WO2015093344A1

WO2015093344A1 - Image capturing apparatus and image capturing method

Info

Publication number: WO2015093344A1
Application number: PCT/JP2014/082531
Authority: WO
Inventors: 博末木; 憲司上山
Original assignee: 株式会社Ｓｃｒｅｅｎホールディングス
Priority date: 2013-12-19
Filing date: 2014-12-09
Publication date: 2015-06-25
Also published as: JP2015118036A; JP6195373B2

Abstract

　Provided is an image capturing apparatus (1) for capturing an image of a biological specimen which is supported, together with a liquid, in a flat-bottomed well (W) provided to a specimen container (WP), wherein the apparatus is provided with: a holding means (11) for holding the specimen container such that the bottom surface of the well is horizontal; an illuminating means (12) for causing illuminating light to be incident on the opening surface of the well from above the specimen container; an image capturing means (134) for receiving light transmitted from the bottom surface of the well, and capturing an image of the biological specimen; and an observation optical system (130) provided between the image capturing means and the specimen container held by the holding means, the observation optical system causing light exiting from the bottom surface of the well to be incident on the image capturing means. The observation optical system has an objective lens (131), and an aperture stop (132) provided between the objective lens and the image capturing means, the aperture stop being arranged closer to the image capture means than is the focal position of the objective lens.

Description

Imaging apparatus and imaging method

The present invention relates to an imaging apparatus and an imaging method for imaging a biological sample carried with a liquid in a sample container.

Cross-reference to related applications The disclosures in the specification, drawings, and claims of the following Japanese application are incorporated herein by reference in their entirety:
Japanese Patent Application No. 2013-262352 (filed on Dec. 19, 2013).

In medical and biological science experiments, for example, each well of a plate-like sample container (called a microplate, microtiter plate, etc.) provided with a large number of depressions called wells is filled with liquid or gel. A fluid (for example, a culture solution, a medium, etc.) is injected, and the cells cultured here are observed and measured as a sample. In recent years, a sample is imaged with a CCD camera or the like and converted into data, and various image processing techniques are applied to the image data for observation and analysis.

In such an imaging apparatus, for example, when illumination light is incident on the sample from the top of the well and the light transmitted from the bottom of the well is received for imaging, there are the following problems. That is, the illumination light incident through the liquid surface is refracted by the meniscus action, so that sufficient illumination light is not incident particularly on the peripheral edge of the well bottom surface, and the brightness of the image is lowered at this portion. As a technique for dealing with this problem, for example, Patent Document 1 discloses a configuration in which illumination light is incident on a sample from an oblique direction by a diaphragm provided in an illumination optical system.

JP 2012-147739 A

Patent Document 1 does not describe in detail the configuration of an observation optical system that receives light transmitted through a sample. However, when an illumination optical system that performs illumination from an oblique direction as in the above-described prior art is used, it is considered that a lens having a large numerical aperture (NA) is required for the observation optical system that collects transmitted light. Such a lens is expensive to manufacture and has a shallow depth of field. For this reason, for the purpose of observing the three-dimensional structure of the sample, the entire object may not be included in the focus range. For this reason, there is a demand for a technique that can capture an image of an imaging object having a three-dimensional structure satisfactorily with a simple structure and sufficient brightness.

The present invention has been made in view of the above problems, and can capture images with sufficient brightness up to the peripheral edge of the sample container while having a simple configuration, and is also effective for a biological sample having a three-dimensional structure. An object is to provide an imaging technique.

One aspect of the present invention is an imaging apparatus for imaging a biological sample provided with a liquid in a recess provided in a sample container and having a flat bottom surface. In order to achieve the above object, the bottom surface of the recess Holding means for holding the sample container so as to be horizontal, illumination means for making illumination light enter the opening surface of the recess from above the sample container, and receiving light transmitted from the bottom surface of the recess The imaging means for imaging the biological sample, the sample container held by the holding means, and the imaging means are provided, and light emitted from the bottom surface of the recess is incident on the imaging means An observation optical system, and the observation optical system is provided on the optical path between the holding means and the imaging means, and on the optical path between the objective lens and the imaging means. An aperture stop that is Mouth aperture is characterized in that disposed on the imaging device side than the focal position of the objective lens.

According to another aspect of the present invention, there is provided a step of holding a sample container carrying a biological sample together with a liquid in a recess having a flat bottom so that the bottom of the recess is horizontal; An image pickup means is disposed so as to receive light transmitted from the bottom surface, an observation optical system is disposed between the sample container and the image pickup means, and an opening surface of the recess from above the sample container. A step of causing illumination light to enter; and a step of causing the light transmitted from the bottom surface of the recess to enter the imaging means by the observation optical system to image the biological sample, the observation optical system including the holding And an objective lens disposed on the optical path between the imaging means and an aperture stop disposed on the optical path between the objective lens and the imaging means. Take the picture above the focal point of the lens. It is characterized by placing the unit side.

As will be described in detail later, in the invention configured as described above, the light whose traveling path is bent by the meniscus effect of the liquid surface and travels outward from the peripheral portion of the bottom surface of the recess is allowed to pass through the objective lens and the aperture stop. It can be incident on the imaging means. For this reason, the light from the peripheral portion can be collected as efficiently as the light from the central portion, and a bright image can be obtained. In addition, since such an effect is obtained by the arrangement of the aperture stop, it is not necessary to increase the numerical aperture (NA) of the optical system. For this reason, since a comparatively deep depth of field can be obtained, it is also suitable for imaging a three-dimensional sample.

