JP6300679B2 - pH determination device, pH determination method, and pH determination program - Google Patents

Description

  The present invention relates to a pH determination apparatus, a pH determination method, and a pH determination program for measuring the pH of a measurement object to which a pH indicator in a container is added, for example, the pH of a medium containing cultured cells.

Conventionally, in the field of cell culture, a method of observing whether or not the culture of a cell placed in a culture vessel is progressing by measuring the pH of a medium containing the cell has been used.
The pH measurement of the medium was originally measured one by one using a contact-type pH sensor, but in recent years, since the number of cell holding recesses (wells) in the culture container has increased, In order to improve working efficiency, there is a demand for a measuring apparatus that can measure the pH of a medium containing cells without using a sensor or the like.

In such a pH measurement device, a pH indicator (for example, phenol red, methyl orange, phenolphthalein, litmus, etc.) is added to a medium containing cells to be measured, and a change in the color of the pH indicator is detected in advance. The pH measurement can be performed by referring to a table showing the relationship between the stored pH value and hue.
For example, Patent Document 1 discloses a method for displaying a change in pH distribution in which the pH of a measurement object is measured in a non-contact manner and displayed as a color image.

JP 2001-343328 A

However, the conventional method has the following problems.
That is, in the method for displaying a change in pH distribution disclosed in the above publication, if variations due to cameras, light sources, optical components, etc. used when acquiring a color image occur, the detected color also varies. May occur and the measurement accuracy may be reduced.
Moreover, there is a possibility that the measurement accuracy may be lowered due to changes in the camera, the light source, the optical components, and the like due to changes in the environmental temperature and deterioration over time.

Furthermore, if the material of the culture container in which the cells are placed is changed or if there is a difference in the production lot or the date and time of manufacture, even if the product number is the same, there will be a difference in the amount of light transmitted or reflected. There is a case. In this case, the color image acquired by the camera is affected, and there is a possibility that the measurement accuracy may be reduced.
An object of the present invention is to provide a pH determination device, a pH determination method, and a pH determination program capable of preventing a decrease in measurement accuracy caused by a variation with time, variation in material of a culture vessel, etc. for each device. There is.

  A pH determination device according to a first aspect of the present invention is a pH determination device for determining the pH of a measurement object to which a pH indicator in a container is added, by image processing, an image acquisition unit, a reference color display unit, and a storage Part and a control part. The image acquisition unit images the container from above and acquires an image including the portion of the measurement object. The reference color display unit is arranged in the vicinity of the container so as to be included in the image acquired by the image acquisition unit together with the container, and indicates a plurality of colors serving as a reference for the color that changes depending on the pH of the measurement object. The storage unit stores a reference pH value corresponding to each color included in the reference color display unit. The control unit obtains a correction value from the difference between the reference pH value stored in the storage unit and the measured pH value of each color of the reference color display unit calculated based on the image acquired in the image acquisition unit, and the correction value Is used to correct the measured pH value.

  Here, in the pH determination device that determines the pH of the measurement object that changes according to the growth state of the cells placed in the container by image processing of an image acquired so as to include the container and the reference color display unit, A difference between a pH value corresponding to a plurality of colors indicated in the reference color display unit stored in advance in the storage unit and a measured pH value calculated by image processing is obtained. Based on this difference, a correction value for correcting the measured pH value is calculated, and the measurement correction value is corrected.

  Here, for example, a pH indicator such as phenol red, methyl orange, phenolphthalein, and litmus is added to the measurement object in which the cells are put, and the color change is determined by image processing. The pH value corresponding to each color can be calculated. In addition, the reference color display unit is provided at a position adjacent to the position where the container is placed, and varies depending on, for example, about 4 to 8 colors according to a stepwise change in pH of the measurement object. The colored part is shown. The storage unit stores the pH value corresponding to each color of the reference color display unit in association with each other.

  Thereby, since the reference color display part is reflected in the image obtained by photographing the container from above with the container, the pH value corresponding to each color of the reference color display part is calculated by image processing at a desired timing. be able to. Then, if there is a difference between the measured pH value and the pH value corresponding to each color stored in advance, a correction value for eliminating the difference is always obtained, so that the measured pH value is always obtained. Correcting the value enables highly accurate pH measurement.

As a result, it is possible to prevent a decrease in measurement accuracy caused by variations in the apparatus, changes over time, container materials, and the like.
A pH determination device according to a second invention is the pH determination device according to the first invention, wherein the control unit is a reference color display unit calculated using an image acquired by a reference image acquisition unit. A correction value is obtained from the difference between the measured pH value of each color and the measured pH value for each device, and the measured pH value for each device is corrected using the correction value.

Here, when the device is shipped, the measured pH value of each color of the reference color display unit calculated by the image acquisition unit (camera etc.) of the master device serving as a reference, and the image acquisition unit (camera etc.) of other devices When there is a difference between the measured pH values of the respective colors of the reference color display unit calculated by the above, a correction value for eliminating the difference is obtained, and the measured pH value of each device is corrected.
Thereby, the dispersion | variation resulting from the individual difference of the structure (for example, image acquisition part (camera etc.), light source, etc.) on the apparatus side can be eliminated, and the measurement precision for every apparatus can be improved.

A pH determination device according to a third invention is the pH determination device according to the first invention, wherein the control unit updates the correction value every time the system is started or every predetermined time has elapsed, and the updated correction value is obtained. Use to correct the measured pH value.
Here, the measured pH value of each color of the reference color display unit calculated by the image acquisition unit (camera, etc.) automatically every time the apparatus is started or every predetermined time elapses, and the reference color stored in advance When there is a difference between the pH values of the respective colors of the display unit, a correction value for eliminating the difference is obtained, and the measured pH value is corrected.

As a result, it is possible to improve the measurement accuracy by eliminating variations caused by changes in each component of the apparatus over time, changes in environmental temperature, and the like.
A pH determination apparatus according to a fourth aspect of the present invention is the pH determination apparatus according to the first aspect of the present invention, wherein the container has a color display unit corresponding to a specific color of the reference color display unit. The control unit obtains a correction value from the difference between the measured pH value corresponding to the color of the color display unit of the reference container and the measured pH value of the color display unit for each container, and uses the correction value for each container. Correct the measured pH value.

Here, a color display unit for displaying a color corresponding to a predetermined pH value is provided in a part of the container, similarly to the reference color display unit. When the image including the container is acquired, the measured pH value corresponding to the color displayed on the color display unit is calculated, and if there is a difference compared with the pH value stored in advance, the difference is eliminated. Make corrections.
Here, the color display portion is, for example, a portion that is provided at a corner portion of the container and shows the same color as a predetermined position indicated on the reference color display portion, and is always changed even if the container changes. It shows a certain color.

Thereby, for example, even if the product number is the same, even if the material, thickness, shape, etc. are partially changed, even if the color of the measurement object held in the image acquired container is affected, the container It is possible to eliminate the measurement variation caused by. Therefore, even if there is a minor change in the container unintentionally, it is possible to prevent the measurement accuracy from deteriorating.
A pH determination device according to a fifth invention is the pH determination device according to any one of the first to fourth inventions, wherein the container has a plurality of recesses into which measurement objects including cells are placed. ing. In the reference color display unit, each color is arranged at a pitch corresponding to the interval at which the plurality of concave portions of the container are arranged.

Here, when the container has a large number of recesses (wells) for containing cells, such as well plates, the reference color array included in the reference color display section is defined as the recess array pitch. They are arranged to match.
As a result, when the image including the container and the reference color display unit is image-processed, the measurement of the reference color display unit is also performed at the same pitch as the pitch at which each concave portion of the container is measured during pH measurement. Can do. Therefore, it is possible to simplify the image processing in the control unit as much as possible and efficiently perform pH measurement.

