JP7006832B2 - Cell analyzer - Google Patents

Description

The present invention relates to a cell analyzer.

In the field of regenerative medicine, research using pluripotent stem cells such as iPS cells and ES cells has been actively conducted in recent years. In research and development of regenerative medicine using such pluripotent stem cells, it is necessary to culture a large amount of undifferentiated cells in a state where pluripotency is maintained. Therefore, it is necessary to select an appropriate culture environment and stably control the environment, and it is also necessary to confirm the state of cells during culture at a high frequency.

Conventionally, in the field of cell culture for regenerative medicine, a phase-contrast microscope has been used exclusively for morphological observation of cells in culture. A phase-contrast microscope is used because cells are generally transparent and difficult to observe well with a normal optical microscope.

On the other hand, in recent years, a device for acquiring an observation image of a cell using a holographic technique has been put into practical use. As disclosed in Patent Documents 1, 2, etc., this device clearly observes cells by performing data processing such as phase recovery and image reconstruction on hologram data obtained by a digital holographic microscope. An easy-to-use phase image (hereinafter referred to as "IHM phase image" because an in-line Holographic Microscopy (IHM) is used) is created. Similar to the phase-contrast microscope image obtained with a phase-contrast microscope, transparent cells can be observed relatively clearly in this IHM phase image.

International Patent Publication No. 2018/158947 International Patent Publication No. 2018/158810 International Patent Publication No. 2016/162945 Japanese Unexamined Patent Publication No. 2016-189701

When culturing cells for regenerative medicine, it is very important to understand the number of cells existing in the culture vessel and the area occupied by the cells on the medium in the culture vessel (hereinafter sometimes referred to as "cell area"). Is. This is because the number of cells and the cell area are important indicators for determining whether or not the culture conditions are appropriate and for determining the timing of subculture passage. When cells form a mass and form a cell colony (hereinafter, simply referred to as "colony"), it is difficult to separate individual cells and count the number of cells, so the cell area is used as an index. Is often done.

In cell culture, various forms such as one or more wells, dishes (Petri dishes), flasks, etc. formed on a culture plate are used as the culture container. As a technique for determining the number of cells existing in such a culture vessel, the method described in Patent Document 3 is conventionally known. In this conventional method, a phase difference image over a predetermined visual field range in a culture vessel in which cells are cultured is acquired, cells are recognized in the image, and the number is counted. The phase-contrast image over the field of view that can be obtained by the phase-contrast microscope is a small part of the entire culture vessel, and the phase-contrast microscope requires focusing every time the field of view is moved. It takes a lot of time and effort to repeatedly acquire a phase-contrast image while moving. Therefore, the number of cells in the entire culture vessel is estimated by multiplying the ratio of the effective area of the entire culture vessel to the area of the visual field range by the count value of the cells obtained by actual measurement as described above.

However, in the above-mentioned prior art, if the cells do not exist in the culture vessel at a substantially uniform density, the accuracy of the number of cells in the entire culture vessel becomes low. In actual cell cultures, for example, the density of cells often differs significantly between the area near the periphery and the center of the well, which is circular in top view, and in such a situation, the accuracy of the cell number is significantly reduced. become. Further, even if the above technique is adopted for calculating the cell area, the same problem arises.

In cell culture, in addition to the number of cells and cell area, features related to the shape of cells and colonies, such as length in the major axis direction, length in the minor axis direction, roundness, and peripheral length. In some cases, it is desired to obtain image features such as quantity, phase difference, optical path length, or differential value, but statistical results such as the average value and dispersion value of these various features are also accurate in the entire culture vessel. There is a problem that it is difficult to obtain high results.

The present invention has been made to solve the above problems, and its main purpose is to occupy cells or colonies existing in a specific region in one or a plurality of culture vessels, or in each of the plurality of culture vessels. It is an object of the present invention to provide a cell analysis apparatus capable of calculating information on an area with high accuracy and presenting the information so that a user can easily evaluate the state of cells.

The first aspect of the present invention is a cell analysis device for analyzing cells in one or more culture vessels.
An image acquisition unit that acquires an observation image of cells over a predetermined imaging target area including one or a plurality of culture containers, and an image acquisition unit.
A specific region extraction unit that extracts a cell region in which cells are present or characteristic cells are present from the entire observation image obtained by the image acquisition unit or a predetermined analysis target region in the image, and a specific region extraction unit.
An index value calculation unit that calculates an index value relating to the size and / or shape of the cell region extracted by the specific region extraction unit for the entire observation image or the analysis target region.
The index values calculated by the image acquisition unit, the specific region extraction unit, and the index value calculation unit were used for cells in the same culture vessel at a plurality of different time points. A display processing unit that creates a graph showing changes in index values over time and displays the graph on the display unit.
A series of processes of the image acquisition unit, the specific region extraction unit, and the index value calculation unit performed on cells in the same one or a plurality of culture vessels at a plurality of different time points, at that time point . Using the index value calculated for each, a graph showing the temporal change of the index value using the index value as a plot point is created, the graph is displayed on the display unit , and the plot points are displayed on the graph. In response to the operation by the instructing user, the observation image of the cell corresponding to the instructed plot point is displayed on the display unit, and the displayed cell observation image is displayed with an image showing the extraction result of the cell region. A display processing unit that arranges and displays an operator for selecting whether or not to superimpose in the display screen of the graph, and a display processing unit.
It is equipped with.

The type of cell to be analyzed in the cell analyzer of the first aspect is not particularly limited, but is typically a pluripotent stem cell such as a human iPS cell or an ES cell. In that case, the characteristic cells can include undifferentiated cells, undifferentiated deviant cells, and the like.

In the cell analysis apparatus of the first aspect of the present invention, the specific region extraction unit is the entire image acquired by the image acquisition unit over a predetermined imaging target region including one or a plurality of culture containers, or in the image thereof. For the entire analysis target region of one or a plurality of cells, for example, a cell region in which cells are present is extracted. Then, the index value calculation unit calculates, for example, as an index value regarding the size of the extracted cell region, the total area of the cell region, the average of the area of the cell region in each of the plurality of culture vessels, and the like. Therefore, according to the first aspect of the present invention, even if the distribution density of cells in the culture vessel is non-uniform, it is not affected by the non-uniformity, and information such as the cell area can be obtained with high accuracy. Can be calculated.