Thus, according to the present invention, it is possible to take an image with sufficient brightness up to the peripheral edge of the bottom surface of the sample container with a simple configuration, and even with a sample having a three-dimensional structure, the image is taken with good image quality. It is possible.

The above and other objects and novel features of the present invention will become more fully apparent when the following detailed description is read with reference to the accompanying drawings. However, the drawings are for explanation only and do not limit the scope of the present invention.

1 is a diagram showing a schematic configuration of a first embodiment of an imaging apparatus according to the present invention. It is a 1st figure which shows typically the light which permeate | transmits a well. It is a 2nd figure which shows typically the light which permeate | transmits a well. It is a figure which shows typically the light which permeate | transmits a well and is received by the imaging device. It is a figure which shows the positional relationship of an observation optical system. It is a figure which shows the observation optical system of the prior art as a comparative example. It is a 1st figure which shows the example of the imaged image. It is a 2nd figure which shows the example of the imaged image. It is a figure which shows 2nd Embodiment of the imaging device concerning this invention. It is a figure which shows 3rd Embodiment of the imaging device concerning this invention. It is a 1st figure for demonstrating the problem accompanying a scanning movement. It is a 2nd figure for demonstrating the problem accompanying a scanning movement. It is a disassembled perspective view which shows the opening shape of the aperture stop in 3rd Embodiment. It is a figure which shows the optical path of the light radiate | emitted from a well peripheral part. It is a 1st figure which shows the example of the imaged image. It is a 2nd figure which shows the example of the imaged image. It is the 1st figure showing a 4th embodiment of an imaging device concerning this invention. It is a 2nd figure which shows 4th Embodiment of the imaging device concerning this invention. It is a 1st figure which shows the example of the imaged image. It is a 2nd figure which shows the example of the imaged image.

<First Embodiment>
FIG. 1 is a diagram showing a schematic configuration of a first embodiment of an imaging apparatus according to the present invention. This imaging apparatus 1 is an apparatus that images a biological sample in a liquid injected into a recess called a well W formed on the upper surface of a well plate WP. Hereinafter, in order to uniformly indicate the directions in the drawings, XYZ orthogonal coordinate axes are set as shown in FIG. Here, the XY plane represents a horizontal plane and the Z axis represents a vertical axis. More specifically, the (+ Z) direction represents a vertically upward direction.

The well plate WP is generally used in the fields of drug discovery and biological science. A plurality of wells W having a substantially circular cross section and a transparent bottom surface are provided on the upper surface of the flat plate WP. Although the number of wells W in one well plate WP is arbitrary, for example, 96 (12 × 8 matrix arrangement) can be used. The diameter and depth of each well W is typically about several mm. Note that the size of the well plate and the number of wells targeted by the imaging apparatus 1 are not limited to these, and may be arbitrary, for example, may be a generally available 384 hole.

A predetermined amount of liquid as a medium is injected into each well W of the well plate WP. Biological samples such as cells and bacteria that are cultured in this liquid under predetermined culture conditions are imaging targets of the imaging device 1. The medium may be one to which an appropriate reagent has been added, or it may be in a liquid state and gelled after being placed in the well W. As will be described later, in the imaging apparatus 1, for example, spheroids (cell agglomerates) cultured on the inner bottom surface of the well W can be set as an imaging target. Commonly used liquid volume is about 50 to 200 microliters.

The imaging apparatus 1 includes a holder 11 that holds a well plate, an illumination unit 12 that is disposed above the holder 11, an imaging unit 13 that is disposed below the holder 11, and a CPU 141 that controls operations of these units. And a control unit 14. The holder 11 is in contact with the peripheral edge of the lower surface of the well plate WP carrying the biological sample together with the liquid in each well W, and holds the well plate WP in a substantially horizontal posture.

The illumination unit 12 emits appropriate diffused light (for example, white light) toward the well plate WP held by the holder 11. More specifically, for example, a combination of a white LED (Light-Emitting-Diode) light source 121 as a light source and a diffusion plate 122 can be used as the illumination unit 12. The illumination unit 12 illuminates the biological sample in the well W provided on the well plate WP from above.

An imaging unit 13 is provided below the well plate WP held by the holder 11. In the imaging unit 13, an objective lens 131 is disposed immediately below the well plate WP. The optical axis of the objective lens 131 is directed in the vertical direction (Z direction), and the aperture stop 132, the imaging lens 133, and the imaging device 134 are further provided in order from the top to the bottom along the optical axis of the objective lens 131. Is provided. The objective lens 131, the aperture stop 132, and the imaging lens 133 are arranged so that their centers are aligned in a line along the vertical direction (Z direction), and these constitute an observation optical system 130 as a unit. In this example, the components constituting the imaging unit 13 are arranged in a line in the vertical direction. However, as long as the distance (optical path length) between the components satisfies the relationship described later, the optical path is folded by a reflecting mirror or the like. May be.

