JP5104637B2 - Observation device - Google Patents

Description

  The present invention employs a surface light source (striped light source) in which a bright part and a dark part are repeatedly arranged for illumination, thereby observing a transparent object such as a living cell in a wide field of view. The present invention relates to a stripe illumination type observation apparatus.

A stripe illumination type observation device (see Patent Document 1) obliquely illuminates a portion of an observation object facing a dark portion of a stripe light source with two bright portions adjacent to the dark portion. Can be obtained. Therefore, if a plurality of images are acquired while shifting the light-dark phase of the stripe light source and only the dark image is synthesized, for example, a dark field image overlooking the cell group scattered in the culture vessel is generated. You can also
JP 2007-178426 A

  However, in the stripe illumination type observation apparatus, a linear stripe pattern may be deformed into an arc shape or the like due to the meniscus effect of the culture medium present in the culture vessel, and in such a case, There has been a problem that it is difficult to extract and synthesize only a dark part image from a plurality of images taken while shifting the phase.

  The present invention has been made to solve such a conventional problem, and an object of the present invention is to provide a stripe illumination type observation apparatus that can easily extract a dark part when a composite image is created.

Observation apparatus of the first invention has a surface light source formed by providing a light and dark portions are alternately illuminating means for transmission illumination the I Rikomu observation object to the surface light source, illuminated by the illuminating means an imaging optical system for imaging light emitted from the object under observation, an imaging unit for obtaining an optical image of said object under observation which imaging optical system is formed, the transmissive illumination light is the object of the illumination means Illumination light correction means for correcting the light image of the bright and dark portions of the surface light source deformed by transmitting the observation object, and the phase of the light and dark portions of the surface light source corrected by the illumination light correction means while shifting And image synthesizing means for acquiring a plurality of light images in the imaging means and generating a composite image from the plurality of light images .

Observation apparatus according to the second invention, in the observation apparatus of the first aspect of the invention, the correction means, light dark light image acquired by the imaging means to correct the lighting conditions of the surface light source so that a straight line It is characterized by that.

The observation device according to a third aspect is the observation device according to the second aspect, wherein the correction means corrects an illumination state of the surface light source based on information about the transparent container, and information about the transparent container is transparent. It is characterized by the type of the container, the material of the transparent container, the type of the culture solution, and the amount of the culture solution.

The observation device according to a fourth aspect is the observation device according to the second aspect , wherein the correction means obtains a shape change of the bright and dark part from an image of the optical image of the observation object acquired by the imaging means, and the imaging The distortion correction factor for each pixel of the means is obtained, the illumination state of the surface light source is corrected, and the surface light source is a transmissive liquid crystal panel .

Fifth monitoring apparatus invention comprises an illuminating means for transmission illumination the I Rikomu observation object surface light source formed by providing a light and dark portions are alternately emitted from the object under observation which is illuminated by said illuminating means An imaging optical system for imaging light, an imaging means for acquiring an image of the object formed by the imaging optical system, and a plurality of sheets on the imaging means while shifting the light / dark phase of the surface light source The control means for acquiring an image, and the distortion of the bright and dark part of the image of the observed object acquired by the imaging means deformed by the transmitted illumination light of the illuminating means being transmitted through the observed object. And a correction means for image processing .

Sixth monitoring apparatus of the present invention is directed to the observation device of the fifth invention, the correcting means, by the imaging means in a state where the light dark portion of the illumination means in a straight line to acquire an image of each image acquired bright dark part and correcting so that a straight line.

Seventh monitoring apparatus of the present invention is directed to the observation apparatus of the sixth aspect of the invention, the correction means after the light dark portion of the acquired image is corrected to be a straight line, back to the original size of the object under observation Correction is performed.

  An observation apparatus according to an eighth invention is the observation apparatus according to any one of the first, fourth to seventh inventions, wherein the transparent container is a well plate having a plurality of wells, and a predetermined one well is corrected. It is characterized by being a well for acquiring only information for use.

