WO2018055762A1

WO2018055762A1 - System for measuring cell state

Info

Publication number: WO2018055762A1
Application number: PCT/JP2016/078263
Authority: WO
Inventors: 靖展伊賀; 仁越後; 朗松下
Original assignee: オリンパス株式会社
Priority date: 2016-09-26
Filing date: 2016-09-26
Publication date: 2018-03-29

Abstract

The system (100) according to the present invention for measuring cell state comprises: an image acquisition unit (2) which is provided with a linear line sensor (21) for detecting light from cells being cultured in a container and acquires a two-dimensional image by moving the line sensor (21) in a direction intersecting with the longitudinal direction of the line sensor (21); a cell state measurement unit (3) which divides the image into a plurality of unit areas and measures the cell state in each of the plurality of unit areas; and a display unit (4) which displays the result of the cell state measurement in each unit area.

Description

Cell state measurement device

The present invention relates to a cell state measuring apparatus.

Conventionally, a method of generating a tiling image of the entire bottom surface of a culture container has been used to observe the distribution of cells and colonies being cultured in the culture container (see, for example, Patent Document 1). The tiling image is generated by acquiring a large number of images with a two-dimensional image sensor while changing the shooting position, and connecting the large number of images.

JP 2012-173725 A

However, in order to generate a wide range of tiling images such as the entire bottom surface of the culture vessel, several tens to several hundreds of images are required, and it takes a long time to acquire many images. Therefore, there is a problem that a difference occurs in image acquisition time, and it is difficult to accurately grasp the cell state.
In addition, when the entire tiling image is displayed on the monitor, individual cells are displayed very small. For example, when a tiling image of the entire bottom surface of a culture dish having a diameter of 100 mm is displayed on the monitor, the size of one cell (diameter about 20 μm) on the monitor is about 40 μm in diameter of about one pixel. Therefore, there is a problem that it is difficult to grasp the state of cells in the entire container from the tiling image.

The present invention has been made in view of the above-described circumstances, and is capable of acquiring an image of a wide range of cells in a short time and can easily grasp the state of a wide range of cells. An object is to provide an apparatus.

In order to achieve the above object, the present invention provides the following means.
One embodiment of the present invention includes a linear line sensor that detects light from cells cultured in a container, and moves the line sensor in a scanning direction that intersects the longitudinal direction of the line sensor. An image acquisition unit that acquires a two-dimensional image by a cell state, and a cell state measurement unit that divides the image acquired by the image acquisition unit into a plurality of unit regions and measures a cell state in each of the plurality of unit regions And a display unit that displays a measurement result of the state of the cell in each of the unit regions measured by the cell state measurement unit.

According to this aspect, in the image acquisition unit, the line sensor moves in the scanning direction with respect to the container while detecting light from the cells in the container, whereby a two-dimensional image of the cells in the container is acquired. In this way, by using the line scanning type image acquisition unit, even in the case of a wide range of images such as the entire bottom surface of the container, compared to a case where a large number of images are acquired using a two-dimensional image sensor, a short time is required. Can be obtained at.

Subsequently, in the cell state measurement unit, the acquired image is divided into a plurality of unit regions, the cell state in each unit region is measured, and the measurement result of each unit region is displayed on the display unit. Thereby, the operator can easily grasp the state of a wide range of cells in the container based on the measurement result of each unit area displayed on the display unit.

In the above aspect, the cell state measurement unit integrates the measurement results of the plurality of unit regions to calculate the cell state in the entire image, and the display unit measures the cell state in the entire image. May be further displayed. The cell state measurement unit may calculate a cell state in the entire image by adding the measurement results of the plurality of unit regions.
By doing in this way, based on the measured value of the whole image, the state of the cell over a wide range or the whole in the container can be further grasped.

In the above aspect, the display unit may display a spatial distribution of measurement results of the plurality of unit regions.
By doing in this way, distribution of the state of the cell in a container can be grasped | ascertained easily based on the displayed spatial distribution.

In the said aspect, the cell type information acquisition part which acquires the information of the cell type of the said cell cultured in the said container is provided, The said cell state measurement part of the cell type acquired by the said cell type information acquisition part The size of the unit area may be determined according to information.
Cell size and colony-forming ability vary depending on the cell type. Especially in the case of cell types that form colonies, by determining the size of each unit area so that the size of each unit area is larger than the size of the colony, the image is converted into a unit area so that the colony spans multiple unit areas. It is possible to prevent division and improve the measurement accuracy of the cell state.