Further, in the cell analysis apparatus of the first aspect of the present invention, the display processing unit uses, for example, the cell area calculated for each of the cells in one or a plurality of culture vessels at a plurality of different time points, and the cell area thereof is used. Create a graph showing changes over time and display it on the display. Therefore, according to the first aspect of the present invention, the user can easily and accurately determine the accuracy of the culture conditions in the cell culture, the timing of the passage of the culture, and the like from the information displayed in this way. As a result, the cell culture work can be efficiently performed and work mistakes can be reduced.

The schematic block block diagram of the cell analysis apparatus which is one Embodiment of this invention. The flowchart which shows the work procedure and process flow in the cell state evaluation work using the cell analysis apparatus of this embodiment. The conceptual diagram for demonstrating the photographing operation and the image reconstruction processing in the cell analysis apparatus of this embodiment. The figure which shows the display example of the information of the culture container used for culture in the cell analysis apparatus of this embodiment. The figure which shows an example of the image pickup unit which takes a picture when the area to be photographed is limited in the cell analysis apparatus of this embodiment. The figure which shows an example of the classifier selection screen in the cell analysis apparatus of this embodiment. The figure which shows an example of the extraction result of the cell area when the inside of a well is designated as the area to be photographed. The figure which shows an example of the extraction result of the cell area by a classifier. The figure which shows an example of the extraction result of the undifferentiated cell region and the undifferentiated deviant cell region by the classifier. The figure which shows an example of the display screen which contains the graph of the time change of a cell area. The figure which shows an example of the display image when the area identification result by a classifier and the cell observation image are superposed.

An embodiment of the cell analysis apparatus according to the present invention will be described with reference to the accompanying drawings.

[Overall configuration of equipment]
FIG. 1 is a schematic block configuration diagram of the cell analysis device of the present embodiment.
This cell analysis device includes a microscopic observation unit 1, a control / processing unit 2, an input unit 3 and a display unit 4 which are user interfaces.
Here, the microscopic observation unit 1 is an in-line holographic microscope, has a light source unit 10 including a laser diode and the like, and an image sensor 11, and a cell (or colony) is provided between the light source unit 10 and the image sensor 11. A culture plate 12 or another culture container in which the 13 is cultured is arranged.

The control / processing unit 2 controls the operation of the microscopic observation unit 1 and processes the data acquired by the microscopic observation unit 1. The imaging control unit 20, the hologram data storage unit 21, and the phase information calculation. Unit 22, image reconstruction unit 23, reconstruction image data storage unit 24, specific area extraction unit 25, index value calculation unit 26, calculation result storage unit 27, display processing unit 28, and measurement / analysis. A condition setting unit 29 and a condition setting unit 29 are provided as functional blocks.

Normally, the entity of the control / processing unit 2 is a personal computer or a higher-performance workstation in which predetermined software (computer program) is installed, or a high-performance computer connected to such a computer via a communication line. Including computer system. That is, the function of each block included in the control / processing unit 2 is stored in the computer or computer system, which is executed by executing software installed in a computer system including a single computer or a plurality of computers. It is embodied by processing using various types of data. Of course, it is also possible to realize some of these functions with a dedicated hardware circuit.

[Cells to be observed and analyzed by this device]
The cells to be observed and analyzed by the cell analyzer of the present embodiment may be of any type as long as they are cells cultured in the culture vessel, but are typically pluripotent such as iPS cells and ES cells. It is a sex stem cell. The culture vessel is a culture plate, dish, flask or the like in which a large number of wells are formed. The size and shape of these culture vessels vary depending on the type or manufacturer. For example, the number of wells provided on one culture plate is 6, 12, 24, 48, 96, 384, and the like. There are many types. Since cells of different strains can be independently cultured in the plurality of wells formed on one culture plate, each well can be regarded as an individual culture container. Therefore, for example, a culture plate in which 6 wells are formed can be regarded as one in which 6 culture containers are connected and integrated.

[Processing operation of cell analysis in this device]
Hereinafter, as an example, in the case where the analysis target is a human iPS cell and the cells are cultured in each of the six wells formed on one culture plate 12, the analysis operation in the cell analysis apparatus of the present embodiment is performed. explain.
FIG. 2 is a flowchart showing a work procedure and a flow of processing in a cell analysis work using the cell analysis device of this embodiment.

<Collection of hologram data>
FIG. 3 is a conceptual diagram for explaining an imaging operation in the microscopic observation unit 1 and an image reconstruction process performed in the control / processing unit 2.
FIG. 3A is a schematic top view of an example of the culture plate 12. Six wells 12a having a circular shape in the top view are formed on the culture plate 12, and cells are cultured on the medium contained in each well 12a. Here, the entire culture plate 12 shown in FIG. 3A, that is, the entire rectangular area including the six wells 12a is the area that can be photographed by the microscopic observation unit 1.

The microscopic observation unit 1 includes four sets of a light source unit 10 and an image sensor 11, and each of the light source unit 10 and the image sensor 11 of each set includes the entire culture plate 12 as 4 mag as shown in FIG. 3A. It is responsible for collecting hologram data of the four divided four-division ranges 81. That is, the four sets of the light source unit 10 and the image sensor 11 share the collection of hologram data over the entire culture plate 12 (that is, the entire imageable area).

As shown in FIG. 3B, the range in which the set of the light source unit 10 and the image sensor 11 can be photographed at one time is a substantially square shape including one well 12a in the four division range 81. It is a range corresponding to one imaging unit 83 obtained by dividing the range 82 into 10 equal parts in the X-axis direction and 12 equal parts in the Y-axis direction. One 4-division range 81 includes 15 × 12 = 180 imaging units 83.

The four light source units 10 and the four image sensors 11 are arranged in the XY plane including the light source unit 10 and the image sensor 11, respectively, near the four vertices of a rectangle having the same size as the four division range 81. At the same time, holograms are acquired for four different imaging units 83 on the culture plate 12. Then, as will be described later, by moving the light source unit 10 and the image sensor 11 in steps by a predetermined distance in the XY plane, the light source unit 10 and the image sensor 11 of each set are combined into 180 image pickup units 83. Hologram data in the corresponding range are acquired in order. In the conventional general phase-contrast microscope, it was necessary to focus at the time of shooting (image acquisition), but in the holographic microscope, the focused image is reconstructed in the process of data processing as described later. It is possible to do. Therefore, it has a feature that it is not necessary to focus at the time of shooting.

