JP2009229276A - Method for analyzing image for cell observation, image processing program and image processor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means capable of quantitatively grasping the motion state of a cell. <P>SOLUTION: An image processing program of a cell observation image includes: steps (S40 and S45) for acquiring a first image, which is photographed by an imaging device and contains a plurality of cells in an observation region, and the second image of the observation region photographed by the imaging device after a predetermined time is elapsed from the photographing time of the first image; the step (S30) for selecting one cell as a cell of interest from a plurality of the cells contained in the first image; the step (S35) for indicating the cell positioned in the periphery of the cell of interest as a peripheral cell; the step (S50) for calculating the speed statistical values of the peripheral cell to the cell of interest on the basis of the values of the relative movements of the cell of interest and the peripheral cells in the first and second images; and the step (S55) for outputting the calculated statistical value of the speed of the peripheral cell to the outside. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an image processing means for cell observation that analyzes a movement state of a cell from an image obtained by performing cell observation every predetermined time.

  Cell division is basic information on cell movement. In general, cell division and the formation of a scaffold are considered to be related to each other, and a clustered cell population can maintain a normal growth rate rather than a single existing cell. On the other hand, some cells secrete growth factors such as PDGF (platelet-derived growth factor) to control proliferation of other populations (see, for example, Non-Patent Document 1). Thus, cell proliferation is controlled by social interaction.

Conventionally, conditions such as cell proliferation and suppression as described above have been determined by performing microscopic observations at regular time intervals, photographing microscopic observation images, and observing a number of microscopic observation images. For example, when observing cells, the situation where cells adhere and colonize is observed as a binary change from single to colony due to cell behavior.
Bruce Albert et al., Translated by Keiko Nakamura and Kenichi Matsubara, “Molecular biology of the Cell (USA)”, 2nd edition, Tokyo Newton Press, November 1993, p. 748-749, p. 1188-1189

  As described above, the behavior of cells grasped by conventional observation methods is only a binary change, when time series changes when cells gather and colonize, and when cell growth is suppressed and degenerates The analysis method when “quantification of change” is necessary for the time series change of the time series and the time series change when the cells become discrete has not been solved.

  Further, in the determination by microscopic observation at regular time intervals, it is necessary to make a determination based on a large number of microscopic observation images taken. For this reason, in a technical field such as drug discovery in which many parameters are changed little by little and the behavior of cells is observed for each combination, it takes a lot of time to analyze the behavior. But it was pointed out as an issue. In addition, the action of drugs includes those that cause cell death of certain cells and those that inhibit proliferation. So far, drugs that inhibit cell proliferation have been generally evaluated using cell division assay reagents, and only values that include the division of other types of cells can be obtained only in an environment where single species are cultured. It was. However, there are actually some cells that are controlled by social action with other types of cells, and the evaluation of these cells has been an issue in terms of accuracy.

  This invention is made | formed in view of the said subject, and it aims at providing the means to grasp | ascertain the movement state of a cell quantitatively.

  According to the first aspect exemplifying the present invention, the first image including a plurality of cells captured in the observation area and the observation area captured by the imaging apparatus a predetermined time before the first image is captured. The second image is acquired, one cell is selected from a plurality of cells included in the first image as a target cell, and a cell located around the target cell is used as a peripheral cell, and attention is paid in the first image and the second image. Based on the relative movement amount of each peripheral cell with respect to the cell, the statistic of the velocity of the peripheral cell with respect to the cell of interest (for example, the attractive repulsion rate and the attractive repulsion amount in the embodiment) is calculated, and the interaction of each peripheral cell with respect to the target cell An image analysis method for cell observation is provided, which is configured to be able to determine the state of the cell.

  According to the second aspect illustrating the present invention, the first image captured by the imaging device and including a plurality of cells in the observation region, and the observation region captured by the imaging device a predetermined time before the first image. Obtaining a second image; selecting a cell as a target cell from a plurality of cells included in the first image; designating a cell located around the target cell as a peripheral cell; Calculating a statistic of the velocity of the peripheral cell with respect to the target cell based on the relative movement amount of the target cell and the peripheral cell in the image and the second image (for example, attractive repulsion speed, attractive repulsion amount in the embodiment); And outputting the statistics of the velocity of each peripheral cell to the outside, and the cell observation characterized in that the state of interaction of each peripheral cell with respect to the cell of interest can be determined The image processing program is provided.

  According to the third aspect exemplifying the present invention, an imaging device that images a cell, a first image that is captured by the imaging device, and a second image that is captured by the imaging device a predetermined time before the first image are obtained. An image storage unit for storing, an image analysis unit for analyzing the state of interaction between a plurality of cells located in the observation region based on the first image and the second image, and an output for outputting analysis data by the image analysis unit A first image and a second image of a target cell selected from a plurality of cells included in the observation field of the first image and a peripheral cell located around the target cell in the image analysis unit. Based on the relative amount of movement of the attention cell and the surrounding cells in the image, the statistic of the speed of the surrounding cells with respect to the attention cell (for example, the attractive repulsion speed, the attractive repulsion amount in the embodiment) is calculated, and the speed calculated in the image analysis unit The statistics, the image processing apparatus of cell observation, characterized by being configured so as to output from the output section is provided.

  According to the cell observation image analysis method, the image processing program, and the image processing apparatus as described above, it is possible to provide means for quantitatively grasping the movement state of the cells.

