JP2012173725A - Image generation device and microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an image generation device capable of generating a wide-range high-definition fluorescence tiling image having high positioning accuracy obtained by a low magnification image and a fluorescence image having magnification higher than that of the low magnification image of a sample and to provide a microscope device having the image generation device.SOLUTION: An image generation device 40 generating a tiling image by processing an image from an imaging apparatus 14 disposed in the imaging position of a microscope 1 includes image generation means 41 generating one fluorescence image obtained by re-sizing a plurality of binarized fluorescence images obtained by binarizing the plurality of high-magnification fluorescence images of a sample 2 obtained through the imaging apparatus and a binarized sample image obtained by binarizing a low magnification image over the whole of the sample obtained through the imaging apparatus to the same magnification and tailing each of the binarized fluorescence images with the binarized sample image as a reference.

Description

  The present invention relates to an image generation apparatus that generates a high-definition fluorescent tiling image and a microscope apparatus having the image generation apparatus.

  Conventionally, in the fluorescence observation of cells, when the field of view is narrow with respect to the specimen, in order to generate a joined image of the fluorescence images representing the entire specimen (so-called tiling processing), it is possible to seamlessly perform adjacent fluorescence images. It is necessary to prepare a certain amount of margin so as to match the images. Here, matching means that the entire specimen is divided into rectangles like a grid, and when the stage is moved to each position to acquire an image, the image is taken with a larger field of view than the grid, and adjacent images are captured. This means that the overlap (hereinafter referred to as “margin”) is positioned using the correlation and difference of the image itself. Such matching is used for the purpose of absorbing a positioning error of the stage (see, for example, Patent Document 1).

JP 2007-065669 A

  However, the margin required for the above matching must always contain some image (for example, a cell), and the margin is treated as extra information to generate the final image of the entire specimen. There is a waste of being. Further, when tiling is performed as a whole, the degree of matching with respect to the margin is not necessarily the same in the upper, lower, left, and right directions, and a problem of balance adjustment also arises. Further, in the fluorescence image, there is a problem that the part that becomes the margin is a dark image without light, and it is very difficult to perform tiling of the fluorescence image because there is no image as a reference for matching.

  The present invention has been made in view of the above problems, and generates a high-definition fluorescent tiling image in a wide range with high positioning accuracy from a low-magnification image of a specimen and a fluorescent image higher in magnification than the low-magnification image. An object of the present invention is to provide an image generating apparatus capable of performing the above and a microscope apparatus having the same.

  In order to solve the above-described problem, the present invention is an image generation device that generates a tiling image by processing an image from an imaging device disposed at an imaging position of a microscope, and is acquired via the imaging device. A plurality of binarized fluorescence images obtained by binarizing a plurality of high-magnification fluorescence images of a specimen and a binarized sample image obtained by binarizing a low-magnification image over the entire specimen are resized to the same magnification, and There is provided an image generating apparatus comprising image generating means for generating one fluorescent image obtained by tiling the binary fluorescent images with reference to a binarized specimen image.

  The present invention also relates to a low-magnification image capturing device that acquires a cell image of the entire bottom of a well in which cells are cultured, a fluorescent image acquisition device that acquires a fluorescent image with a higher magnification than the low-magnification image acquisition device, and the cell Using the first cell image obtained by extracting the cells from the image and the second cell image obtained by extracting the cells from the fluorescence image, template matching of the second cell image with respect to the first cell image is performed. And an image generation device that determines a position of the fluorescence image with respect to the cell image.

  According to the present invention, an image generating apparatus capable of generating a high-definition fluorescent tiling image in a wide range with high positioning accuracy from a low-magnification image of a specimen and a fluorescent image higher in magnification than the low-magnification image, A microscope apparatus can be provided.