A pH determination apparatus according to a sixth aspect of the present invention is the pH determination apparatus according to any one of the second to fourth aspects of the present invention, wherein the control unit includes L * (brightness), C * (saturation), h The measured pH value is corrected using the h value of the L * C * h color system consisting of (hue angle).
Here, the measured pH value is corrected using the value h representing the hue of the L * C * h color system.

Thereby, it is possible to prevent the measurement accuracy from being lowered due to other factors such as luminance, and to perform more accurate pH measurement.
A pH determination device according to a seventh aspect of the present invention is the pH determination device according to any one of the first to sixth aspects, wherein the light source unit is disposed below the container and irradiates the container with light. And an optical system having telecentricity disposed between the container and the image acquisition unit.

Here, in the pH determination apparatus, the light source unit, the container, the optical system having telecentricity, and the image acquisition unit are arranged in this order from the bottom.
Here, the telecentric optical system includes, for example, a Fresnel lens. The light source unit includes, for example, a white LED.
Thereby, for example, in a state where white light is irradiated from under the container, the image of the container viewed from directly above can be acquired by an image acquisition unit such as a camera by the Fresnel lens arranged on the upper surface side of the container. .

Therefore, the change in the pH value of the measurement object in the container can be determined more accurately.
A pH determination device according to an eighth invention is the pH determination device according to the seventh invention, wherein the optical system having telecentricity acquires an image in a range including the container and the reference color display unit in the image acquisition unit. The optical center position is arranged at a position shifted to the container side.

Here, the optical center position of an optical system having telecentricity such as a Fresnel lens is disposed at a position shifted from the center of the range including the container and the reference color display unit toward the container.
Thereby, in the image acquisition unit, it is possible to acquire an image in which the distortion on the container side that may affect the measurement accuracy is minimized as compared with the reference color display unit. Therefore, based on the acquired image, the pH value of the measurement object held in the container can be determined more accurately.

  A pH determination method according to a ninth aspect of the present invention is a pH determination method for determining, by image processing, the pH of a measurement object to which a pH indicator in a container is added, an acquisition step, a storage step, and a correction value calculation step. And a correction step. In the acquisition step, the container is photographed from above, and an image including the portion of the measurement object is acquired. The storage step is arranged in the vicinity of the container so as to be included in the image together with the container, and corresponds to each color included in the reference color display unit indicating a plurality of colors serving as a reference of the color that changes depending on the pH of the measurement object. The reference pH value to be stored is stored. In the correction value calculating step, a correction value is obtained from a difference between the reference pH value and the measured pH value of each color of the reference color display unit calculated based on the image. In the correction step, the measured pH value is corrected using the correction value.

  Here, in the pH determination device that determines the pH of the measurement object that changes according to the growth state of the cells placed in the container by image processing of an image acquired so as to include the container and the reference color display unit, A difference between a pH value corresponding to a plurality of colors indicated in the reference color display unit stored in advance and a measured pH value calculated by image processing is obtained. Based on this difference, a correction value for correcting the measured pH value is calculated, and the measurement correction value is corrected.

  Here, for example, a pH indicator such as phenol red, methyl orange, phenolphthalein, and litmus is added to the measurement object in which the cells are put, and the color change is determined by image processing. The pH value corresponding to each color can be calculated. In addition, the reference color display unit is provided at a position adjacent to the position where the container is placed, and varies depending on, for example, about 4 to 8 colors according to a stepwise change in pH of the measurement object. The colored part is shown. Then, pH values corresponding to the respective colors of the reference color display unit are associated and stored in advance.

  Thereby, since the reference color display part is reflected in the image obtained by photographing the container from above with the container, the pH value corresponding to each color of the reference color display part is calculated by image processing at a desired timing. be able to. Then, if there is a difference between the measured pH value and the pH value corresponding to each color stored in advance, a correction value for eliminating the difference is always obtained, so that the measured pH value is always obtained. Correcting the value enables highly accurate pH measurement.

As a result, it is possible to prevent a decrease in measurement accuracy caused by variations in the apparatus, changes over time, container materials, and the like.
A pH determination method according to a tenth aspect of the invention is the pH determination method according to the ninth aspect of the invention, wherein, in the correction value calculation step, a reference color display calculated using an image acquired by a reference image acquisition unit The correction value is obtained from the difference between the measured pH value of each color of the unit and the measured pH value of each device.

Here, when the device is shipped, the measured pH value of each color of the reference color display unit calculated by the image acquisition unit (camera etc.) of the master device serving as a reference, and the image acquisition unit (camera etc.) of other devices When there is a difference between the measured pH values of the respective colors of the reference color display unit calculated by the above, a correction value for eliminating the difference is obtained, and the measured pH value of each device is corrected.
Thereby, the dispersion | variation resulting from the individual difference of the structure (for example, image acquisition part (camera etc.), light source, etc.) on the apparatus side can be eliminated, and the measurement precision for every apparatus can be improved.

A pH determination method according to an eleventh aspect of the present invention is the pH determination method according to the ninth aspect of the present invention, wherein, in the correction value calculation step, the correction value is updated every time when starting up or every predetermined time. In the correction step, the measured pH value is corrected using the updated correction value.
Here, the measured pH value of each color of the reference color display unit calculated by the image acquisition unit (camera, etc.) automatically every time the apparatus is started or every predetermined time elapses, and the reference color stored in advance When there is a difference between the pH values of the respective colors of the display unit, a correction value for eliminating the difference is obtained, and the measured pH value is corrected.

As a result, it is possible to improve the measurement accuracy by eliminating variations caused by changes in each component of the apparatus over time, changes in environmental temperature, and the like.
A pH determination method according to a twelfth aspect of the present invention is the pH determination method according to the ninth aspect of the present invention, wherein the container has a color display unit corresponding to a specific color of the reference color display unit. In the correction value calculating step, a correction value is obtained from the difference between the measured pH value corresponding to the color of the color display portion of the container serving as a reference and the measured pH value of the color of the color display portion for each container.

Here, a color display unit for displaying a color corresponding to a predetermined pH value is provided in a part of the container, similarly to the reference color display unit. When the image including the container is acquired, the measured pH value corresponding to the color displayed on the color display unit is calculated, and if there is a difference compared with the pH value stored in advance, the difference is eliminated. Make corrections.
Here, the color display portion is, for example, a portion that is provided at a corner portion of the container and shows the same color as a predetermined position indicated on the reference color display portion, and is always changed even if the container changes. It shows a certain color.

Thereby, for example, even if the product number is the same, even if the material, thickness, shape, etc. are partially changed, even if the color of the measurement object held in the image acquired container is affected, the container It is possible to eliminate the measurement variation caused by. Therefore, even if there is a minor change in the container unintentionally, it is possible to prevent the measurement accuracy from deteriorating.
A pH determination method according to a thirteenth aspect of the present invention is the pH determination method according to any one of the ninth to twelfth aspects of the present invention, wherein the container has a plurality of recesses into which measurement objects including cells are placed. In the reference color display section, each color is arranged at a pitch corresponding to the interval at which the plurality of concave portions of the container are arranged. In the correction value calculating step, the pH determination is performed using the image including the reference color display unit and the container while detecting the measurement points at the same pitch.

