JP5655608B2 - Observation device and image correction program - Google Patents

Description

  The present invention relates to an observation apparatus and an image correction program that acquire an observation image of an observation target while illuminating the observation target with illumination light.

  In the field of cell observation using an observation apparatus such as a microscope apparatus, it is generally performed to capture an image of an observation target such as a cell and perform image analysis. In this cell observation, it is desirable to use illumination light with a uniform illumination distribution for the observation target, but in reality, illumination light with a non-uniform light amount distribution is used depending on the observation environment and the characteristics of the light source. Yes. For this reason, an image obtained by imaging (hereinafter referred to as an observation image) is an image in which illumination unevenness of illumination light occurs. In order to remove the illumination unevenness generated in the observation image, the obtained observation image is subjected to image processing such as shading correction processing, or an image in a state where the observation target is not installed in the observation apparatus (hereinafter, non-observation). Observation image) is acquired in advance, and image correction is performed to subtract the non-observation image from the observation image.

JP 2009-151163 A

  The shading correction process described above is correction for increasing the luminance of the low-frequency component region included in the image. For example, if the area occupied by the cell to be observed is large and the area is subjected to the shading correction process for an observation image that extends from the high frequency component to the low frequency component, the cell executed after the shading correction process In the region extraction process, there is a problem that the cell region cannot be accurately extracted. In addition, when the non-observation image is acquired in advance, there is a drawback that it is not possible to deal with the case where the illumination unevenness between the acquisition of the non-observation image and the acquisition of the observation image changes.

  Accordingly, an object of the present invention is to provide an observation apparatus and an image correction program that can appropriately remove the illumination unevenness that occurs in the observation image by specifying the illumination unevenness that occurs when the observation image is acquired. And

In order to solve the above-described problem, an observation apparatus according to the present invention includes a light source that emits illumination light that illuminates an observation target , and an imaging range that includes a part of the observation target that is illuminated by the illumination light. an imaging device that performs an imaging, a moving mechanism for moving the light source on a plane orthogonal to the optical axis of the illumination light from a first image obtained by the imaging device in a state that is not moving the light source, the A setting unit that fixes a position of the background area with respect to the imaging range after setting an area that does not include an observation target as a background area; and a second direction orthogonal to the first direction and the first direction in the background area And determining the amount of movement, the direction of movement, and the number of movements when moving the light source based on the number of pixels and the number of pixels in the first direction and the second direction in the effective pixel region of the image sensor. A movement control unit that controls the movement mechanism based on the movement amount, the movement direction, and the number of movements determined by the determination unit, and changes a relative position of the light source and the observation target; An extraction unit that extracts each of the background regions from an image and a plurality of second images obtained by the imaging element while changing the relative positions of the light source and the observation target, and the background extracted from the first image With the region as a reference, each of the background regions extracted from the plurality of second images is arranged based on the movement amount and the movement direction to obtain an illumination distribution of the illumination light, and the imaging range in the imaging range An illumination unevenness specifying unit that specifies illumination unevenness of illumination light is provided.

In addition, the observation apparatus of the present invention includes a light source that emits illumination light that illuminates an observation target, an imaging element that performs imaging with an area including a part of the observation target illuminated by the illumination light as an imaging range, and The observation target is included from a moving mechanism for moving the observation target on a plane orthogonal to the optical axis of the illumination light, and a first image obtained by the imaging element without moving the observation target. After setting a non-existing region as a background region, a setting unit for fixing the position of the background region with respect to the observation target, the first direction in the background region and the number of pixels in the second direction orthogonal to the first direction, A determination unit that determines a movement amount, a movement direction, and a number of movements when the observation target is moved based on the number of pixels in the first direction and the second direction in the effective pixel region of the imaging element; A movement control unit that controls the movement mechanism based on the movement amount, the movement direction, and the number of movements determined by the determination unit, and changes a relative position of the light source and the observation target; the first image; An extraction unit that extracts each of the background regions from a plurality of second images obtained by the imaging element while changing the relative positions of the light source and the observation target, and the background region extracted from the first image as a reference As described above, an illumination distribution of the illumination light is obtained by arranging each of the background regions extracted from the plurality of second images based on the movement amount and the movement direction, and the illumination light in the imaging range is obtained. An illumination unevenness identifying unit that identifies illumination unevenness is provided.

Further, the pixel value of the pixel included in the background region extracted from the first image and the plurality of second images illuminates the imaging range of the imaging element among the illumination light irradiated to the observation target. It is preferable to consist of the luminance value of the illumination light.