  Prior to specific description of the embodiment of the present invention, the basic principle constituting this embodiment will be described below. The present embodiment presents a new feature amount for estimating a cell state by quantifying the attractive force / repulsive force between cells, which is one of social interactions.

2, and one cell (target cell) C _i, is a conceptual diagram of a motion analysis model illustrating a modeled other cells (peripheral cells) C _j of its periphery. With respect to the time-series images of cells, the areas and positions of the cell of interest C _i and the surrounding cells C _{j in} the image at a certain time t (the center of gravity with respect to the cell region and the position of the cell nucleus), and the previous time t−1 The area and position of the same cell in the image of (2) is detected by image measurement. For the cell of interest C _i , the distance d _j between the cells at time t−1, the angle θ _j when the moving direction is viewed from the cell of interest with reference to the line segment L connecting the cells, and time t The amount of movement of the surrounding cells at time t from −1 (the amount of relative movement of the surrounding cells C _j with respect to the cell of interest C _i ) v _j is calculated. At this time, the attractive repulsion velocity V _ij to the peripheral cell C _{j of the} cell of interest C _i is _calculated using the movement amount v _j ,
It becomes. However, when the distance d _j is far away, considering weighting in consideration of reliability, if the weight function is W (d _j ), the attractive repulsion speed considering the distance weight function is
It becomes. If the surrounding cells is a M-number of attraction repulsion rate sum (hereinafter referred to as "attraction repulsion amount") V _i is
It is expressed. A statistic obtained by summing up the attractive repulsive force V _i for all cells in the image as i = 1, ..., N in the cell of interest C _i (the total velocity statistic in the claims, hereinafter referred to as “total attractive force”). Called repulsive force)
And calculate.

The sign of the attractive force repulsion speed changes depending on the angle θ _j , and the sign is reversed between the attractive force and the repulsive force. Accordingly, when the attractive force is generated between the cells, the total attractive force repulsion amount V _all is a positive large value, and when the repulsive force is large, the total attractive repulsion amount V _all is a negative large value. . Therefore, by analyzing the images at time t-1 and time t in the cell observation process and calculating the total attractive force repulsion amount of the cells in the observation region, it is possible to quantitatively grasp the movement state of the cells. Become. For the weighting W (d _j ) corresponding to the distance d _j , various weights such as secondary weighting or Gaussian can be considered in addition to weighting such as 1 / d _j . These are preferably determined after observing the behavior between cells.

In the above, we have seen the attraction and repulsion of cells throughout the image.
Also, the maximum value, minimum value, and histogram are also easy-to-understand indicators that quantitatively represent the movement state of cells. Therefore, by analyzing a time-series image based on such a basic principle, it becomes possible to observe a time-series change that an attractive force or a repulsive force is acting between cells in the cell observation process.

  Next, modes for carrying out the present invention will be described with reference to the drawings. As an example of a system to which the image processing apparatus for cell observation of the present embodiment is applied, a schematic configuration diagram and a block diagram of a culture observation system are shown in FIGS. 3 and 4, respectively.

  This culture observation system BS is broadly divided into a culture chamber 2 provided at the top of the housing 1, a shelf-like stocker 3 that accommodates and holds a plurality of culture containers 10, and a sample in the culture container 10. From an observation unit 5 for observation, a transport unit 4 for transporting the culture vessel 10 between the stocker 3 and the observation unit 5, a control unit 6 for controlling the operation of the system, an operation panel 7 equipped with an image display device, etc. Composed.

The culture room 2 is a room for forming and maintaining a culture environment according to the type and purpose of the cells to be cultured, and is kept sealed after the sample is charged in order to prevent environmental changes and contamination. Along with the culture chamber 2, a temperature adjustment device 21 for raising and lowering the temperature in the culture chamber, a humidifier 22 for adjusting the humidity, a gas supply device 23 for supplying a gas such as CO ₂ gas and N ₂ gas, and the culture A circulation fan 24 for making the environment of the entire chamber 2 uniform, an environmental sensor 25 for detecting the temperature, humidity and the like of the culture chamber 2 are provided. The operation of each device is controlled by the control unit 6, and the culture environment defined by the temperature, humidity, carbon dioxide concentration, etc. of the culture chamber 2 is maintained in a state that matches the culture conditions set on the operation panel 7.

  The stocker 3 is formed in a shelf shape that is partitioned into a plurality of parts in the front-rear direction and the vertical direction in FIG. A unique address is set for each shelf. For example, when the front-rear direction is A to C rows and the vertical direction is 1 to 7 rows, the A-row 5 rows shelf is set as A-5. The

  The culture vessel 10 has a type such as a flask, a dish, and a well plate, a form such as a round shape and a square shape, and a size, and an appropriate one can be selected and used according to the type and purpose of the cell to be cultured. . In the present embodiment, a configuration using a dish is illustrated. Samples such as cells are injected into the culture vessel 10 together with a liquid medium containing a pH indicator such as phenol red. The culture container 10 is assigned a code number and is stored in association with the designated address of the stocker 3. The culture container 10 is housed and held on each shelf in a state where a container holder for transportation formed according to the type and form of the container is mounted.