1 is a schematic configuration diagram of a microscope apparatus according to an embodiment. The flowchart which produces | generates a tiling image. (A) is an image acquired by the low-magnification image acquisition device, and (b) is an image obtained by binarizing the image shown in (a). (A) is the fluorescence image acquired from the fluorescence image acquisition apparatus, (b) is the fluorescence image which binarized the fluorescence image shown to (a). (A) shows a fluorescence tiling image obtained by performing template matching on the binarized image shown in FIG. 3 (b) using the binarized fluorescent image shown in FIG. 4 (b), and (b) Indicates an example of a tiling image.

  Hereinafter, a microscope apparatus having an image generation apparatus according to an embodiment of the present application will be described with reference to the drawings. The following embodiments are only for facilitating the understanding of the invention, and excluding additions and substitutions that can be performed by those skilled in the art without departing from the technical idea of the present invention. It is not intended.

  As shown in FIG. 1, a microscope apparatus 1 having an image generation apparatus 40 according to the present embodiment includes a stage 3 on which a well plate 10 is placed, and a low-magnification illumination optical system 4 disposed above the stage 3. , A low-magnification imaging optical system 5 including a low-magnification imaging optical system 5 disposed below the stage 3, an excitation optical system 24 disposed in exchange for the low-magnification imaging optical system 5, and a low-magnification imaging optical system A camera 14 that picks up an image of the imaging optical system that is disposed at the imaging position of the system 5 or the fluorescence imaging optical system 20 and is inserted in the optical axis, and processes the acquired image to generate a tiling image to be described later The image generation apparatus 40 is provided. The stage 3, the low-magnification illumination optical system 4, the low-magnification imaging optical system 5, and the fluorescence imaging optical system 20 are not connected to a microscope column (not shown) having a base portion (not shown) below. The position in the vertical direction is supported via a support member shown in the figure so as to be changeable.

  In the present embodiment, a sample 2 that is a phase object such as a cell is injected into each well 10a of the well plate 10 together with a culture solution (not shown) as shown in FIG.

  As shown in FIG. 1, the low-magnification imaging optical system 5 is arranged for observing a sample (cell) 2 in an arbitrary well 10a illuminated by the low-magnification illumination optical system 4. The low-magnification imaging optical system 5 includes, in order from the stage 3 side, a low-magnification objective lens 12 having a high numerical aperture and a lens barrel 13, and includes a fluorescence coupling including an excitation optical system 24 on the optical axis of the camera 14. The optical system 20 can be selectively inserted into and removed from the image optical system 20.

  The low-magnification objective lens 12 is an objective lens having a high numerical aperture (NA) for condensing light emitted from the bottom surface of the well 10a with almost no leakage. In this embodiment, the objective lens 12 has a diameter of about 6.4 mm. In order to realize the field of view, one having a magnification of 1.25 times and a numerical aperture of 0.25 or more is used.

  The fluorescence imaging optical system 20 includes a revolver 23 for exchanging a plurality of objective lenses 21 from low magnification to high magnification with an optical axis, a lens barrel portion 25 coupled to an excitation optical system 24, and a sample 2. It is comprised from the excitation light source 22 for irradiating excitation light. A dichroic mirror 26 used at the time of fluorescence observation and a filter cube 28 including an emission filter 27 are arranged at the coupling portion between the excitation optical system 24 and the lens barrel portion 25 so as to be detachable from the optical axis.

  A 2/3 inch CCD camera is used as the camera 14 disposed at the imaging position of the low-magnification imaging optical system 5 selectively inserted on the optical axis or the imaging position of the fluorescence optical system 20. The camera 14 is connected to an image generation device 40.

  The image generation apparatus 40 includes, for example, a personal computer (hereinafter simply referred to as a PC) 41, a monitor 42, and an input / output device 43 such as a keyboard and a mouse, which are image processing means acquired by the camera 14. The PC 41 has a built-in memory 44 for storing the acquired image, and is equipped with hardware and software necessary for image processing and image generation. The memory 44 can use an external storage device such as an external hard disk.