Furthermore, the illumination unevenness specifying unit, by the least squares method using pixel values of pixels in the background area that will be extracted from the first image and the plurality of second images, identifies the illumination unevenness of the illumination light It is preferable to do.

In addition to the first image and the plurality of second images , the imaging element can capture an observation image, and using the observation image and illumination unevenness of the illumination light, It is preferable that an image correction unit that corrects the observation image is provided.

In addition, the image correction program of the present invention performs an imaging process in which an illumination process that emits illumination light to illuminate an observation target and an area that includes a part of the observation target that is illuminated by the illumination light is included in an imaging range. Thus, after setting the first imaging step for acquiring the first image, and from the acquired first image, an area that does not include the observation target as a background area, the position of the background area with respect to the imaging range A first step in the background region and the number of pixels in the second direction orthogonal to the first direction, and the first direction and the second direction in the effective pixel region of the image sensor that performs the imaging. A determination step of determining a movement amount, a movement direction, and a number of movements when moving the illumination range of the illumination light based on the number of pixels, and based on the determined movement amount, the movement direction, and the number of movements. Moving the illumination range of the illumination light, changing the illumination range of the illumination light and the relative position of the observation object, and changing the illumination range of the illumination light and the relative position of the observation object, A second imaging step of imaging a plurality of second images, an extraction step of extracting the background regions from the first image and the plurality of second images, respectively, and the background regions extracted from the first images The illumination distribution of the illumination light is obtained by arranging each of the background regions extracted from the plurality of second images based on the movement amount and the movement direction with reference to the illumination area, and the illumination in the imaging range It is possible to cause the computer to execute an illumination unevenness specifying step for specifying light illumination unevenness.
In addition, the image correction program of the present invention performs an imaging process in which an illumination process that emits illumination light to illuminate an observation target and an area that includes a part of the observation target that is illuminated by the illumination light is included in an imaging range. Thus, after setting the first imaging step for acquiring the first image and the area that does not include the observation target as the background area from the acquired first image, the position of the background area with respect to the observation target A first step in the background region and the number of pixels in the second direction orthogonal to the first direction, and the first direction and the second direction in the effective pixel region of the image sensor that performs the imaging. A determination step of determining a movement amount, a movement direction, and the number of movements when moving the observation target based on the number of pixels, and based on the determined movement amount, the movement direction, and the number of movements Moving the observation object to change the illumination range of the illumination light and the relative position of the observation object, and changing the illumination range of the illumination light and the relative position of the observation object, and a plurality of second images With reference to the second imaging step of capturing the image, the extraction step of extracting the background region from each of the first image and the plurality of second images, and the background region extracted from the first image as a reference An illumination distribution of the illumination light is obtained by arranging each of the background regions extracted from a plurality of second images based on the movement amount and the movement direction, and uneven illumination of the illumination light in the imaging range is obtained. It is possible to cause the computer to execute the illumination unevenness identification process to be identified.

  According to the present invention, it is possible to appropriately remove the illumination unevenness that occurs in the observation image by specifying the illumination unevenness that occurs when the observation image is acquired.

It is a figure which shows an example of a structure of the observation apparatus of this invention. It is a figure which shows an example of the background area | region set from an imaging range and an imaging range. It is a figure which shows the illumination range, imaging range, and background area | region of the illumination light before moving a light source. It is a figure which shows the illumination range, imaging range, and background area | region of illumination light when a light source is moved to -X direction with a light source movement mechanism. It is a figure which shows the illumination range of illumination light, and the position of the illumination distribution in the imaging range obtained when a light source is moved 5 times in a -X direction, 5 times in a -Y direction, and a total of 25 times. It is a flowchart which shows the flow of a process at the time of performing image correction with respect to the observation image data obtained when observing a test object with an observation apparatus.

  The configuration of the observation apparatus of the present invention is shown in FIG. As shown in FIG. 1, the observation apparatus 10 includes an observation unit 11 and a computer (PC) 12, which are electrically connected by wire such as a connection cable. Thereby, various image data can be output from the observation unit 11 toward the computer 12, and an operation signal can be output from the computer 12 toward the observation unit 11. In the present embodiment, the observation apparatus 10 includes the observation unit 11 and the computer 12. However, the present invention is not limited to this, and the observation unit 11 of the present invention is an observation apparatus. May be an observation system that is connected by wire or the like. In FIG. 1, only the main part of the observation unit 11 is shown.

  The observation apparatus 10 displays an image (hereinafter referred to as a reference image) for specifying illumination unevenness of illumination light when observing an image of the test object 35 to be observed as a relative position between the light source 21 and the test object 35. Before the change, and after changing the relative positions of the light source 21 and the test object 35 a plurality of times. Below, the case where the relative position of the light source 21 and the test object 35 is changed by moving the light source 21 is demonstrated. In addition, after acquiring these reference images, the observation device 10 returns the relative positions of the light source 21 and the test object 35 to an initial state, and displays an image for observing the test object 35 (hereinafter, an observation image). get.