The imaging unit 13 can be moved in the XYZ directions by a mechanical drive unit 146 provided in the control unit 14. Specifically, based on a control command from the CPU 141, the mechanical drive unit 146 integrally connects the objective lens 131, the aperture stop 132, the imaging lens 133, and the imaging device 134 that constitute the imaging unit 13 in the X direction and the Y direction. Move to. Thereby, the imaging unit 13 moves in the horizontal direction with respect to the well W. When the imaging object in one well W is imaged, the mechanical drive unit 146 positions the imaging unit 13 in the horizontal direction so that the optical axis of the observation optical system 130 coincides with the center of the well W.

Further, the mechanical drive unit 146 performs the focusing of the imaging unit 13 with respect to the imaging target by moving the imaging unit 13 in the Z direction. Specifically, the mechanical drive unit 146 includes the objective lens 131, the aperture stop 132, the imaging lens 133, and the imaging so that the objective lens 131 is focused on the inner bottom surface of the well W where the biological sample that is the imaging target is present. The device 134 is moved up and down integrally.

The mechanical drive unit 146 moves the illumination unit 12 integrally with the imaging unit 13 in the XY direction when moving the imaging unit 13 in the XY direction. That is, the illumination unit 12 is arranged so that the optical center thereof substantially coincides with the optical axis of the observation optical system 130. When the imaging unit 13 including the objective lens 131 moves in the XY direction, the illumination unit 12 is interlocked with this. Move in the XY direction. Thus, regardless of which well W is imaged, the center of the well W and the optical center of the illumination unit 12 are always located on the optical axis of the observation optical system 130. Thereby, the illumination conditions for each well W can be made constant and the imaging conditions can be maintained well.

The biological sample in the well W is imaged by the imaging unit 13. Specifically, light emitted from the illumination unit 12 and incident on the liquid from above the well W illuminates the imaging target, and light transmitted downward from the bottom surface of the well W is collected by the objective lens 131. An image of the imaging object is finally formed on the light receiving surface of the imaging device 134 via the aperture stop 132 and the imaging lens 133, and this is received by the light receiving element 1341 of the imaging device 134. The light receiving element 1341 is a two-dimensional image sensor, and converts a two-dimensional image of the imaging object formed on the surface thereof into an electrical signal. As the light receiving element 1341, for example, a CCD sensor or a CMOS sensor can be used.

The distance between the bottom surface of the well W (more precisely, the inner bottom surface) and the objective lens 131 is the focal length f1 of the objective lens 131, while the distance d between the objective lens 131 and the aperture stop 132 is the focal length of the objective lens 131. It arrange | positions so that it may become longer than f1. Further, the distance between the aperture stop 132 and the imaging lens 133 and the distance between the imaging lens 133 and the imaging device 134 are all the focal length f2 of the imaging lens 133. The reason for this arrangement will be described in detail later.

The image signal output from the light receiving element 1341 is sent to the control unit 14. That is, the image signal is input to an AD converter (A / D) 143 provided in the control unit 14 and converted into digital image data. The CPU 141 executes image processing as appropriate based on the received image data. The control unit 14 further includes an image memory 144 for storing and storing image data, and a memory 145 for storing and storing programs to be executed by the CPU 141 and data generated by the CPU 141. It may be integral.

In addition, the control unit 14 is provided with an interface (I / F) 142. The interface 142 receives an operation input from the user, presents information such as a processing result to the user, and exchanges data with an external apparatus connected via a communication line.

2A and 2B are diagrams schematically showing light transmitted through the well. FIG. 3 is a diagram schematically showing light that passes through the well and is received by the imaging device. As shown in FIG. 2A, the cylindrical well W provided in the well plate WP has a holder so that the central axis of the cylinder coincides with the vertical axis, that is, the opening surface St and the bottom surface Sb are substantially horizontal. 11 is held. At this time, the opening surface St and the bottom surface Sb of the well W are concentric in the vertical direction. The opening surface St and the bottom surface Sb of the well W do not have to be completely the same size. For example, even if the well side wall surface has an inverted truncated cone shape and the bottom surface Sb is slightly smaller than the opening surface St. Good.

The liquid L is injected into the well W to be imaged, and a generally downward meniscus is formed on the liquid surface. If it is assumed that no liquid is injected or the liquid level is flat, as shown by a solid line arrow in FIG. 2A, light incident from the opening surface St of the well W travels through the well W as it is. From the bottom surface Sb of the lens, and is condensed by the objective lens 131 toward the imaging device 134.

However, the light is refracted by the meniscus on the liquid level as indicated by the dotted arrow. Therefore, in particular, the illumination light passing through the peripheral portion (hereinafter referred to as “well peripheral portion”) We of the well bottom surface Sb travels in a direction away from the optical axis of the objective lens 131. Therefore, in a conventional imaging apparatus using an observation optical system that does not consider this point, light emitted from the well peripheral portion We cannot be sufficiently incident on the imaging device, and as a result, in the well peripheral portion We. The image becomes dark.

The observation optical system 130 of the present embodiment has a configuration that can solve this problem. The concept will be described below. FIG. 2B is an optical path diagram showing a typical path of light passing through the well W. FIG. 3 is an optical path diagram showing a course until these lights are finally received by the imaging device 134. In these drawings, as indicated by solid lines and dotted lines, light emitted from various portions of the illumination unit 12 passes through the well peripheral edge We. Of these, attention is focused on light rays passing through the center of the opening surface St of the well W indicated by a solid line. This light beam corresponds to the principal ray of the aperture when the opening surface St of the well W is regarded as a kind of aperture.

