WO2006051813A1 - 細胞培養装置、画像処理装置及び細胞検出システム - Google Patents
細胞培養装置、画像処理装置及び細胞検出システム Download PDFInfo
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- WO2006051813A1 WO2006051813A1 PCT/JP2005/020529 JP2005020529W WO2006051813A1 WO 2006051813 A1 WO2006051813 A1 WO 2006051813A1 JP 2005020529 W JP2005020529 W JP 2005020529W WO 2006051813 A1 WO2006051813 A1 WO 2006051813A1
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- image acquisition
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- 238000012545 processing Methods 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 claims description 33
- 238000004113 cell culture Methods 0.000 claims description 29
- 238000000605 extraction Methods 0.000 claims description 15
- 238000012258 culturing Methods 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 3
- 238000012937 correction Methods 0.000 claims description 2
- 238000010191 image analysis Methods 0.000 claims description 2
- 230000002093 peripheral effect Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 97
- 238000010586 diagram Methods 0.000 description 15
- 239000002609 medium Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000004891 communication Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009499 grossing Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 239000004973 liquid crystal related substance Substances 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
- G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T5/00—Image enhancement or restoration
- G06T5/50—Image enhancement or restoration using two or more images, e.g. averaging or subtraction
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/10—Segmentation; Edge detection
- G06T7/11—Region-based segmentation
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/10—Segmentation; Edge detection
- G06T7/174—Segmentation; Edge detection involving the use of two or more images
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
- G06V20/00—Scenes; Scene-specific elements
- G06V20/60—Type of objects
- G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
- G06V20/695—Preprocessing, e.g. image segmentation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10056—Microscopic image
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10141—Special mode during image acquisition
- G06T2207/10148—Varying focus
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/20—Special algorithmic details
- G06T2207/20212—Image combination
- G06T2207/20224—Image subtraction
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/30—Subject of image; Context of image processing
- G06T2207/30004—Biomedical image processing
- G06T2207/30024—Cell structures in vitro; Tissue sections in vitro
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S128/00—Surgery
- Y10S128/92—Computer assisted medical diagnostics
- Y10S128/922—Computer assisted medical diagnostics including image analysis
Definitions
- Cell culture device image processing device, and cell detection system
- the present invention relates to a cell culture device, an image processing device, and a cell detection system that can extract a cell portion from a plurality of pieces of image data acquired during cell culture.
- a threshold value is calculated based on the distribution of pixel values, and pixels that are greater than or less than the threshold value are extracted as cells.
- the image may become whitish depending on the intensity of light applied to the cell, and it may be detected that the cell exists more than the actual cell amount.
- filtering such as noise removal, smoothing filter, and edge enhancement was essential.
- Patent Document 1 a change in brightness is monitored in image data, a trigger signal is generated at a specific threshold value, and a camera is repeatedly imaged after a predetermined delay time to obtain an addition average. What you did is listed.
- Patent Document 1 Japanese Patent Laid-Open No. 2000-275539
- Non-patent document 1 describes a technique that enables observation.
- the filter processing prior to the cell extraction processing has had a great influence on the cell extraction accuracy as in the past.
- this filtering process it is possible to remove to some extent the difference in brightness between the central part and the peripheral part of the image, noise contained in the image, etc.
- the one described in Patent Document 1 also removes noise contained in the image to some extent. be able to.
- the effect may be small, or the cell outline may be unclear.
- Non-Patent Document 1 since the technique described in Non-Patent Document 1 is to simply photograph cells in the incubator, individual cells can be separated and identified at the initial stage of culture, but the culture process proceeds. As a result, it was thought that it was difficult to separate and identify the cells here.
- the present invention has been made in view of the above points, and an object of the present invention is to provide a cell culture device capable of observing the progress of cell culture without taking out the cells outside the device. It is a thing.
- the present invention also provides a cell culture apparatus and image that can extract cells as an image without being affected by color change of the medium, change in light quantity, brightness difference between the center and periphery of the image, noise, etc. It is in providing a processing apparatus and a cell detection system.
- the cell culture device has a container capable of forming a space that is shielded from the external atmosphere, a culture container for culturing cells, and a cell in the culture process in the culture container.
- a light source that irradiates light to the vesicle and an image acquisition device that is installed on the back of the culture vessel with respect to the light source and captures an image of cells in the culture process are disposed in the vessel.
- the cell culture apparatus of the present invention includes an incubator means for culturing cells, an image acquisition means for acquiring an image of the cell from the incubator means, and either one of the image acquisition means and the incubator means.