  The observation unit 11 uses the illumination light from the light source 21 to illuminate the test object 35 placed on the stage (not shown) from below, and the light transmitted through the test object 35 is transmitted to the image sensor 27. By receiving light in this manner, it is composed of a so-called transmission type observation unit that acquires an observation image of the test object. Here, examples of the test substance include culture containers such as preparations, petri dishes containing flasks to be cultured, flasks, petri dishes, and well plates. In addition, in FIG. 1, it has described about the case where a preparation is made into a test object.

  The observation unit 11 includes a light source 21, a light source moving mechanism 22, a condensing optical system 23, an objective optical system 24, a lens driving mechanism 25, an imaging optical system 26, an image sensor 27, an A / D converter 28, and a buffer memory 29. , A controller 30 and an interface (I / F) 31.

  As the light source 21, for example, a lamp, an LED, a laser light oscillator, or the like is used. The light source 21 emits illumination light toward a test object 35 placed on a stage (not shown). This illumination light is light whose optical axis L is parallel to the Z direction. The light source moving mechanism 22 moves the light source 21 on a plane orthogonal to the optical axis L, in other words, on the XY plane. Note that the method of moving the light source 21 by the light source moving mechanism 22 and the amount of movement of the light source 21 will be described later.

  The condensing optical system 23 converts the illumination light from the light source 21 into parallel light. The illumination light converted into the parallel light illuminates the test object 35 from below the stage. Part of the illumination light that illuminates the test object 35 from below the test object 35 passes through the test object 35 and enters the objective optical system 24. The illumination light emitted from the objective optical system 24 enters the imaging optical system 26. The imaging optical system 26 images the illumination light emitted from the objective optical system 24 on the imaging surface of the imaging element 27. The lens driving mechanism 25 moves the objective optical system 24 along the optical axis L during focus adjustment.

  As the image sensor 27, for example, a CCD image sensor or a CMOS image sensor is used. The imaging element 27 receives the illumination light imaged by the imaging optical system 26 to acquire an optical image of the test object 35. The imaging range in the imaging element 27 is an observation visual field range when observing the test object 35. An image signal based on the optical image obtained by the image sensor 27 is output to the A / D converter 28.

  The A / D converter 28 converts the analog image signal output from the image sensor 27 into a digital image signal. The image signal converted into a digital image signal by the A / D conversion unit 20 is written in the buffer memory 29.

  The controller 30 performs lighting / extinguishing control of the light source 21, driving control of the light source moving mechanism 22 and the lens driving mechanism 25, and imaging control in the imaging element 27. The interface 31 is electrically connected to the interface 50 of the computer 12 by wire such as a connection cable.

  The computer 12 is provided to execute various operations by the observation unit 11. The computer 12 includes a CPU 41, a memory 42, a buffer memory 43, an image memory 44, an image processing circuit 45, an image correction circuit 46, a display device 47, a display control circuit 48, an operation unit 49, an interface (I / F) 50, and the like. Composed. Note that the CPU 41, the memory 42, the buffer memory 43, the image memory 44, the image processing circuit 45, the image correction circuit 46, the display control circuit 48, and the interface 50 are electrically connected via the bus 51.

  The CPU 41 controls each part of the computer 12 based on a control program stored in the memory 42. Further, the CPU 41 outputs an operation signal based on the operation program stored in the memory 42 to the observation unit 11. Here, although details are omitted, the CPU 41 uses the position data of the background area stored in the memory 42 and the reference image data stored in the image memory 44 to transmit the illumination light that illuminates the test object 35. Illumination distribution data indicating the state of the luminance distribution is generated. The procedure for setting the background region and the procedure for generating the illumination distribution data will be described later.

  The memory 42 stores the above-described control program, operation program, and the like, as well as background area position data, which will be described later. The buffer memory 43 stores observation image data output from the observation unit 11, illumination distribution data, observation image data subjected to image correction (shading correction), and image data after image processing. The image data after image processing includes, for example, reference image data obtained immediately after the observation is started, and image data after image processing is performed on the observation-corrected observation image data. The image memory 44 stores a plurality of reference image data output from the observation unit 11. The reference image data stored in the image memory 44 is accompanied by the position data of the light source 21.