Consider the light Le that is emitted from the point P of the illumination unit 12 and passes through the center of the well opening surface St and passes through the well peripheral portion We. At this time, an angle between the direction of the light Le emitted from the well bottom surface Sb and the optical axis direction of the observation optical system 130 indicated by a one-dot chain line in FIG. Due to refraction at the liquid surface, this angle θ becomes larger than the incident angle θ1 when entering the aperture surface St.

The inventor of the present application performed a light distribution simulation by changing the amount of liquid to be injected into the well W in various ways using a commercially available general 96-well well plate WP having a well diameter of 7 mm. As a result, the angle θ formed by the light distribution center of gravity with respect to the optical axis of the observation optical system was in the range of 14 degrees to 18 degrees. Further, in the case of a 384 well and a square well with a side of 3.5 mm, the value of the angle θ was in the range of 8 to 15 degrees. In other various well plates, the value of the angle θ is estimated to be approximately 10 degrees.

According to the knowledge of the inventor of the present application, as shown in FIG. 3, by installing the aperture stop 132 at a position where the light Le passes through the center of the aperture stop 132, the light emitted from the well peripheral edge We is efficiently captured. It is possible to lead to. That is, the aperture stop 132 may be installed at a position where the optical path of the light Le intersects the optical axis of the observation optical system 130. At this time, the principal ray of light emitted from the well peripheral portion We and incident on the aperture stop 132 via the objective lens 131 is the principal ray of light incident from the opening surface St of the well W and passing through the well peripheral portion We. Will match. Thereby, the light emitted from the well peripheral portion We can be effectively guided to the imaging device 134. In order to achieve such a state, the aperture stop 132 is provided farther from the objective lens 131 than its focal length f1, that is, at a position closer to the imaging device 134 than the focal position of the objective lens 131. That is, in FIG. 3, the relationship of d> f1 is established.

As for the aperture size of the aperture stop 132, the lights L1 and L2 having the largest incident angle to the well aperture surface St among the light incident on the well aperture surface St from the illumination unit 12 and emitted from the well peripheral portion We. The size can be passed. By doing so, all of the light that illuminates the well peripheral portion We passes through the aperture stop 132 and reaches the imaging device 134, and the brightness of the image at the well peripheral portion We can be maximized. However, if the aperture size of the aperture stop 132 is increased, the depth of field may become shallower or the image formation state may deteriorate, so it is actually necessary to consider the balance between image brightness and image quality.

FIG. 4 is a diagram showing the positional relationship of the observation optical system. The focal length of the objective lens 131 is denoted by f1, the distance between the objective lens 131 and the aperture stop 132 is denoted by d, the distance from the rear focal point of the objective lens 131 to the aperture stop 132 is denoted by d2, and the focal length of the imaging lens 133 is defined. This is represented by reference numeral f2. Further, the distance from the apparent well opening surface St to the well bottom surface Sb when viewed from the objective lens 131 side through the liquid surface of the liquid L in the well W is represented by reference sign d1. Further, the radius of the well bottom surface Sb is represented by the symbol R.

As a relationship for the light Le to pass through the center of the aperture stop 132, from the distance of each part and the Newton imaging formula shown in FIG.
d2 = f1 × f1 / d1 = f1 × f1 / (R / tan θ) (Equation 1)
Is obtained. Therefore, the distance d between the objective lens 131 and the aperture stop 132 is expressed by the following formula:
d = f1 + d2 = f1 + f1 × f1 / (R / tan θ) (Expression 2)
It is represented by Here, as representative dimensions of each part, for example,
f1 = 45mm
2R = 6.5mm
If θ = 18 degrees, the distance d is about 247 mm.

Although the distance from the aperture stop 132 to the imaging lens 133 and the distance from the imaging lens 133 to the imaging device 134 are arbitrary, for example, both are made to coincide with the focal length f2 of the imaging lens 133. be able to. By doing so, a telecentric optical system can be configured between the aperture stop 132 and the imaging device 134, and the image of the imaging target imaged on the imaging device 134 is sharpened, and the image quality is improved. can do.

FIG. 5 is a diagram showing a conventional observation optical system as a comparative example. In this observation optical system, an illumination unit LS is provided above the well W, and an image of the well bottom surface Sb is formed on the light receiving surface of the imaging device IM at the same magnification by the camera lens CL disposed below the well W. It is something to be made. In such an observation optical system, the distance from the well bottom surface Sb to the camera lens CL and the distance from the camera lens CL to the imaging device IM are each set to twice the focal length f0 of the camera lens CL.

6A and 6B are diagrams showing examples of captured images. FIG. 6A is an example of an image captured using the conventional observation optical system shown in FIG. 5, and the image is dark in a region corresponding to the peripheral edge of the well. In contrast, FIG. 6B is an example of an image captured using the observation optical system 130 of the present embodiment, and it can be seen that a bright image is obtained up to the peripheral edge of the well.