Here, when the container has a large number of recesses (wells) for containing cells, such as well plates, the reference color array included in the reference color display section is defined as the recess array pitch. They are arranged to match.
As a result, when the image including the container and the reference color display unit is image-processed, the measurement of the reference color display unit is also performed at the same pitch as the pitch at which each concave portion of the container is measured during pH measurement. Can do. Therefore, it is possible to simplify the image processing in the control unit as much as possible and efficiently perform pH measurement.

A pH determination method according to a fourteenth invention is the pH determination method according to the tenth to twelfth inventions, wherein the control unit is based on L * (lightness), C * (saturation), and h (hue angle). The measured pH value is corrected using the h value of the L * C * h color system.
Here, the measured pH value is corrected using the value h representing the hue of the L * C * h color system.

Thereby, it is possible to prevent the measurement accuracy from being lowered due to other factors such as luminance, and to perform more accurate pH measurement.
A pH determination program according to a fifteenth aspect of the present invention is a pH determination program for causing a computer to execute a pH determination method for determining, by image processing, the pH of a measurement object to which a pH indicator in a container has been added. A computer is caused to execute a pH determination method including a storage step, a correction value calculation step, and a correction step. In the acquisition step, the container is photographed from above, and an image including the portion of the measurement object is acquired. The storage step is arranged in the vicinity of the container so as to be included in the image together with the container, and corresponds to each color included in the reference color display unit indicating a plurality of colors serving as a reference of the color that changes depending on the pH of the measurement object. The reference pH value to be stored is stored. In the correction value calculating step, a correction value is obtained from a difference between the reference pH value and the measured pH value of each color of the reference color display unit calculated based on the image. In the correction step, the measured pH value is corrected using the correction value.

  Here, in the pH determination device that determines the pH of the measurement object that changes according to the growth state of the cells placed in the container by image processing of an image acquired so as to include the container and the reference color display unit, A difference between a pH value corresponding to a plurality of colors indicated in the reference color display unit stored in advance and a measured pH value calculated by image processing is obtained. Based on this difference, a correction value for correcting the measured pH value is calculated, and the measurement correction value is corrected.

  Here, for example, a pH indicator such as phenol red, methyl orange, phenolphthalein, and litmus is added to the measurement object in which the cells are put, and the color change is determined by image processing. The pH value corresponding to each color can be calculated. In addition, the reference color display unit is provided at a position adjacent to the position where the container is placed, and varies depending on, for example, about 4 to 8 colors according to a stepwise change in pH of the measurement object. The colored part is shown. Then, pH values corresponding to the respective colors of the reference color display unit are associated and stored in advance.

  Thereby, since the reference color display part is reflected in the image obtained by photographing the container from above with the container, the pH value corresponding to each color of the reference color display part is calculated by image processing at a desired timing. be able to. Then, if there is a difference between the measured pH value and the pH value corresponding to each color stored in advance, a correction value for eliminating the difference is always obtained, so that the measured pH value is always obtained. Correcting the value enables highly accurate pH measurement.

As a result, it is possible to prevent a decrease in measurement accuracy caused by variations in the apparatus, changes over time, container materials, and the like.
A pH determination program according to a sixteenth aspect of the present invention is the pH determination program according to the fifteenth aspect of the present invention, wherein, in the correction value calculation step, a reference color display calculated using an image acquired by a reference image acquisition unit The correction value is obtained from the difference between the measured pH value of each color of the unit and the measured pH value of each device.

Here, when the device is shipped, the measured pH value of each color of the reference color display unit calculated by the image acquisition unit (camera etc.) of the master device serving as a reference, and the image acquisition unit (camera etc.) of other devices When there is a difference between the measured pH values of the respective colors of the reference color display unit calculated by the above, a correction value for eliminating the difference is obtained, and the measured pH value of each device is corrected.
Thereby, the dispersion | variation resulting from the individual difference of the structure (for example, image acquisition part (camera etc.), light source, etc.) on the apparatus side can be eliminated, and the measurement precision for every apparatus can be improved.

A pH determination program according to a seventeenth aspect of the present invention is the pH determination program according to the fifteenth or sixteenth aspect of the present invention, and in the correction value calculating step, the correction value is updated every time when starting up or every predetermined time. In the correction step, the measured pH value is corrected using the updated correction value.
Here, the measured pH value of each color of the reference color display unit calculated by the image acquisition unit (camera, etc.) automatically every time the apparatus is started or every predetermined time elapses, and the reference color stored in advance When there is a difference between the pH values of the respective colors of the display unit, a correction value for eliminating the difference is obtained, and the measured pH value is corrected.

As a result, it is possible to improve the measurement accuracy by eliminating variations caused by changes in each component of the apparatus over time, changes in environmental temperature, and the like.
A pH determination program according to an eighteenth aspect of the present invention is the pH determination program according to the fifteenth aspect of the present invention, wherein the container has a color display unit corresponding to a specific color of the reference color display unit. In the correction value calculating step, a correction value is obtained from the difference between the measured pH value corresponding to the color of the color display portion of the container serving as a reference and the measured pH value of the color of the color display portion for each container.

Here, a color display unit for displaying a color corresponding to a predetermined pH value is provided in a part of the container, similarly to the reference color display unit. When the image including the container is acquired, the measured pH value corresponding to the color displayed on the color display unit is calculated, and if there is a difference compared with the pH value stored in advance, the difference is eliminated. Make corrections.
Here, the color display portion is, for example, a portion that is provided at a corner portion of the container and shows the same color as a predetermined position indicated on the reference color display portion, and is always changed even if the container changes. It shows a certain color.

Thereby, for example, even if the product number is the same, even if the material, thickness, shape, etc. are partially changed, even if the color of the measurement object held in the image acquired container is affected, the container It is possible to eliminate the measurement variation caused by. Therefore, even if there is a minor change in the container unintentionally, it is possible to prevent the measurement accuracy from deteriorating.
A pH determination program according to a nineteenth aspect of the present invention is the pH determination program according to any one of the fifteenth to eighteenth aspects of the present invention, wherein the container has a plurality of recesses into which measurement objects including cells are placed. In the reference color display section, each color is arranged at a pitch corresponding to the interval at which the plurality of concave portions of the container are arranged. In the correction value calculating step, the pH determination is performed using the image including the reference color display unit and the container while detecting the measurement points at the same pitch.

Here, when the container has a large number of recesses (wells) for containing cells, such as well plates, the reference color array included in the reference color display section is defined as the recess array pitch. They are arranged to match.
As a result, when the image including the container and the reference color display unit is image-processed, the measurement of the reference color display unit is also performed at the same pitch as the pitch at which each concave portion of the container is measured during pH measurement. Can do. Therefore, it is possible to simplify the image processing in the control unit as much as possible and efficiently perform pH measurement.

A pH determination program according to a twentieth invention is a pH determination program according to the sixteenth to eighteenth inventions, wherein the control unit determines from L * (brightness), C * (saturation), and h (hue angle). The measured pH value is corrected using the h value of the L * C * h color system.
Here, the measured pH value is corrected using the value h representing the hue of the L * C * h color system.

  Thereby, it is possible to prevent the measurement accuracy from being lowered due to other factors such as luminance, and to perform more accurate pH measurement.

  According to the pH determination apparatus according to the present invention, it is possible to prevent a decrease in measurement accuracy caused due to variations in the apparatus, changes over time, material of the culture vessel, and the like.