  The image processing circuit 45 performs image processing such as white balance processing, color interpolation processing, and gradation conversion processing on the reference image data and observation image data acquired immediately after the start of observation. Since the contents of each of these image processes are well known, details are omitted here. Image data that has been subjected to image processing by the image processing circuit 45 is stored in the buffer memory 43.

  The image correction circuit 46 corrects the observation image data temporarily stored in the buffer memory 43 using the illumination distribution data. This correction is a process of subtracting the pixel value (luminance value) of each pixel of the illumination distribution data from the pixel value (luminance value) of each pixel of the observation image data. Note that the observation image data subjected to the image correction is stored in the buffer memory 43 again.

  The display device 47 is composed of, for example, an LCD panel or an organic EL panel. The display device 47 displays a reference image, an observation image, a setting image, and the like acquired immediately after the observation is started. Display control in the display device 47 is executed by a display control circuit 48.

  The operation unit 49 includes, for example, a keyboard and a mouse. When these keyboard and mouse are operated, operation signals based on these operations are input to the CPU 41. Note that the operation unit 49 is operated, for example, when a background area is designated for a reference image displayed on the display device 47. The interface 50 is electrically connected to the interface 31 of the observation unit 11 by wire such as a connection cable.

  Next, setting of the background area will be described. For example, when the reference image is acquired by imaging with the imaging device 27 that is executed immediately after the observation is started, the reference image is displayed on the display device 47. The user operates the operation unit 49 to designate the background area. If the operation unit 49 is a mouse, the mouse is operated to move the mouse pointer displayed on the display device 47 onto the reference image, and then the mouse is dragged to specify the background area. In FIG. 2, an area 57 surrounded by a frame 56 with respect to the reference image 55 is an area designated by a mouse drag operation.

  In response to this operation, the CPU 41 sets a rectangular area 57 associated with the mouse drag operation as a background area, and stores the set position data of the background area in the memory 42. The position data of the background area is composed of pixel position data of the reference image corresponding to the start point of the mouse drag operation and position data of the pixel of the reference image corresponding to the end point of the mouse drag operation. As a method for setting the background region 57, mouse drag operation by the user is mentioned, but the present invention is not limited to this, and the background region 57 can be automatically set from the reference image 55. .

  Next, the setting of the movement amount when moving the light source 21 will be described. As described above, the background area is set by operating the operation unit 49 on the reference image displayed on the display device 47. With this background area setting, the memory 42 stores the position data of the background area. The CPU 41 reads the position data of the background area stored in the memory 42. The CPU 41 obtains the number of pixels in each direction of the X direction and the Y direction in the background area from the read position data of the background area. Then, the CPU 41 determines the amount of movement of the light source 21 in the X direction and the amount of movement in the Y direction based on the obtained number of pixels in the X direction and Y direction in the background region.

  Further, the CPU 41 determines the number of times the light source 21 is moved in the X direction (number of times of movement in the X direction) and the number of times of movement in the Y direction. For example, since the number of pixels in each of the X and Y directions in the effective pixel area of the image sensor 27 is set in advance, the CPU 41 determines the number of pixels in each of the X and Y directions in the background area, and the image sensor. The number of movements of the light source 21 in the X direction and the number of movements in the Y direction are obtained from the number of pixels in each of the X and Y directions in the 27 effective pixel regions. Then, the CPU 41 outputs to the controller 30 the amount of movement of the light source 21 in the X direction, the amount of movement of the light source 21 in the Y direction, the number of movements of the light source 21 in the X direction, and the number of movements in the Y direction. The controller 30 receives the drive signal from the computer 12 and executes movement control of the light source 21 while referring to these data. As the movement control of the light source 21, for example, the operation of moving the light source 21 in the -X direction is performed a plurality of times, the light source 21 is returned to the initial position in the X direction, and then the light source 21 is moved in the -Y direction. After performing the control of moving the light source 21 in the −X direction a plurality of times or performing the operation of moving the light source 21 in the −X direction a plurality of times, the light source 21 is moved in the Y direction and the light source 21 is moved in the X direction. For example, a control for performing the operation of moving a plurality of times. In conjunction with the movement control of the light source 21, the controller 30 controls the image sensor 21 and causes the image sensor 21 to execute an image capturing process.

  Finally, a procedure for generating illumination distribution data will be described. The CPU 41 reads the position data of the background area stored in the memory 42. Then, the CPU 41 reads the reference image data stored in the image memory 44 and extracts the pixel value of the pixel corresponding to the position data of the background area from each reference image data. Next, the CPU 41 arranges the background area of the reference image data with reference to the position data of the light source attached to the reference image data, the amount of movement of the light source 21 in the X direction, and the amount of movement in the Y direction. .