As described above, the sizes and shapes of the culture vessels are very diverse, and even inside the culture vessel, not all of them are regions for culturing cells. When acquiring a cell observation image, it is meaningless to acquire an image of a region other than the region where cells are cultured, which is a waste of measurement time. On the other hand, after acquiring the cell observation image, the region that the user actually wants to analyze (for example, to know the cell area) is not not all the region where the cell exists, but may be only a part of the region. Therefore, in the cell analysis apparatus of the present embodiment, it is possible to limit the imaging target area for actual imaging and the analysis target region for actual analysis as follows.

That is, when the user inputs information such as the measurement date and time from the input unit 3 before executing the measurement (shooting), the user inputs the information about the culture container such as the manufacturer and model number of the culture container to be used, and the area to be photographed. The scan conditions for determining the scan conditions and the analysis conditions for determining the analysis target area are input from the input unit 3. Then, the culture plate 12 in which the cells 13 to be observed and analyzed are being cultured is set at a predetermined position of the microscopic observation unit 1, and the measurement execution is instructed (step S1).

Information about the culture vessel and scanning conditions specified by the user can be confirmed on the measurement target information display screen 100 as shown in FIG. In FIG. 4, "manufacturer model number" 101 is information indicating the manufacturer and model number of the culture plate input by the user, and "plate shape" 102 is information indicating the shape of the entire culture plate, "well shape" 103. Is information indicating the shape of one well, and these are uniquely determined by the manufacturer's model number and the like. The "plate reference point" 104 is information regarding the installation position of the culture plate, and the "well position" 105 is information indicating the center position coordinates of each of the six wells, which was taken by the camera attached to the microscopic observation unit 1. Calculated from optical images. The "scan condition" 106 is information indicating the range to be excluded from the imaging target, and the "analysis condition" 107 is information indicating the range to be excluded from the analysis target such as extraction of the cell region described later. Input is set.

The imaging control unit 20 has a container information storage unit in which information including the shape and size of various culture containers is registered in advance. The imaging control unit 20 collates the information (manufacturer model number) about the culture container set from the input unit 3 with the container information storage unit, and reads out information such as the plate shape and the well shape displayed in FIG. Further, the plate reference point, the position coordinates of the well, and the like are specified based on the image obtained by optically taking the mounted culture plate 12. Then, from the input information and the position information acquired based on the optical image, the region in which the cells can be cultured is calculated in the imageable region, and the imaging target region is determined according to the calculation result (step). S2). The details of the shooting target area will be described later.

When the imaging target area is determined, the imaging control unit 20 controls each unit of the microscopic observation unit 1 to perform imaging and collect hologram data (step S3).
That is, one light source unit 10 irradiates a predetermined region (one imaging unit 83) of the culture plate 12 with coherent light having a spread of a minute angle of about 10 °. The coherent light (object light 15) transmitted through the culture plate 12 and the cell 13 reaches the image sensor 11 while interfering with the light (reference light 14) transmitted through the region close to the cell 13 on the culture plate 12. The object light 15 is light whose phase has changed when it passes through the cell 13, while the reference light 14 is light that does not pass through the cell 13 and therefore does not undergo the phase change caused by the cell 13. Therefore, on the detection surface (image surface) of the image sensor 11, an interference image of the object light 15 whose phase has been changed by the cell 13 and the reference light 14 whose phase has not changed, that is, a hologram is formed, and this hologram is formed. The two-dimensional light intensity distribution data (hologram data) corresponding to the above is output from the image sensor 11.

As described above, coherent light is emitted from the four light source units 10 toward the culture plate 12 almost simultaneously, and the four image sensors 11 acquire hologram data of regions corresponding to different imaging units 83 on the culture plate 12. Will be done. Each time the measurement at one measurement position is completed, the light source unit 10 and the image sensor 11 are moved in the X-Y axis direction and the Y-axis direction by a distance corresponding to one image pickup unit 83 in the XY plane due to a moving unit (not shown). It is moved sequentially in steps. As a result, measurements are performed with 180 or less imaging units 83 included in the 4-division range 81, and the entire culture plate 12 or a specific imaging target area is used for the entire four sets of light source units 10 and the image sensor 11. Measurement will be performed. As described above, since it is not necessary to perform focusing at the time of imaging with a holographic microscope, the time required for imaging (measurement) at one measurement position can be shortened. As a result, shooting can be completed in a relatively short time even if the shooting target area is wide. As described above, the hologram data obtained by the four image sensors 11 of the microscopic observation unit 1 is sent to the control / processing unit 2 and stored in the hologram data storage unit 21 together with the attribute information such as the measurement date and time.

As shown in FIGS. 3A and 3B, the microscopic observation unit 1 can acquire hologram data of the entire rectangular imageable area including the six wells 12a, but some of them. The imaging unit 83 is the outer part of the well 12a in which no cells are present. Even if hologram data is acquired for such an imaging unit, the data is useless. Therefore, in step S2, the imaging control unit 20 determines whether or not each imaging unit 83 includes the inner region of the well 12a (that is, the outer region of the well 12a) by the calculation based on the shape of the well 12a and the information of the position thereof. Whether or not it contains only) is judged. Then, only the imaging unit 83 determined to include the inner region of the well 12a is determined as the imaging target region.

FIG. 5 is an example of dividing a 10 × 12 imaging unit 83 including one well 12a into an imaging target region and a region other than the imaging target region. In this example, of the total 120 imaging units 83, 48 imaging units 83B shown by diagonal lines are out of the imaging target area, and the remaining 72 imaging units 83A form an imaging target area. .. The image pickup unit 83B outside the shooting target area is skipped during the shooting operation as described above, and hologram data is not acquired. As a result, it is possible to shorten the measurement execution time and reduce the amount of hologram data for the imaging target area. This is effective in saving the memory capacity in the hologram data storage unit 21 and reducing the amount of data transfer from the microscopic observation unit 1 to the control / processing unit 2.

Of course, at the time of shooting, the entire holographic area including the part where significant hologram data cannot be obtained is photographed (measured) to collect hologram data, and the imaging unit is out of the imaging target area during the analysis described later. May be excluded from the analysis target.