  The low-magnification illumination optical system 4 is for transmitting and illuminating the sample 2 in the well 10a. A condenser lens, an aperture stop, a movable stop, a collector lens, and a light source (not shown) are sequentially provided from the stage 3 side. Have. The low magnification illumination optical system 4 is used not only when the low magnification imaging optical system 5 is inserted into the optical axis but also for transmission illumination when the fluorescence imaging optical system 20 is inserted into the optical axis. Is possible.

  A movable diaphragm (not shown) is a diaphragm member for generating low NA illumination light for illuminating the bottom surface of the well 10a substantially uniformly. The movable diaphragm (not shown) has a diaphragm diameter, a position on the optical axis, and the like so that the NA of illumination light emitted from the illumination optical system 4 (hereinafter referred to as “illumination NA”) is 0.1 or less. A previously designed throttle member is used. The movable diaphragm also plays a role of narrowing the illumination light irradiation region so that the luminous flux of the illumination light is smaller than the inner diameter of the well 10a in order to prevent the illumination light from being reflected or transmitted by the wall surface of the well 10a. Yes.

  Further, the movable diaphragm is provided with a movement mechanism (not shown) for moving the movable diaphragm in the optical axis direction and a direction perpendicular to the optical axis, and a diaphragm diameter adjustment mechanism (not shown) for changing the diaphragm diameter. Yes. Thereby, the angle of the chief ray of the illumination light to the sample 2 can be changed by moving the movable diaphragm in the optical axis direction by the moving mechanism. Further, the effective NA of the illumination light can be changed by changing the stop diameter of the movable stop by the stop diameter adjusting mechanism. Furthermore, the irradiation position of the illumination light emitted from the illumination optical system 4 on the sample 2 can be changed in the direction perpendicular to the optical axis by moving the movable diaphragm in the direction perpendicular to the optical axis by the moving mechanism. it can.

  The condenser lens (not shown) is provided with a movement mechanism (not shown) for moving the condenser lens in the optical axis direction. Thereby, the condenser lens is moved in the optical axis direction by the moving mechanism, whereby the condensing position of the illumination light can be changed in the optical axis direction, and shading in the field of view can be removed.

  As a light source (not shown), a red LED (light emitting diode) with high coherence suitable for observation of a phase object is used. Thereby, the uniformity and long life of illumination can be ensured. In addition, nutrients such as phenol red in the culture solution change color with the deterioration of the culture solution, and this absorbs light in the visible range, so the brightness of the observed image changes depending on the culture state. Can be resolved.

  In the microscope apparatus 1 according to the present embodiment, the illumination light emitted from the light source of the low magnification illumination optical system 4 passes through the movable aperture after passing through the collector lens. The movable diaphragm can change the direction of the principal ray depending on the position on the optical axis, and the effective NA of the illumination light can be changed by changing the size of the aperture or the position of the aperture to a position away from the optical axis. Can be changed. The illumination light passes through the aperture stop and is collected by the condenser lens, thereby forming the effective NA of the necessary illumination light and the direction of the principal ray. The illumination light thus formed is applied to the sample 2 in an arbitrary well 10a in the 96-well plate 10 on the stage 3 through the culture solution. As described above, the illumination optical system 4 can realize illumination in which the convergence angle of the light beam condensed at one point on the sample 2 and the direction of the principal ray thereof are controlled.

  Here, the surface of the culture solution in the well 10a has a concave surface in the vicinity of the wall surface of the well 10a due to surface tension, and a lens effect (meniscus effect) occurs. In the microscope apparatus 1, illumination light having an effective NA smaller than the numerical aperture of the low-magnification objective lens 12 constituting the low-magnification imaging optical system 5 is set by sufficiently increasing the numerical aperture of the low-magnification objective lens 12. By irradiating the concave surface of the culture solution, even if the direction of the illumination light is refracted toward the outside of the low-magnification objective lens 12 due to the concave surface of the culture solution, it is less affected by the lens effect due to the liquid surface of the culture solution. Yes. At this time, the direction of the principal ray of the illumination light is directed to the optical axis in the direction opposite to the direction of the refracting in consideration of the direction of refracting the principal ray of the illumination light by the liquid surface of the culture solution. Thus, it is possible to prevent the refracted illumination light from being reflected or transmitted by the wall surface of the well 10a, that is, the illumination light beam travels only in the well 10a, and thereby the bottom surface of the well of the well 10a is uniformly distributed. Illumination can be performed substantially uniformly.