In the above aspect, the cell state measurement unit measures at least one of the cell density and the cell number in each unit region as the cell state, and the display unit displays the cell density in each unit region and At least one of the cell numbers may be displayed.
By doing in this way, the spatial distribution and density of the cells in the container can be easily grasped based on the spatial distribution of the measurement values displayed on the display unit.

In the above aspect, the cell state measurement unit further measures at least one of the cell density and the number of cells in the entire image as the cell state, and the display unit displays the cell density and cells in the entire image. At least one of the numbers may be further displayed.
By doing in this way, the whole state of the cell in a container can be grasped | ascertained easily based on the measured value of the whole image displayed on the display part.

In the above aspect, the image acquisition unit acquires a plurality of the time-series images at time intervals, and the display unit calculates each of the plurality of images calculated by the cell state measurement unit. You may display the time-dependent change of the measured value of a unit area.
By doing in this way, the time-dependent change of the state of the cell in each unit field in a container can be grasped easily based on the time-dependent change of the displayed measured value.

According to the present invention, it is possible to acquire an image of a wide range of cells in a short time and to easily grasp the state of a wide range of cells.

It is a block diagram which shows the whole structure of the cell state measuring apparatus which concerns on one Embodiment of this invention. It is a longitudinal cross-sectional view of the housing | casing of the cell state measuring apparatus of FIG. 1, and the container mounted on this housing | casing. It is a figure which shows an example of the distribution map produced | generated by the cell state measurement part of FIG. It is a figure which shows the modification of the display on the display part of FIG. It is a figure which shows the modification of the distribution map produced | generated by the cell state measurement part of FIG. It is a functional block diagram of the modification of the cell state measuring apparatus of FIG. It is a figure explaining the method of dividing | segmenting an image into a several unit area | region by the cell state measurement part of the cell state measuring apparatus of FIG.

A cell state measuring apparatus 100 according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIGS. 1 and 2, the cell state measurement device 100 according to the present embodiment acquires an image that acquires a two-dimensional image of the bottom surface 1 a by scanning the line sensor 21 with respect to the bottom surface 1 a of the container 1. Unit 2, a cell state measuring unit 3 that analyzes the image acquired by the image acquiring unit 2 and measures the state of the cells A distributed on the bottom surface 1 a, and displays the measurement result by the cell state measuring unit 3 And a display unit 4.

Moreover, the cell state measuring apparatus 100 includes a casing 5 made of a substantially rectangular parallelepiped-shaped sealed container having a height, a width, and a depth. The image acquisition unit 2 is accommodated in the housing 5, and the cell state measurement unit 3 and the display unit 4 are arranged outside the housing 5. Transmission /

reception units

6 and 7 are provided inside and outside the housing 5, respectively, and data of a two-dimensional image is transmitted from the image acquisition unit 2 to the cell state measurement unit 3 via the transmission /

reception units

6 and 7. ing.

As shown in FIG. 2, the top plate of the housing 5 provided on one side in the height direction (vertical direction in FIG. 2) is made of a flat plate-like member arranged horizontally, and the container 1 is placed thereon. The stage 5a is configured. The stage 5a is made of an optically transparent material such as glass so as to transmit illumination light from the illumination unit 23 described later.

The container 1 is a closed container formed of an entirely optically transparent material, such as a cell culture flask or dish, and contains cells A and a medium B. The container 1 has an upper plate 1b and a bottom plate 1c facing each other, and the upper plate 1b is provided with a reflecting surface for reflecting illumination light downward.

The image acquisition unit 2 includes a linear line sensor 21 arranged along the depth direction of the housing 5 (in a direction perpendicular to the paper surface in FIG. 2) substantially parallel to the stage 5a, the line sensor 21 and the stage 5a. A plurality of objective lenses 22, an illumination unit 23 that illuminates the field of view of the plurality of objective lenses 22, and a scanning mechanism 24 that moves the line sensor 21.