  According to the present invention, it is possible to provide a stripe illumination type observation apparatus that can easily extract a dark part when creating a composite image.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
(First embodiment)
FIG. 1 shows a first embodiment of the observation apparatus of the present invention. This observation apparatus includes a transmissive liquid crystal panel 11, a stage (specimen stage) 13, an imaging optical system 15, an image sensor 17, a panel controller 19, a controller 21, and a display 23.

  A transparent culture vessel (96 well plate in the figure) 25 is placed on the stage 13. In the well 25a of the culture vessel 25, for example, an object to be observed such as unstained cells is accommodated.

  The transmissive liquid crystal panel 11 has a backlight and illuminates substantially the entire area of the culture vessel 25. The portion of the stage 13 on which the culture vessel 25 is placed is made of a transparent member such as glass or is hollow so that the illumination light is not obstructed.

  The visual field of the imaging optical system 15 is sufficiently large, and light emitted from substantially the entire area of the culture vessel 25 is captured by the imaging optical system 15 and forms an image on the imaging surface of the imaging element 17. The imaging element 17 acquires an image (luminance distribution) on the imaging surface in response to an instruction from the controller 21. The image is taken into the controller 21 and displayed on the display 23 after image processing. The controller 21 can store an image before image processing and an image after image processing as necessary. In addition, an image before image processing can be displayed on the display 23 as necessary.

  In response to an instruction from the controller 21, the panel controller 19 displays a stripe pattern in which bright portions and dark portions are repeatedly arranged on the transmissive liquid crystal panel 11. At this time, a striped surface light source is formed on the transmissive liquid crystal panel 11.

  FIG. 2 is a block diagram showing the configuration of the controller 21. The controller 21 includes an input unit 27, a stage control unit 29, a panel control unit 31, an illumination control unit 33, an image sensor control unit 35, and a CPU 37.

  The input unit 27 inputs an instruction from the user. Information that can be input by the user via the input unit 27 includes various instructions such as start of observation. In a second embodiment to be described later, the type of the culture vessel 25, the material of the culture vessel 25, the type of the culture solution (medium), the amount of the culture solution (medium), and the like are input.

  The stage control unit 29 controls the movement of the stage 13 and sets the position of the stage 13 in the optical axis direction (Z direction position) and the position in the direction perpendicular to the optical axis direction (XY direction position).

  The panel control unit 31 moves the transmissive liquid crystal panel 11 to set a position in the optical axis direction (Z direction position) and a position in the direction perpendicular to the optical axis direction (XY direction position). .

  The illumination control unit 33 controls the shape of the stripe pattern displayed on the transmissive liquid crystal panel 11 by controlling the panel controller 21.

  The image sensor control unit 35 performs drive control of the image sensor 17.

  The CPU 37 controls the stage control unit 29, panel control unit 31, illumination control unit 33, and image sensor control unit 35.

  FIG. 3 is a diagram for explaining the principle of the observation apparatus described above. FIG. 3 shows a state where the focusing plane P1 of the imaging lens 4 of the imaging optical system 15 is in the vicinity of the bottom surface of the well 25a of the culture vessel 25 and the cells 3a attached to the bottom surface are observed. In FIG. 3, reference numeral 1d indicates one dark part of the stripe pattern 1, and reference numeral 1b indicates bright parts located on both sides of the dark part 1d.

  Focusing on the partial region 3d facing the dark portion 1d in the well 25a of the culture vessel 25, the partial region 3d is illuminated from an oblique direction by the light Lb emitted from the two bright portions 1b, but light is emitted from the dark portion 1d. Since it does not inject, the partial area 3d is not illuminated from the front. That is, the partial area 3d is obliquely illuminated by the light Lb. Therefore, a dark field image 5d of the partial region 3d is formed in a region conjugate with the partial region 3d in the image sensor 17. In this dark field image 5d, an image of the cell 3a existing in the partial region 3d appears. This image (cell image) is not an image by direct light transmitted through the cell 3a but an image by scattered light scattered at the edge of the cell 3a.