  The transfer unit 4 is provided inside the culture chamber 2 so as to be movable in the vertical direction and is moved up and down by the Z-axis drive mechanism. The transfer unit 4 is attached to the Z stage 41 so as to be movable in the front-rear direction and is moved by the Y-axis drive mechanism. The Y stage 42 that is moved back and forth, the X stage 43 that is attached to the Y stage 42 so as to be movable in the left-right direction and is moved left and right by the X-axis drive mechanism, etc. A support arm 45 for lifting and supporting the culture vessel 10 is provided on the distal end side. The transport unit 4 has a moving range in which the support arm 45 can move between the entire shelf of the stocker 3 and the sample table 15 of the observation unit 5. The X-axis drive mechanism, the Y-axis drive mechanism, and the Z-axis drive mechanism are configured by, for example, a servo motor with a ball screw and an encoder, and the operation thereof is controlled by the control unit 6.

  The observation unit 5 includes a first illumination unit 51, a second illumination unit 52, and a third illumination unit 53, a macro observation system 54 that performs macro observation of the sample, a micro observation system 55 that performs micro observation of the sample, and an image processing apparatus. 100 or the like. The sample stage 15 is made of a material having translucency, and a transparent window portion 16 is provided in the observation region of the microscopic observation system 55.

  The first illumination unit 51 is a surface-emitting light source provided on the lower frame 1 b side, and backlight-illuminates the entire culture vessel 10 from the lower side of the sample stage 15. The second illumination unit 52 includes a light source such as an LED and an illumination optical system including a phase ring, a condenser lens, and the like. The second illumination unit 52 is provided in the culture chamber 2 and receives light from the microscope observation system 5 from above the sample stage 15. Illuminate the sample in the culture vessel along the axis. The third illumination unit 53 includes a plurality of light sources such as LEDs and mercury lamps that emit light having a wavelength suitable for epi-illumination observation and fluorescence observation, and the light emitted from each light source as an optical axis of the microscopic observation system 55. And an illumination optical system composed of a beam splitter, a fluorescent filter, and the like to be superposed, disposed in the lower frame 1b located on the lower side of the culture chamber 2, and from the lower side of the sample stage 15 to the microscopic observation system 5. Illuminate the sample in the culture vessel along the optical axis.

  The macro observation system 54 includes an observation optical system 54a and an imaging device 54c such as a CCD camera that takes an image of the sample imaged by the observation optical system. The macro observation system 54 is located above the first illumination unit 51 and is a culture chamber. 2 is provided. The macro observation system 54 captures a whole observation image (macro image) from above the culture vessel 10 that is backlit by the first illumination unit 51.

  The microscopic observation system 55 includes an observation optical system 55a composed of an objective lens, an intermediate zoom lens, a fluorescent filter, and the like, and an imaging device 55c such as a cooled CCD camera that takes an image of a sample imaged by the observation optical system 55a. And disposed inside the lower frame 1b. A plurality of objective lenses and intermediate zoom lenses are provided, and are configured to be set to a plurality of magnifications using a displacement mechanism such as a revolver or a slider (not shown in detail). For example, zooming is possible in the range of 2 to 80 times. The microscopic observation system 55 is transmitted light that has been illuminated by the second illumination unit 52 and transmitted through the cell, reflected light that has been illuminated by the third illumination unit 53 and reflected by the cell, or illuminated by the third illumination unit 53. A microscopic image (micro image) obtained by microscopic observation of fluorescence emitted by the cells is taken.

  The image processing apparatus 100 performs A / D conversion on signals input from the macro observation system imaging device 54c and the micro observation system imaging device 55c, and performs various image processing to generate an image of the entire observation image or the micro observation image. Generate data. Further, the image processing apparatus 100 performs image analysis on the image data of these observation images, and performs generation of a time-lapse image, calculation of the amount of cell movement, analysis of the movement state of the cell, and the like. Specifically, the image processing device 100 is constructed by executing an image processing program stored in the ROM of the control device 6 described below. The image processing apparatus 100 will be described in detail later.

  The control unit 6 includes a CPU 61, a ROM 62 in which data for controlling the operation of the culture observation system BS and data for controlling each unit are set and stored, a RAM 63 in which image data and the like are temporarily stored, and the like. They are connected by a data bus. The input / output port of the control unit 6 includes a temperature adjustment device 21 in the culture chamber 2, a humidifier 22, a gas supply device 23, a circulation fan 24 and an environmental sensor 25, and X, Y, Z stages 43, 42 in the transfer device 4. 41, the first, second, and third illumination units 51, 52, and 53 in the observation unit 5, the macro observation system 54 and the microscopic observation system 55, the operation panel 71 and the display panel 72 in the operation panel 7, and the like. Is connected. Detection signals are input to the CPU 61 from the above-described units, and the above-described units are controlled in accordance with a control program preset in the ROM 62.

  The operation panel 7 includes an operation panel 71 provided with input / output devices such as a read / write device for reading and writing information from a keyboard, a sheet switch, a magnetic recording medium, an optical disk, and the like, various operation screens, image data, and the like. And a display panel 72 for displaying the information, and setting the observation program (operating conditions), selecting conditions, operating commands, and the like on the operation panel 71 while referring to the display panel 72, thereby culturing through the CPU 61. Each part of the observation system BS is operated. That is, the CPU 61 adjusts the environment of the culture chamber 2 according to the input from the operation panel 71, transports the culture vessel 10 in the culture chamber 2, observes the sample by the observation unit 5, analyzes the acquired image data, and displays the display panel. Display to 72 is executed. On the display panel 72, in addition to input screens for operation commands, condition selection, and the like, numerical values of environmental conditions of the culture chamber 2, analyzed image data, a warning when an abnormality occurs, and the like are displayed. The CPU 61 can transmit and receive data to and from an externally connected computer or the like via a communication unit 65 configured in accordance with a wired or wireless communication standard.