The front view which shows the structure of the pH determination apparatus which concerns on one Embodiment of this invention. The perspective view which shows the state which took out the main structures of the pH determination apparatus of FIG. 1 from the housing | casing part. The front view of the pH determination apparatus of FIG. The top view of the pH determination apparatus of FIG. The figure which shows the positional relationship of the culture container (well plate) set to the pH determination apparatus of FIG. 1, and a master chart. (A), (b) is the top view and side view which show the structure of the Fresnel lens contained in the pH determination apparatus of FIG. The top view which shows the structure of the culture container (well plate) used with the pH determination apparatus of FIG. (A)-(c) is a figure which shows the process of correct | amending the individual difference for every pH determination apparatus by the pH determination apparatus of FIG. 1, for example at the time of shipment of an apparatus. (A), (b) is a figure which shows the process of correct | amending the error of the measured pH value of the master chart accompanying a time-dependent change (environment temperature) by the pH determination apparatus of FIG. (A), (b) is a figure which shows the process of correct | amending the error resulting from the individual difference of a culture container (well plate) by the pH determination apparatus of FIG. The flowchart which shows the flow of a process of the pH determination method by the pH determination apparatus of FIG. (A)-(e) is a figure which shows the positional relationship of the culture container (well plate) set to the pH determination apparatus which concerns on other embodiment of this invention, and a master chart.

The pH determination device 10 according to an embodiment of the present invention will be described below with reference to FIGS.
(Configuration of pH determination device 10)
The pH determination apparatus 10 according to the present embodiment is adapted to change the pH of a medium that changes according to the growth state of cells C (see FIG. 7) placed in a well plate (culture container) 20 (see FIG. 7). As shown in FIG. 1 and FIG. 2, the housing unit 11, the mount 12, the light source unit 13, the container mounting unit 14, and the master chart 15 And a Fresnel lens 16 (optical system having telecentricity), a camera (image acquisition unit) 17, a control unit 18 (see FIG. 4), and a storage unit 19 (see FIG. 4).

(Case 11)
The casing 11 has two doors (not shown) with double doors (not shown) on the front side of the box shape, and forms a dark room inside by closing the two doors. As shown in FIGS. 1 and 2, the housing 11 includes main components such as a mounting base 12, a light source unit 13, a container mounting unit 14, a master chart 15, a Fresnel lens 16, and a camera 17. Store.

In the present embodiment, the pH is measured in a state where the main part that measures pH is housed in the casing 11. Thereby, while being able to implement pH measurement without being influenced by ambient light, the power of the light source unit 13 can also be saved. Furthermore, by carrying out pH measurement in a dark box, it is possible to suppress changes in temperature and CO ₂ concentration due to the influence of wind.

As in the present embodiment, when the main configuration of the pH determination device 10 is installed in a dark box and a power supply device, a USB hub, or the like is disposed under the casing unit 11, the power supply device or the like is cooled. Therefore, the arrangement may be devised so that the cooling air does not flow in the direction of the well plate 20.
In the present embodiment, by detecting the change in pH of the medium containing the cells C held in the well plate 20 using the light irradiated from the light source unit 13 in the dark room formed in the housing unit 11, A highly accurate pH determination can be performed without being affected by ambient light.

(Mounting base 12)
As shown in FIGS. 2 and 3, the mounting table 12 is a table for mounting the light source unit 13, the container mounting unit 14, the master chart 15, the camera 17, and the like, and includes a base unit 12a, a support unit 12b, and two pieces. Column 12c, 12c and a camera mounting portion 12d.
The base portion 12a is a flat member that is placed on the installation surface on which the pH determination device 10 is installed and forms the base portion of the pH determination device 10, and the light source portion 13 is disposed therein. ing. Further, the master chart 15 and the well plate 20 are placed on the portion of the base portion 12a where the light source unit 13 is disposed.

The support portion 12b is a flat plate member that is attached above the upper surface of the base portion 12a via a predetermined gap, and is formed in a through hole formed in a portion facing the master chart 15 and the well plate 20. The Fresnel lens 16 is installed.
As shown in FIGS. 2 and 4, the two support columns 12 c and 12 c are erected along a substantially vertical direction on the side opposite to the front side where the well plate 20 is introduced in the base portion 12 a. . Moreover, the camera support part 12d to which the camera 17 is attached to the upper end part is fixed to the two support columns 12c and 12c.

The direction in which the well plate 20 is introduced may be from not only the front surface of the housing 11 but also from the side surface.
As shown in FIGS. 2 and 4, the camera mounting portion 12d is a substantially T-shaped member in plan view, and is supported by the upper ends of the two columns 12c and 12c. A camera 17 arranged in the direction of the master chart 15 and the well plate 20 is attached to the substantially T-shaped tip portion of the camera attachment portion 12d.

(Light source unit 13)
The light source part 13 is comprised by white LED, and irradiates light toward the master chart 15 and the well plate 20 which are arrange | positioned upwards from the base part 12a.
As the light source unit 13, other light sources such as a halogen lamp may be used. However, for example, when a halogen lamp is used as the light source, the environmental temperature rises due to heat generation, and the temperature in the housing 11 also rises. In this case, there is a possibility that the cells placed in each well 20a of the well plate 20 will die in a high temperature environment.

Therefore, as the light source unit, it is preferable to use an LED having a small calorific value as in the present embodiment.
(Container placement part 14)
The container mounting part 14 is a flat plate-like member having a through hole installed on the base part 12a, and the well plate 20 is placed in this through part. The penetration part of the container mounting part 14 is formed according to the size of the well plate 20.

Thus, when the well plate 20 is placed on the container placement portion 14, the well plate 20 is placed in a state of being aligned with a predetermined location on the base portion 12a. Therefore, alignment of the well plate 20 with respect to the master chart 15 arranged at a position adjacent to the container placement unit 14 can be performed at the same time.
(Master chart 15)
As shown in FIGS. 4 and 5, the master chart 15 is disposed adjacent to the back side (the side opposite to the front surface) of the container mounting portion 14 in the base portion 12a. In addition, the master chart 15 is configured to include, for example, color swatches 15a to 15h that change in eight steps as a reference when performing pH determination.

  In the 8-color swatches 15a to 15h, pH values corresponding to the hues actually measured using a contact-type pH sensor or the like are stored in the storage unit 19 to be described later, and from the hue (h value). The value converted into pH value is set to change by 0.05. Specifically, the color look 15a is 6.85, the color look 15b is 6.90, the color look 15c is 6.95, the color look 15d is 7.00, the color look 15e is 7.04, and the color look 15f is 7. 0.09, color shade 15g is set to 7.12 and color shade 15h is set to a color corresponding to the pH value of 7.15 (see FIG. 8B, etc.).

Each trials 15a~15h of 8 colors of the master chart 15 are arranged at the same pitch intervals d _1. This interval d ₁ is set to be the same as the interval between the wells 20 a of the well plate 20.
Note that the pH values corresponding to the eight color shades 15a to 15h of the master chart 15 are based on a table indicating the correspondence between the hue and the pH value corresponding to each of the color shades 15a to 15h stored in the storage unit 19 in advance. Is calculated. In this correspondence, first, solutions having various pH values are prepared (for example, a diluent is added to a medium to obtain an arbitrary pH value solution), and a pH indicator for color development (for example, phenol red) is used. ) Is added. Next, by measuring the pH value with a commercially available pH meter and photographing the color development, the correspondence between the hue and the pH value can be determined.