  FIG. 3 shows an imaging range in the imaging device 27 and an illumination range of illumination light that are executed before the light source 21 is moved. In FIG. 3, the imaging range in the imaging device 27 is indicated by a frame 61 with respect to the illumination range 60 of the illumination light. An area 63 surrounded by the frame 62 is a background area.

  If the light source 21 is moved in the −X direction with the state shown in FIG. 3 as an initial state, the illumination range 60 of the illumination light also moves in the −X direction. The test object 35 and the image sensor 27 are fixed with respect to the movement of the light source 21 in the −X direction. FIG. 4 shows the positional relationship between the illumination range of illumination light when the light source 21 is moved in the −X direction, and the imaging range and background region of the imaging device 27. In FIG. 4, the range 61 surrounded by the alternate long and short dash line is the imaging range of the imaging device 27 before moving the light source 21, and the range 61 ′ surrounded by the solid line is imaging when the light source 21 is moved in the −X direction. This is the imaging range of the element 27. An area 63 surrounded by a dotted line is a background area before the light source 21 is moved, and an area 63 'surrounded by a solid line is a background area when the light source 21 is moved in the -X direction. Here, when the illumination range 60 that moves with the movement of the light source 21 is considered as a reference, when the light source 21 moves in the −X direction, the imaging range 61 and the background region 63 of the image sensor 21 move in the X direction. Will be.

  The reference image data obtained by the image sensor 27 when the light source 21 is moved in the X direction or the Y direction is image data in which the pixel value of each pixel is a luminance value. For this reason, when background regions extracted from a plurality of reference image data obtained when the light source 21 is moved a plurality of times in the −X direction or the −Y direction are arranged, the image pickup device 27 captures an image when the light source 21 is fixed. It becomes the illumination distribution of illumination light in the range. In FIG. 5, a range indicated by a frame 65 is an imaging range of the imaging device 27, and a plurality of areas 63 included in the imaging range 65 are background areas extracted from each reference image data. The CPU 41 generates illumination distribution data by collecting pixel values of pixels in each background area when the background areas extracted from each reference image data are arranged as one image data. In FIG. 5, the light source 21 is moved 5 times in the −X direction and 5 times in the −Y direction, for a total of 25 times, as an example.

  Next, the flow of processing when performing image correction on observation image data obtained when observing a test object with the observation apparatus 10 will be described based on the flowchart of FIG.

  Step S101 is processing for acquiring reference image data. The CPU 41 outputs an imaging signal for acquiring reference image data to the controller 30. In response to this, the controller 30 executes an imaging process in the imaging device 27. Thereby, the reference image data when the light source 21 is in the initial position is acquired. The reference image data is output from the observation unit 11 toward the computer 12 by the controller 30. When the reference image data is output to the computer 12, the CPU 41 stores the reference image data in the image memory 44. At this time, the CPU 41 attaches the position data of the light source 21 to the reference image data. The reference image data stored in the image memory 44 is read by the image processing circuit 45 and image processing is executed. The reference image data that has been subjected to image processing by the image processing circuit 45 is temporarily stored in the buffer memory 43.

  Step S102 is processing for setting a background area. As described above, when the reference image data is acquired by the process of step S101, the obtained reference image data is output to the computer 12. When the process of step S102 is executed, the display control circuit 48 reads the image-processed reference image data temporarily stored in the buffer memory 43, and displays the reference image based on the read reference image data on the display device 47. To do. Upon receiving the display of the reference image, the user operates the operation unit 49 to designate a background area from the reference image. When a background region is designated based on the operation of the operation unit 49, the CPU 41 stores position data (background region position data) based on the operation of the operation unit 49 in the memory 42. Thereby, the background area in the reference image is set.

  Step S103 is processing for calculating the movement amount and the number of movements of the light source. By executing the processing in step S102, the background area in the reference image is set. The CPU 41 reads the position data of the background area set from the memory 42 and obtains the number of pixels in each direction of the X direction and the Y direction in the background area. Based on the number of pixels in the X direction and Y direction in the background region, the CPU 41 determines the amount of movement of the light source 21 in the X direction and the amount of movement in the Y direction. Further, the CPU 41 uses the number of pixels in each of the X and Y directions in the imaging range of the image sensor 27 and the number of movements of the light source 21 in the X direction using the number of pixels in the X and Y directions in the background area. And the number of movements in the Y direction. The CPU 41 outputs the calculated movement amount and number of movements of the light source to the controller 30 of the observation unit 11.

  Step S104 is processing to move the light source. The CPU 41 outputs a drive signal toward the controller 30. In response to this, the controller 30 refers to the movement amount and the number of movements of the light source 21 calculated in step S103. Then, the controller 30 drives the light source moving mechanism 22 to move the light source 21 by the amount of movement determined in step S103.