  In the well 25a of the culture vessel 25, when attention is paid to the partial region 3b that faces the bright portion 1b, the partial region 3b is illuminated from the front by the bright portion 1b. Therefore, a bright field image 5b of the partial region 3b is formed in a region conjugate with the partial region 3b in the image sensor 17. In the bright field image 5b, an image of the cell 3a existing in the partial region 3b does not appear.

  Therefore, a stripe image in which the dark field image 5d including the cell image and the bright field image 5b not including the cell image are repeatedly arranged is formed on the image sensor 17. Therefore, if the imaging element 17 repeatedly performs imaging while shifting the phase of the stripe pattern displayed on the transmissive liquid crystal panel 11 (phase shift), and the dark field images 5d of a plurality of images obtained thereby are connected, the culture A composite image can be obtained in which the cells scattered throughout the container 25 can be viewed.

  For example, as shown in FIGS. 4A, 4B, and 4C, an image is picked up by the image pickup device 17 while shifting the phase of the stripe pattern by the shift pitch Δ, and the image shown in FIG. If the effective areas of the three images shown in A), (B), and (C) (the portion of width Ba ′ in FIG. 4) are connected, as shown in FIG. A composite image with a panoramic view of the cell group can be obtained.

  FIG. 7 is a flowchart showing the operation of the CPU 37 of the controller 21 relating to the observation of the object to be observed. This flow starts when the user inputs an observation start to the input unit 27.

  Step S1: The CPU 37 displays a linear stripe pattern as shown in FIG.

  Step S2: The CPU 37 captures an image of the observation object in the culture vessel 25 and acquires an image.

  FIG. 8 shows an image G <b> 1 acquired by the image sensor 17. This image G1 is an image of one well 25a when a 96-well plate is used as the transparent culture vessel 25. The straight stripe pattern displayed on the transmissive liquid crystal panel 11 is deformed into an arc shape due to the meniscus effect of the culture solution accommodated in the well 25a. Thus, when a linear stripe pattern is deformed into an arc shape or the like and imaged, only the dark part image is extracted and synthesized from a plurality of images imaged while shifting the phase as shown in FIG. It becomes difficult to do.

  That is, as shown in FIG. 9, when the culture solution 41 is stored in the well 25a of the 96-well plate, the outer peripheral portion of the surface of the culture solution 41 rises due to the surface tension. Then, due to the meniscus effect caused by the rise of the surface of the culture solution 41, the linear stripe pattern imaged on the image sensor 17 via the imaging optical system 15 is deformed into an arc shape. In the 96-well plate, the diameter of the well 25a is 6 mm, and the surface shape of the culture solution 41 is close to an arc-shaped lens shape. Therefore, the magnification of the object to be observed is maintained at a substantially uniform magnification.

  Step S3: The CPU 37 obtains the shape change of the dark part from the acquired image G1. This shape change is performed by recognizing the acquired image G1 and comparing it with the stripe pattern of the transmissive liquid crystal panel 11.

  Step S4: The CPU 37 calculates the distortion correction rate between the shape change of the dark part B1 of the acquired image G1 and the required linear pattern for each pixel, and applies this to apply the correction of the dark part of the acquired image. A correction shape is obtained so that the shape becomes a straight line. FIG. 10 shows the obtained corrected shape H1.

  Step S5: The CPU 37 displays the obtained corrected shape H1 on the transmissive liquid crystal panel 11. The correction shape H1 may be displayed only on the portion corresponding to the well 25a being observed on the 96 well plate, or may be displayed on the portion corresponding to the whole well 25a of the 96 well plate. good.

  Step S6: The CPU 37 repeats a series of processes including image acquisition and phase shift, and acquires m (for example, three) images set in advance.

