Image pickup apparatus

Abstract

An image capturing apparatus for a phase object includes: an image capturing unit that captures an image of the phase object through an image capturing lens; a light irradiation unit including a collimation unit that converts the light emitted from the light source into substantially parallel light and a branch prism that branches and outputs the substantially parallel light, branched substantially parallel light rays in a plurality of different directions that are not parallel to the optical axis of the imaging lens irradiate the phase object. And a placement unit on which the phase object is placed in a region where the light rays output in the plurality of different directions by the light irradiation unit initially intersect each other.

Description

Technical Field

[0001] The present disclosure relates to an imaging device for phase objects. Background Art

[0002] When observing cells in cell culture, it is important to obtain not only the morphological information of the cells but also the positional information of the cells. Since it is difficult to visualize cells as transparent phase objects using ordinary bright field microscopes, phase contrast microscopes that solve this problem are widely used. Phase contrast microscopes visualize cells by setting an annular slit in the transmitted illumination unit and a phase plate for the objective lens. However, there are limitations on the lenses that phase plates are applicable to, and low-magnification lenses cannot be used.

[0003] On the other hand, there is a technique called oblique illumination, which visualizes a phase object to be observed by illuminating the phase object with transmitted light at a slight angle to the optical axis. Through this technique (disclosed in Japanese Patent Application Laid-Open No. 2010-216920), a technique for visualizing widely existing cells by using a low-magnification lens, which cannot be achieved by a phase contrast microscope, has also been developed.

[0004] In Japanese Patent Laid-Open No. 2010-216920, in oblique illumination that illuminates in one direction, a deviation in the shadow around the cell may be caused depending on the angle and direction of light irradiation. This deviation may prevent the shape of the cell from being determined at the time of image processing, etc. In addition, in oblique illumination, the position of the drawn cell may vary depending on the angle and direction of light irradiation. Therefore, it may be difficult to accurately determine the position and shape of the cell by using only the oblique illumination technique. Summary of the invention

[0005] The present disclosure provides an imaging device capable of accurately obtaining information such as the position and shape of a phase object such as a widely existing cell.

[0006] The imaging device according to the present disclosure is an imaging device for phase objects, comprising: an imaging unit, which images the phase object through an imaging lens; a light irradiation unit, which includes a collimating unit and a branching prism, wherein the collimating unit converts the light emitted from the light source into substantially parallel light, and the branching prism branches and outputs the substantially parallel light, so that the branched substantially parallel light in multiple different directions that are not parallel to the optical axis of the imaging lens illuminate the phase object; and a placement unit, on which the phase object is placed in an area where the light output by the light irradiation unit in the multiple different directions initially intersects each other.

[0007] Further features of the present disclosure will become apparent from the following description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 is a diagram illustrating a configuration of an image pickup apparatus according to an embodiment of the present disclosure.

[0009] Figure 2 is a diagram illustrating an image pickup apparatus according to a first embodiment of the present disclosure.

[0010] Figure 3A and Figure 3B is a diagram illustrating an example of light distribution before and after substantially parallel light rays pass through a branching prism according to an embodiment of the present disclosure.

[0011] Figure 4 is a diagram illustrating an image pickup apparatus according to a second embodiment of the present disclosure.

[0012] Figure 5A and Figure 5B is a diagram illustrating an example of the size and position of illumination light in the imaging range of the imaging apparatus according to the second embodiment of the present disclosure.

[0013] Fig. 6A and Figure 6B is a diagram illustrating an example of substantially parallel light rays output from a plurality of substantially parallel light output ports according to an embodiment of the present disclosure.

[0014] Fig. 7A and Figure 7B is a diagram illustrating an image pickup apparatus according to a third embodiment of the present disclosure. DETAILED DESCRIPTION

[0015] An imaging apparatus according to an embodiment of the present disclosure will be described below with reference to the drawings.

[0016] Camera equipment

[0017] The image pickup apparatus 100 according to the present embodiment includes an image pickup unit 007 that picks up an image of a phase object 006 through an image pickup lens 008 .

[0018] The imaging device 100 according to the present embodiment includes a light irradiation unit 030. The light irradiation unit 030 includes a collimating unit 002 that converts the light emitted from the light source 001 into substantially parallel light and a branching prism 003 that branches and outputs the substantially parallel light, so that the substantially parallel light after branching in multiple directions that are not parallel to the optical axis 020 of the imaging lens 008 irradiates the phase object 006.