Further, the master chart 15 has a central portion and end portions on the back side of the well plate 20 so that the image including the well plate 20 is acquired by the camera 17 to be described later, so that the master chart 15 is included in the image together with the well plate 20. The distance between the center portion of the wells located in the vicinity of each other and the distance between the wells is 3 times the distance d ₂ that is the pitch of each well.
As described above, in the pH determination apparatus 10 of the present embodiment, the arrangement interval of the master chart 15 is set with reference to the intervals d ₁ and d ₂ between the wells 20a of the well plate 20 in both the horizontal direction and the vertical direction. ing. Thereby, when performing image processing in the control unit 18, the portions of the wells 20 a of the well plate 20 and the portions of the color charts 15 a to 15 h of the master chart 15 can be measured at the same interval. As a result, the processing at the time of image processing by the control unit 18 can be simplified, so that the processing load can be reduced.

However, as will be described later, when the number of wells in the well plate increases, it is only necessary to have a certain number of types of color viewing. Therefore, it is possible to take a method other than making each color viewing and each well face each other one to one. it can.
(Fresnel lens 16)
As shown in FIGS. 2 and 3, the Fresnel lens 16 is disposed between the well plate 20 placed on the base 12a and the camera 17 in the vertical direction, and is a lens having telecentricity. Thus, the optical axis and the chief ray can be regarded as parallel at the position of the camera 17 where the image is formed.

Normally, the well located at a position far from the optical axis of the camera due to parallax can only observe the side surface portion of the well and cannot see the central portion of the well. However, in the pH determination device 10 of the present embodiment, Using the characteristics of the Fresnel lens 16, an image as if each well 20a of the well plate 20 was viewed from directly above can be acquired by the camera 17.
In addition, as shown in FIGS. 6A and 6B, the Fresnel lens 16 has a plurality of irregularities formed on the surface, and forms an image at a position away from the surface having no irregularities by a focal length.

In the present embodiment, the Fresnel lens 16 takes in the light irradiated upward from the light source unit 13 to the well plate 20 and the master chart 15, and the position of the lens of the camera 17 installed at a position separated by the focal length. To form an image.
Further, in the present embodiment, the Fresnel lens 16 is disposed at a position where the optical center X is shifted toward the well plate 20 from the dimensional center O in a range including the master chart 15 and the well plate 20. (Since the Fresnel lens is a substantially flat lens, it can be cut out as such.)

  It is desirable that the optical center X of the Fresnel lens 16 coincides with the center of the well plate 20. In FIG. 4, the optical center L of the camera 17 is located between the dimensional center O and the optical center X of the Fresnel lens 16. It is desirable that the optical center L of 17 coincides with the optical center X of the Fresnel lens 16.

Thereby, it is possible to acquire an image without distortion when the wells 20a of the well plate 20 are viewed from directly above. On the other hand, with respect to the master chart 15 where the optical center X of the Fresnel lens 16 is separated, the measurement portions of the color swatches 15a to 15h are formed vertically, so that even if some distortion occurs, the measurement is hardly affected. Absent.
(Camera 17)
The camera 17 is an imaging device capable of acquiring a color image, and is fixed above the Fresnel lens 16 while being supported by the camera mounting portion 12d of the mounting base 12 described above, as shown in FIGS. Has been.

Further, the camera 17 is arranged at a position away from the upper surface of the Fresnel lens 16 by a predetermined distance so that the range including the master chart 15 and the well plate 20 can be photographed via the Fresnel lens 16. .
Further, the camera 17 sends the acquired color image to the control unit 18.
(Control unit 18)
The control unit 18 performs image processing on the pH measurement site (well 20a and color portions 15a to 15h) included in the color image acquired by the camera 17, and digitizes the hue of each pH measurement site. Thereafter, the control unit 18 calculates a pH value corresponding to the hue of each measurement site based on a table indicating the relationship between the stored hue and the pH value stored in the storage unit 19 in advance.

Thereby, the pH value of the medium which changes according to the growth state of the cell C can be detected by the change in the hue of the medium containing the pH indicator (phenol red).
Specifically, for example, when the growth state of the cells C is good, the medium changes to the acidic side within a certain range. On the other hand, when the growth state of the cells C is not good, the medium does not change to the acidic side, and in many cases, the pH value does not change or is small.

  Therefore, the pH determination device 10 digitizes the hue of the pH measurement site, and refers to the table stored in the storage unit 19 to obtain the pH value corresponding to the hue value, whereby each well 20a of the well plate 20 is obtained. The growth state of the cell C can be determined. As a result, when the pH value is low, that is, when the medium is acidified, the cells C of the well 20a are dispensed into the other well 20a and placed again in the culture space in the incubator. The culture of the cell C can be promoted.

  On the other hand, if the pH value is not low as a result of pH measurement, that is, if the medium is not acidified, it means that the growth of the cells C in the well 20a has not progressed. Therefore, in this case, operations such as medium replacement can be performed in order to eliminate a factor that can be considered as the cause of the cell C not growing. As a result, by performing the treatment so that the growth of the cells C is promoted, the growth of the cells C in all the wells 20a can be advanced.

  Note that the hue represents the position or range of the color in the color space, and various color systems can be used as the color space. It has been found that it is most effective to use the h value of the L * C * h color system consisting of * (lightness), C * (saturation), and h (hue angle). This h value is shown as an angle in the L * C * h color system.

More specifically, the hue angle h (θ) can be obtained by the following relational expression (1).
h (θ) = tan ⁻¹ (b * / a *) (1)
Here, a * and b * indicate the chromaticity of the L * a * b * color system, and C * of the L * C * h color system is obtained by the following relational expression (2).

また、制御部１８は、カメラ１７から送信されたマスタチャート１５とウェルプレート２０とを含むカラー画像を受け取って、画像処理を実施し、画像処理によって得られた補正値を用いて測定ｐＨ値の補正を行う。
具体的には、制御部１８は、カメラ１７において取得されたカラー画像に含まれるマスタチャート１５の各色見１５ａ〜１５ｈの部分の色相に関する情報をそれぞれ取得し、予め記憶部１９に記憶された色相とｐＨ値との関係を示すテーブルに基づいて、各色見１５ａ〜１５ｈに対応するｐＨ値を算出する。

Further, the control unit 18 receives the color image including the master chart 15 and the well plate 20 transmitted from the camera 17, performs image processing, and uses the correction value obtained by the image processing to measure the measured pH value. Make corrections.
Specifically, the control unit 18 acquires information regarding the hue of each of the color views 15 a to 15 h of the master chart 15 included in the color image acquired by the camera 17, and the hue stored in the storage unit 19 in advance. Based on a table showing the relationship between the pH value and the pH value, the pH value corresponding to each of the colors 15a to 15h is calculated.

  Here, the control unit 18 compares the measured pH value with the actual pH values corresponding to the color swatches 15a to 15h stored in the storage unit 19 in advance. If there is a difference, the control unit 18 eliminates the difference. A correction value for obtaining the value is obtained. Then, the control unit 18 stores the correction value in the storage unit 19 and actually measures each well 20a of the well plate 20, and then corrects the measured pH value of each well 20a with the correction value.

Thereby, for example, measurement errors due to changes over time, environmental changes, and the like can be corrected to improve measurement accuracy.
The correction process by the control unit 18 will be described in detail later.
(Storage unit 19)
As shown in FIG. 4, the storage unit 19 is connected to the control unit 18, and stores information related to the reference pH values corresponding to the color charts 15 a to 15 h of the master chart 15 in advance.

The storage unit 19 also stores correction values calculated by the control unit 18 described above.
The correction value eliminates, for example, individual differences between apparatuses caused by the camera 17 and the light source unit 13, measurement errors caused by changes over time, measurement errors caused by material changes of the well plate 20, and the like. Therefore, it is only required to be stored until updated to a new correction value.