As described above, in this embodiment, the aperture stop 132 disposed behind the objective lens 131 when viewed from the well bottom surface Sb on which the biological sample as the imaging target is distributed is further rearward than the focal point of the objective lens 131 ( Arranged on the imaging device side). As a result, light spreading in a direction away from the optical axis of the observation optical system 130 due to refraction at the liquid surface in the well W can be collected more efficiently and guided to the imaging device 134. Therefore, in this embodiment, an image having the same brightness as that of the well central portion can be taken even in the peripheral portion of the well.

Further, the numerical aperture (NA) of the observation optical system 130 does not need to be increased as in the conventional technique in which the illumination light described in the above-described patent document is incident obliquely. Therefore, a special lens design with good imaging performance is not required. In addition, since the depth of field of the observation optical system can be increased, even an imaging object having a three-dimensional structure such as a spheroid Sp can be captured clearly. In particular, since the appearance of the shadow of the object to be imaged in the image is almost the same in the entire visual field, it is suitable for imaging for the purpose of observing a biological sample having a three-dimensional structure.

As described above, it is most preferable that the light Le incident on the center of the well opening surface St and passing through the well peripheral edge We passes through the center of the aperture stop 132 and the relationship of the above (Formula 2) is satisfied. . Therefore, strictly speaking, it is necessary to adjust the position of the aperture stop 132 in accordance with the size of the well and the type and amount of the liquid to be injected. However, the types of well plates WP actually used in this type of culture experiment and the range of the amount of the culture solution are limited to some extent. If the position of the aperture stop 132 is set in accordance with standard culture conditions, it is possible to obtain a sufficient image quality improvement effect at least as compared with the prior art.

As described above, under practical culture conditions, the spread angle θ of the light Le emitted from the well peripheral edge We is about 8 degrees to 18 degrees. According to the experiment of the present inventor, if the aperture stop 132 is arranged so that the incident angle φ (FIG. 4) of the light emitted from the well peripheral portion We and incident on the aperture stop is about 5 degrees to 30 degrees, For biological samples prepared under these general culture conditions, it is possible to perform imaging with sufficient brightness up to the peripheral edge of the well. Naturally, the incident angle φ is more preferably in the range of 8 degrees to 18 degrees, and more ideally, the position of the aperture stop 132 is desirably adjusted in order to obtain an angle corresponding to the sample.

Second Embodiment
Next, a second embodiment of the imaging apparatus according to the present invention will be described. As described above, in the imaging apparatus according to the present invention, the aperture stop is provided at a position closer to the imaging device side than the focal position of the objective lens. By doing so, light spreading in a direction away from the optical axis of the observation optical system due to refraction at the liquid surface is efficiently collected, and a bright image is obtained up to the peripheral edge of the well. In order to perform imaging under the best conditions, it is desirable to adjust the position of the aperture stop according to the sample. In the first embodiment, the position of the aperture stop is set in accordance with a standard sample. However, in the second embodiment described below, the position of the aperture stop is variable, and the imaging condition corresponding to the sample is set. Can be optimized.

FIG. 7 is a diagram showing a second embodiment of the imaging apparatus according to the present invention. The imaging device 2 of this embodiment is partially different from that of the first embodiment in the configuration of the imaging unit. More specifically, the imaging unit 23 of this embodiment is configured to be able to change the distance between the objective lens and the aperture stop in the observation optical system. Except for this point, the apparatus configuration and operation are the same as those in the first embodiment. Therefore, here, components having the same functions as those of the first embodiment are denoted by the same reference numerals, description thereof is omitted, and portions different from those of the first embodiment will be mainly described.

In this embodiment, the objective lens 231, the aperture stop 232, the imaging lens 233, and the imaging device 234 that constitute the imaging unit 23 are integrally moved up and down by the mechanical drive unit 146 to adjust the focus to the well bottom surface Sb. It is executable. In addition to this, only the aperture stop 232, the imaging lens 233, and the imaging device 234 can be integrally moved up and down by the mechanical drive unit 146 while the objective lens 231 is fixed.

Therefore, in this embodiment, the distance between the objective lens 231 and the aperture stop 232 can be changed while maintaining the telecentric optical system including the aperture stop 232, the imaging lens 233, and the imaging device 234. As a result, the position of the aperture stop 232 can be adjusted according to the sample, and the brightness of the image at the periphery of the well can be equally maintained even between different samples. In addition, raising / lowering of the aperture stop 232 may be performed manually by the user. At this time, if the image picked up by the image pickup device 234 is displayed in real time, the user can efficiently perform the adjustment work while checking the brightness of the image.

<Third Embodiment>
Next, a third embodiment of the imaging apparatus according to the present invention will be described. In the imaging devices of the first and second embodiments described above, a two-dimensional image sensor is used as the imaging device. Alternatively, it is possible to perform imaging using a linear image sensor that captures a one-dimensional image. That is, a two-dimensional image can be acquired by scanning and moving the linear image sensor in a direction crossing the longitudinal direction.

In this case, it is also possible to replace only the imaging device from the two-dimensional image sensor to the linear image sensor in the imaging apparatus of the first or second embodiment described above. In that case, the linear image sensor is scanned and moved while the observation optical system is positioned below the well W to be imaged. Therefore, it is necessary that the observation optical system and the imaging device can be moved individually in the horizontal direction.