- a focus position adjusting means for adjusting the focus of the image acquisition means so as to be positioned before and after the cells in culture of the incubator means, and at a plurality of focus positions set by the focus position adjusting means
- Extraction means for performing difference processing on a plurality of pieces of image data having different focal positions acquired by the image acquisition means, and extracting only the cell portion by binarizing the difference processed image; It is characterized by having
- a plurality of images taken by changing the focal position of the camera have medium values and noise components having almost the same pixel values. Therefore, by performing differential processing on multiple image data shot at different focal points as in the present invention, the effects of color change in the medium, change in light intensity, contrast between the center and the periphery of the image, noise, etc. Disappears and only the cell part is extracted.
- the cell culture device provides a focus position of the image acquisition means for acquiring an optimal image for extracting only a cell portion by the extraction means in the cell culture device.
- the image acquired at the calculated focus position is an image suitable for cell extraction processing. Then, by positioning the image acquisition means to the calculated focus position at the next measurement, it is possible to always focus with the same accuracy.
- the cell culture device is provided with a position correction unit that corrects the positions of a plurality of pieces of image data acquired by the image acquisition unit. This corrects the positional deviation in the X and Y directions and the rotational direction of the image that occurs when the focus is changed by image processing, thereby preventing a decrease in cell extraction accuracy due to the positional deviation of multiple images. be able to.
- An image processing apparatus includes an image acquisition unit that acquires an image of an object scattered on a plane and having a thickness in a predetermined range, and the focal point of the image acquisition unit before and after the thickness direction of the object.
- difference processing is performed on a plurality of pieces of image data taken at different focal points using image acquisition means, such as color change of medium, change of light intensity, difference in brightness between the center and periphery of the image, noise, etc. Only the cell part can be extracted without being affected.
- the cell detection system includes an image acquisition unit that acquires an image of a cell extending on a plane, and a direction orthogonal to the plane of the cell that extends the focal point of the image acquisition unit on the plane. Difference processing is performed on a plurality of pieces of image data having different focal positions acquired by the image acquiring means at a plurality of focal positions set by the focal position adjusting means and the focal position adjusting means. And an extraction means for extracting only the cell portion by application.
- FIG. 1 is a block diagram showing a configuration of a cell culture device, an image processing device, and a cell detection system according to this embodiment.
- FIG. 2 is a diagram showing an outline of the arrangement of each constituent means in the culture apparatus.
- FIG. 3 is a diagram showing details of the image processing unit in FIG. 1.
- FIG. 4 is a flowchart showing an example of cell extraction processing executed by the image processing unit.
- FIG. 5 is a diagram showing the relationship between the moving distance of a camera and the sum of differences between adjacent pixel values.
- FIG. 6 is a diagram showing an example of an image when the focal position of the objective lens is located on the bottom surface of the incubator.
- FIG. 7 is a diagram showing an example of an image when the focal position of the objective lens is on the front side (camera side) of the bottom surface of the incubator.
- FIG. 8 is a diagram showing an example of an image when the focal position of the objective lens is on the rear side (light source side) from the bottom surface of the incubator.
- FIG. 9 is a diagram showing an image when the difference 'positioning process and the binarization process are performed on the images of FIGS. 7 and 8.
- FIG. 1 is a block diagram showing the configuration of the cell culture device, the image processing device, and the cell detection system according to this embodiment.
- the cell culture device 11 has a box-type structure in which the inside is cut off from the outside space, and inside the incubator 12 for culturing cells, and for photographing the cells in the incubator 12
- the CCD camera 13, the converter 15 for transferring the image data obtained from the CCD camera 13 to the image processing unit 14, and the CCD camera 13 or the incubator 12 are moved.
- the incubator 12 has a transparent bottom surface, and the CCD camera 13 is configured to photograph the light emitted from the light source 18 and transmitted through the bottom surface of the incubator 12. .
- the CCD camera 13 is provided with a CCD device having about 400,000 pixels, and the light source 18 is preferably an LED or miniature bulb that emits diffuse light instead of parallel light.
- FIG. 2 is a diagram showing an outline of the arrangement of each constituent means in the culturing apparatus 11.
- the incubator 12 when moving the CCD camera 13 in the culturing apparatus 11, the camera 'incubator drive unit 16.
- FIG. 4 is a diagram showing the arrangement of a moving guide 21 and an objective lens 22 respectively.
- a light source 18 is attached to the upper surface of the culture apparatus 11.
- An incubator 12 is disposed below the light source 18, and a CCD camera 13 having an objective lens 22 is disposed below the center of the incubator 12.
