Imaging device

Abstract

To provide an imaging device with which it is possible to acquire, with good accuracy, information regarding the position and the shape of a phase object such as a cell existing in a wide range.SOLUTION: Provided is an imaging device for phase objects including an imaging unit for capturing a phase object via an imaging lens. The imaging device comprises: a light irradiation unit including a collimate unit for converting a light ray emitted from a light source into a substantially parallel light rays and a branch prism for branching and emitting the substantially parallel light rays so that the substantially parallel light rays are not parallel to an optical axis of the imaging lens and the phase object is irradiated with the substantially parallel light rays from a plurality of mutually different directions; and a placement unit on which the phase object is placed in a region where the light rays from the plurality of mutually different directions emitted from the light irradiation unit first intersect each other.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an apparatus for imaging a phase object. [Background technology]

[0002] When observing cells in cell culture, it is important to obtain not only morphological information but also positional information of the cells. Cells are transparent phase objects, so it is difficult to visualize them in a normal bright field. Phase contrast microscopes, which solve this problem, are widely used. Phase contrast microscopes visualize cells by installing a ring slit in the transmitted illumination section and a phase plate in the objective lens. However, there are restrictions on the lenses that can be used to install the phase plate, and low magnification lenses cannot be used.

[0003] On the other hand, there is a technique called oblique illumination, which visualizes a phase object by irradiating the phase object to be observed with transmitted light at a slight angle from the optical axis. Using this technique, a technique for visualizing a wide range of cells using a low-magnification lens, which could not be realized with a phase contrast microscope, has also been developed (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2010-216920 A Summary of the Invention [Problem to be solved by the invention]

[0005] In Patent Document 1, oblique illumination from one direction can cause bias in the shadows around the cells depending on the angle and direction of the light irradiation. This bias can be an obstacle when identifying the shape of the cell by image processing, etc. In addition, with oblique illumination, the position of the cell depicted may change depending on the angle and direction of the light irradiation. For these reasons, it may be difficult to accurately determine the position and shape of the cell just by adopting oblique illumination technology.

[0006] The present invention has been made in consideration of the above problems, and aims to provide an imaging device that can accurately acquire information such as the position and shape of phase objects such as cells that exist over a wide range. [Means for solving the problem]

[0007] The imaging device of the present invention is an imaging device for a phase object having an imaging unit that images a phase object through an imaging lens, and includes a collimating unit that converts light emitted from a light source into approximately parallel light, a light irradiation unit having a branching prism that branches and emits the approximately parallel light so that the approximately parallel light is non-parallel to the optical axis of the imaging lens and is irradiated onto the phase object from a plurality of different directions, and an installation unit that installs the phase object in a region where the light from the plurality of different directions emitted from the light irradiation unit first intersect. Effect of the Invention

[0008] It is possible to provide an imaging device that can accurately acquire information such as the position and shape of phase objects, such as cells, that exist over a wide range. [Brief description of the drawings]

[0009] [Figure 1] 1 is a diagram illustrating a configuration of an imaging apparatus according to an embodiment of the present invention. [Diagram 2] 1 is a diagram showing an imaging device according to a first embodiment of the present invention. [Diagram 3] 5A to 5C are diagrams illustrating an example of a light distribution before and after approximately parallel light passes through a branching prism in the embodiment of the present invention. [Figure 4] FIG. 4 is a diagram showing an imaging device according to a second embodiment of the present invention. [Diagram 5] 13A and 13B are diagrams illustrating an example of the size and position of illumination light in an imaging range of an imaging device according to a second embodiment of the present invention. [Figure 6] 5A and 5B are diagrams illustrating an example of how substantially parallel light is emitted from a plurality of substantially parallel light exit ports in the embodiment of the present invention. [Figure 7]FIG. 11 is a diagram showing an imaging device according to a third embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] An imaging device according to an embodiment of the present invention will now be described with reference to the drawings.

[0011] (Imaging device) The imaging device 100 according to this embodiment has an imaging section 007 that captures an image of a phase object 006 via an imaging lens 008 .