<Creation of IHM phase image>
When the series of measurements (shooting) described above is completed, the phase information calculation unit 22 sequentially reads hologram data from the hologram data storage unit 21 and performs light wave propagation calculation processing to perform phase information and amplitude at each two-dimensional position. Restore information. Since the spatial distribution of this information is obtained for each imaging unit 83, if the phase information and amplitude information of all the imaging units 83 are obtained, the image reconstruction unit 23 takes a picture based on the phase information and the amplitude information. A phase image of the entire target region, that is, an IHM phase image is formed (step S4).

That is, the image reconstruction unit 23 reconstructs the IHM phase image of each imaging unit 83 based on the spatial distribution of the phase information calculated for each imaging unit 83. Then, by performing a tiling process (see FIG. 3D) for connecting the IHM phase images in a narrow range, the IHM phase image for the imaging target region, that is, the entire culture plate 12, or a specific imaging target region thereof. To form. The data constituting the IHM phase image is stored in the reconstructed image data storage unit 24. In the tiling process, it is advisable to perform an appropriate correction process so that the IHM phase images at the boundary of the imaging unit 83 are smoothly connected.

Further, the reconstructed image obtained by normal processing is the highest resolution IHM phase image obtained in principle from the acquired hologram data, but not only that, binning processing or the like is performed based on the highest resolution IHM phase image. Creates IHM phase images with multiple resolutions with reduced resolution. Then, the data constituting the IHM phase image having such various resolutions is stored in the reconstructed image data storage unit 24. As a result, when displaying IHM phase images of various resolutions later selectively, the display processing unit 28 should read the reconstructed image data of the required resolution from the reconstructed image data storage unit 24 and display it. Images can be formed quickly.

Further, the phase information calculation unit 22 calculates not only the phase information but also the intensity information and the pseudo phase information obtained by merging the phase information and the intensity information based on the hologram data, and the image reconstruction unit 23 calculates. It is also possible to create a reproduced image (IHM intensity image, IHM pseudo-phase image) based on these. These images may be used in place of the IHM phase image. Further, when creating an IHM phase image or the like based on hologram data, it is possible to create an IHM phase image or the like at a plurality of focal positions. This makes it possible to obtain an IHM phase image at the in-focus position without focusing at the time of shooting.

<Extraction of cell regions, etc.>
Next, the specific region extraction unit 25 executes a process of extracting a cell region in which cells exist, a cell region in which specific cells exist, or other characteristic regions from the IHM phase image corresponding to the region to be imaged. (Step S5). Here, region extraction is performed using a classifier learned by a machine learning algorithm such as deep learning using a multi-layer neural network, or more simply, using a technique of image segmentation by deep learning.

Specifically, for example, a large number of IHM phase images in which the person in charge determines the background region in which cells do not exist and the cell region in which cells exist and labels them as binary values are prepared as teacher data, and the teacher data. To create a discriminative model (discriminative model) by training with a multi-layer neural network. This classifier takes the data constituting the IHM phase image as an input, and outputs the image segmented according to the binary label as the discrimination result. The specific area extraction unit 25 has this classifier, and inputs the IHM phase image to be analyzed created as described above to the classifier as input data. Then, as the output of the classifier, a segmentation image in which the pixels contained in the background region and the cell region are labeled is obtained.

Only one type of classifier may be used, but in the cell analysis apparatus of the present embodiment, a plurality of types of classifiers are prepared in the specific region extraction unit 25, and are different for each of the six wells 12a depending on the manual selection by the user. The area extraction process using the classifier can be performed.

FIG. 6 is a diagram showing an example of the classifier selection screen 110. When the user performs a predetermined operation on the input unit 3 at an appropriate time before the execution of step S5, the measurement / analysis condition setting unit 29 displays the classifier selection screen 110 on the display unit 4. A well-compatible classifier list 111 is arranged on the classifier selection screen 110. In this list 111, the analysis parameter indicates a discriminator, and in the example of FIG. 6, it means that a discriminator named "Cell Analysis_01" is used for the well whose well number is "1". .. When the down arrow symbol 113 at the right end of the list 111 is clicked, a list of registered classifiers is displayed in the down menu. You can choose the vessel.

Further, even if the user looks at the name of the classifier, it may not be possible to determine what kind of analysis the classifier is suitable for, what kind of area extraction is to be performed, and the like. Therefore, a comment field may be added to each line of the well-compatible classifier list so that the pre-filled comment about the classifier selected at that time is displayed. Of course, these comments may be displayed in a pop-up when the mouse cursor hovers over the classifier name. Further, in FIG. 6, different classifiers are selected depending on the well number, but the user selects the name of one of the classifiers and then clicks the "Apply to all wells" button 114. Then, the classifier can be set for all wells.

In general, discriminators that are different from each other have the same discrimination algorithm (for example, deep learning using the same multi-layer neural network), but different teacher data at the time of learning. That is, even if the labeling is the same (for example, a binary label of a cell region and a background region), if the IHM image itself to be trained is different, the discriminator will be different. Not only when the cell type is different, but also when the cell type is the same but the culture conditions are different, the cell shape and the like may be different. Therefore, by creating discriminators using IHM images of cells cultured under different culture conditions as teacher data and selecting discriminators according to the culture conditions for analysis, the differences in culture conditions are achieved. It is possible to reduce the influence of the above and make more accurate identification.

In addition, those with different labeling are, of course, different as discriminators. Specifically, in addition to the classifier created by learning IHM images labeled corresponding to the two regions of the background region and the cell region as teacher data, the background region, the undifferentiated cell region, and the undifferentiated cell region. A discriminator created by learning labeled IHM images corresponding to three (or more) regions of the differentiation-developed cell region as teacher data can be used. Furthermore, not only the cell region but also the region where foreign matter such as dust exists and the region where some characteristic pattern exists on the image such as interference fringes are labeled as the characteristic region. You can create a classifier. Such different classifiers can be used properly according to the purpose of analysis, the state of cells, and the like.

Further, in the specific region extraction unit 25, when extracting a cell region or a characteristic region from an IHM phase image for a certain one or a plurality of wells, a plurality of discriminators different from each other may be used in combination. For example, the region extraction result obtained from the first discriminator that discriminates between the cell region and the background region, and the region extraction obtained from the second discriminator that discriminates between the undifferentiated deviated cell region and the other region. By combining the results, specifically, logical operations such as logical sum, logical product, and exclusive OR for each pixel in different segmentation images, it is possible to obtain the extraction result of the cell region that is not undifferentiated. Can be done.