  In the RAM 63, operating conditions of the observation program set on the operation panel 71, for example, environmental conditions such as temperature and humidity of the culture chamber 2, observation schedule for each culture vessel 10, observation type and observation position in the observation unit 5, observation Observation conditions such as magnification are recorded. In addition, the management data of the culture container 10 such as the code number of each culture container 10 accommodated in the culture chamber 2, the storage address of the stocker 3 in which the culture container 10 of each code number is accommodated, and various data used for image analysis are stored. To be recorded. The RAM 63 is provided with an image data storage area (image storage unit 110 to be described later) for recording image data photographed by the observation unit 5, and each image data includes an index including a code number of the culture vessel 10 and a photographing date and time. -Data is recorded in association with each other.

  In the culture observation system BS schematically configured as described above, the CPU 61 controls the operation of each part based on the control program stored in the ROM 62 according to the setting conditions of the observation program set on the operation panel 7, and the culture vessel 10 The sample inside is automatically captured. That is, when the observation program is started by a panel operation on the operation panel 71 (or a remote operation via the communication unit 65), the CPU 61 reads each condition value of the environmental conditions stored in the RAM 63, and from the environment sensor 25. The environmental state of the culture chamber 2 to be input is detected, and the temperature adjustment device 21, the humidifier 22, the gas supply device 23, the circulation fan 24, etc. are operated according to the difference between the condition value and the actual measurement value. Feedback control is performed on the culture environment such as temperature, humidity, and carbon dioxide concentration.

  Further, the CPU 61 reads the observation conditions stored in the RAM 63, operates the driving mechanism of each axis of the X, Y, and Z stages 43, 42, and 41 of the transport unit 4 based on the observation schedule, and the observation target from the stocker 3. The culture container 10 is transported to the sample stage 15 of the observation unit 5 and observation by the observation unit 5 is started. For example, when the observation set in the observation program is macro observation, the culture vessel 10 transported from the stocker 3 by the transport unit 4 is positioned on the optical axis of the macro observation system 54 and placed on the sample stage 15. Then, the light source of the first illumination unit 51 is turned on, and the entire observation image is taken by the imaging device 54c from above the culture vessel 10 that is backlit. The signal input from the imaging device 54c to the control device 6 is processed by the image processing device 100 to generate a whole observation image, and the image data is recorded in the RAM 63 together with index data such as the shooting date and time.

  When the observation set in the observation program is micro observation of the sample at a specific position in the culture container 10, the specific position of the culture container 10 that has been transported by the transport unit 4 is set to the optical axis of the microscopic observation system 55. Positioned above and placed on the sample stage 15, the light source of the second illumination unit 52 or the third illumination unit 53 is turned on, and the microscopic observation image by transmitted illumination, epi-illumination, and fluorescence is photographed by the imaging device 55c. A signal photographed by the imaging device 55c and inputted to the control device 6 is processed by the image processing device 100 to generate a microscopic observation image, and the image data is recorded in the RAM 63 together with index data such as photographing date and time. .

  The CPU 61 performs the observation as described above for the samples of the plurality of culture containers accommodated in the stocker 3 according to the observation schedule with a time interval of about 30 minutes to 2 hours based on the observation program. Shoot sequentially. In this embodiment, the photographing time interval may be constant or different. The image data of the photographed whole observation image and microscopic observation image is recorded in the image data storage area (image storage unit 110) of the RAM 63 together with the code number of the culture vessel 10. The image data recorded in the RAM 63 is read from the RAM 63 in response to an image display command input from the operation panel 71, and an entire observation image or a microscopic observation image (single image) at a specified time or an entire observation in a specified time region. An image or a time-lapse image of a microscopic observation image is displayed on the display panel 72 of the operation panel 7.

  In the culture observation system BS configured as described above, the image processing apparatus 100 has an image analysis function for analyzing the movement state of the cells in the observation region in addition to the basic functions such as the generation of the time-lapse image described above. I have. Hereinafter, in describing the specific contents of the image analysis performed by the image processing apparatus 100, first, a method for quantitatively calculating the movement state of a cell from two time-series images will be described.

(Preprocessing)
FIG. 5 is a diagram schematically showing a microscopic observation image of a cell photographed at a certain time t. First, for this image, individual cell regions are extracted and segmented. For example, there are methods such as binarization using luminance values, binarization using variance values, and a dynamic contour extraction method such as Snakes or Level Set method. As a result, as shown in FIG. 6, the segmented cell region is labeled, and a technique for measuring the label of surrounding cells for each cell label is taken.

  Next, as shown in FIG. 7, the correspondence between the cells of the image taken at time t (cells indicated by solid lines) and the cells at the time t−1 taken before a predetermined time (cells indicated by dotted lines). In other words, cell tracking is performed. Here, the cell region labels at time t and time t−1 that are closest to each other and similar in shape are selected. Thereby, the movement vector from time t-1 to t of each cell is calculated.