- the CCD camera 13 is moved up and down along the moving guide 21 by the drive control of the camera incubator driving device 16 operated by the command output from the CPU 32, and the incubator 12 is changed in the vertical direction by changing the focal position.
- the imaging device combining the CCD camera 13 and the objective lens 22 has a minute area near the bottom located near the center of the incubator 12, for example, a minute area of about 1.5 mm (vertical) X 2.0 mm (horizontal). It is desirable to be able to shoot.
- FIG. 3 is a diagram showing details of the image processing unit 14 of FIG.
- the image processing unit 14 includes a CPU 32 that performs arithmetic processing via the data bus 31, a main memory 33 that the CPU 32 temporarily uses as a storage area, an external storage device 34 that stores image data and position information, It comprises a communication port 35 for communicating with the motor controller 17, a monitor 36 for displaying an image after cell extraction, and a keyboard 37 for receiving user input.
- the image processing unit 14 takes in an image from the CCD camera 13 via the converter 15 and performs various image processing.
- FIG. 4 is a flowchart showing an example of a cell extraction process executed by the image processing unit. Hereinafter, the cell extraction processing steps shown in FIG. 4 will be described.
- the series of steps described in FIG. 4 is performed by pre-installed software.
- the units shown in FIGS. 1 to 3 are executed under the control of the units sequentially in accordance with commands output from the CPU 32 at predetermined time intervals in the cell culture process, for example, once / day. is there. Note that the time required to repeatedly execute the series of steps described in FIG. 4 at a predetermined time interval can be obtained by using the output of the clock generator provided in the CPU 32.
- the CPU 32 issues a command to the motor controller 17 to execute an image acquisition operation for taking images at a predetermined pitch interval, for example, 27 zm while moving the CCD camera 13 in the vertical direction.
- the plurality of image data acquired by this image acquisition operation is stored in the external storage device 34 via the converter 15.
- step S42 after all the images have been taken, an image selection process is executed.
- this image selection process at least two images with clear cell edges are selected from a plurality of images stored in the external storage device 34.
- the pixel value changes more than in an unclear image. Therefore, the absolute value of the difference between adjacent pixel values is calculated, and the sum of them is obtained and stored in the main memory 33.
- FIG. 5 is a diagram showing the relationship between the moving distance of the camera and the sum of the differences between adjacent pixel values. In FIG. 5, there are two peaks, and the image showing these peak values is an image with clear cell edges. 6 to 8 show images at the focal positions of typical cameras in FIG. Here, FIG.
- FIG. 6 is a diagram showing an example of an image when the focal position of the objective lens 22 is located on the bottom surface of the incubator 12, and FIG. 7 shows the focal position of the objective lens 22 from the bottom surface of the incubator 12.
- FIG. 8 is a diagram showing an example of an image when the objective lens 22 is on the rear side (light source side) with respect to the bottom surface of the incubator 12. It is a figure which shows an example. Since these images are images obtained by setting the focal length to a position where the force is not very different, the density difference between these images is almost the same. As is clear from these figures, it is invented that the focal position of the objective lens 22 is shifted either forward or backward with respect to the bottom surface of the incubator 12, and that it is possible to acquire an image with clear cell edges. It became clear by the experiment of the person. [0022] [Step S43]
- step S43 whether or not two or more images corresponding to the two peaks described above could be selected, that is, whether the image with clear cell edges shown in FIGS. 7 and 8 was acquired. (A determination is made as to whether or not the position has been calculated by the CPU 32. If yes, the process proceeds to the next step S44, and if no, the process returns to step S42.
- the image stored in the device 34 is subjected to image processing using profile processing, Fourier transform, differentiation, etc., and the outline of the cell image is extracted, and an image with a clear outline of the cell image is obtained. This includes determining the position of the CCD camera 13. The position of the CCD camera 13 from which the cell image is clearly obtained is stored in the main memory 33 and used for the next cell extraction process.
- step S44 for the two images selected in steps S42 to S43, a small image area of 256 pixels x 256 pixels is set at the center of the image, and the small image area of one image is set to the other image.
- a difference / alignment process is performed to align the cells in both images by shifting the difference by one pixel in the X (horizontal) direction and the Y direction from the small image area of the image.
- the reason that the alignment step of both images is included in the embodiment is that when the CCD camera 13 moves in the direction of the incubator 12, the force depends on whether the assembly accuracy of the apparatus is good or not. This is because a slight misalignment may occur. Therefore, an alignment step can be omitted in an apparatus that does not cause misalignment of a captured image even when the CCD camera 13 is moved.