FIG. 8 is a diagram showing an example of an image of the result of extracting a cell region (performing segmentation) using a discriminator capable of discriminating between a background region and a cell region. In this image, the background area and the cell area are shown in different display colors. In the image of the extraction result, the region where the cells and colonies are present is well extracted. FIG. 9 is a diagram showing an example of an image of the result of performing segmentation using a discriminator capable of discriminating between a background region, an undifferentiated cell region, and an undifferentiated deviant cell region. The background region, the undifferentiated cell region, and the undifferentiated deviant cell region are shown in different display colors. In the image of the extraction result, the cell region and the background region are well distinguished, and the undifferentiated cells and the undifferentiated deviant cells are well distinguished even in the same cell region.

FIG. 7 is a diagram showing an IHM phase image (right side) near the peripheral edge of the well and an image (left side) of the result of extracting a cell region using the entire IHM phase image as an analysis target region. From FIG. 7, it can be seen that the inside of the well peripheral portion, which has a substantially circular shape, is defined as the imaging target region, and the cells (colonys) in the imaging target region can be appropriately detected.

<Calculation of index values such as cell area>
The index value calculation unit 26 performs a predetermined arithmetic process on the cell region or characteristic region extracted as described above, and is related to, for example, the cell area of each well or the entire culture plate including a plurality of wells. The index value to be calculated is calculated (step S6). Then, the calculated index value is stored in the calculation result storage unit 27.

As an example, by integrating the number of pixels of the cell region extracted on the IHM phase image for each well and multiplying the number of pixels by the actual area value in the well corresponding to one pixel, for each well. The cell area can be calculated. Further, from the cell area in one well and the internal area of the well obtained in this way, the ratio of the region occupied by the cells in the internal region of the well and the cell area per unit area in the internal region of the well can be calculated. Further, it is possible to calculate statistics such as the total cell area of one culture plate as a whole and the average and dispersion of cell areas in each of a plurality of wells in one culture plate.

Further, when the undifferentiated deviated cell region and the undifferentiated cell region are extracted, the ratio of the area occupied by the undifferentiated deviated cells to the area occupied by the undifferentiated cells can be calculated for each well. In addition, when a region where foreign matter is present or a region where some characteristic pattern is present on the image such as interference fringes is extracted as a characteristic region, the area of those characteristic regions and the area of the characteristic region are extracted. The ratio between the area inside the well and the area inside the well, or the ratio between the area of the characteristic region and the area of the cell region can be calculated.

Furthermore, the index value calculation unit 26 can also calculate various index values related to the size and shape of the cell region other than the area of the cell region. Specifically, since the extracted cell region is the shape of an individual cell or the shape of a colony which is a mass of a plurality of cells, the length in the major axis direction and the length in the minor axis direction for each region, Characteristic index values that reflect the size and shape, such as peripheral length and roundness, can be calculated. Thereby, statistics such as the average and dispersion of the peripheral length of the cell region can be calculated for each well. Furthermore, image features such as the luminance value on the image in the cell region or feature region, the gradient of the luminance value, and the differential value indicating the spatial change of the luminance value, which are not directly related to the size and shape of the cell. It may be possible to calculate the numerical value indicating the above and calculate the characteristic amount of the cell (or colony) region or the characteristic region based on the numerical value. Further, a new index value may be calculated by combining different index values such as the above-mentioned area value. For the calculation of such an index value, for example, an existing method described in Patent Document 4 or the like can be used.

<Display of the status of temporal changes in index values>
After the analysis processing as described above is completed, when the user performs a predetermined operation in the input unit 3, the display processing unit 28 is based on the latest analysis result and the past analysis result for the same culture plate 12. , A graph showing the temporal change of the analysis result is created and displayed on the display unit 4 (step S7). FIG. 10 is an example of displaying a graph showing the temporal change of the cell density (ratio of the cell area to the internal area of the well) for each well.

As shown in FIG. 10, on the analysis result display screen 120, a culture container selection instruction field 121 for selecting a culture plate and a well to be displayed and a time lapse graph 122 are arranged. In the culture vessel selection instruction field 121, the user selects and puts a check mark in the culture plate and well to be displayed on the time lapse graph 122. Further, the index value used for the graph to be displayed is selected in the display graph selection field 123. In the example of FIG. 10, Confluency is selected.

When the above selection is performed, the display processing unit 28 reads out the calculation result of the specified index value associated with the specified plate name stored in the calculation result storage unit 27, and reads it out. The cell culture period is assigned to the horizontal axis (or vertical axis), the index value is assigned to the vertical axis (or horizontal axis), and the values at each measurement time point are plotted for each designated well to create a graph. Then, the time lapse graph 122 is displayed in the analysis result display screen 120. The lines on the graph 122 corresponding to each well are displayed in different colors. As a result, the user can easily evaluate the change with time of the index value in each well and the difference in the change with time of the index value for each well.

When the user changes the selection of the index value in the display graph selection field 123, the display processing unit 28 changes the index value, creates a new graph, and updates the displayed graph in response to the change operation. Thereby, it is possible to confirm the change with time for various index values in order. Further, as shown in FIG. 10, the time-lapse graph 122 shows not only the index value for a plurality of wells on one culture plate but also the time-dependent change of the index value for the wells on another culture plate. can. Thereby, for example, it is possible to easily evaluate the difference from the reference culture plate or well.

Further, when the user clicks on one of the plots indicating each measurement time point on the time passage graph 122, the display processing unit 28 corresponds to the designated plot point, that is, corresponds to the measurement time point, the plate, and the well. Identify the attached IHM phase image. Then, the data constituting the IHM phase image is acquired from the reconstructed image data storage unit 24 to form an image, and the image is displayed on the screen of the display unit 4. The image may be displayed on a part of the analysis result display screen 120, or may be displayed in another newly opened window. The displayed image allows the user to confirm and examine the state of cells in any well at any time of measurement.

On the analysis result display screen 120, when displaying the IHM phase image as described above, a switching button 124 for selecting whether or not to superimpose the segmentation image which is the region extraction result on the IHM phase image is arranged. Has been done. Depending on the selection by the switching button 124, only the IHM phase image is displayed on a screen different from the analysis result display screen 120, or as shown in FIG. 11 in which a translucent segmentation image is superimposed on the IHM image. It is possible to switch whether to display a composite image. After the user makes such a selection on the analysis result display screen 120, the user can efficiently display and confirm the target cell observation image by instructing the selection of an arbitrary plot point on the time-lapse graph 122. Can be done.