(Calculation of attractive force and repulsive force)
Respect to the moving vector of each cell obtained in the previous processing, to calculate a statistical amount of speed by attraction or repulsion with respect to the target cells C _i and one C _j of surrounding cells. Typical examples of the statistic of velocity include the attractive repulsion velocity and the attractive repulsion amount of the surrounding cell C _j with respect to the cell of interest C _i . As shown in FIG. 2, the contour inner area of the cell of interest C _i, wherein the cell area at t from time t-1 of one C _j of surrounding cells to s _j · · · peripheral cells C _j movement line connecting the quantity v _j · · · · cell of interest relative movement distance of surrounding cells C _j for C _i d _j · · · · · noted cell C _i and the distance angle theta _j · · · · cells near the cell C _j It is assumed that the angle is when the moving direction is viewed with the minute L as a reference. First, if the cells share some kind of ionic substance and the attractive force or repulsive force works depending on the concentration, energy that is inversely proportional to the distance d _j between the cells can be considered, and in this case the weight function W (d _j ) is ,
If it is related to the area,
It becomes. Or if you can determine the weighting range within a certain range,
Here, D may be a range of cell interaction (interaction): specified by the user.
If we think more probabilistically, the normal distribution as a probability density function
σ may be defined as a standard deviation.
At this time, the attractive repulsion velocity V _ij exerted by the cell of interest C _i on one peripheral cell C _j is
It becomes.
When you apply this to all of the surrounding cells (M number), attraction repulsion amount V _i possessed by the cell of interest C _i is,
It becomes. This for all cells in the image, the total attraction repulsion amount summed by replacing cells of interest as i = 1 to N in the target cell C _i
Is calculated. As described above, when the attractive force between the cells is large, the total attractive force repulsive amount V _all is a positive large value. When the repulsive force is large, the total attractive repulsive force amount V _all is a large negative value. It becomes. Therefore, in the cell observation process, by analyzing the two images at time t-1 and time t and calculating the total attractive force repulsion amount V _all of the cells in the observation region, whether the movement state of the cells is attractive or not. It is possible to quantitatively grasp whether it is repulsive or not.

Further, with respect to time-series data that causes state changes as shown in FIGS. 8 (1) → (2), the values of the total attractive force values calculated every predetermined time are displayed side by side in time series, for example, as shown in FIG. Thus, by plotting on the graph with the horizontal axis as the time axis, a change graph in which the change of the attractive force is visualized can be obtained. This represents a quantitative property in which cells that were initially separated from each other clump together and transformed into a colony, and the change state of the cell group can be grasped quantitatively and easily.
In addition, the average value of each cell, maximum value, minimum value,
Furthermore, time-series changes in statistics such as histograms are also interesting as an index representing the movement state of cells. It is also conceivable that only a positive value (attraction) or only a negative value (repulsion) is handled and observed separately from attraction and repulsion.

  The following example is given as an observation application for obtaining such speed statistics by image analysis and observing interaction between cells.

(A: Measurement cell designation)
The observer designates one or a plurality of cells to be measured, and calculates the sum of the attraction and repulsion speeds for the designated measurement cells. For example, as illustrated in FIGS. 10 (1) and 10 (2), a specific cell is designated as a measurement cell C _s from a large number of cells included in the image, and an effective distance (range) of attractive force / repulsive force is set. Then, the total velocity of the attractive force / repulsive force for the designated measurement cell C _s is calculated. In addition, the attractive repulsion speed of the surrounding cells to one cell C _s to be measured is displayed as shown in FIG. As a result, it is possible to know the individual movement state of the measurement target cell C _s , the cell in which the most attractive force or repulsive force is working, and the like.

(B: Speed statistics calculation and time series change)
As shown in FIG. 12, the measurement cell C _s designated by A is sequentially replaced with another cell to obtain the attractive force repulsion speed of each cell, and the sum of the obtained attractive force repulsion speeds is calculated for each cell. When the amount of attractive repulsive force is displayed for a cell, it is possible to know the most influential cell in the field of view from the maximum value. In addition, as a statistic of the total velocity of each cell, the average value, maximum value, minimum value, total attractive force repulsion amount, and dispersion can be displayed to know the overall situation. Furthermore, by displaying these time-series data in a graph along the time axis, changes in the time domain can be easily grasped. The measurement cell C _s own speed specified by A, the size (area), shape can be displayed for the time-series variation of such (including circularity and complexity), thereby the cell state of motion Can be grasped in detail.

  Next, a specific application of image analysis executed in the image processing apparatus 100 will be described by taking as an example the case of analyzing a microscopic observation image (phase difference image) of a living cell. FIG. 13 is a block diagram illustrating a schematic configuration of an image analysis portion that performs image analysis of a cell movement state in the image processing apparatus 100.

  The image processing apparatus 100 includes an image storage unit 110 that stores a first image photographed by the imaging devices (54c, 55c) and a second image photographed at a time t-1 that is a predetermined time before the first image. An image analysis unit 120 that analyzes an interaction state (attraction state of attractive force or repulsive force) acting between a plurality of cells located in the observation region based on the first image and the second image, and an image analysis unit 120 An output unit 130 that outputs analysis data, and the image analysis unit 120 calculates the attractive repulsion rate of each cell based on the relative position and relative movement amount of the cell of interest and the surrounding cells in the first image and the second image. These are summed to calculate the attractive force of the target cell, and then the total attractive force of the entire cell is calculated by summing up the attractive force obtained by sequentially replacing the target cell. To, configured so as to display example on the display panel 72.