Then, light from the sample 2 illuminated with illumination light having an NA smaller than the numerical aperture of the low-magnification objective lens 12 constituting the imaging optical system 5 enters the low-magnification objective lens 12 of the imaging optical system 5. . Here, since the low-magnification objective lens 12 is an objective lens having a high numerical aperture as described above, light emitted from the bottom surface of the well 10a (including light from the sample 2 illuminated with the above-described refracted illumination light is included. ) Can be collected almost without leakage. The light condensed by the low-magnification objective lens 12 in this way forms an image of the sample 2 on the imaging surface of the camera 14 via an imaging lens (not shown). Specifically, the bottom surface of the well 10a is substantially reduced while suppressing the generation of reflected light, that is, noise light, on the wall surface of the well 10a by the illumination light having the principal ray direction and the effective NA of the illumination light as described above. By uniformly illuminating, the NA of light (direct light) from the background of the sample 2 is reduced, so that the direct light and the diffracted light diffracted by the sample 2 interfere with each other and the sample has good contrast Two observation images are formed. The image of the sample 2 formed in this way is taken by the camera 14, displayed on the monitor 42, and observed by the user.
Here, there is a demand for observing the entire field of view of high-definition fluorescent images of cells cultured in regenerative medicine research and the like. As will be described later, the image generation apparatus 40 of the present application generates a fluorescence tiling image obtained by tiling a high-magnification fluorescence image with reference to a cell image acquired at a low magnification. For this reason, in the present application, in order to perform observation over the entire field of view while taking into consideration the influence of the lens effect of the culture medium when acquiring low-magnification cell images, the low-magnification objective lens 12 has a numerical aperture equivalent to 0.25 and 1.25 The magnification is about double.

  In addition, as described above, the microscope apparatus 1 according to the present embodiment has an effective NA of illumination light of 0.1 or less, a magnification of the objective lens 12 of 1.25, and a numerical aperture of 0.25 or more. As a result, observation can be performed in the entire field of view (actual field of view of about 6.4 mm). Further, as described above, since the inner diameter of the well 10a in the 96 well plate 10 is about Φ6.4 mm, the microscope apparatus 1 according to the present embodiment uses the camera 14 which is a 2/3 inch CCD camera to the 96 well plate 10. The entire bottom surface of the well 10a can be taken as a single image at a time. A low-magnification image of the sample 2 in which the influence of the lens effect due to the concave surface of the culture solution is eliminated can be acquired.

  Further, as described above, the magnitude of the lens effect of the culture solution varies depending on the type of the culture vessel, the composition of the culture solution, the material of the culture vessel, the amount of the culture solution, and the like. In particular, the diameter of the concave surface of the culture medium varies depending on the type of the culture vessel, and as a result, the radius of curvature of the concave surface changes greatly, and the lens effect also changes greatly.

  Therefore, the microscope apparatus 1 according to the present embodiment moves the movable diaphragm in the optical axis direction or changes the diaphragm diameter of the movable diaphragm as described above according to the change in the lens effect of the culture solution. The effective NA of the illumination light and the direction of the chief ray of the illumination light can be changed. As a result, even when the lens effect of the culture solution changes, the bottom surface of the culture vessel can be illuminated almost uniformly by the low NA illumination light corresponding to this, and the magnification of the sample 2 in which the influence of the lens effect is eliminated is low. Images can be acquired.

  FIG. 3A is an example in which an image 30 of the sample (cell) 2 obtained over the entire bottom surface of the well 10 a acquired by the camera 14 is displayed on the monitor 42. The acquired image 30 is stored in the memory of the PC 41 together with information on the well 10a.

  Next, a case where a fluorescent image from the sample 2 is acquired using the high-magnification objective lens 21 will be described.