Further, on the analysis result display screen 120, a CSV file output button, a screen capture button, and an output instruction button 125 such as a PC output button are arranged. When the user clicks such a button, the time elapsed graph 122 confirmed on the analysis result display screen 120 is displayed as either a CSV file (text file), a screen capture, or a screen file (for example, a JPEG file). It can be output with. When outputting as a CSV file, it is preferable to output the type of the classifier used for the area extraction process, the calculation type of the feature amount, and the like for each measurement plot on the graph 122. Thereby, for each plot point on the graph 122, information on the type of the classifier used for the area extraction and the type of calculation of the feature amount can be specified.

As described above, in the cell analysis apparatus of the present embodiment, the cell region is based on the image (IHM image) acquired so that the cells can be sufficiently observed in the entire culture vessel or the entire culture vessel. Etc. can be satisfactorily extracted, and index values such as the area occupied by the extracted cell region can be calculated. Therefore, even when the cell density is not uniform in the culture vessel, the index value such as the cell area can be accurately obtained. Further, in the cell analysis apparatus of the present embodiment, it is possible to confirm at a glance on the graph the temporal change of the index value such as the cell area for the cells being cultured in one culture container. Thereby, for example, the suitability of the culture conditions and the timing of the passage of the culture can be appropriately and easily determined.

Further, the image quality of the IHM phase image near the peripheral edge of the culture vessel such as a dish may not be sufficient due to the influence of light reflection or the like by the wall surface of the vessel. Further, depending on the type of cells, the method of seeding, the culture conditions, etc., the cells may be biased toward the peripheral portion of the culture vessel, or conversely, biased to the central portion. In this way, depending on measurement restrictions, cell types, culture conditions, etc., it may be desirable to exclude a specific range of the culture vessel from the imaging target or analysis target. On the other hand, in the cell analysis apparatus of the present embodiment, a specific range can be excluded from photography and analysis by appropriately setting the scan condition or the analysis condition in the measurement target information display screen 100 shown in FIG. can. As a result, unnecessary shooting and analysis can be omitted, work efficiency can be improved, and accurate analysis results can be obtained according to the purpose.

In the apparatus of the present embodiment, the range excluded from the analysis target is set on the measurement target information display screen 100, but the measurement target information display screen 100 does not provide the item of "analysis condition" 107. An item that can set the range to be excluded from the analysis target may be provided in the screen for setting the analysis condition such as the classifier selection screen. In that case, the classifier and the range excluded from the analysis target may be set and specified for each well.

[Various modifications of the apparatus of the above embodiment]
In addition to the above description, the cell analysis apparatus of the above embodiment can be modified in various ways as follows.

<Modification 1>
In the cell analysis apparatus of the above embodiment, the specific region extraction unit 25 executes the process of extracting the cell region and the characteristic region only for the analysis target region, and at that time, the range included in the analysis target region in the IHM phase image. The area extraction process may be performed using only the data, but other than that, the following may be performed.

That is, as one method, mask processing is performed on a part of the IHM phase image outside the area to be analyzed, and the data extracted from the masked part is treated as invalid data or zero data. do. As a result, even if the region extraction process is executed including the region outside the analysis target region, the cell region and the characteristic region can be substantially extracted only for the analysis target region. As another method, the area extraction process is performed even outside the area of the analysis target area in the IHM phase image (for example, the area extraction process is performed for the entire imaging target area), but the extraction result is analyzed. Mask the outside of the target area or use the data of only the analysis target area. This also makes it possible to obtain extraction results of cell regions and characteristic regions substantially only for the analysis target region.

<Modification 2>
In the cell analysis apparatus of the above embodiment, a discriminant created by using deep learning, which is a method of machine learning, is used to extract a cell region or the like, but discriminant analysis by another machine learning method is used. Alternatively, regression analysis may be used. Specifically, a support vector machine, a random forest, or the like may be used. Further, instead of using the classifier created by machine learning, a method of extracting a characteristic region by using the luminance value of the IHM phase image, the texture image, or the like can also be used. In any case, any method may be used as long as it can detect and identify a characteristic region on the image from a certain image information.

<Modification 3>
Further, in the cell analysis apparatus of the above embodiment, it is possible to use a different discriminator for each well, and the region obtained by using a plurality of discriminators for one well (IHM phase image). It is also possible to obtain one area extraction result by combining the extraction results. On the other hand, for one IHM phase image, the area extraction process is executed by different types of classifiers, the results are saved, and the plurality of area extraction results are arranged and switched according to the instruction from the user. , Or it may be possible to display them in an overlapping manner. This makes it possible to easily compare the differences in the region extraction results of different types of classifiers. It is also possible to select a more appropriate classifier based on the comparison result.

<Modification example 4>
Further, in the cell analysis apparatus of the above embodiment, all the processing is performed by the control / processing unit 2, but in general, a huge amount of calculation is performed for the calculation of the phase information based on the hologram data and the image reconstruction processing. is necessary. In addition, the load of the cell state determination process is large. Therefore, a personal computer connected to the microscopic observation unit 1 is used as a terminal device, and a computer system in which this terminal device and a server, which is a high-performance computer, are connected via a communication network such as the Internet or an intranet is used. Complicated calculations and processing such as are performed by a high-performance computer, and the roles are divided so that the control of the microscopic observation unit 1 and the display processing using the processed data are executed by a relatively low-performance personal computer. May be good.

Furthermore, it is clear that the above-described embodiment and each modification are examples of the present invention, and even if further modifications, modifications, and additions are made as appropriate within the scope of the present invention, they are included in the scope of the claims of the present application. Is.

[Various aspects]
The first aspect of the present invention is a cell analysis device for analyzing cells in one or more culture vessels.
An image acquisition unit that acquires an observation image of cells over a predetermined imaging target area including one or a plurality of culture containers, and an image acquisition unit.
A specific region extraction unit that extracts a cell region in which cells are present or characteristic cells are present from the entire observation image obtained by the image acquisition unit or a predetermined analysis target region in the image, and a specific region extraction unit.
An index value calculation unit that calculates an index value relating to the size and / or shape of the cell region extracted by the specific region extraction unit for the entire observation image or the analysis target region.
A series of processes of the image acquisition unit, the specific region extraction unit, and the index value calculation unit performed on cells in the same one or a plurality of culture vessels at a plurality of different time points, at that time point . Using the index value calculated for each, a graph showing the temporal change of the index value using the index value as a plot point is created, the graph is displayed on the display unit , and the plot points are displayed on the graph. In response to the operation by the instructing user, the observation image of the cell corresponding to the instructed plot point is displayed on the display unit, and the displayed cell observation image is displayed with an image showing the extraction result of the cell region. A display processing unit that arranges and displays an operator for selecting whether or not to superimpose in the display screen of the graph, and a display processing unit.
It is equipped with.