  The image processing apparatus 100 is configured by an image processing program set and stored in advance in the ROM 62 being read by the CPU 61 and processing based on the image processing program being executed by the CPU 61.

  The image analysis processing of the present embodiment can be performed not only on a time-series image that has already been taken at predetermined time intervals based on an observation program and stored in the image storage unit 110, but also the movement of cells at the time of observation. It is possible to analyze the state and monitor in real time. Therefore, in the present embodiment, for this real-time image analysis processing, referring to the flowchart of the image processing program GP shown in FIG. 1 and the display image of the cultured cell motion analysis interface displayed on the display panel 72 shown in FIG. explain.

  In this interface, a “dish selection” frame 721 is displayed on the display panel 72 and the code number of the culture vessel stored in the stocker 3 is displayed. In step S10, the culture vessel 10 to be observed is selected. FIG. 14 shows a state in which the cultured cell dish (culture vessel) having the code number Cell-0002 is selected by the cursor provided on the operation panel 71.

  When the observation target is selected in step S <b> 10, the CPU 61 operates the driving mechanism of each axis of the transport unit 4 to transport the observation target culture container 10 from the stocker 3 to the observation unit 5. Then, the whole observation image by the macro observation system 54 or the micro observation image by the micro observation system 55 is photographed by the imaging device (54c, 55c), and the image is displayed in the “observation position” frame 722.

  In step S20, an observation position is set as to which region is the observation region. FIG. 14 shows a state in which the observer designates a shaded area from the center right using a mouse or the like provided on the operation panel 71. At this time, the image of the set observation region is immediately subjected to segmentation processing in the image analysis unit 120, and an image obtained by superimposing the cell contour on the phase difference image or a schematic diagram (segmentation image) based on the cell contour is “observation image”. Is displayed in a frame 723. At this time, a “motion analysis option” frame 724 including “designation cell designation” 724a and “designation of surrounding cells” 724b is displayed on the display panel 72.

In step S30, the observer selects a cell to be measured on the schematic diagram displayed in the “observation image” frame 723 by using the mouse, and presses the “Specify measurement cell” 724a determination button to perform measurement. to determine the cell C _s. Next, in step S35, whether to designate the peripheral cell C _j manually or by specifying the distance (radius) from the measurement cell C _{s is} selected by a selection button provided in the “designate peripheral cell” 724b. When manual is selected, the peripheral cells _Cj are individually selected with the mouse and confirmed by pressing the enter button. When the radius specification is selected, the radius value is a numerical value (unit is pixel, μm, etc.) Or input with the mouse.

With this as an initial setting, the observation image is captured (step S40) and the observation image is stored (step S45) according to the observation schedule for each predetermined time set in the observation program, and time-lapse observation is performed. The measurement cells C _s and the surrounding cells C _j in the observation region designated in Step S30 and Step S35 are subjected to cell image segmentation and tracking in the image analysis unit 120 at predetermined time intervals set in the observation schedule. In S50, the attractive repulsion speed of each cell is calculated.

As for the contents of the attractive repulsion rate calculation (step S50) of each cell, as already described in detail, the flow in the right column in FIG. 1 (steps S51 to S55) summarizes this briefly in the flowchart. That is, the attractive force repulsion speed calculation of each cell in the image analysis unit 120 is shown in FIG. 6 for the observation image (first image) at time t and the observation image (second image) at time t-1 a predetermined time before. Cell outermost shell contour extraction processing (segmentation processing: step S51), the measurement cell C _s and the peripheral cell C _j shown in FIG. 7 between these two images (step S52), and obtained by this association Calculation of the movement vector of the peripheral cell C _j with respect to the measured cell C _s obtained (step S53), and the attractive force of each peripheral cell C _{j with} respect to the measurement cell C _s obtained based on the movement vector by the method described in the movement model of FIG. It performed the calculation of repulsion rate (step S54), the calculation of the attraction repulsion velocity of each cell, i.e., the attraction repulsion amount V _i related cell of interest C _i for the M surrounding cells is calculated At the same time, the attractive repulsion amount of each cell when the target cell is replaced is calculated, and these calculation results are output from the output unit 130 (step S55).

In step S60, as shown in FIG. 11, the attractive repulsion speed of each peripheral cell C _{j and} the attractive repulsive force amount of each cell as shown in FIG. 12 are displayed in the “observation image” frame 723 for each observation. The value of the attractive force repulsion speed of each cell that is displayed in real time on the screen is accumulated in the RAM 63. Further, in step S70, the measurement cell C _s as the target cell C _i, the sum of the attractive force repulsion of whole cells by replacing a cell of interest as i = 1 to N in C _i included in the observation area (total attraction repulsion amount) The calculation of V _all is executed, and these calculation results are stored in the RAM 63. In the interface, the display of the attractive repulsion speed of each peripheral cell shown in FIG. 11 or the display of the attractive repulsive force amount shown in FIG. 12 can be selected and displayed by the selection button provided in the “observation image” frame 723. It has become.

During execution of the time lapse observation, this is displayed in real time as a graph in a “time series change graph” frame 725. In this graph, as described above with reference to FIG. 12, changes in the time axis such as the maximum and minimum values of the attractive force of each cell, the total attractive force, the average, and the variance are displayed. In addition, it is possible to display the change over time of the area and shape change (circularity, complexity, etc.) of the measurement cell C _s , and the display can be switched by selecting the display item of the time series change graph. Further, the output unit 130 is provided with an output terminal and is connected to the communication unit 65, and the calculation result can be exported to a computer or the like externally connected via the communication unit 65.