  Step S105 is processing to acquire reference image data. When the process of step S104 is executed, the light source 21 is moved. The controller 30 executes an imaging process in the image sensor 27. Thereby, the reference image data when the light source 21 moves by a predetermined amount from the initial position is acquired. The controller 30 outputs the acquired reference image data to the computer 12. When the reference image data is output to the computer 12, the CPU 41 stores the reference image data in the image memory 44. At this time, the CPU 41 attaches the position data of the light source 21 to the reference image data.

  Step S106 is processing for determining whether or not the movement of the light source is completed. By executing the process of step S103, the CPU 41 calculates the number of times the light source 21 is moved in the X direction and the number of times the light source 21 is moved in the Y direction. The CPU 41 calculates the number of movements of the light source 21 from these numbers. Then, the CPU 41 determines whether or not the movement of the light source 21 is completed by counting the number of reference image data stored in the image memory 44. If the number of reference image data stored in the image memory 44 has reached the calculated number of movements of the light source 21, the CPU 41 determines Yes in step S106. In this case, the process proceeds to step S107. If the determination process in step S106 is Yes, the CPU 41 outputs an operation signal for moving the light source 21 to the initial position (the position of the light source 21 in step S101) toward the controller 30. In response to this, the controller 30 drives the light source moving mechanism 22 to move the light source 21 to the initial position.

  On the other hand, when the number of reference image data stored in the image memory 44 has not reached the calculated number of movements of the light source 21, the CPU 41 determines No in step S106. In this case, the process returns to step S104. That is, the process of acquiring the reference image data is repeatedly performed while moving the light source 21 until the calculated number of movements of the light source 21 is reached.

  Step S107 is processing for specifying illumination unevenness. A plurality of reference image data is stored in the image memory 44 by executing the processes of step S104 and step S105 a plurality of times. The CPU 41 reads the position data of the background area from the memory. Then, the CPU 41 extracts pixel values of pixels included in the background area of each reference image data based on the position data of the background area. Since the reference image data is attached with the position data of the light source 21, the CPU 41 refers to the position data of the light source 21, and the pixels of the pixels included in the background area of each extracted reference image data The values are arranged in the buffer memory 43.

  As described above, since the background area is an area that does not include the observation target, the pixel value of each pixel included in the background area indicates the luminance value of the illumination light. For this reason, by arranging the background regions extracted from the respective reference image data, the illumination distribution (luminance distribution) of the illumination light that illuminates the imaging range of the imaging device 27 is obtained. After the pixel values of the pixels included in the background area of each extracted reference image data are arranged in the buffer memory 43, the CPU 41 collects the pixel values of the arranged pixels as one image data. Thereby, illumination distribution data is generated. That is, by generating this illumination distribution data, the illumination unevenness of the illumination light in the imaging range of the image sensor 27 is specified. The illumination distribution data is stored in the buffer memory 43.

  Step S108 is processing for acquiring observation image data. By executing the process of step S108, imaging by the imaging element 27 is executed, and observation image data is acquired. The observation image data is output from the controller 30 toward the computer 12. The CPU 41 stores this observation image data in the buffer memory 43.

  Step S109 is processing for correcting observation image data. The image correction circuit 46 reads observation image data and illumination distribution data stored in the buffer memory 43, respectively. The image correction circuit 46 corrects the observation image data using the illumination distribution data. In addition, what is necessary is just to subtract the pixel value of each pixel of illumination distribution data from the pixel value of each pixel of observation image data as correction | amendment of observation image data, respectively. As a result, correction (shading correction) is performed on the observation image data. The corrected observation image data is stored in the buffer memory 43. By executing the processing in step S109, observation image data from which illumination unevenness has been removed is acquired.

  Step S110 is image processing. The image processing circuit 45 performs image processing on the corrected observation image data stored in the buffer memory 43. The observation image data subjected to this image processing is written in the buffer memory 43.

  Step S111 is a process for displaying an image. The display control circuit 48 reads the image-processed observation image data stored in the buffer memory 43 and displays an observation image based on the observation image data. Note that the user can perform various operations using the operation unit 49 on the displayed observation image. For example, if the operation of the operation unit 49 is an operation for storing the observation image displayed on the display device 47 as it is, the CPU 41 omits illustration of the observation-processed observation image data such as a hard disk drive and a memory card. Stored in the storage medium. In addition, if the operation of the operation unit 49 is an operation for performing a process of extracting a cell region on the observation image displayed on the display device 47, the CPU 41 is displayed on the image processing circuit 45. What is necessary is just to perform the process which extracts the area | region of a cell, for example using a high-pass filter with respect to the observation image data used as the basis of an observation image.