  Step S7: The CPU 37 creates one composite image by arranging the effective areas of the acquired m images in the order of acquisition and connecting them.

  Step S8: The CPU 37 displays the composite image created in step S7 on the display 15 and ends the flow. Prior to the display, the contour of the cell image may be emphasized by performing contour extraction processing on the composite image.

In this embodiment, the image G1 is acquired by the imaging device 17 in a state where the dark portion of the transmissive liquid crystal panel 11 is a straight line, and the light and dark shape of the transmissive liquid crystal panel 11 is such that the dark portion B1 of the acquired image G1 is a straight line. Therefore, it is possible to easily extract a dark part when creating a composite image.
(Second Embodiment)
FIG. 11 is a flowchart showing the operation of the second embodiment of the observation apparatus of the present invention. In this embodiment, the same elements as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  Step S1: The CPU 37 determines whether or not information related to the culture vessel 25 is input to the input unit 27. The information regarding the culture vessel 25 includes the type of the culture vessel 25, the material of the culture vessel 25, the type of the culture solution (medium) 41, and the amount of the culture solution (medium) 41.

  Step S2: The CPU 37 obtains a correction pattern that makes the shape of the dark part of the acquired image a straight line based on the information input to the input unit 27.

As shown in FIG. 12, when the culture vessel 25 is a 96-well plate or the like, the curvature radius R of the surface of the culture solution 41 in the well 25a is approximately,
R = 2 · γ / g · h · ρ
It becomes.

  Here, γ is the surface tension and can be determined from the material of the culture vessel 25. g is a gravitational constant. h is the minimum surface height of the culture solution 41 and can be determined from the amount of the culture solution 41. ρ is the density of the culture solution 41 and can be obtained from the type of the culture solution 41. In FIG. 12, D is the inner diameter of the well 25a and can be determined from the type of the culture vessel 25.

  When the culture vessel 25 is placed on the stage 13, it is equivalent to inserting a lens having a radius of curvature R and a diameter D in the optical path of the normal imaging optical system 15 at a distance h from the bottom surface. Become an optical system. Therefore, the CPU 37 obtains the curvature radius R by the above-described equation, and obtains a correction pattern in which the dark portion captured when the curvature radius R is influenced by the lens having the diameter D becomes a straight line.

  That is, the CPU 37 determines the surface tension γ from the material of the culture vessel 25 input from the input unit 27, the minimum surface height h of the culture solution 41 from the amount of the culture solution 41, and the type of the culture solution 41 from the type of the culture solution 41. The density ρ is obtained and the curvature radius R is calculated. Then, the inner diameter D of the well 25a is obtained from the type of the culture vessel 25, and the distortion function (distortion) which is the aberration of the lens that is assumed that the bright and dark stripe pattern (FIG. 4) is influenced by the lens having the radius of curvature R and the diameter D. ) Specifically, this is a distortion function of aberration determined from a lens having a diameter D and the optical performance of the imaging optical system 15 of the image sensor 17. The correction pattern is a light / dark pattern formed by the inverse function of the obtained distortion function.

  FIG. 13A shows the obtained correction pattern H2. This correction pattern H2 is approximate to FIG. When this correction pattern H2 is displayed on the transmissive liquid crystal panel 11, the dark part B2 of the image G2 imaged by the image sensor 17 becomes a straight line as shown in FIG.

  Step S3: The CPU 37 displays the obtained correction pattern H2 on the transmissive liquid crystal panel 11.

  Step S4: The CPU 37 repeats a series of processes including image acquisition and phase shift, and acquires m (for example, three) images set in advance.

  Step S5: The CPU 37 creates one composite image by arranging and connecting the effective areas of the acquired m images in the order of acquisition.

  Step S6: The CPU 37 displays the composite image created in step S5 on the display 15, and ends the flow.