  The user changes the low-magnification imaging optical system 5 and inserts the fluorescence optical system 20 into the optical axis of the camera 14 and also installs a filter cube 28 including a dichroic mirror 26 and an emission filter 27 corresponding to the acquired fluorescence image. Arranged in the excitation optical system 24.

  Excitation light from the excitation light source 22 is reflected by the dichroic mirror 26 in the filter cube 28 toward the objective lens 21 and is irradiated to the sample 2 in the well 10 a through the objective lens 21.

  The sample 2 is preliminarily introduced with a substance that emits a predetermined fluorescence, and emits a fluorescence having a predetermined wavelength by the irradiated excitation light. The fluorescence emitted from the sample 2 is collected by the objective lens 21, travels through the lens barrel 25 toward the dichroic mirror 26, passes through the dichroic mirror 26, and further passes through the emission filter 27 to form an image (not shown). The lens forms an image on the imaging surface of the camera 14. The fluorescence image of the sample 2 imaged on the imaging surface of the camera 14 is captured by the camera 14 and stored in the memory 44 in the PC 41 together with the information of the well 10a and displayed on the monitor 42.

  The fluorescent image 35 shown in FIG. 4A is an example of an image obtained by resizing the fluorescent image of the circular field obtained in this way into a square having a predetermined size (A side is A).

  The PC 41 acquires a high-magnification fluorescent image 35 of the square of one side A and the entire bottom surface of the well 10a, and stores each fluorescent image 35 in the memory 44 together with the XY coordinates of the stage 3 at the time of acquisition.

  In this way, the image generating device 40 has the image 30 (see FIG. 3A) of the entire bottom surface of the well 10a acquired by the low-magnification imaging optical system 5 and a plurality of fluorescence acquired by the fluorescence imaging optical system 20. The image 35 (see FIG. 4A) is stored in the memory 44 with the respective information.

  Next, FIG. 2 shows a procedure for generating a fluorescence tiling image 135 (see FIG. 5) of the high-magnification fluorescence image 35 using the image 30 obtained at the low magnification and the plurality of fluorescence images 35 obtained at the high magnification. This will be described with reference to a flowchart.

(Step S1)
The PC 41 of the image generation apparatus 40 reads the image 30 (see FIG. 3A) acquired from the memory 44 at a low magnification.

(Step S2)
The PC 41 corrects the luminance unevenness of the image 30 and generates a corrected image. For luminance unevenness correction, a correction image of the entire bottom surface of each well 10a is acquired and stored in the memory 44 in a state where only the culture medium for culturing cells is injected into each well 10a of the well plate 10. The PC 41 performs a difference process between the acquired cell image 30 and a correction image corresponding to the image 30 to generate a corrected image in which luminance unevenness of the cell image 30 is corrected. The luminance unevenness correction is not limited to the above-described difference processing, but can be performed by applying a low-pass filter (for example, a Gaussian filter) having a sufficiently wide band to the cell image 30 to obtain a difference from the image 30. .

(Step S3)
The PC 41 binarizes the correction image corrected for luminance unevenness, and generates a binarized cell image 30a (see FIG. 3B). Thereby, the cell 2 becomes a binarized cell image 2a with a uniform intracellular structure. Specifically, when extracting the cells, the brightness of the region that clearly becomes the background is sampled from the brightness unevenness corrected image in step S2, and the noise components, that is, the average value μ1 of the background brightness and the standard deviation σ1 are calculated. The luminance value that is not a noise component in the background portion is assumed to be a cell, and the luminance unevenness correction image is binarized using (μ1−m1 × σ1) as a threshold value. Here, m1 is an appropriate magnification. Since the cell image in the luminance unevenness corrected image is dark, the luminance value is small. Therefore, a process is performed in which a pixel having a luminance value exceeding the threshold (μ1−m1 × σ1) is set to luminance value = 0 (background), and an image having a luminance value lower than the threshold is set to luminance value = 1 (cell). A valued cell image 30 a is generated and stored in the memory 44. The binarized cell image 30a is an image in which the cells 2 are bright and the background is dark.