On the other hand, the imaging apparatus according to the third embodiment described below has a configuration capable of performing imaging by scanning and moving the observation optical system and the imaging device integrally in the horizontal direction. That is, a mechanism for moving the imaging device independently of the observation optical system is not required.

FIG. 8 is a diagram showing a third embodiment of the imaging apparatus according to the present invention. In this embodiment as well, the same components as those in the first embodiment are denoted by the same reference numerals and description thereof is omitted. In the imaging unit 33 of the imaging apparatus 3 according to the third embodiment, the imaging device 334 includes a linear image sensor. More specifically, the imaging device 334 includes a linear image sensor 3341 that captures a one-dimensional image along the X direction as a longitudinal direction. In the imaging unit 33, the objective lens 331, the aperture stop 332, the imaging lens 333, and the imaging device 334 are integrally scanned and moved in the Y direction with respect to the well plate WP by the mechanical drive unit 146 of the control unit 14. . Therefore, in this embodiment, the position of the optical axis itself of the observation optical system 330 changes with respect to the well W to be imaged.

9A and 9B are diagrams for explaining problems associated with scanning movement. In the first and second embodiments described above, the aperture shape of the aperture stop can be, for example, circular. However, when the aperture of the aperture stop 332 is similarly circular in the third embodiment, the following problem occurs. is there. That is, as shown in FIG. 9A, when the optical axis (indicated by the alternate long and short dash line) of the observation optical system 330 passes near the center of the well W, most of the light emitted from the well bottom surface Sb passes through the objective lens 331. Can pass through the aperture stop 332. Note that the light emitted from the well peripheral edge We also passes through the aperture stop 332 at this time, but does not enter the light receiving surface of the linear image sensor 3341.

On the other hand, as shown in FIG. 9B, as the optical axis of the observation optical system 330 approaches the well peripheral edge We, the incident direction of the light emitted from the well bottom surface Sb to the objective lens 331 is different from that in FIG. 9A. come. For this reason, the light from the well peripheral portion We is shielded by the aperture stop 332. That is, the image becomes dark at the well peripheral edge We.

10A and 10B are exploded perspective views showing the aperture shape of the aperture stop according to the third embodiment. Here, in order to facilitate understanding, the well plate WP in which only one well W is virtually formed is illustrated. As shown in FIG. 10A, the observation optical system 330 including the objective lens 331, the aperture stop 332, and the imaging lens 333, and the imaging device 334 are integrally formed in a predetermined scanning direction Ds (here, coincides with the Y direction). Move to. Although not shown, the same applies to the illumination unit 12. The imaging device 334 includes a linear image sensor 3341 whose longitudinal direction is the X direction orthogonal to the scanning direction Ds (that is, the Y direction), and captures a two-dimensional image by scanning and moving the imaging target in the Y direction. .

Here, the aperture stop 332 has a slit-like opening extending thinly with the scanning direction Ds of the linear image sensor 3341, that is, the Y direction as the longitudinal direction. Therefore, as shown in FIG. 10B, even when the optical axis (one-dot chain line) of the observation optical system 330 is close to the well peripheral portion We, the light emitted from the well peripheral portion We passes through the aperture stop 332 and the imaging device. 334. This improves the problem that the image becomes dark at the periphery of the well.

FIG. 11A and FIG. 11B are diagrams showing examples of captured images. FIG. 11A shows, as a comparative example, an image captured using an aperture stop having a small aperture size shown in FIG. 9A. Images are darker at both ends in the scanning direction of the imaging device 334 than at the center. On the other hand, in the imaging result using the aperture stop 332 opened in the slit shape of this embodiment, sufficient brightness is obtained at both ends in the scanning direction as shown in FIG. 11B.

<Fourth embodiment>
12A and 12B are views showing a fourth embodiment of the imaging apparatus according to the present invention. In the first to third embodiments described above, the spheroid Sp that is three-dimensionally cultured in the liquid injected into the well W of the well plate WP, more specifically, the inner bottom surface of the well W, is the imaging target. Since the spheroid Sp has a three-dimensional structure, diffused light is used as illumination light, and the numerical aperture (NA) of the observation optical system is set to be relatively small and the depth of field is large. On the other hand, as an imaging target to be imaged using this type of imaging apparatus, there is one in which cells C are flat-cultured so as to spread thinly along the inner bottom surface of the well W as shown in FIG.

According to the experiments by the inventors of the present application, in imaging of a biological sample that has been cultured in this manner, there is a case where a clearer image is obtained by illumination with light close to parallel light than by illumination with diffused light. The imaging device 4 according to the fourth embodiment shown in FIG. 12B can be suitably applied to such a case. In FIG. 12B, the same components as those in the first embodiment are denoted by the same reference numerals and description thereof is omitted.

In this imaging apparatus 4, the illumination unit 42 is disposed at a position far away from the upper surface of the well plate WP, and an illumination stop 422 is provided between the light source 421 and the well plate WP. For this reason, as shown in the optical path diagram of FIG. 12B, the light incident on the well W is reduced in diffusivity by the diaphragm 422 and is close to parallel light. The configuration of the imaging unit 13 is the same as that of the first embodiment.