  Further, the storage unit 19 associates a numerical value indicating the hue of each well 20a of the well plate 20 included in the color image acquired by the camera 17 with a pH value corresponding to each hue indicated in stages. Stores the table. Therefore, the control unit 18 digitizes the hue of each well 20a of the well plate 20, and then refers to the table to calculate a pH value corresponding to the numerical value indicating the hue.

(Well plate 20)
Moreover, in the pH determination apparatus 10 of this embodiment, as shown in FIG. 7, each well 20a of the well plate 20 having a total of 48 wells (recesses) 20a in the horizontal 8 columns and 6 vertical rows is formed on the surface. Cell C and medium are added to culture the cells.
The well plate 20 is a culture container for culturing cells C in a high temperature and high humidity culture space formed in an incubator (not shown), and resin injection molding is performed so that 48 wells 20a are formed on the surface. Is formed by.

Each well 20a contains cells C together with a medium containing phenol red as a pH indicator.
Further, as shown in FIG. 5, the well plate 20 is placed in a state in which the well plate 20 is fitted into a penetrating portion of the container placement portion 14 formed in the base portion 12 a of the mounting base 12. The well plate 20, with each well 20a in the transverse direction at intervals d ₁ are arranged, each well 20a at intervals d ₂ in the vertical direction is disposed.

Further, as described above, when the well plate 20 is placed on the penetration portion of the container placement portion 14 of the base portion 12a, the alignment is made with respect to the master chart 15 arranged in the vicinity of the container placement portion 14. Is done.
As a result, the pH measurement site (well 20a and color portions 15a to 15h) included in the color image acquired by the camera 17 is image-processed while being detected at the same pitch, thereby measuring the pH value of each pH measurement site. Can be calculated. Therefore, it is possible to reduce the processing load on the control unit 18 and efficiently measure the pH value and correct the measured pH value.

<Method for correcting measured pH value by pH determination device 10>
In the pH determination device 10 of the present embodiment, after acquiring the above-described measured pH value, the control unit 18 performs correction for eliminating measurement errors caused by various factors as follows.
(Correction of measurement error due to individual differences at the time of shipment)
In the pH determination apparatus 10 according to the present embodiment, in order to eliminate measurement errors caused by individual differences for each apparatus, a correction value for eliminating the measurement error for each apparatus is calculated before the apparatus is shipped. And stored in the storage unit 19 of each device.

Here, the individual difference for each apparatus may be a difference in luminance of light emitted from the light source unit 13 such as a white LED, a difference in sensitivity of the camera 17, an individual difference due to an optical system, or the like. Such an individual difference for each device is a factor that causes variations in the measured value even when the same pH value is measured under the same conditions. Therefore, it is desirable to eliminate the difference before shipping.
In the pH determination device 10 of the present embodiment, first, a reference pH value corresponding to each of the color references 15a to 15h shown in the master chart 15 acquired by the camera 17a of the master device shown in FIG. 8A is stored. Stored in the unit 19.

Here, when collated with the reference pH value stored in the storage unit 19, as described above, the color viewing 15a is 6.85, the color viewing 15b is 6.90, the color viewing 15c is 6.95, and the color viewing 15d. Is 7.00, color viewing 15e is 7.04, color viewing 15f is 7.09, color viewing 15g is 7.12, and color viewing 15h is 7.15.
Then, as shown in FIG. 8B and FIG. 8C, images of the portion of the master chart 15 showing the same color views 15a to 15h are acquired by the camera 17b of another pH determination device 10 to be corrected. Then, the pH value of each color viewing portion is measured by image processing.

At this time, the pH value obtained by performing image processing on the images acquired by the camera 17b and the camera 17c has the following results.
That is, in the image acquired by the camera 17b, the color viewing 15a is 6.88, the color viewing 15b is 6.93, the color viewing 15c is 6.99, the color viewing 15d is 7.03, and the color viewing 15e is 7.07. The color viewing 15f is 7.12, the color viewing 15g is 7.15, and the color viewing 15h is 7.18.

In the image acquired by the camera 17c, the color viewing 15e is 6.97, the color viewing 15f is 7.01, the color viewing 15g is 7.09, and the color viewing 15h is 7.12.
From the above results, the pH determination device 10 equipped with the camera 17b is calculated as a pH value that is about 0.03 higher than the reference pH value set by the pH determination device 10 serving as a master. A correction value (−0.03) for elimination is calculated and stored in the storage unit 19.

On the other hand, in the pH determination device 10 equipped with the camera 17c, the pH value is calculated as 0.03 lower than the reference pH value set by the master pH determination device 10, so that this error is eliminated. The correction value (+0.03) is calculated and stored in the storage unit 19.
Thereby, in the stage before shipment, the reference pH value set by the pH determination device 10 serving as a master is compared with the measurement result of the master chart 15 measured by each device. By calculating a correction value for eliminating the error and storing the correction value in the storage unit 19 of each device, it is possible to eliminate the measurement error caused by the individual difference for each device. Therefore, the pH determination apparatus 10 with higher measurement accuracy than the conventional one can be obtained.

(Correction of measurement error due to changes over time)
In the pH determination device 10 of the present embodiment, in order to eliminate measurement errors caused by changes over time such as aging deterioration due to repeated use of the device, the measurement error is measured at the start-up of the device or every predetermined time. A correction value for canceling is calculated and stored in the storage unit 19.
Here, as the time-dependent change in the pH determination device 10, an increase in the environmental temperature after a predetermined time has elapsed immediately after the power is turned on, aged deterioration of components such as the light source unit 13, the optical system, and the camera 17 can be considered.

Such a change with time causes a measurement error, so that it is desirable to eliminate it by correction every certain period.
In the pH determination device 10 of the present embodiment, first, as shown in FIG. 9A, each of the color views 15a to 15 shown in the master chart 15 acquired by the camera 17 in the state where the environmental temperature is 25 ° C. immediately after the power is turned on. A reference pH value corresponding to 15 h is stored in the storage unit 19.

Here, the reference pH values stored in the storage unit 19 are the same as described above, the color viewing 15a is 6.85, the color viewing 15b is 6.90, the color viewing 15c is 6.95, and the color viewing 15d is 7 .00, color look 15e is 7.04, color look 15f is 7.09, color look 15g is 7.12, color look 15h is 7.15.
Then, as shown in FIG. 9 (b), measured pH values corresponding to the respective colors 15a to 15h shown in the master chart 15 acquired by the camera 17 in a state where the environmental temperature becomes 40 ° C. after a predetermined time has elapsed. The color viewing 15a is 6.82, the color viewing 15b is 6.87, the color viewing 15c is 6.93, the color viewing 15d is 6.97, the color viewing 15e is 7.01, the color viewing 15f is 7.06, The color viewing 15g is 7.09 and the color viewing 15h is 7.12.

From the above results, the pH determination device 10 equipped with the camera 17 is calculated as a pH value that is about 0.03 lower than the reference pH value stored in the storage unit 19 in advance, so that this error is eliminated. The correction value (+0.03) is calculated and stored in the storage unit 19.
Thereby, when the well plate 20 is actually set and the pH value of the portion of each well 20a is measured, the result of correcting the measured pH value using the correction value (+0.03) is measured. It can be calculated as a pH value.

Therefore, by calculating a correction value every time a predetermined time elapses and updating it to a new correction value, the occurrence of measurement errors due to changes over time, including changes in environmental temperature and aging of parts, is eliminated. Thus, it is possible to obtain the pH determination device 10 capable of performing measurement with higher accuracy.
(Correction of measurement error due to culture vessel material)
In the pH determination apparatus 10 of the present embodiment, in order to eliminate a measurement error caused by a change in the material or the like of the well plate 20 in which the cells C are placed, a correction value for eliminating the measurement error is calculated, and a storage unit 19 is stored.