The line sensor 21 has a plurality of light receiving elements arranged in the longitudinal direction, detects light incident on the plurality of light receiving elements, and acquires an image for one line at a time. It is preferable that the line sensor 21 extends over substantially the entire length of the depth dimension of the housing 5 so that substantially the entire range in the depth direction of the stage 5a is included in the imaging range by the line sensor 21.

The plurality of objective lenses 22 are arranged so that the optical axis is along the direction orthogonal to the stage 5a, and collects the light transmitted through the stage 5a. The plurality of objective lenses 22 are arranged in a line along the longitudinal direction of the line sensor 21 and form an optical image on the same surface. A line sensor 21 is arranged on the image plane of the plurality of objective lenses 22, and an optical image connected on the image plane by the plurality of objective lenses 22 is acquired by the line sensor 21. The focal point of the objective lens 22 is adjusted by a focus adjustment mechanism (not shown) so as to match the bottom surface 1a. The objective lens 22 having a large depth of field may be used so that the adjustment of the focal position is not necessary.

The illumination unit 23 is arranged side by side with the image acquisition unit 2 in the width direction of the housing 5 (lateral direction in FIG. 2), and emits illumination light upward. The illumination light emitted from the illumination unit 23 passes through the stage 5a and the bottom plate 1c of the container 1, and is reflected downward on the reflection surface of the upper plate 1b of the container 1. Thereby, the field of view of the plurality of objective lenses 22 is illuminated from above, and the illumination light transmitted through the cell A, the bottom plate 1c, and the stage 5a is incident on the objective lens 22.

The scanning mechanism 24 scans the line sensor 21, the objective lens 22, and the illumination unit 23 integrally with the longitudinal direction of the line sensor 21 by a linear actuator (not shown), for example (that is, the width direction of the housing 5). Is moved one-dimensionally. The scanning mechanism 24 includes the line sensor 21 and the objective over the entire length of the width direction from one end to the other end of the casing 5 so that substantially the entire range of the stage 5a in the width direction is included in the imaging range of the line sensor 21. It is preferable to move the lens 22 and the illumination unit 23. The line sensor 21 acquires a wide range of two-dimensional images, preferably substantially the same as the stage 5a, by repeatedly acquiring images line by line while being moved in the scanning direction by the scanning mechanism 24.

When the cell state measurement unit 3 acquires a two-dimensional image from the image acquisition unit 2 via the transmission /

reception units

6 and 7, the cell state measurement unit 3 divides the image into a plurality of unit regions Q, and the cell A in each unit region Q A measurement process for measuring the state and a map generation process for generating a spatial map (spatial distribution) of the obtained measurement values are executed.

In the dividing step, the image P is equally divided into a predetermined number (5 × 5 in the example shown in FIG. 3) in the vertical and horizontal directions. Instead of dividing into a predetermined number, the image P may be divided so that each unit region Q has a predetermined size.
Next, in the measurement step, the state of the cell A in each unit region Q is measured. Specifically, by extracting cells A from each unit region Q using known image processing, and counting the number of cells A in each unit region Q, at least one of the number of cells and the cell density is a cell. Measured as A state.

Next, in the map generation step, the measurement value of the state of the cell A in each unit region Q calculated in the measurement step is superimposed and displayed on the corresponding unit region Q in the image P, as shown in FIG. Next, a distribution map representing the spatial distribution of the measurement values in the image P is created. FIG. 3 shows a cell density distribution map as an example. In this distribution map, the cell density represents a value when the density of the cells in the confluent state is 100%. In order to make it easier to visually recognize the spatial distribution of measurement values, the unit region Q in the distribution map may be color-coded according to the size of the measurement values.
The created distribution map is transmitted from the cell state measuring unit 3 to the display unit 4 and displayed on the display unit 4.

Such a cell state measuring unit 3 is realized by, for example, a computer arranged outside the housing 5. The computer includes a central processing unit (CPU) and a storage device that stores a cell state measurement program. The function of the cell state measurement unit 3 is realized by the CPU executing the above-described processing according to the cell state measurement program.

Next, the operation of the cell state measuring apparatus 100 configured as described above will be described.
The housing 5 of the cell state measuring apparatus 100 according to the present embodiment is disposed in the incubator with the container 1 placed on the stage 5a with the bottom plate 1c facing downward. The image acquisition unit 2 in the housing 5 executes photographing in an incubator according to a command signal transmitted by the operator or a preset program. The command signal is transmitted from the input device (not shown) outside the housing 5 to the image acquisition unit 2 via the transmission /

reception units

6 and 7.