In this embodiment, since the bright and dark shape of the transmissive liquid crystal panel 11 is corrected so that the dark part B2 of the acquired image G2 is a straight line based on the information about the culture vessel 25, the dark part is extracted when the composite image is created. It can be done easily.
(Third embodiment)
FIG. 14 is a flowchart showing the operation of the third embodiment of the observation apparatus of the present invention. In this embodiment, the same elements as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  Step S1: The CPU 37 displays a linear stripe pattern as shown in FIG.

  Step S2: The CPU 37 images the calibration well 25a for acquiring correction information and acquires an image. For example, in the case of a 96 well plate, one well 25a out of 96 wells 25a is preset as a calibration well 25a. In this well 25a, only the culture solution 41 of the same amount as the well 25a in which the observation object is accommodated is accommodated, and the observation object is not accommodated. Therefore, calibration can be accurately performed without being affected by the object to be observed.

  FIG. 15A shows an image G <b> 3 acquired by the image sensor 17. This image G3 is an image of the well 25a when a 96-well plate is used as the transparent culture vessel 25, and is a straight line displayed on the transmissive liquid crystal panel 11 by the meniscus effect of the culture solution 41 accommodated in the well 25a. The stripe pattern is deformed in an arc shape.

  Step S3: The CPU 37 obtains correction data such that the dark part B3 of the acquired image G3 is a straight line. This correction data is performed by recognizing the acquired image G3 and comparing it with a straight stripe pattern. FIG. 15B shows a linear stripe pattern P3 displayed on the transmissive liquid crystal panel 11, and the width of the dark portion B4 is a predetermined width W. Further, it has a predetermined pitch P.

  Step S4: The CPU 37 repeats a series of processes including image acquisition and phase shift for the wells 25a other than the calibration well 25a, and acquires m images (for example, three images) set in advance.

  Step S5: The CPU 37 corrects so that the dark part of the acquired m images becomes a straight line. This correction is performed using the correction data obtained in step S3. This correction causes an error in the size of the object to be observed.

  Step S6: The CPU 37 creates one composite image by arranging the effective areas of the acquired m images in the order of acquisition and connecting them.

  Step S7: The CPU 37 restores the size of the object to be observed in the composite image created in step S6. This correction is performed using the correction data obtained in step S3. This correction can eliminate the size error of the object to be observed. If high accuracy is not required for the size of the object to be observed, this step may be omitted.

  Step S8: The CPU 37 displays the composite image created in step S7 on the display 15 and ends the flow.

In this embodiment, the image G3 is acquired by the imaging device 17 with the dark part of the transmissive liquid crystal panel 11 being a straight line, and the dark part B3 of the acquired image G3 is corrected to be a straight line. Sometimes dark areas can be easily extracted.
(Supplementary items of the embodiment)
As mentioned above, although this invention was demonstrated by embodiment mentioned above, the technical scope of this invention is not limited to embodiment mentioned above, For example, the following forms may be sufficient.

  (1) In the above-described embodiment, the transmissive liquid crystal panel 11 with a backlight is used to generate the surface light source. However, other devices may be used as long as the same surface light source can be generated. . For example, a large number of small light sources such as LEDs may be prepared, and these may be densely arranged in an array to form a surface light source, and a mask member having a stripe-shaped opening may be disposed on the surface light source.

  (2) In the above-described embodiment, the example in which the present invention is applied to the observation of the culture container 25 composed of a 96-well plate has been described. However, the present invention can also be applied to a culture container composed of a flask or the like. For example, at the corner of the flask, the linear stripe pattern is relatively largely deformed by the meniscus effect, but can be processed in the same manner as in the above-described embodiment.

  (3) In the second embodiment described above, an example in which the correction pattern is set by a calculation formula has been described. However, for example, it corresponds to the type of the culture vessel, the material of the culture vessel, the type of the culture solution, the amount of the culture solution, and the like. Then, the correction pattern may be set by a reference table that can be set.