- the next determination is made based on the result of the difference 'positioning process in the previous step S44. That is, when the cell image drawn in the small image area is greatly inclined in the vertical direction, it is determined whether or not the number of cells is minimum. The reason for this is that if the cells are tilted greatly, if the alignment is not accurate, the cells will be doubled due to the difference, and it will appear that there are many cells. When the cell image drawn in the small image area is greatly inclined in the horizontal direction, it is determined whether or not the total length of the cells is the minimum. The reason is that the cells are lined up sideways The alignment is not accurate. This is because the cells can be seen horizontally longer than the actual difference.
- step S46 If the result of the determination is yes, the difference between the two images at that position is completed, and the process proceeds to step S46. If no, the process returns to step S44. Difference / alignment processing is performed until the determination in step S 45 is yes.
- the two images with different focal positions caused by light emitted from a point light source such as an LED lamp have different information on the light scattering state on the cell surface. The inventors infer that this is the case.
- the images subjected to the difference processing have a density difference of about 19% as shown in FIG. This makes it possible to eliminate almost all the effects such as the brightness difference between the center and the periphery of the image and the noise.
- step S46 the image is binarized to facilitate analysis of the length, number, and shape of the cells.
- Fig. 9 shows the binarization process for the difference image with the smallest number of cells and the smallest total cell length as a result of the difference and registration process for the images in Figs. 7 and 8. It is a figure which shows the image of the result of giving. In this way, by binarizing the difference image in which the number of cells is the smallest and the sum of the lengths of the cells is minimized, individual cells are clearly depicted as white portions.
- the binarization process in step S46 facilitates the subsequent handling of the data by a computer, for example, counting of the number of cells described later, measuring the distance between cells, measuring the cell size, etc. If not, you can skip this step S46.
- the proliferation rate of the cell can be estimated by counting the number of pixels, and the cell interval, that is, the black width is measured. In other words, the cell growth rate can be estimated. Furthermore, the degree of cell growth can be estimated by measuring the length of the cells.
- a box-type structure whose interior is blocked from the external space.
- the cell culture device is configured to include the light source 18 installed on the upper part of the CCD camera 13, it is possible to observe the degree of cell proliferation without taking out the cells in the culture process outside the device.
- a monitor display such as a liquid crystal display is connected to the image unit 14 and image information is supplied to the monitor display, so that an image of a cell photographed by a camera and a difference process can be performed.
- the performed image can be displayed.
- the CPU32 can measure the cell size (length) and the cell distribution density in the incubator on the image, and display the measurement results on the monitor display. .
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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EP05806165A EP1811017B1 (en) | 2004-11-09 | 2005-11-09 | Cell cultivating device, image processing device and cell detecting system |
JP2006544915A JP4921978B2 (ja) | 2004-11-09 | 2005-11-09 | 細胞培養装置、画像処理装置及び細胞検出システム |
US11/718,807 US8064661B2 (en) | 2004-11-09 | 2005-11-09 | Cell culture device, image processing device and cell detecting system |
CN2005800372380A CN101048492B (zh) | 2004-11-09 | 2005-11-09 | 细胞培养装置、图像处理装置和细胞检测*** |
Applications Claiming Priority (2)
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JP2004324456 | 2004-11-09 | ||
JP2004-324456 | 2004-11-09 |
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WO2006051813A1 true WO2006051813A1 (ja) | 2006-05-18 |
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US (1) | US8064661B2 (ja) |
EP (1) | EP1811017B1 (ja) |
JP (1) | JP4921978B2 (ja) |
CN (1) | CN101048492B (ja) |
WO (1) | WO2006051813A1 (ja) |
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JP2006230333A (ja) * | 2005-02-28 | 2006-09-07 | Hitachi Medical Corp | フローサイトメータ、細胞の解析方法、細胞解析プログラム、蛍光検出器の感度設定方法および陽性率判定法における基準ゲート設定方法 |
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JPWO2019065144A1 (ja) * | 2017-09-29 | 2020-02-27 | 富士フイルム株式会社 | 観察装置、観察方法および観察プログラム |
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Also Published As
Publication number | Publication date |
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JPWO2006051813A1 (ja) | 2008-05-29 |
CN101048492A (zh) | 2007-10-03 |
EP1811017A1 (en) | 2007-07-25 |
EP1811017B1 (en) | 2013-01-09 |
EP1811017A4 (en) | 2010-12-01 |
US8064661B2 (en) | 2011-11-22 |
JP4921978B2 (ja) | 2012-04-25 |
CN101048492B (zh) | 2013-01-09 |
US20080273786A1 (en) | 2008-11-06 |
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