The illumination light emitted from the illuminating unit 23 is transmitted through the stage 5a and the bottom plate 1c of the container 1, is reflected downward on the upper plate 1b, is transmitted through the cells A on the bottom surface 1a, the bottom plate 1c and the stage 5a, The light is condensed by a plurality of objective lenses 22 and an optical image of the bottom surface 1 a is formed on the line sensor 21. The optical image is taken by the line sensor 21, and an image for one line is acquired. The image acquisition unit 2 repeats photographing the bottom surface 1 a line by line sensor 21 while scanning the line sensor 21, the objective lens 22 and the illumination unit 23 in the scanning direction with respect to the bottom surface 1 a by the operation of the scanning mechanism 24. Thereby, the two-dimensional image P of the bottom surface 1a of the container 1 is acquired. The acquired image P is transmitted to the cell state measuring unit 3 arranged outside the incubator.

In the cell state measurement unit 3, the image P is divided into a plurality of unit regions Q, the state of the cells A in each unit region Q is measured, and the measurement value of the state of the cells A is superimposed on each unit region Q in the image P. A distribution map is created by being displayed. The distribution map is displayed on the display unit 4. Thereby, the operator can observe the image of the bottom face 1a of the container 1 in the incubator outside the incubator.

In this case, according to the present embodiment, by using the line scanning type image acquisition unit 2 that acquires the two-dimensional image P by scanning the line sensor 21, a wide range 2 such as the entire bottom surface 1 a of the container 1 can be obtained. The dimension image P is acquired in a short time for one scanning of the line sensor 21. Thereby, since the cells A distributed over a wide range are photographed with a slight time difference, there is an advantage that the state of the cells A changing with time can be accurately measured. Further, there is an advantage that the operator can easily grasp the state of the cell A in the wide range of the bottom surface 1a based on the measurement value of each unit region Q displayed on the image P.

In the present embodiment, the cell state measurement unit 3 further calculates a measurement value of the state of the cell A in the entire image P, and the display unit 4 displays the measurement value of each unit region Q as shown in FIG. In addition, the measurement value of the entire image P may be further displayed. FIG. 4 shows the average cell density and the total number of cells as the measurement values of the entire image P. Thus, by displaying the measured values of the entire image P on the display unit 4, the overall state of the cells A in the container 1 such as the total number and average density of the cells A can be easily grasped.

The measurement value of the entire image P may be calculated by measuring the state of the cell A of the entire image P separately from the measurement of the unit region Q, or may be calculated by integrating the measurement values of the unit region Q. Good. For example, the average value of the cell densities of all unit regions Q may be calculated as the average cell density of the entire image P. Alternatively, the sum of the numbers of cells in all unit regions Q may be calculated as the total number of cells in the entire image P.

In the present embodiment, as shown in FIG. 5, the cell state measurement unit 3 uses an image P that has been subjected to image processing for color-coding the region where the cell A exists and the region where the cell A does not exist as a distribution map. May be used.
By doing in this way, the operator can grasp | ascertain the area of the area | region where the cell A exists visually more easily.

In this embodiment, as shown in FIG. 6, the cell state measuring unit 3 further includes a cell type information acquiring unit 8 that acquires cell type information of the cell A to be measured that is cultured in the container 1. However, the size of the unit region Q may be determined based on the cell type information acquired by the cell type information acquisition unit 8, and the image P may be divided into the unit regions Q having the determined size.
The cell type information acquisition unit 8 is configured to acquire cell type information based on, for example, an operator's input operation. The cell type information acquired by the cell type information acquisition unit 8 is transmitted to the cell state measurement unit 3.

The cell state measuring unit 3 holds a table in which cell types and the sizes of the unit areas Q are associated with each other. The size of the unit region Q is set for each cell type. For example, the cell type forming the colony C is set such that the size of one side of the unit region Q is larger than the diameter of the colony C as shown in FIG. In FIG. 7, a round dish-like container 1 is shown.