It is explanatory drawing which shows 1st Embodiment of the observation apparatus of this invention. It is a block diagram which shows the controller of the observation apparatus of FIG. It is explanatory drawing explaining the background luminance distribution of a dark field image. It is explanatory drawing which shows the stripe pattern displayed during a phase shift. It is explanatory drawing which shows the image acquired during the phase shift. It is explanatory drawing which shows the dark field image formed by synthesize | combining each effective area | region of FIG. It is explanatory drawing which shows operation | movement of CPU regarding observation. It is explanatory drawing which shows the image of the stripe pattern acquired by the image pick-up element. It is explanatory drawing for demonstrating the meniscus effect by a culture solution. It is explanatory drawing which shows the correction | amendment shape of a stripe pattern. It is explanatory drawing which shows operation | movement of 2nd Embodiment of the observation apparatus of this invention. It is explanatory drawing which shows the curvature radius of the surface of a culture solution. It is explanatory drawing which shows the correction | amendment shape of a stripe pattern. It is explanatory drawing which shows operation | movement of 3rd Embodiment of the observation apparatus of this invention. It is explanatory drawing which shows the image of the stripe pattern acquired by the image pick-up element.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 ... Transmission-type liquid crystal panel, 13 ... Stage, 15 ... Imaging optical system, 17 ... Imaging element, 21 ... Controller, 25 ... Culture container, 25a ... Well.

Claims (8)

Has a surface light source formed by providing alternating between bright and dark, and illuminating means for transmitting illuminating the O Rikomu observation object to the surface light source,
An imaging optical system that forms an image of light emitted from the object illuminated by the illumination means;
An imaging means for acquiring a light image of the observation object formed by the imaging optical system;
Illumination light correction means for correcting a light image of a bright and dark part of the surface light source deformed by transmission of the illumination light of the illumination means through the object to be observed;
Image synthesizing means for acquiring a plurality of light images in the imaging means while shifting the phase of the bright and dark portions of the surface light source corrected by the illumination light correcting means, and generating a synthesized image from the plurality of light images;
An observation apparatus comprising: The observation apparatus according to claim 1,
Said correction means, an observation apparatus bright dark light image acquired by the imaging means and corrects the illumination state of the surface light source so that a straight line. The observation apparatus according to claim 2,
The correction means corrects the illumination state of the surface light source based on information on the transparent container,
The information regarding the transparent container is the type of the transparent container, the material of the transparent container, the type of the culture solution, and the amount of the culture solution. The observation apparatus according to claim 2 ,
The correction means obtains the shape change of the bright and dark part from the image of the optical image of the observation object acquired by the imaging means, obtains the distortion correction rate for each pixel of the imaging means, and determines the illumination state of the surface light source Correct ,
The observation apparatus , wherein the surface light source is a transmissive liquid crystal panel . Illuminating means for transmission illumination the I Rikomu observation object surface light source formed by providing alternating between bright and dark,
An imaging optical system that forms an image of light emitted from the object illuminated by the illumination means;
Imaging means for acquiring an image of the object to be observed formed by the imaging optical system;
Control means for causing the imaging means to acquire a plurality of images while shifting the light-dark phase of the surface light source;
Correction means for performing image processing so as to correct distortion of a bright and dark part of the image of the object acquired by the imaging means deformed by transmission of the transmitted illumination light of the illumination means through the object ;
An observation apparatus comprising: The observation apparatus according to claim 5, wherein
It said correction means, observation apparatus obtains an image by the image pickup means while the bright dark portion in a straight line, light dark portion of each image acquired and correcting so that the straight line of the illumination means. The observation apparatus according to claim 6, wherein
Wherein the correction means, the observation light dark portion of the acquired image after correcting to a straight line, and performs a correction to restore the size of the object under observation device. The observation device according to any one of claims 1 and 4 to 7,
The transparent device is a well plate having a plurality of wells, and a predetermined one well is a well that acquires only correction information.

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