In the cell analysis apparatus of the first aspect, after acquiring an observation image of cells over a predetermined imaging target region including one or a plurality of culture containers, the entire observation image or the entire predetermined analysis target region in the image is obtained. The cell region is extracted. That is, for the analysis target region for which the user wants to confirm the cell area, the cell region is extracted for the entire analysis target region, not for a region obtained by sampling a part of the analysis target region. Then, based on the extraction result, the area of cells (or colonies) and the like are calculated, and index values such as the total and average of the areas and the ratio of the cell area to the internal area of the culture vessel are calculated. Therefore, according to the first aspect, even if the distribution density of cells in the culture vessel is non-uniform, the information such as the cell area is calculated with high accuracy without being affected by the non-uniformity. Can be done.

Further, in the cell analysis apparatus of the first aspect of the present invention, the display processing unit uses, for example, the cell area calculated for each of the cells in one or a plurality of culture vessels at a plurality of different time points, and the cell area thereof is used. Create a graph showing changes over time and display it on the display. Therefore, according to the first aspect, the user can easily and accurately determine the accuracy of the culture conditions in the cell culture, the timing of the passage of the culture, and the like from the information displayed in this way. As a result, the cell culture work can be efficiently performed, and work mistakes related to the cell culture can be reduced. Further, according to the first aspect, the state of the cells at the culture time of interest on the graph can be immediately confirmed by the observation image.

In the second aspect of the present invention, in the cell analyzer of the first aspect,
The image acquisition unit is a holographic microscope that acquires hologram data over a region to be imaged, and either phase information, intensity information, or information including both elements by a predetermined arithmetic process using the hologram data. It may include an image reconstruction unit that creates an image showing a spatial distribution.

With a holographic microscope, focusing is not required at the time of imaging, and imaging of a certain two-dimensional range (acquisition of hologram data) can be completed in a short time. Furthermore, when creating a phase image or the like in the image reconstruction unit, it is possible to create a fine image at the in-focus position, and if necessary, create a low-resolution image with a reduced resolution. can do. Thereby, in the second aspect, the measurement can be efficiently completed in a short time even when the imaging target area is wide, for example, the entire culture plate in which a large number of wells are formed, or the entire large-capacity flask. can do. Therefore, it is possible to shorten the time required to take out the culture vessel in which the cells are cultured from the incubator and reduce the influence on the cell culture. In addition, the throughput of cell culture work can be improved.

In the third aspect of the present invention, in the cell analyzer of the first or second aspect,
In addition to the cell region, the specific region extraction unit has a substance other than cells from the entire image obtained by the image acquisition unit or a predetermined analysis target region in the image, or is characterized on the image. The feature area may be extracted, and the index value calculation unit may calculate an index value relating to the size or shape of the feature area.

According to the third aspect, in addition to information related to cells such as cell area, information related to foreign substances such as dust or information derived from the image itself such as interference fringes appearing in the image at the time of photographing is also collected. Can be done.

In the fourth aspect of the present invention, in the cell analyzer according to any one of the first to third aspects, the index value relating to the size and / or shape of the cell region is an index value related to the area of the cell region. Can be assumed to be.

In the fifth aspect of the present invention, in the cell analyzer of the fourth aspect,
The area to be photographed includes a plurality of culture containers, and the area to be photographed contains a plurality of culture containers.
The index value calculation unit shall calculate the total area of the cell region in each of the plurality of culture vessels and / or the average of the area of the cell region in each of the plurality of culture vessels as the index value. Can be done.

According to the fourth and fifth aspects, useful information can be obtained for determining the appropriateness of the culture conditions of cells (particularly colonies) and the timing of passage.

In the sixth aspect of the present invention, in the cell analyzer according to any one of the first to fifth aspects, the index value regarding the size and / or shape of the cell region is the major axis direction of each cell region. It can be at least one of length, length in the minor axis direction, roundness, or peripheral length.

Further, in the seventh aspect of the present invention, in the cell analyzer according to any one of the first to sixth aspects,
The index value calculation unit further obtains an index value for characterizing the cell region and / or the region other than the cell region by using the brightness value of each pixel on the image for the cell region and / or the region other than the cell region. It can be calculated.

According to the sixth and seventh aspects, information useful for determining the state of cells and colonies, information useful for evaluating the state of the culture medium, and the like can be obtained.

In the eighth aspect of the present invention, in the cell analyzer of the second aspect,
The container information acquisition unit that acquires information about the culture vessel used,
Based on the information about the culture vessel, the region in which the cells can be cultured is specified in the region that can be photographed by the holographic microscope, and the region to be imaged is determined so as to cover the region in which the cells can be cultured. An imaging control unit that controls the holographic microscope to perform imaging,
Can be further provided.

According to the eighth aspect, it is possible to omit the execution of imaging in the region where cells are known to be absent in the imageable region. As a result, the measurement time can be shortened, the amount of collected hologram data can be reduced, the storage capacity of the storage unit for storing the data can be saved, and the data transfer time can be shortened. ..

In the ninth aspect of the present invention, in the cell analyzer according to any one of the first to eighth aspects,
Further provided with an analysis condition setting unit for the user to specify the analysis target area in the area in the culture vessel.
The specific region extraction unit can extract the cell region only from the analysis target region designated by the analysis condition setting unit.

According to the ninth aspect, the region extraction process is performed only on the region of interest to the user, or the region excluding the region in which cells are substantially absent even in the culture vessel. Extraction processing can be carried out. As a result, the processing time of the area extraction processing can be shortened, and the amount of data of the processing result can also be reduced.

In the tenth aspect of the present invention, in the cell analyzer according to any one of the first to ninth aspects,
The specific region extraction unit can extract a cell region using a discriminator created by a supervised machine learning method.

The method of supervised machine learning is not particularly limited, and various methods such as deep learning using a multi-layer neural network and a support vector machine can be used.
According to the tenth aspect, for example, by performing learning using a large number of images appropriately labeled with and without cells as teacher data, a classifier with high discrimination accuracy is created, for example, cells. It is possible to extract the area where the label exists with high accuracy. Thereby, the accuracy of the index value such as the area of the cell region can be improved.