  Here, the change in the material and the like for each culture vessel (well plate 20) means that, in the field of cell culture, if the performance as a culture vessel is not affected, even if it is the same product number, part of the material and shape May have been changed. For this reason, even when measurement is performed under the same conditions, there may be a measurement error caused by individual differences in the culture container, such as the transmission of the light emitted from the light source unit 13. Such individual differences for each culture vessel can cause variations in the measured value even when the same pH value is measured under the same conditions, so it is corrected whenever the lot number of the culture vessel is changed. It is desirable to resolve the problem by updating the value.

In the pH determination apparatus 10 of the present embodiment, an image is acquired by the camera 17 so as to include the master chart 15 and the well plate 20 as shown in FIG. At this time, master charts (color display portions) 20ba and 20bb are provided at corner portions of the well plate 20 in order to eliminate measurement errors caused by changes in the material and the like of the well plate 20.
In the present embodiment, the master charts 20ba and 20bb both indicate colors based on 7.00. Note that the master chart attached to the well plate 20 is not based on 7.00 but may be based on other numerical values.

Therefore, as shown in FIG. 10A, when the measured pH value obtained as a result of image processing of the portion of the master chart 20ba attached to the well plate 20 is 6.98, it becomes a reference. Since the value is calculated as 0.02 lower than 00, it is necessary to correct this error 0.02 by correction.
On the other hand, as shown in FIG. 10B, when the measured pH value obtained as a result of image processing of the portion of the master chart 20bb attached to the well plate 20 is 6.95, it becomes a reference. Since it is calculated as a value 0.05 lower than 00, it is necessary to correct this error 0.05 by correction.

As a result, the result of correcting the measurement result of the pH value of each well 20a portion of the well plate 20 using the correction values (+0.02) and (+0.05) is calculated as the measured pH value. can do.
Therefore, each time the production lot of the culture container changes or every time a predetermined time elapses, the correction value is calculated and updated to a new correction value, resulting in a change in the material of the culture container such as the well plate 20 Thus, it is possible to obtain the pH determination device 10 that can eliminate the occurrence of measurement error and can perform measurement with higher accuracy.

<Flow of correction by pH determination device 10>
Here, regarding the correction process of the measured pH value by the pH determination device 10 of the present embodiment, the flow when performing the correction process for eliminating the measurement error due to the above-described change over time is a flowchart shown in FIG. The explanation will be as follows.
That is, in the pH determination device 10 of the present embodiment, as shown in FIG. 11, when the device is started in step S11, the correction process is started.

Next, in step S <b> 12, after the apparatus is activated, as soon as the camera 17 is ready, an image is acquired by the camera 17 so as to include the well plate 20 and the master chart 15.
Next, in step S13, a reference pH value corresponding to each of the color portions 15a to 15h of the master chart 15 included in the image acquired in step S12 is stored. In addition, this step S13 may be memorize | stored in the memory | storage part 19 previously in the step before this flow is started.

Next, in step S14, the reference pH value corresponding to each of the color views 15a to 15h stored in advance in the storage unit 19 is compared with the measured pH value calculated by the image processing to determine whether there is a difference. Check. If there is a difference, it is determined that there is a measurement error, and a correction value for eliminating the measurement error is calculated.
Next, in step S15, the pH value of the medium containing the cells C actually held in each well 20a of the well plate 20 is measured, and the measurement result is corrected using the correction value calculated in step S14. .

Accordingly, it is possible to provide a pH determination device 10 capable of measuring pH with higher accuracy by preventing occurrence of measurement errors due to changes over time such as environmental temperature and aging deterioration of various parts such as the camera 17. Can do.
Next, in step S16, it is determined whether or not a predetermined time has elapsed since the start of the apparatus or the previous correction process. Here, when the predetermined time has elapsed, by repeating steps S12 to S15 again, the latest correction value is calculated and stored in the storage unit 19, so that the measurement accuracy is always high. Can be maintained.

[Other Embodiments]
As mentioned above, although one Embodiment of this invention was described, this invention is not limited to the said embodiment, A various change is possible in the range which does not deviate from the summary of invention.
(A)
In the said embodiment, the example which applied this invention with respect to the pH determination apparatus was given and demonstrated. However, the present invention is not limited to this.
For example, you may implement this invention as a pH determination program which makes a computer perform the pH determination method performed according to the control flow (refer FIG. 11) mentioned above.
An effect similar to that obtained by the pH determination device 10 can be obtained by a PC or the like in which the pH determination program is introduced.

(B)
In the above-described embodiment, an example has been described in which the hue of each color viewing portion of the master chart 15 obtained by image processing is converted to a pH value, and correction is performed based on the pH value. However, the present invention is not limited to this.
For example, the h value (hue) of the L * C * h color system composed of L * (lightness), C * (saturation), and h (hue angle) before being converted to a pH value using a table is used. The measured pH value may be corrected. Alternatively, the measured pH value may be corrected by an element other than the h value (hue).

(C)
In the embodiment described above, an example in which the well plate 20 including a plurality of wells (concave portions) 20a is used as a container for storing cells has been described. However, the present invention is not limited to this.

For example, a petri dish or the like may be used as the culture container.
However, the cell culture efficiency can be improved by putting cells in a plurality of wells like a well plate and confirming the growth state of the cells, so that the cell culture efficiency can be improved as in the above-described embodiment. Moreover, it is more preferable to use a culture vessel capable of performing cell culture at a plurality of locations.

(D)
In the above embodiment, the arrangement interval (pitch) of the plurality of wells (concave portions) 20a of the well plate 20 and the arrangement interval of each color of the master chart 15 are arranged to be the same in both the vertical direction and the horizontal direction. And explained. However, the present invention is not limited to this.
For example, the structure arrange | positioned so that either the pitch of a vertical direction or a horizontal direction may become the same.

(E)
In the above-described embodiment, an example in which the number of wells (recesses) 20a is 48 columns × 6 rows in total 48 well plates 20 has been described. However, the present invention is not limited to this.

For example, a well plate including a smaller number or a larger number of wells such as 4 columns × 3 rows in total 12 wells and 40 columns × 90 rows in total 3600 wells may be used.
For example, when the number of wells is 384 of 24 columns × 16 rows and 8 types of color viewing are used, 24 columns of the well plate are made to correspond to the horizontal direction in FIG. 5 and the same pitch as the horizontal well pitch. As shown in FIG. 12A, the color swatches 31a may be arranged so as to correspond to the eight columns from the left of the well plate 32 as shown in FIG.

Or as shown in FIG.12 (b) and FIG.12 (c), you may arrange | position the color schemes 31b and 31c in a jump from the left or two.
Furthermore, as shown in color schemes 31d and 31e shown in FIGS. 12D and 12E, the widths may be created and arranged corresponding to the 2 pitches or 3 pitches of the wells.
Even in this case, it is possible to efficiently determine the pH regardless of the number of wells by acquiring an image with a camera or the like disposed above the well plate.

(F)
In the above embodiment, an example in which a Fresnel lens is used as the lens of the telecentric optical system has been described. However, the present invention is not limited to this.
For example, instead of the Fresnel lens, another telecentric optical system may be used.

(G)
In the said embodiment, the example which performs pH determination in the dark room formed inside the housing | casing part 11 was given and demonstrated. However, the present invention is not limited to this.
For example, the pH determination may be performed in a room where the brightness is constant in consideration of measurement errors due to ambient brightness.