When the size of the unit region Q is smaller than the diameter of the colony C, since one colony C extends over the plurality of unit regions Q, the number and size of the colonies C cannot be accurately measured. On the other hand, by making the size of one side of the unit region Q larger than the diameter of the colony C, the frequency with which one colony C extends over the plurality of unit regions Q is reduced, and the entire one colony C is the same. The image P can be divided so as to exist in the unit region Q.
Thus, the measurement accuracy of the state of the cell A can be improved by determining the size of the unit region Q according to the cell type of the cell A cultured in the container 1.

In the present embodiment, the image acquisition unit 2 may acquire a plurality of time-series two-dimensional images P at predetermined time intervals.
In this case, the cell state measurement unit 3 obtains a plurality of time-series measurement values for the unit region Q at the same position by measuring the state of the cell A in each unit region Q in each of the plurality of images. Instead of or in addition to the distribution map, the display unit 4 displays the change over time of the measurement value of the unit region Q at the same position, for example, as a graph. Thereby, the operator can easily grasp the change with time of the state of the cell A over a wide area or the whole of the container 1 based on the change with time displayed on the display unit 4.

In the present embodiment, the cell number and the cell density are given as examples of the state of the cell A, but other indicators used for evaluating the state of the cell A may be measured. For example, in the case of cells that form colonies, the size, number, or density of the colonies may be measured.

In the present embodiment, the measurement result calculated by the cell state measurement unit 3 is superimposed on the image P and displayed on the display unit 4. Instead, the measurement result and the image P are displayed on the display unit 4. May be displayed side by side in separate areas.

In the present embodiment, the illumination unit 23 is provided in the housing 5, but instead, an illumination unit may be provided outside the housing 5. For example, an illumination unit separate from the housing 5 may be provided above the container in the incubator. Alternatively, the illumination unit may be fixed to the side plate or the upper plate of the container 1.

In the present embodiment, the light from the cells detected by the line sensor 21 is light by illumination light from the illuminating unit, but instead, light by fluorescence or light emission phenomenon generated in the cells. It may be.

DESCRIPTION OF SYMBOLS 100 Cell state measuring device 1 Container 1a Bottom surface 2 Image acquisition part 21 Line sensor 22 Objective lens 23 Illumination part 24 Scanning mechanism 3 Cell state measurement part 4 Display part 5

Case

6, 7 Transmission / reception part 8 Cell type information acquisition part

Claims

It has a linear line sensor that detects light from cells cultured in a container, and acquires a two-dimensional image by moving the line sensor in a scanning direction that intersects the longitudinal direction of the line sensor. An image acquisition unit to
A cell state measurement unit that divides the image acquired by the image acquisition unit into a plurality of unit regions and measures the state of cells in each of the plurality of unit regions;
A cell state measurement apparatus comprising: a display unit that displays a measurement result of the state of the cell in each unit region measured by the cell state measurement unit.
The cell state measurement unit calculates the cell state in the entire image by integrating the measurement results of the plurality of unit regions,
The cell state measurement device according to claim 1, wherein the display unit further displays a measurement result of a cell state in the entire image.
The cell state measuring device according to claim 2, wherein the cell state measuring unit adds measurement results of the plurality of unit regions.
The cell state measurement device according to any one of claims 1 to 3, wherein the display unit displays a spatial distribution of measurement results of the plurality of unit regions.
A cell type information acquisition unit for acquiring information on the cell type of the cells cultured in the container;
The cell state measuring device according to any one of claims 1 to 4, wherein the cell state measuring unit determines a size of the unit region according to information on a cell type acquired by the cell type information acquiring unit.
The cell state measurement unit measures at least one of the density and the number of cells in each unit region as the state of the cells,
The cell state measuring device according to any one of claims 1 to 5, wherein the display unit displays at least one of a cell density and a cell number in each unit region.
The cell state measurement unit further measures at least one of the density and the number of cells in the entire image as the state of the cells,
The cell state measurement apparatus according to claim 6, wherein the display unit further displays at least one of a cell density and a cell number in the entire image.
The image acquisition unit acquires a plurality of time-series images at time intervals,
The cell state measurement according to any one of claims 1 to 7, wherein the display unit displays a time-dependent change in a measurement value of each unit region calculated from each of the plurality of images by the cell state measurement unit. apparatus.