In the eleventh aspect of the present invention, in the cell analyzer according to any one of the first to tenth aspects,
The display processing unit may further display the graph and the observation image of the cells on the display unit as separate screens.
The display processing unit may display the observation image of the cell corresponding to the designated plot point on the display unit as a screen different from the graph.

According to the first aspect, since the observation images of the cells are displayed on a screen different from the graph, it is possible to display the observation images at a plurality of culture times and compare them.

In the first and second aspects of the present invention, in the cell analyzer according to any one of the first to eleventh aspects,
The display processing unit further displays an image in the same screen as the graph, or separately from the graph, in an aspect in which the cell region extracted by the specific region extraction unit and the other regions can be visually distinguished. The screen can be displayed on the display unit.

The visually identifiable aspect referred to here is typically a display with a different display color. As a result, the user can immediately confirm the state of the observation area at the culture time of interest on the graph.

In the thirteenth aspect of the present invention, in the cell analyzer according to any one of the first to the first and second aspects , the characteristic cell can be an undifferentiated cell or an undifferentiated deviant cell. ..
This makes it possible to grasp at a glance, for example, how many undifferentiated deviant cells to be removed are present when culturing pluripotent stem cells.

1 ... Microscopic observation unit 10 ... Light source unit 11 ... Image sensor 12 ... Culture plate 12a ... Well 13 ... Cell 14 ... Reference light 15 ... Object light 2 ... Control / processing unit 20 ... Imaging control unit 21 ... Hologram data storage unit 22 ... Phase information calculation unit 23 ... Image reconstruction unit 24 ... Reconstruction image data storage unit 25 ... Specific area extraction unit 26 ... Index value calculation unit 27 ... Calculation result storage unit 28 ... Display processing unit 29 ... Measurement / analysis condition setting unit 3 ... Input unit 4 ... Display unit 100 ... Measurement target information display screen 110 ... Discriminator selection screen 111 ... Well-compatible classifier list 120 ... Analysis result display screen 121 ... Culture container selection instruction field 122 ... Time lapse graph 123 ... Display graph selection Column 124 ... Switching button

Claims (14)

A cell analyzer for analyzing cells in one or more culture vessels.
An image acquisition unit that acquires an observation image of cells over a predetermined imaging target area including one or a plurality of culture containers, and an image acquisition unit.
A specific region extraction unit that extracts a cell region in which cells are present or characteristic cells are present from the entire observation image obtained by the image acquisition unit or a predetermined analysis target region in the image, and a specific region extraction unit.
An index value calculation unit that calculates an index value relating to the size and / or shape of the cell region extracted by the specific region extraction unit for the entire observation image or the analysis target region.
A series of processes of the image acquisition unit, the specific region extraction unit, and the index value calculation unit performed on cells in the same one or a plurality of culture vessels at a plurality of different time points, at that time point . Using the index value calculated for each, a graph showing the temporal change of the index value using the index value as a plot point is created, the graph is displayed on the display unit , and the plot points are displayed on the graph. In response to the operation by the instructing user, the observation image of the cell corresponding to the instructed plot point is displayed on the display unit, and the displayed cell observation image is displayed with an image showing the extraction result of the cell region. A display processing unit that arranges and displays an operator for selecting whether or not to superimpose in the display screen of the graph, and a display processing unit.
A cell analyzer equipped with. The image acquisition unit is a holographic microscope that acquires hologram data over a region to be imaged, and either phase information, intensity information, or information including both elements by a predetermined arithmetic process using the hologram data. The cell analysis apparatus according to claim 1, further comprising an image reconstruction unit that creates an image showing a spatial distribution. In addition to the cell region, the specific region extraction unit has a substance other than cells from the entire image obtained by the image acquisition unit or a predetermined analysis target region in the image, or is characterized on the image. The cell analysis apparatus according to claim 1, wherein a characteristic region is extracted, and the index value calculation unit calculates an index value relating to the size or shape of the characteristic region. The cell analysis apparatus according to claim 1, wherein the index value relating to the size and / or shape of the cell region is an index value related to the area of the cell region. The area to be photographed includes a plurality of culture containers, and the area to be photographed contains a plurality of culture containers.
4. The index value calculation unit calculates the total area of cell regions in each of the plurality of culture vessels and / or the average area of cell regions in each of the plurality of culture vessels as the index value. The cell analyzer according to. The index value relating to the size and / or shape of the cell region is at least one of the length in the major axis direction, the length in the minor axis direction, the roundness ratio, or the peripheral length of each cell region. The cell analysis apparatus according to 1. The index value calculation unit further obtains an index value for characterizing the cell region and / or the region other than the cell region by using the brightness value of each pixel on the image for the cell region and / or the region other than the cell region. The cell analysis apparatus according to claim 1, which is calculated. The container information acquisition unit that acquires information about the culture vessel used,
Based on the information about the culture vessel, the region in which the cells can be cultured is specified in the region that can be photographed by the holographic microscope, and the region to be imaged is determined so as to cover the region in which the cells can be cultured. An imaging control unit that controls the holographic microscope to perform imaging,
The cell analysis apparatus according to claim 2, further comprising. Further provided with an analysis condition setting unit for the user to specify the analysis target area in the area in the culture vessel.
The cell analysis apparatus according to claim 1, wherein the specific region extraction unit extracts a cell region only from an analysis target region designated by the analysis condition setting unit. The cell analysis apparatus according to claim 1, wherein the specific region extraction unit extracts a cell region using a discriminator created by a supervised machine learning method. The cell analysis device according to claim 1, wherein the display processing unit displays an observation image of cells corresponding to the designated plot points on the display unit as a screen different from the graph. The display processing unit further displays an image in the same screen as the graph, or separately from the graph, in an embodiment in which the cell region extracted by the specific region extraction unit and the other regions can be visually distinguished. The cell analysis apparatus according to claim 1, wherein the screen is displayed on the display unit. The cell analysis apparatus according to claim 1, wherein the characteristic cell is an undifferentiated cell or an undifferentiated deviant cell. The index value calculation unit calculates the area of the cell region in the whole of one or a plurality of culture vessels as the index value, and the display processing unit displays a graph showing the temporal change of the area of the cell region. The cell analysis apparatus according to claim 4.

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