(Step S4)
Next, the fluorescent image 35 is processed. The PC 41 reads the fluorescence image 35 (see FIG. 4A) acquired at a high magnification stored in the memory 44.

(Step S5)
The PC 41 corrects luminance unevenness of the fluorescent image 35 read from the memory 44 and generates a luminance unevenness corrected fluorescent image. The method for correcting luminance unevenness uses the same method as in step S2. The PC 41 calculates the noise component of the background portion, that is, the average value μ2 of the background luminance and the standard deviation σ2 using the luminance unevenness corrected fluorescent image.

(Step S6)
The PC 41 performs binary processing on the luminance unevenness-corrected fluorescent image by the same process as in step S <b> 3, generates a binary fluorescent image 35 a (see FIG. 4B), and stores it in the memory 44. At this time, assuming that the cell portion of the fluorescent image 35 is bright, the luminance unevenness corrected fluorescent image obtained in step S5 is binarized using (μ2 + m2 × σ2) as a threshold value. Here, m2 is an appropriate magnification. Since the cell image in the luminance unevenness fluorescence correction image is bright, the luminance value is large. Therefore, binarization is performed by setting a pixel having a luminance value exceeding the threshold (μ2 + m2 × σ2) to luminance value = 1 (cell) and an image having a luminance value lower than the threshold being luminance value = 0 (background). A fluorescent image 35 a is generated and stored in the memory 44. The PC 41 performs the processing from step S4 to step S6 over all of the plurality of high-magnification fluorescent images 35 acquired in the well 10a. As a result, the memory 44 stores a low-magnification binarized cell image 30a binarized over the entire bottom surface of the well 10a and a plurality of binarized fluorescent images 35a binarized from the plurality of high-magnification fluorescent images 35, respectively. Is done.

(Steps S7, S8)
In order to make the image magnifications of the binarized cell image 30a formed in steps S1 to S6 and the binarized fluorescent images 35a the same, the PC 41 uses the binarized cell image 30a and the plural binarized cell images 30a based on the respective magnification differences. Each of the binarized fluorescent images 35a is resized. Thereby, the cell 2a in each binarized image is resized to substantially the same size. FIG. 5A shows the binarized cell image 130a resized to the same magnification obtained in this way, and FIG. 5B shows the binarized fluorescent image 135a (template image resized to the same magnification). ) Respectively.

(Steps S9 and S10)
The PC 41 executes template matching processing on the resized binarized cell image 130a using the resized binarized fluorescent image 135a as a template. Since the images used for matching are both binarized images, the difference value between the two images is used as the binarized cell image matching evaluation value. The position of the template image is determined when the difference value is the smallest, and the tiling position is determined.

(Step S11)
By performing such template matching processing on all the high-magnification fluorescent images 35 that have been acquired, it is possible to generate a fluorescence tiling image in which the high-magnification fluorescent images over the entire bottom surface of the well 10a are arranged. If this template matching process is performed as it is, it takes time for calculation in the PC 41. Therefore, using the XY coordinates of the stage 3 when the fluorescent image 35 stored in the memory 44 is acquired, the low-magnification cell image 30 is displayed. The template matching processing time can be shortened by obtaining an approximate position and determining a predetermined search range based on the obtained position.

  As described above, the image generating apparatus according to the present embodiment does not evaluate and tile the matching degree of the pattern image existing in the marginal portion as in the past (in the fluorescent image, the marginal portion is dark and the matching degree is high). There is no pattern image to evaluate), and by using the binarized image of the entire sample image obtained at low magnification as a reference, it is obtained at high magnification by evaluating the degree of matching of the emission pattern of the cells of the fluorescence image obtained at high magnification The tiling of the fluorescent image can be performed. In addition, since the margin portion is not used as in the prior art, it is not necessary to consider the margin portion when acquiring a high-magnification fluorescent image, and the tiling error that occurs in the margin portion can be suppressed to a low level.