(H)
In the embodiment described above, an example in which the master chart 15 indicating eight reference colors is used as the reference color display unit has been described. However, the present invention is not limited to this.
For example, the number of reference colors indicated by the master chart is not limited to eight colors, and may be smaller or larger than eight colors such as four colors, six colors, or twelve colors.

(I)
In the above embodiment, in order to recognize a change in pH as a change in color, an example in which phenol red is used as a pH indicator added to a medium containing cells has been described. However, the present invention is not limited to this.
For example, in addition to phenol red, a pH indicator such as methyl orange, phenolphthalein or litmus may be used.

  The pH determination device of the present invention has an effect of preventing a decrease in measurement accuracy caused by variation over time, variation in the material of the culture vessel, etc. for each device. On the other hand, it is widely applicable.

DESCRIPTION OF SYMBOLS 10 pH determination apparatus 11 Case part 12 Mounting base 12a Base part 12b Support part 12c Support | pillar 12d Camera mounting part 13 Light source part 14 Container mounting part 15 Master chart (reference color display part)
16 Fresnel lens (optical system with telecentricity)
17 Camera (image acquisition unit)
18 Control unit 20 Well plate (container)
20a well (concave)
20ba, 20bb Master chart (color display part)
31a, 31b, 31c, 31d, 31e Master chart 32 Well plate C cell O Optical center of Fresnel lens X Dimensional center of well plate L Optical center of camera

Claims (20)

A pH determination device for determining the pH of a measurement object to which a pH indicator in a container is added by image processing,
An image acquisition unit that images the container from above and acquires an image including a part of the measurement object;
A reference color display unit that is arranged in the vicinity of the container so as to be included in the image acquired by the image acquisition unit together with the container, and that indicates a plurality of colors that serve as a reference for the color that varies depending on the pH of the measurement object When,
A storage unit for storing a reference pH value corresponding to each color included in the reference color display unit;
A correction value is obtained from the difference between the reference pH value stored in the storage unit and the measured pH value of each color of the reference color display unit calculated based on the image acquired in the image acquisition unit, and the correction value A controller for correcting the measured pH value using
A pH determination device. The control unit obtains a correction value from a difference between the measured pH value of each color of the reference color display unit calculated using the image acquired by the image acquiring unit serving as a reference and the measured pH value of each device. The measured pH value for each apparatus is corrected using the correction value.
The pH determination apparatus according to claim 1. The control unit updates the correction value every time of starting or every predetermined time, and corrects the measured pH value using the updated correction value.
The pH determination apparatus according to claim 1. The container has a color display unit corresponding to a specific color of the reference color display unit,
The control unit obtains a correction value from the difference between the measured pH value corresponding to the color of the color display unit of the container serving as a reference and the measured pH value of the color of the color display unit for each container, and the correction value Correct the measured pH value for each container using
The pH determination apparatus according to claim 1. The container has a plurality of recesses into which measurement objects including cells are placed,
The reference color display section has each color arranged at a pitch corresponding to the interval at which the plurality of recesses of the container are arranged.
The pH determination apparatus according to any one of claims 1 to 4. The control unit corrects the measured pH value using the h value of the L * C * h color system composed of L * (lightness), C * (saturation), and h (hue angle).
The pH determination apparatus according to any one of claims 2 to 4. A light source unit disposed below the container and irradiating light to the container;
An optical system having telecentricity disposed between the container and the image acquisition unit,
The pH determination apparatus according to any one of claims 1 to 6. The telecentric optical system is arranged at a position where the image acquisition unit can acquire an image in a range including the container and the reference color display unit, and the optical center position is on the container side. Arranged at a shifted position,
The pH determination apparatus according to claim 7. A pH determination method for determining the pH of a measurement object to which a pH indicator in a container is added by image processing,
An acquisition step of photographing the container from above and acquiring an image including a part of the measurement object;
It is arranged in the vicinity of the container so as to be included in the image together with the container, and corresponds to each color included in the reference color display unit indicating a plurality of colors serving as a reference of the color that changes depending on the pH of the measurement object. A storage step for storing a reference pH value;
A correction value calculating step for obtaining a correction value from a difference between the reference pH value and a measured pH value of each color of the reference color display unit calculated based on the image;
A correction step of correcting the measured pH value using the correction value;
A pH determination method comprising: In the correction value calculating step, a correction value is calculated from a difference between a measured pH value of each color of the reference color display unit calculated using an image acquired by the image acquiring unit serving as a reference and a measured pH value for each device. Seeking
The pH determination method according to claim 9. In the correction value calculating step, the correction value is updated every time when starting or every predetermined time has elapsed,
In the correction step, the measured pH value is corrected using the updated correction value.
The pH determination method according to claim 9. The container has a color display unit corresponding to a specific color of the reference color display unit,
In the correction value calculating step, a correction value is obtained from a difference between a measured pH value corresponding to the color of the color display portion of the container serving as a reference and a measured pH value of the color of the color display portion for each container,
The pH determination method according to claim 9. The container has a plurality of recesses into which objects to be measured including cells are placed, and the reference color display unit has each color arranged at a pitch corresponding to an interval in which the plurality of recesses of the container are arranged. ,
In the correction value calculating step, using an image including the reference color display unit and the container, pH determination is performed while detecting measurement points at the same pitch.
The pH determination method according to any one of claims 9 to 12. In the correction step, the measured pH value is corrected using the h value of the L * C * h color system consisting of L * (lightness), C * (saturation), and h (hue angle).
The pH determination method according to any one of claims 10 to 12. A pH determination program for causing a computer to execute a pH determination method for determining the pH of a measurement object to which a pH indicator in a container is added by image processing,
An acquisition step of photographing the container from above and acquiring an image including a part of the measurement object;
It is arranged in the vicinity of the container so as to be included in the image together with the container, and corresponds to each color included in the reference color display unit indicating a plurality of colors serving as a reference of the color that changes depending on the pH of the measurement object. A storage step for storing a reference pH value;
A correction value calculating step for obtaining a correction value from a difference between the reference pH value and a measured pH value of each color of the reference color display unit calculated based on the image;
A correction step of correcting the measured pH value using the correction value;
A pH determination program for causing a computer to execute a pH determination method comprising: In the correction value calculating step, a correction value is calculated from a difference between a measured pH value of each color of the reference color display unit calculated using an image acquired by the image acquiring unit serving as a reference and a measured pH value for each device. Seeking
The pH determination program according to claim 15. In the correction value calculating step, the correction value is updated every time when starting or every predetermined time has elapsed,
In the correction step, the measured pH value is corrected using the updated correction value.
The pH determination program according to claim 15 or 16. The container has a color display unit corresponding to a specific color of the reference color display unit,
In the correction value calculating step, a correction value is obtained from a difference between a measured pH value corresponding to the color of the color display portion of the container serving as a reference and a measured pH value of the color of the color display portion for each container,
The pH determination program according to claim 15. The container has a plurality of recesses into which objects to be measured including cells are placed, and the reference color display unit has each color arranged at a pitch corresponding to an interval in which the plurality of recesses of the container are arranged. ,
In the correction value calculating step, using an image including the reference color display unit and the container, pH determination is performed while detecting measurement points at the same pitch.
The pH determination program according to any one of claims 15 to 18. In the correction step, the measured pH value is corrected using the h value of the L * C * h color system consisting of L * (lightness), C * (saturation), and h (hue angle).
The pH determination program according to any one of claims 16 to 18.

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