  Thus, by acquiring data indicating the illumination distribution of illumination light from the background region included in the plurality of reference images immediately before observing the test object, it is possible to easily identify illumination unevenness of illumination light during cell observation. be able to. In addition, even if the area occupied by the test object is large relative to the observation visual field range, in other words, if the background area is included in the imaging range, illumination light illumination using the background area is performed. Since data indicating the distribution can be acquired, appropriate shading correction can be performed on the observation image data.

  In the present embodiment, a case has been described in which when the background areas extracted from a plurality of obtained reference image data are arranged, the size of all arranged background areas matches the size of the imaging range. However, in practice, the size of the entire background area arranged may not match the imaging range. Thus, if the entire background area to be arranged and the imaging range do not match, the reference image data is acquired in advance so that the area of the entire background area to be arranged is larger than the imaging range, When the background regions extracted from the respective reference image data are arranged, the pixel values of the pixels included in the imaging range can be collected as illumination distribution data.

  In addition, when a background area extracted from a plurality of reference image data obtained by a plurality of times of imaging is arranged, the size of the arranged background area may be smaller than the imaging range. For example, considering that the illumination distribution of illumination light can be approximated to a spherical surface, among the pixels included in the imaging range, the image values of the pixels corresponding to the places where the background region is not arranged are the pixel values of the pixels included in the plurality of background regions. Can be obtained by the method of least squares using

  In the present embodiment, the same length as the set width of the background area is set as the amount of movement of the light source 21 in the X direction, and the same length as the height of the background area is set as the amount of movement of the light source 21 in the Y direction. There is no need to be limited to this, and the length of the background region that is a predetermined multiple of the width of the light source 21 is the amount of movement in the X direction, and the length of the background region is a predetermined amount of the height of the light source 21 in the Y direction. Also good. In this case, when background areas obtained from the respective reference image data are arranged, the background areas are thinned out and arranged. Also in this case, considering that the illumination distribution of illumination light can be approximated to a spherical surface, the background area is not arranged in the imaging range by the least square method using the pixel values of the pixels included in the arranged background areas. The pixel values of the pixels included in the region can be obtained.

  In the present embodiment, the background area is extracted from each reference image data after all the obtained reference image data is acquired. However, the present invention is not limited to this, and the background area is acquired immediately after each reference image data is acquired. The image data based on the background region can be stored in the computer 12.

  In this embodiment, the case where the light source 21 is moved is described as an example of changing the relative position of the light source 21 and the test object 35. However, the present invention is not limited to this, and only the test object 35 is moved. Alternatively, the relative position between the light source 21 and the test object 35 can be changed by moving the test object 35 and the image sensor 27.

  In the present embodiment, the observation apparatus 10 is taken as an example, but an image correction program executed by a computer 12 that drives and controls the observation unit 11 may be used. In this case, any image correction program that can execute the processing flow shown in the flowchart of FIG. The image correction program is preferably stored in a computer-readable storage medium such as a memory card, an optical disk, or a magnetic disk.

  DESCRIPTION OF SYMBOLS 10 ... Observation apparatus, 11 ... Observation unit, 12 ... Computer, 21 ... Light source, 22 ... Light source moving mechanism, 27 ... Image sensor, 41 ... CPU, 42 ... Memory, 44 ... Image memory, 45 ... Image processing circuit, 46 ... Image correction circuit, 47... Display device, 49.

Claims (7)