  In the above description, the low-magnification image of the entire well bottom surface has been described for the case where the high-magnification fluorescent image is subjected to template matching processing using the bright-field transmission image. It is also possible to use low-magnification fluorescent images. In this case, in FIG. 1, the low-magnification illumination optical system 4 and the low-magnification imaging optical system 5 are configured to acquire a low-magnification fluorescent image and a high-magnification fluorescent image by the fluorescence imaging optical system 20 without necessity. good. Further, in the binarization process of step S3, the luminance unevenness correction image is binarized using (μ1 + m1 × σ1) as a threshold value as in step S6. In the low-magnification fluorescent image, since the cell image in the luminance unevenness correction image is bright, the luminance value is large. Therefore, the processing is changed so that the pixel having the luminance value exceeding the threshold (μ1 + m1 × σ1) is set to luminance value = 1 (cell) and the pixel having the luminance value lower than the threshold is set to luminance value = 0 (background). . And other processes can show the same operation and effect by performing the same processes as described above.

  As described above, the image generation apparatus according to the present embodiment can template-match a high-magnification fluorescent image with high positional accuracy by using a high-magnification fluorescent image as a template based on the low-magnification cell image. As a result, a high-magnification fluorescent image arranged with high positional accuracy over the entire bottom surface of the well can be obtained.

1 Microscope device 2 Sample (cell)
3 Stage 4 Low-magnification illumination optical system 5 Low-magnification imaging optical system 10 Well plate 12 Low-magnification objective lens 13, 25 Lens barrel part 14 Camera 20 Fluorescence imaging optical system 21 Objective lens 22 Excitation light source 23 Revolver 24 Excitation optical system 26 Dichroic mirror 27 Emission filter 28 Filter cube 30 Low-magnification cell image 30a Binarized cell image 35 High-magnification fluorescence image 35a Binarized fluorescence image 40 Image generation device 41 PC
42 monitor 43 input device 44 memory 130a fluorescent tiling image 135a template

Claims (7)

An image generation device that generates a tiling image by processing an image from an imaging device disposed at an imaging position of a microscope,
A plurality of binarized fluorescent images obtained by binarizing a plurality of high-magnification fluorescent images of a specimen and a binarized specimen image obtained by binarizing the low-magnification image over the entire specimen, obtained through the imaging device, An image generation apparatus comprising: an image generation unit that generates a single fluorescence image that is resized to a magnification and tiles each of the binarized fluorescence images with the binarized sample image as a reference. The low-magnification image and the plurality of high-magnification fluorescent images each have a position coordinate value at the time of acquisition,
The image generation means, after preliminary tiling with reference to the position coordinates, each binarized fluorescent image at a position where a difference value between the binarized sample image and the binarized fluorescent image is minimum. The image generating apparatus according to claim 1, wherein tiling is performed.   The image generation apparatus according to claim 1, further comprising a storage unit that stores the low-magnification image, the fluorescence image, and the tiling image.   The image generation apparatus according to claim 1, wherein the low-magnification image is a low-magnification fluorescent image. A low-magnification image capturing device that acquires a cell image of the entire bottom surface of a well in which cells are cultured;
A fluorescence image acquisition device for acquiring a fluorescence image having a higher magnification than the low magnification image acquisition device;
A template for the first cell image of the second cell image using a first cell image obtained by extracting the cell from the cell image and a second cell image obtained by extracting the cell from the fluorescence image. An image generating apparatus that performs matching and determines a position of the fluorescent image with respect to the cell image.   The said low-magnification image acquisition apparatus suppresses the meniscus effect by the deformation | transformation of the culture medium surface of the well which culture | cultivates the said cell, and acquires the said cell image of the bottom part whole surface of the said well. Microscope device. The low-magnification image acquisition device includes an illumination optical system and an imaging optical system,
The microscope apparatus according to claim 5 or 6, wherein an effective NA of illumination light of the illumination optical system is smaller than a numerical aperture of an objective lens of the imaging optical system.

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