection rate of 10% and 20%, respectively.
! The image in which living cells and dead cells were randomly present was inputted in the same manner as above, and the number of living cells and the number of ! dead cells were measured. As for the living cells, the .
, *Trade mark ) :, . , . - . , .
..
L3~5475 visually counted number was 47 and the number measured by the image processing was 50, an error of 6.4%. As for the dead cells, the visually counted number was 453 and the number measured by the image processing was 453, an error of 0~. Further, the error of the measured number of total cells (the number of living cells and the number of the dead cells) had a standard deviation of +10.2~ in one sigma for 39 samples. The error was determined according to the following equation:
Error =
; (The number measured by image processing) -(The visually counted number) The visually counted number Example 3 The procedure as in Example 2 was repeated except that the binary digitizing threshold based on the actual number of living cells was used instead of the binary digitizing threshold based on the visually counted number of ~' living cells. When the image was filcered with the spatial filter having the same shape and coefficients as used in Example 2, the detection rate was 73 % and 82 ~ in the erroneous detection rate of 10 % and 20 %, respectively.
When the binary digitized image was subjected to 8-neighbor expansion processing once, the error caused by `~ converting the number of pixels to the number of , living cells was 8 ~. It was 17 % when the image had not been subjected to the expansion processing.
~' .
, .. , :. . . :
; . . :,. . .
: . .
Claims (12)
1. A method of monitoring cell culture comprising the steps of:
digitizing an image of cultured cells which have been input to image processing equipment through a TV camera connected with observing means;
extracting only living cells by processing with a spatial filter having a round shape, a designated size and positive coefficients in circumferential area wherein said round shape and designated size is determined from an average pattern of cell brightness distribution patterns, and measuring the number of living cells, wherein the cell culture comprises attachment-dependent eukaryotic cells.
digitizing an image of cultured cells which have been input to image processing equipment through a TV camera connected with observing means;
extracting only living cells by processing with a spatial filter having a round shape, a designated size and positive coefficients in circumferential area wherein said round shape and designated size is determined from an average pattern of cell brightness distribution patterns, and measuring the number of living cells, wherein the cell culture comprises attachment-dependent eukaryotic cells.
2. A method of monitoring cell culture comprising the steps of:
digitizing an image of cultured cells which have been input to image processing equipment through a TV camera connected with observing means;
extracting living cells and dead cells by processing with a spatial filter having a round shape, a designated size and positive coefficients in circumferential area wherein said round shape and designated size is determined from an average pattern of cell brightness distributed patterns, and measuring the number of living cells and the number of dead cells, wherein the cell culture comprises attachment-dependent eukaryotic cells.
digitizing an image of cultured cells which have been input to image processing equipment through a TV camera connected with observing means;
extracting living cells and dead cells by processing with a spatial filter having a round shape, a designated size and positive coefficients in circumferential area wherein said round shape and designated size is determined from an average pattern of cell brightness distributed patterns, and measuring the number of living cells and the number of dead cells, wherein the cell culture comprises attachment-dependent eukaryotic cells.
3. The method of monitoring cell culture according to claim 1 or 2, wherein a focus is displaced from an entirely focused state and the image of the cultured cells is input at a focus point where center area of cultured living cells is brighter and circumferential area thereof is darker so that an image has a clear profile.
4. The method of monitoring cell culture according to claim 1 or 2, wherein the image of cultured cells is input at the focus point where the focus is displaced in a distance of +5 to micrometers from the entirely focused state.
5. The method of monitoring cell culture according to claim 4, wherein the spatial filter has a size of n x n pixels (n ? 5).
6. The method according to claim 5, wherein the image of cultured cells is input in such magnification that one pixel has a size of N/n (wherein N (µm) is an average diameter of the cultured cells).
7. The method of monitoring cell culture according to claim 1 or 2, wherein the image processed with the spatial filter is binary digitized and the number of cultured living cells is calculated according to the following equation:
The number of the living cells =
The number of the living cells =
8. The method of monitoring cell culture according to claim 7, wherein binary digitized thresholds in plural images are determined so that the visually measured number or actual number of living cells in one image is the same as the number of masses which are considered to have the living cells and then the binary digitizing is carried out by using an average value of the binary digitized thresholds as a fixed binary digitized threshold.
9. The method of monitoring cell culture according to claim 7, wherein the spatially filtered image is binary digitized and subjected to a 4-neighbor expansion processing, 8-neighbor expansion processing or combination thereof one or more times and then the number of living cells is calculated from that of pixels.
10. The method of monitoring cell culture according to claim 1 or 2, wherein the spatially filtered image is binary digitized with a threshold lower than the threshold which is used to determine the number of living cells and then inverted, and the number of dead cells is calculated according to the following equation:
The number of the dead cells =
The number of the dead cells =
11. The method of monitoring cell culture according to claim 10, wherein the spatially filtered image is binary digitized with the threshold for determining the number of dead cells and then inverted and a binary digitized image which is binary digitized with the threshold for determining the number of living cells or a binary digitized image which is subjected to an expansion processing a suitable number of times is deducted from said inverted image on the display, thereby parts corresponding to the dead cells are extracted.
12. The method of monitoring cell culture according to claim 10, wherein the binary digitized image in which parts corresponding to the dead cells are extracted is subjected to a 4-neighbor expansion processing, 8-neighbor expansion processing or combination thereof one or more times before the number of the dead cells is calculated.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/JP1987/000750 WO1989003431A1 (en) | 1987-10-06 | 1987-10-06 | Cell culture monitor method |
| CA000584597A CA1325475C (en) | 1987-10-06 | 1988-11-30 | Method of monitoring cell culture |
| US07/900,791 US5270173A (en) | 1987-10-06 | 1992-06-22 | Method of monitoring cell culture |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/JP1987/000750 WO1989003431A1 (en) | 1987-10-06 | 1987-10-06 | Cell culture monitor method |
| CA000584597A CA1325475C (en) | 1987-10-06 | 1988-11-30 | Method of monitoring cell culture |
| US07/900,791 US5270173A (en) | 1987-10-06 | 1992-06-22 | Method of monitoring cell culture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1325475C true CA1325475C (en) | 1993-12-21 |
Family
ID=25672265
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000584597A Expired - Fee Related CA1325475C (en) | 1987-10-06 | 1988-11-30 | Method of monitoring cell culture |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1325475C (en) |
-
1988
- 1988-11-30 CA CA000584597A patent/CA1325475C/en not_active Expired - Fee Related
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