FIG. 13A and FIG. 13B are diagrams showing examples of captured images. FIG. 13A is an example of an image obtained by capturing an image of a well W including cells that are two-dimensionally cultured on the bottom surface as a biological sample, using the imaging device 1 of the first embodiment, and a partially enlarged view thereof. It can be seen that sufficient brightness is obtained at both the central portion and the peripheral portion of the image, but the image contrast of the biological sample is low and is not necessarily suitable for observation of the sample. On the other hand, FIG. 13B is an example of an image in which the same well W is imaged by the imaging device 4 of the fourth embodiment, and a partially enlarged view thereof. An image is obtained in which two-dimensionally cultured cells are captured with higher image contrast.

<Others>
As described above, in each of the above embodiments, the well plate WP corresponds to the “sample container” of the present invention, and each well W corresponds to the “concave portion” of the present invention. In these embodiments, the holder 11 functions as the “holding means” of the present invention. The

illumination units

12 and 42 all function as “illumination means” of the present invention, and the

imaging devices

134, 234 and 334 all function as “imaging means” of the present invention. The objective lens 131, the aperture stop 132, and the imaging lens 133 are integrated to function as the “observation optical system” of the present invention. In the second embodiment, the mechanical drive unit 146 functions as the “position changing means” of the present invention.

Note that the present invention is not limited to the above-described embodiment, and various modifications other than those described above can be made without departing from the spirit of the present invention. For example, in each of the above embodiments, an imaging lens is provided in the observation optical system. However, even in an observation optical system that does not use an imaging lens, it is possible to obtain the same effects as in the above-described embodiment by applying the present invention with respect to the arrangement of the objective lens and the aperture stop.

Also, the aperture stop position adjusting mechanism in the second embodiment can be applied to the third or fourth embodiment. Similarly, the imaging unit 33 in the third embodiment and the illumination unit 42 in the fourth embodiment can be combined.

In the imaging device 4 of the fourth embodiment, the light source 421 of the illumination unit 42 is arranged away from the well plate WP in order to bring the illumination light close to parallel light. However, the light source 421 is light with low diffusivity. May be disposed closer to the well plate WP as long as it can emit light. For example, it is possible to switch between the first embodiment and the fourth embodiment by configuring the illumination unit 12 of the first embodiment with a combination of a light source that emits light with low diffusivity and a diffusion plate. . That is, if a diffusing plate is disposed between a light source and a well plate and the illumination unit 12 is used as a diffusing light source, an imaging device suitable for imaging a biological sample having a three-dimensional structure such as a spheroid can be configured. it can. On the other hand, if the illuminating unit 12 is used as a parallel light source by retracting the diffusion plate from between the light source and the well plate, an imaging device suitable for imaging a biological sample cultured in a plane can be configured.

In the above embodiment, the sample carried in the well W provided in the well plate WP is imaged, but is carried together with the liquid in another sample container, for example, a sample container called “dish” having a larger diameter and a shallower shape. The present invention can also be applied to imaging of a biological sample.

As described above, the image pickup apparatus according to the present invention includes, for example, a position changing unit that moves the aperture stop in the direction along the optical axis of the objective lens, as described in the specific embodiment. Also good. According to such a configuration, the position of the aperture stop can be adjusted according to the sample, and the imaging conditions can be optimized for various samples.

Further, for example, the observation optical system may be configured to include an imaging lens that converges the light that has passed through the aperture stop onto the image pickup unit on the optical path between the aperture stop and the image pickup unit. According to such a configuration, the image quality can be improved by adjusting the incident direction of the light focused on the imaging means.

For example, the illumination means may be configured to allow diffused light to enter the opening surface of the recess. According to the knowledge of the inventors of the present application, particularly when a biological sample to be imaged has a three-dimensional structure, a favorable imaging result is obtained when diffused light is used as illumination light in this way.

Alternatively, for example, the illuminating means may include a light source and a reduction unit that reduces the degree of diffusion of light incident on the opening surface of the recess from the light source. In particular, when the biological sample to be imaged is distributed in a planar manner, for example, along the bottom surface of the depression, a favorable imaging result can be obtained by using illumination light with a suppressed diffusivity.

Further, for example, the imaging unit may include a linear image sensor as a light receiving element, and the observation optical system and the linear image sensor may be configured to scan and move relative to the sample container held by the holding unit. . The imaging means in this invention is either an area image sensor (also referred to as an area sensor) that captures a two-dimensional image or a linear image sensor (also referred to as a linear sensor or a line sensor) that captures a one-dimensional image as a light receiving element. Is applicable. When a linear image sensor is used, imaging can be performed in a state where the optical axis of the observation optical system coincides with the center of the linear image sensor by scanning the linear image sensor together with the observation optical system.

On the other hand, in this case, as the observation optical system moves, the positional relationship between the light emitted from the bottom surface of the recess and the aperture stop changes. For this reason, for example, in an aperture stop having a circular opening, the amount of light incident on the linear image sensor decreases and the brightness of the image decreases particularly as the horizontal position of the linear image sensor moves away from the position directly below the center of the bottom of the recess. To do. In order to eliminate this, for example, the aperture stop may be configured to have a slit-like opening whose longitudinal direction is the scanning direction of the linear image sensor.