[0019] The image pickup apparatus 100 according to the present embodiment further includes a placement unit 004 on which a phase object is placed in a region 005 where light rays outputted in a plurality of different directions by the light irradiation unit 030 initially intersect each other.

[0020] Since the imaging device 100 according to the present embodiment illuminates the phase object 006 such as a cell with light from multiple different directions as described above, the shadow deviation of the phase object is reduced compared to illuminating the phase object with oblique light from only one direction. Therefore, information such as the position and shape of the phase object can be accurately obtained.

[0021] Furthermore, since there is the placement unit 004 that can place the phase object 006 in the region 005 where the light rays outputted in a plurality of different directions by the light irradiation unit 030 initially intersect each other, the image pickup apparatus 100 can be reduced in size.

[0022] Subject

[0023] The subject mainly assumed by the imaging device according to this embodiment is a phase object. In this embodiment, the phase object is a colorless and transparent object, and the intensity of light passing through the phase object does not change, but only the phase of light passing through the phase object changes. Examples of phase objects include cells, suspension solutions containing cells, etc.

[0024] When the phase object is a cell, the subject may be a liquid containing the cell provided in a transparent container. An example of the container is a laboratory culture dish made of commercially available glass or polystyrene, and an example of the liquid is a solution that maintains cell survival during cell culture, such as a culture medium, a physiological saline solution, or a solution equivalent to a physiological saline solution. The surface of the container facing the light irradiation unit and the imaging unit needs to be optically transparent to the illumination light irradiating the phase object, but the other surfaces do not need to be optically transparent.

[0025] Placement Unit

[0026] The placement unit 004 according to this embodiment is a placement table for placing a phase object or a container having a phase object, and the surface facing the light irradiation unit and the imaging unit needs to be optically transparent to the illumination light for irradiating the phase object. Figure 1 The inverted imaging device shown may have a hole formed in a portion of the placement table close to the imaging unit. In addition, a fixing portion for the container may be provided to prevent the container containing the cells from moving due to a slight collision.

[0027] Light irradiation unit

[0028] The light irradiation unit according to the present embodiment irradiates the subject with substantially parallel light rays (also referred to as collimated light rays) in multiple directions. The substantially parallel light rays only need to be parallel to the extent that the effects of the present disclosure can be obtained. The light irradiation unit includes a collimating member and a branching prism, the collimating member makes the light rays from the light source parallel, the light source is a radiation light source like a point light source, and the branching prism converts the collimated light rays into a plurality of collimated light rays.

[0029] Light that has passed through a pinhole provided between the light source and the sample may be used, but it is also effective to use light from the exit end of an optical fiber bundle to which light from any light source has been input.

[0030] Collimation unit

[0031] The collimating unit according to the present embodiment converts the light emitted from the light source into substantially parallel light (collimated light). A convex lens, a telecentric lens, etc. can be used as a collimator unit (also referred to as a collimator). It should be noted that the optical axis of the collimated light converted by the collimating member can be concentric with the optical axis of the imaging unit.

[0032] Branching prism

[0033] The branch prism according to the present embodiment can be a conical prism or a polygonal conical prism. For example, when a square conical prism is used, the collimated light is incident on the bottom surface of the square cone. The incident light is converted into four collimated light beams that are bent toward the center at equal inclination angles along the optical axis. Then, the area where these four light beams intersect each other is illuminated with collimated light beams incident at the same inclination angle in four directions. The area illuminated by the light beams in these four directions is the shooting area.

[0034] It should be noted that when the branching prism is a polygonal prism, when the number of sides of the polygon of the bottom surface of the prism increases, the number of collimated light rays after branching increases. This means that the subject is illuminated with collimated light rays in more directions, but the area of ​​the shooting area that is evenly illuminated is reduced. When the number of sides of the polygon becomes infinite, the polygonal prism becomes a circular cone prism, and the circular cone prism is called an axicon lens. When an axicon lens is used as a branching prism, the subject is illuminated with collimated light rays in all directions, but a Bessel beam is formed in the intersection area, and the area of ​​the shooting area that is evenly illuminated becomes very narrow. Therefore, the branching prism can be selected according to the size and characteristics of the phase object to be photographed.

[0035] The light from the branching prism can be incident at the same angle relative to the optical axis (oblique incidence). To achieve this, a regular polygonal prism or an axicon lens can be used as a branching prism. In addition, the irradiation angle of the light can be roughly equivalent to the numerical aperture (NA) of the camera unit. It should be noted that as long as the optical sheet exhibits the above-mentioned effect, it is effective to use an optical sheet or the like as a branching prism.