A light source that emits illumination light to illuminate the observation target;
An imaging element that performs imaging with an imaging range of a region including a part of the observation target illuminated by the illumination light; and
A moving mechanism for moving the light source on a plane orthogonal to the optical axis of the illumination light;
A setting for fixing the position of the background area with respect to the imaging range after setting an area that does not include the observation target as a background area from the first image obtained by the imaging element without moving the light source And
Based on the number of pixels in the first direction and the second direction orthogonal to the first direction in the background region, and the number of pixels in the first direction and the second direction in the effective pixel region of the imaging device, the light source A determination unit for determining a movement amount, a movement direction, and the number of movements when moving
A movement control unit that controls the movement mechanism based on the movement amount, the movement direction, and the number of movements determined by the determination unit, and changes a relative position of the light source and the observation target;
An extraction unit that extracts each of the background regions from the first image and a plurality of second images obtained by the imaging device while changing the relative positions of the light source and the observation target;
Illumination of the illumination light by arranging each of the background regions extracted from the plurality of second images based on the movement amount and the movement direction with the background region extracted from the first image as a reference An illumination unevenness specifying unit that acquires distribution and specifies illumination unevenness of the illumination light in the imaging range ;
An observation apparatus comprising: A light source that emits illumination light to illuminate the observation target;
An imaging element that performs imaging with an imaging range of a region including a part of the observation target illuminated by the illumination light; and
A moving mechanism for moving the observation object on a plane orthogonal to the optical axis of the illumination light;
An area not including the observation target is set as a background area from the first image obtained by the imaging element without moving the observation target, and then the position of the background area with respect to the observation target is fixed. A setting section;
Based on the number of pixels in the first direction and the second direction orthogonal to the first direction in the background region, and the number of pixels in the first direction and the second direction in the effective pixel region of the imaging device, the observation A determination unit that determines a movement amount, a movement direction, and a number of movements when moving the object;
A movement control unit that controls the movement mechanism based on the movement amount, the movement direction, and the number of movements determined by the determination unit, and changes a relative position of the light source and the observation target;
An extraction unit that extracts each of the background regions from the first image and a plurality of second images obtained by the imaging device while changing the relative positions of the light source and the observation target;
Illumination of the illumination light by arranging each of the background regions extracted from the plurality of second images based on the movement amount and the movement direction with the background region extracted from the first image as a reference An illumination unevenness specifying unit that acquires distribution and specifies illumination unevenness of the illumination light in the imaging range;
Observation device comprising the. In the observation device according to claim 1 or 2,
A pixel value of a pixel included in the background region extracted from the first image and the plurality of second images illuminates an imaging range of the imaging element among the illumination light irradiated to the observation target. An observation apparatus comprising a luminance value of illumination light . The observation device according to claim 3 ,
The illumination unevenness specifying unit specifies the illumination unevenness of the illumination light by a least square method using pixel values of pixels included in the background region extracted from the first image and the plurality of second images. An observation apparatus characterized by. In the observation device according to any one of claims 1 to 4,
In addition to the first image and the plurality of second images, the imaging element can capture an observation image.
An observation apparatus comprising: an image correction unit that corrects the observation image using the observation image and illumination unevenness of the illumination light .   An illumination process for illuminating an observation target by emitting illumination light;
  A first imaging step of acquiring a first image by performing imaging in an imaging range of a region including a part of the observation target illuminated by the illumination light;
  A setting step of fixing a position of the background region with respect to the imaging range after setting the region not including the observation target as a background region from the acquired first image;
  Based on the first direction in the background region and the number of pixels in the second direction orthogonal to the first direction, and the number of pixels in the first direction and the second direction in the effective pixel region of the imaging element that performs the imaging. A determination step for determining a movement amount, a movement direction, and a number of movements when moving the illumination range of the illumination light;
  A movement step of moving the illumination range of the illumination light based on the determined movement amount, the movement direction, and the number of movements, and changing the illumination range of the illumination light and the relative position of the observation target;
  A second imaging step of imaging a plurality of second images while changing the illumination range of the illumination light and the relative position of the observation target;
  An extraction step of extracting each of the background regions from the first image and the plurality of second images;
  Illumination of the illumination light by arranging each of the background regions extracted from the plurality of second images based on the movement amount and the movement direction with the background region extracted from the first image as a reference An illumination unevenness specifying step of acquiring a distribution and specifying the illumination unevenness of the illumination light in the imaging range;
  Correction program capable of causing a computer to execute.   An illumination process for illuminating an observation target by emitting illumination light;
  A first imaging step of acquiring a first image by performing imaging in an imaging range of a region including a part of the observation target illuminated by the illumination light;
  From the acquired first image, after setting an area that does not include the observation object as a background area, a setting step of fixing the position of the background area with respect to the observation object;
  Based on the first direction in the background region and the number of pixels in the second direction orthogonal to the first direction, and the number of pixels in the first direction and the second direction in the effective pixel region of the imaging element that performs the imaging. A determination step for determining a movement amount, a movement direction, and the number of movements when moving the observation object;
  A movement step of moving the observation object based on the determined movement amount, the movement direction, and the number of movements, and changing the illumination range of the illumination light and the relative position of the observation object;
  A second imaging step of imaging a plurality of second images while changing the illumination range of the illumination light and the relative position of the observation target;
  An extraction step of extracting each of the background regions from the first image and the plurality of second images;
  Illumination of the illumination light by arranging each of the background regions extracted from the plurality of second images based on the movement amount and the movement direction with the background region extracted from the first image as a reference An illumination unevenness specifying step of acquiring a distribution and specifying the illumination unevenness of the illumination light in the imaging range;
  Correction program capable of causing a computer to execute.