As will be described later, in the present invention, it is most preferable that the light incident on the center of the opening surface of the recess and emitted from the peripheral edge of the bottom surface is incident on the center of the aperture stop through the objective lens. That is, it is desirable that the aperture stop be disposed at a position where the optical path of light that enters the center of the opening surface of the recess and passes through the peripheral edge of the bottom surface of the recess intersects the optical axis of the objective lens.

From this point, strictly speaking, it is desirable to adjust the position of the aperture stop according to the sample, that is, according to the size of the recess, the amount of liquid to be injected, and the like. However, it has been found that sufficiently good imaging results can be obtained if the aperture stop is disposed within a certain range from the optimum position. As a result of evaluation using a plurality of typical sample containers used for this type of purpose, for example, the optical path of light that enters the center of the opening surface of the recess and passes through the bottom peripheral edge of the recess is the objective. If the angle formed with respect to the optical axis direction of the lens is 5 degrees to 30 degrees, generally good imaging results can be obtained for various samples.

According to the present invention, in order to prevent the edge portion from becoming dark due to the meniscus of the liquid surface, the aperture stop provided between the objective lens and the imaging means is closer to the imaging means than the focal position of the objective lens. Be placed. Thereby, although it is a simple structure, it can image brightly to the edge part of a sample container. In addition, since it is not necessary to increase the numerical aperture of the observation optical system, good image quality can be obtained even when the biological sample has a three-dimensional structure.

Although the invention has been described with reference to specific embodiments, this description is not intended to be construed in a limiting sense. Reference to the description of the invention, as well as other embodiments of the present invention, various modifications of the disclosed embodiments will become apparent to those skilled in the art. Accordingly, the appended claims are intended to include such modifications or embodiments without departing from the true scope of the invention.

The present invention can be particularly suitably applied to a field that requires imaging of a sample containing a living organism, such as a well on a well plate used in the medical / biological science field. It is not limited to the medical / biological science field.

1, 2, 3, 4 Imaging device 11 Holder (holding means)
12, 42 Illumination part (illumination means)
13, 23, 33 Imaging unit 14 Control unit 131 Objective lens (observation optical system)
132 Aperture stop (observation optical system)
133 Imaging lens (observation optical system)
134, 234, 334 Imaging device (imaging means)
146 Mechanical drive (position changing means)
421 Light source 422 Diffuser (reduction part)
W well
WP well plate (sample container)

Claims

In an imaging device for imaging a biological sample that is provided in a sample container and is carried with a liquid in a recess having a flat bottom surface,
Holding means for holding the sample container so that the bottom surface of the recess is horizontal;
Illumination means for making illumination light incident on the opening surface of the recess from above the sample container;
Imaging means for receiving light transmitted from the bottom surface of the recess and imaging the biological sample;
An observation optical system that is provided between the sample container held by the holding unit and the imaging unit and that makes light emitted from the bottom surface of the recess enter the imaging unit;
The observation optical system includes an objective lens provided on the optical path between the holding unit and the imaging unit, and an aperture stop provided on the optical path between the objective lens and the imaging unit. ,
The image pickup apparatus, wherein the aperture stop is disposed closer to the image pickup means than a focal position of the objective lens.
The imaging apparatus according to claim 1, further comprising a position changing unit that moves the aperture stop in a direction along an optical axis of the objective lens.
The imaging apparatus according to claim 1, wherein the observation optical system includes an imaging lens that converges the light that has passed through the aperture stop on the image pickup unit on an optical path between the aperture stop and the image pickup unit. .
The imaging device according to any one of claims 1 to 3, wherein the illuminating unit causes diffused light to enter the opening surface of the recess.
The imaging device according to any one of claims 1 to 3, wherein the illumination unit includes a light source and a reduction unit that reduces the diffusivity of light incident on the opening surface of the recess from the light source.
The imaging device includes a linear image sensor as a light receiving element, and the observation optical system and the linear image sensor integrally scan and move relative to the sample container held by the holding means. The imaging device according to any one of 5.
The imaging apparatus according to claim 6, wherein the aperture stop has a slit-like opening whose longitudinal direction is the scanning direction of the linear image sensor.
The angle formed by the optical path of light incident on the center of the opening surface of the recess and passing through the peripheral edge of the bottom surface of the recess with respect to the optical axis direction of the objective lens is 5 to 30 degrees. The imaging device according to claim 1.
Holding a sample container carrying a biological sample together with a liquid in a recess having a flat bottom so that the bottom of the recess is horizontal;
Arranging an imaging unit so as to receive light transmitted from the bottom surface of the recess, and arranging an observation optical system between the sample container and the imaging unit;
A step of causing illumination light to enter the opening surface of the recess from above the sample container;
Imaging the biological sample by allowing the light transmitted from the bottom surface of the recess to enter the imaging means by the observation optical system,
The observation optical system includes an objective lens disposed on the optical path between the holding unit and the imaging unit, and an aperture stop disposed on the optical path between the objective lens and the imaging unit. ,
An imaging method, wherein the aperture stop is arranged closer to the imaging means than the focal position of the objective lens.
The aperture stop is disposed at a position where an optical path of light that enters the center of the opening surface of the recess and passes through a peripheral portion of the bottom surface of the recess intersects the optical axis of the objective lens. The imaging method described.