[0036] light source

[0037] An LED light source, a halogen light source, or the like can be used as the light source in this embodiment.

[0038] Camera unit

[0039] The imaging unit according to the present embodiment acquires optical information from the subject and creates an image of the acquired optical information. The imaging unit may be a digital camera including a CCD or CMOS sensor as an imaging element. The size of the imaging element included in the imaging device may be larger, and the number of pixels may be larger.

[0040] A higher number of pixels increases the resolution of an object based on digital zoom and improves the ability to depict the morphology of a microscopic object such as a cell. An example of an imaging lens included in the imaging unit according to the present embodiment is a telecentric lens, and a double-sided telecentric lens among the telecentric lenses can be used.

[0041] Even when the imaging lens is not a telecentric lens, imaging can be performed, but a telecentric lens can be used because the unevenness of the transmitted illumination can be suppressed.

[0042] In a lens with a wide angle of view, the longer the distance between the light irradiation unit and the lens, the smaller the area of ​​the light irradiation unit in the field of view, and the area of ​​the smaller light irradiation unit captured becomes a bright image. On the other hand, in a telecentric lens with a narrow angle of view, when the light irradiation unit is larger than the imaging area, a bright image can be acquired over the entire area regardless of the distance between the light irradiation unit and the lens.

[0043] Illumination with collimated light in multiple directions has the same effect as oblique illumination in multiple directions at the same angle relative to the optical axis of the camera unit by using multiple collimated light sources. However, the use of branching prisms is advantageous.

[0044] Fig. 6A and Figure 6B Illumination with collimated light from multiple collimated light output ports is illustrated. Fig. 6A The figure shows the case where the distance between the collimated light emission ports is longer. Figure 6B The diagram shows a case where the distance is shorter. Fig. 6AThe distance from the position where the sample stage can be placed (area 005) is Figure 6B The distance in is long.

[0045] In wide-range photography, the illumination range is also widened, and the width of the collimated light output port needs to be widened. Therefore, the distance from the exit end to the position where the multiple illumination light beams completely intersect each other becomes longer.

[0046] In addition, the NA of a low-magnification lens for wide-area shooting is smaller, and the oblique angle of the illumination light required to visualize the phase object also becomes smaller. When the oblique angle is smaller, the distance from the exit end to the position where multiple beams of illumination light intersect each other becomes longer. In multi-directional oblique illumination for wide-area shooting, the distance from the exit end to the subject is likely to become longer, and the camera equipment also becomes larger. Therefore, the distance between multiple collimated light output ports can be smaller.

[0047] The use of a branching prism sets the distance between the collimated light output ports to zero, and can suppress upsizing of the imaging apparatus.

[0048] Placement Unit

[0049] In this embodiment, as described above, the placement unit for placing the phase object is provided in the region where the light beams outputted from the light irradiation unit in a plurality of different directions initially intersect each other. In this structure, the size of the imaging apparatus can be reduced.

[0050] Change Unit

[0051] The imaging device according to this embodiment may include a crossing region changing unit that changes the region where light rays cross each other by shifting at least one of the above-mentioned light source, collimating unit, and axicon lens. That is, the changing unit according to this embodiment may change the illumination position of the phase object.

[0052] The above-mentioned imaging device can accurately acquire the position information of cells which are transparent phase objects without a phase plate on the lens.

[0053] First embodiment

[0054] Will refer to Figure 2 The structure of an image pickup apparatus according to a first embodiment of the present disclosure is described. Figure 2 In, with Figure 1 The same components as those in the drawings are denoted by the same reference numerals and their description is omitted.

[0055] A typical observation object of the imaging apparatus according to the present embodiment is a cell. Cells cultured in a 6-well polystyrene cell culture dish were provided as the object. It should be noted that the observation object may be a floating cell.

[0056] The attachment to which the universal 6-hole culture dish can be attached is arranged on the placement table, and a leaf spring that pressurizes the culture dish in the horizontal direction of the two axes is arranged, and the culture dish is fixed to the placement table by setting the culture dish in the placement table. The placement table includes an upper member and a lower member. The upper member in contact with the culture dish has an opening, which extends over a wide range including all six holes at the center of the culture dish, and only the peripheral portion of the culture dish contacts the member. The lower member has an opening that is opened only in the observation area. In the upper member, the entire culture dish can be moved in the horizontal direction with the 2-axis stage, and the lower member is fixed in the horizontal direction. The 2-axis stage adopts a manual stage with a rack and pinion structure, but an automatic stage operated by a PC or the like can be adopted. The hole portion to be observed is moved to the opening portion of the lower member, and moved to the observation position by the 2-axis stage.