  Therefore, according to such an image analysis means (an image analysis method, an image processing program, an image processing apparatus), during the execution of the time lapse observation, the momentary movement state of the cell to be observed is expressed by a specific numerical value or graph. Displayed in real time, the movement state of the cell can be grasped quickly and quantitatively.

In the above embodiment, the measurement cell C _s and the surrounding cell C _j are designated when starting the time lapse observation, and the image analysis is performed in parallel during the time lapse observation, and the analysis result is displayed in real time. However, the image analysis of the present invention can be performed on a time-series image stored in the image storage unit 110 after execution of time-lapse observation for a certain time (during execution) or after the time-lapse observation is completed. is there. Therefore, hereinafter, the flow of image analysis in such a case will be briefly described with reference to FIGS. 14 and 1 together.

  First, in step S10, a culture container having a code number to be observed (for example, a cultured cell dish having the code number Cell-0002 described above) is selected from the list of culture containers displayed in the “dish selection” frame 721. The time-series image to be observed selected here is already stored in the image storage unit 110 (that is, steps S40 and S45 in the image processing program shown in FIG. 1 are executed before step S10). . Therefore, when an observation target is selected in step S10, an observation image (entire observation image or microscopic observation image) of the selected culture container at time t is read from the image storage unit 110 and displayed in the “observation position” frame 722. Is displayed. Note that it is possible to select an image to be read at which time during the observation period. For example, it is possible to select an observation start time (second data), or an intermediate time or an end time of the observation period. .

  In step S20, an observation area is set as to which area to observe from the image displayed in the “observation position” frame. The image of the set observation region is immediately subjected to segmentation processing in the image analysis unit 120, and an image obtained by superimposing the cell outline on the phase difference image or a schematic diagram based on the cell outline is displayed in the “observation image” frame. The segmentation process is performed for two sheets of time t and time t-1 that is a predetermined time before, and the schematic diagram is displayed only at time t or both can be displayed in time series.

Next, in step S30, the measurement target cell is selected on the schematic diagram displayed in the “observation image” frame 723, and the measurement cell C _s is determined by pressing the “measurement cell designation” 724a determination button. In step S35, the peripheral cell C _j is designated manually or by a distance (radius) from the measurement cell C _s and is confirmed by pressing the enter button. These selection settings are the same as described above.

When the setting of the measurement cell C _s and the peripheral cell C _j is confirmed in step S30 and step S35, the image analysis unit 120 first performs step S50 on the images at time t and time t−1 that are first read from the image storage unit 110. The calculation of the repulsive force velocity and the repulsive force amount of each cell and the total attractive force amount of all the cells in step S70 is executed and recorded in the RAM 63. Further, the observation images (step S40, step S45) that have already been taken with a predetermined observation schedule and stored in the image storage unit 110 are read out by sequentially moving the time t and set in step S20 from the read images. The image of the observation area is cut out, segmented and tracked, and the calculations in steps S50 and S70 are sequentially executed and recorded in the RAM 63.

  Then, according to the selection of the selection button provided in the “observation image” frame 723, the attractive repulsion speed of the surrounding cells shown in FIG. 11, or the attractive repulsive force amount and the total attractive repulsive force amount of all the cells shown in FIG. Is displayed. In addition, these displays can display the calculation result of the specific time arbitrarily selected. In addition, when time-lapse observation is being performed, a change in the attractive force amount (maximum value, minimum value, average value, etc.) up to the present time is displayed as a graph in the “time series change graph” frame 725, and after the observation is completed. Displays the change in the amount of attractive force from the start to the end of the observation.

  As described above, according to the image processing program GP of the present invention, the image analysis method configured by executing the image processing program, and the image processing apparatus 100, the cell observation image photographed at a predetermined time. From this, the total attractive force of the whole cell at the time of photographing is calculated. For this reason, a means for quickly and quantitatively grasping the movement state of the whole cell can be provided.

  As mentioned above, social interaction is important for cell proliferation. In the present invention, by calculating the interaction between cells, that is, the action state of attraction and repulsion, it is possible to quantitatively evaluate the process of attracting other cells to form a scaffold and the invasion process to other types of cells. it can. That is, it can be used for screening of a reagent that is isolated from other cells and suppresses normal growth, and a reagent for suppressing invasion to other cells.

It is a flowchart of the image processing program shown as an Example of this invention. It is a conceptual diagram of the movement analysis model which shows a cell of interest and surrounding cells as a model. It is a general | schematic block diagram of the culture observation system shown as an example of application of this invention. It is a block diagram of the said culture observation system. It is the schematic diagram which showed typically the microscopic observation image of the cell image | photographed at the time t. FIG. 6 is a schematic diagram showing a state where the microscopic observation image at time t shown in FIG. 5 is segmented. It is explanatory drawing for demonstrating matching with the cell (cell shown as a continuous line) of the image image | photographed at the time t, and the cell (cell shown with a dotted line) of the image image | photographed at the time t-1. It is a schematic diagram which illustrates the state of a time-sequential change of a cell group, (1) It is a schematic diagram which shows the state from which the cell which was initially separated changes to a colony like (2). When a cell group colonizes, it is a change graph of the total attractive force repulsive quantity when the value of the total attractive force repulsive quantity calculated for every predetermined time is plotted on the graph which made the horizontal axis the time axis. It is a schematic diagram for demonstrating designation | designated of the measurement cell used as measurement object. It is a structural example of the screen which displays the attractive repulsion speed of the surrounding cell with respect to a measurement cell. It is a structural example of the screen which displayed the amount of attractive forces. 1 is a block diagram illustrating a schematic configuration of an image processing apparatus. It is a structural example of the display image of the cultured cell movement analysis interface displayed on a display panel when an image analysis program is executed.