[0057] It should be noted that even culture dishes having different shapes, such as 35 mm culture dishes, can be fixed to the placement table by providing dedicated attachments.

[0058] A high-brightness LED light source with a wavelength of 565nm, a bundled optical fiber, and a telecentric lens are used as the light source. The LED light source is incident on the optical fiber bundle, and the telecentric lens is attached to the outlet end of the optical fiber bundle to achieve uniform collimated light. The telecentric lens is placed in a manner to output light in a vertical direction above the placement table. The telecentric lens is used to output a circular uniform light with a diameter of approximately 50mm as the output light.

[0059] An acrylic square pyramid having a square base with a side length of approximately 50 mm is used as a branch prism. The angles of the four surfaces of the square pyramid are approximately 10 degrees, and the collimated light incident vertically can be polarized 5 degrees toward the optical axis. It should be noted that any material used for a transparent resin prism or a glass prism can be used.

[0060] The placement table is located approximately 140 mm away from the branching prism. Figure 3A The figure shows the distribution of the illumination light before it is incident on the branching prism. Figure 3B The diagram shows the illumination light distribution near the placement table. Figure 3A The dotted lines in the figure indicate the positions of the four sides of the branch prism. Figure 3A The four fan-shaped parts divided by the dotted lines in the figure are tilted at an angle of 5 degrees toward the optical axis at the center. Figure 3B Lighting pattern shown.

[0061] At this time, the region where the illumination lights in four directions intersect each other is a square with a side length of approximately 18 mm.

[0062] A commercially available mirrorless single-lens camera including a full-size (36mm×24mm) 8K pixel color CMOS sensor is used as the imaging unit. A commercially available 2× telecentric lens that can be placed in the imaging unit is used as the imaging telecentric lens. It should be noted that when the mount of the digital camera is different from the mount of the imaging telecentric lens, only a conversion adapter is required. The imaging telecentric lens faces the vertical upper side so as to be able to image the opening position of the lower member of the placement table. The imaging area has a size of 18mm×12mm at this time, and falls within the area (18mm×18mm) where the illumination lights in four directions intersect each other, and the entire imaging area falls within the range of the same multi-directional illumination.

[0063] The imaging unit and imaging telecentric lens have a single-axis stage 009 for vertical movement and for focus adjustment. In addition, a monitor for real-time display of the image acquired by the digital camera of the imaging unit is provided.

[0064] The above-mentioned imaging device can be used to provide a system for accurately acquiring position information of cells, which are transparent phase objects widely present in culture dishes.

[0065] Second embodiment

[0066] Will refer to Figure 4 as well as Figure 5A and Figure 5B The structure of the image acquisition device according to the second embodiment of the present disclosure is described. Figure 4 In, with Figure 1 The same components as those in the drawings are denoted by the same reference numerals and their description is omitted.

[0067] The device structure is similar to that of the present invention except that an axicon lens is used as a branch prism and an illumination light scanning mechanism for scanning an illumination position in a horizontal direction is attached to the illumination unit. Figure 2 The image pickup apparatus according to the first embodiment shown is basically the same. Only the differences from the image pickup apparatus according to the first embodiment will be described below.

[0068] A glass axicon lens of approximately 50 mm diameter was used as the branching prism. The angle was 10 degrees, and the specification was that the incident collimated light was tilted approximately 5 degrees toward the optical axis. Figure 5A The size and position of the illumination light in the imaging range are shown. A Bessel beam region 010 is formed in the region where the light from both sides of the axicon lens intersects each other. This Bessel beam causes only a portion of the central portion of the image to be illuminated by many collimated light rays in many directions. In addition, the cell image is displayed only in this portion.

[0069] Reference numeral 011 denotes an illumination light scanning mechanism for moving the illumination position. Figure 5BAs shown, the subject in the imaging area is uniformly scanned using illumination light. During this scanning, the shutter of the camera of the imaging unit should remain open. This process causes the entire imaging area to be illuminated by collimated light in many directions, and the image of the drawn cell can be obtained. It should be noted that the scanning function can also be driven by moving the collimating member and the axicon lens horizontally and vertically (these components remain horizontal and not tilted). In addition, it is also effective to obtain the cell image in the entire imaging area by providing an image processing unit, acquiring an image each time the illumination light moves, and combining all images.

[0070] The above-described imaging device according to the present embodiment can be used to accurately acquire position information of cells, which are transparent phase objects widely present in a culture dish.

[0071] Third embodiment

[0072] Will refer to Fig. 7A and Figure 7B The structure of an image pickup apparatus according to a third embodiment of the present disclosure is described. In these drawings, the same components are denoted by the same reference numerals and description thereof is omitted.

[0073] You can refer to Fig. 7A and Figure 7B The structure of the imaging device according to the present embodiment is described. The structure of the imaging device is basically the same as that according to the present embodiment, except that a fluorescent illumination unit 012 for acquiring a fluorescent image of a subject is attached. Figure 2 The configuration of the image pickup apparatus of the first embodiment shown in is the same as that of the first embodiment. Only the differences will be described below.

[0074] The fluorescence illumination unit irradiates the subject with light at a different angle from the light irradiation unit, and can obtain the fluorescence characteristics of the subject by irradiating with the excitation light. When the fluorescence characteristics of the subject are obtained, the fluorescence information is obtained by irradiating with the incident excitation light to improve the SN of the image. For example, Fig. 7A and Figure 7B As shown, the subject can be irradiated with oblique excitation light from the imaging unit side. In addition, when the irradiation light is uniform, substantially collimated light, the amount of light on the irradiation surface of the oblique irradiation becomes uniform, and is effective for the homogenization of the image and the relative evaluation in the picture. As in the above-mentioned light irradiation unit, such uniform, substantially collimated light enters a pinhole or an optical fiber bundle from a light source, and can be achieved by installing a collimator such as a convex lens or a telecentric lens. By using an imaging device that performs oblique irradiation, the lens can be a commercially available telecentric lens. It should be noted that a filter (for fluorescence) that removes the excitation light and allows only fluorescence to pass can be placed between the subject and the imaging unit.

[0075] The wavelength of the excitation light is selected in a manner suitable for the excitation characteristics of a fluorescent marker, and the type of light source is not limited as long as the light source has the wavelength. In addition, a combination of a wide-area xenon lamp and an excitation filter can also be used. It is also necessary to select a filter (for fluorescence) based on the balance between the wavelength of the fluorescence from the subject and the wavelength of the excitation light. The excitation light and the filter (for fluorescence) can be easily replaced.

[0076] It should be noted that when the subject is, for example, a cell contained in a container, oblique incident light may cause stray light by illuminating the container wall. In order to reduce this stray light, Figure 7B As shown, the light from the second light irradiation unit can be incident on the side of the telecentric lens, and coaxial incident illumination can be achieved by a dichroic mirror 014 arranged in the telecentric lens. The dichroic mirror desirably has a wavelength characteristic of reflecting the excitation light and passing the transmitted illumination light and the fluorescence generated by the subject. In addition, a filter (for fluorescence) 013 can also be provided.

[0077] The above-mentioned imaging device can be used to accurately acquire position information of cells, which are transparent phase objects widely present in a culture dish, and acquire fluorescent staining results of the cells.

[0078] An imaging apparatus can be provided that can accurately acquire information including the position and shape of phase objects such as widely existing cells.

[0079] While the present disclosure has been described with reference to exemplary embodiments, it is to be understood that the present disclosure is not limited to the disclosed exemplary embodiments. The scope of the following claims is to be accorded the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.

Claims

1. A camera device for phase objects, comprising: An imaging unit, which captures an image of a phase object through an imaging lens; a light irradiation unit, comprising a collimating unit and a branching prism, wherein the collimating unit converts the light emitted from the light source into substantially parallel light, and the branching prism branches and outputs the substantially parallel light, so that the branched substantially parallel light in a plurality of different directions that are not parallel to the optical axis of the imaging lens irradiates the phase object; and A placement unit on which the phase object is placed in a region where the light beams outputted from the light irradiation unit in the plurality of different directions initially intersect each other.

2. The imaging device according to claim 1, in, The branch prism is a polygonal pyramid prism.

3. The imaging device according to claim 1, in, The branch prism is an axicon lens.

4. The imaging device according to claim 3, further comprising: A crossing area changing unit changes an area where the light rays cross each other by shifting at least one of the light source, the collimating unit, and the axicon lens.

5. The imaging device according to claim 1, in, The phase objects include cells.

6. The imaging device according to claim 1, in, The collimating unit includes a convex lens or a telecentric lens.

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