Explanation of symbols

BS Culture observation system GP Image processing program
C _i attention cell C _j peripheral cell
C _s measurement cell (attention cell)
54 Macro observation system 54c Imaging device 55 Microscopic observation system 55c Imaging device 100 Image processing device 110 Image storage unit 120 Image analysis unit 130 Output unit

Claims (14)

A first image captured by the imaging device and including a plurality of cells in the observation region, and a second image of the observation region captured by the imaging device a predetermined time before the first image;
Selecting one cell from a plurality of cells included in the first image as a target cell;
A cell located around the cell of interest is a peripheral cell,
Calculating a statistic of the velocity of each of the surrounding cells relative to the cell of interest based on the relative amount of movement of the cell of interest and the surrounding cell in the first image and the second image;
An image analysis method for cell observation, characterized in that the state of interaction of each of the surrounding cells with respect to the cell of interest can be determined. The total velocity statistic of the whole cell in the observation region is calculated by summing the statistic of the velocity obtained by sequentially replacing the selected one target cell with another cell located in the observation region,
The cell analysis image analysis method according to claim 1, wherein the state of interaction between cells in the observation region can be determined based on the total velocity statistic.   The image analysis method for cell observation according to claim 1 or 2, wherein the velocity statistics are weighted according to the distance between the cell of interest and the surrounding cells.   The cell analysis image analysis method according to any one of claims 1 to 3, wherein the state of interaction is an action state of attraction or repulsion. Acquiring a first image captured by the imaging device and including a plurality of cells in the observation region, and a second image of the observation region captured by the imaging device a predetermined time before the first image;
Selecting one cell from the plurality of cells included in the first image as a target cell;
Designating cells located around the cell of interest as peripheral cells;
Calculating a statistic of the speed of each of the surrounding cells with respect to the cell of interest based on a relative amount of movement of the cell of interest and the surrounding cell in the first image and the second image;
Outputting the calculated statistics of the velocity of each of the surrounding cells to the outside,
An image processing program for cell observation, characterized in that the state of interaction of each of the surrounding cells with respect to the cell of interest can be determined. Calculating a total velocity statistic of all the cells in the observation region by summing up the statistics of the velocity obtained by sequentially replacing the selected one target cell with another cell located in the observation region; ,
Outputting the calculated total speed statistic to the outside,
6. The cell observation image processing program according to claim 5, wherein the state of interaction between cells in the observation region can be determined by the total velocity statistic.   The image processing program for cell observation according to claim 5 or 6, wherein the speed statistics are weighted according to the distance between the cell of interest and the surrounding cells.   The cell processing image processing program according to any one of claims 5 to 7, wherein the state of interaction is an action state of attraction or repulsion. An image display device that calculates the total speed statistic for each predetermined time and sequentially arranges the captured images as the first image for three or more images captured at the predetermined time by the imaging device. With steps to display
The cell processing image processing program according to any one of claims 6 to 8, wherein a temporal change in the total speed statistic can be grasped. An imaging device for imaging cells;
An image storage unit that stores a first image taken by the imaging device and a second image taken by the imaging device a predetermined time before the first image;
An image analysis unit that analyzes the state of interaction between the plurality of cells located in the observation region based on the first image and the second image;
An output unit for outputting analysis data by the image analysis unit,
In the image analysis unit, the first image and the second image of one target cell selected from a plurality of cells included in the observation region of the first image and a peripheral cell positioned around the target cell. Calculating the statistic of the velocity of each of the surrounding cells relative to the cell of interest based on the relative amount of movement of the cell of interest and the surrounding cell in
An image processing apparatus for cell observation, characterized in that the speed statistic calculated in the image analysis unit is output from the output unit. The total velocity statistic of the entire cells in the observation region is calculated by summing the statistic of the velocity obtained by sequentially replacing the selected one target cell with another cell located in the observation region,
The cell processing image processing apparatus according to claim 10, wherein the total velocity statistic calculated by the image analysis unit is configured to be output from the output unit.   The image processing apparatus for cell observation according to claim 10 or 11, wherein the velocity statistics are weighted according to a distance between the cell of interest and the surrounding cells.   The image processing apparatus for cell observation according to any one of claims 10 to 12, wherein the state of interaction is an action state of attraction or repulsion. An image display device for displaying images;
In the image analysis unit, for the three or more images captured every predetermined time by the imaging device, the total speed statistic for each predetermined time is calculated using the captured images as the first image sequentially,
The total speed statistic for each predetermined time is output from the output unit to the image display device and displayed in time series on the image display device, so that temporal changes in the total speed statistic can be grasped visually. The image processing apparatus for cell observation according to any one of claims 11 to 13, wherein the image processing apparatus is configured.