[0012] The imaging device 100 according to this embodiment has a light irradiation unit 030. The light irradiation unit 030 has a collimator unit 002 that converts light emitted from a light source 001 into approximately parallel light, and a branching prism 003 that branches and emits the approximately parallel light so that the approximately parallel light is non-parallel to the optical axis 020 of the imaging lens 008 and is irradiated onto a phase object 006 from a plurality of directions different from each other.

[0013] Furthermore, the imaging device 100 according to this embodiment has a mounting unit 004 for mounting a phase object in a region 005 where the light beams emitted from the light irradiation unit 030 and coming from a plurality of different directions first intersect.

[0014] As described above, the imaging device 100 according to this embodiment irradiates the phase object 006 such as a cell with oblique light from a plurality of different directions, which reduces the bias of the shadow of the phase object compared to the case where the phase object is irradiated with oblique light from one direction. Therefore, it is possible to accurately acquire information such as the position and shape of the phase object.

[0015] Furthermore, since the imaging device 100 has the installation unit 004 that allows the phase object 006 to be installed in the region 005 where the light from a plurality of different directions emitted from the light irradiation unit 030 first intersects, the imaging device 100 can be made compact.

[0016] (subject) A phase object is mainly assumed as a subject of the imaging device according to this embodiment. In this embodiment, a phase object is a colorless and transparent object in which the intensity of light passing through the phase object does not change, and only the phase of light passing through the phase object changes. Examples of phase objects include cells and suspensions containing cells.

[0017] When the phase object is a cell, the subject may be a transparent container in which a liquid and cells are placed. An example of the container is a commercially available petri dish made of glass or polystyrene, and an example of the liquid is a liquid for maintaining the survival of cells during cell culture, such as a culture medium, physiological saline, or a solution equivalent to physiological saline. The surface of the container facing the light irradiation unit side or the imaging unit side needs to be optically transparent to the illumination light irradiated to the phase object, but the other surfaces do not need to be optically transparent.

[0018] (Installation part) The installation unit 004 in this embodiment is an installation stand on which a phase object or a container in which a phase object is provided is installed, and the surface facing the light irradiation unit side or the imaging unit side needs to be optically transparent to the illumination light irradiated to the phase object. For example, in the case of an inverted imaging device as shown in FIG. 1, a hole can be formed on the imaging unit side of the installation stand. In addition, a fixing part for the container may be provided so that the container in which the cells are placed will not move even if it is subjected to a small impact.

[0019] (Light irradiation part) The light irradiation unit in this embodiment irradiates the subject with approximately parallel light (which can also be called collimated light) from multiple directions. The approximately parallel light may be parallel enough to obtain the effects of the present invention. The light irradiation unit is configured to include a collimating means for converting light from a light source, which is a radiation light source close to a point light source, into parallel light, and a branching prism for converting the collimated light into multiple collimated lights.

[0020] Although a pinhole may be placed in front of the light source, it is also effective to use light from the output end of a bundle fiber that captures light from an arbitrary light source.

[0021] (Collimation part) The collimator unit in this embodiment converts the light emitted from the light source into approximately parallel light (collimated light). A convex lens, a telecentric lens, or the like can be used as the collimator unit (which can also be called a collimator). It is preferable that the optical axis of the collimated light converted by the collimator means is coaxial with the optical axis of the imaging unit.

[0022] (Diverging Prism) In this embodiment, the branching prism is preferably a pyramidal prism, and more preferably a polygonal pyramid prism. For example, when a regular square pyramid prism is used, collimated light is incident from the bottom side of the regular square pyramid. The incident light is converted into four collimated lights that are bent toward the center at equal inclination angles along the optical axis. The area where these four lights intersect is illuminated by the collimated lights that are incident at equal inclinations from four directions. The area illuminated from these four directions becomes the shooting area.

[0023] In addition, when the branching prism is a polygonal pyramid prism, the number of branched collimated light beams increases as the number of sides of the polygon at the base of the prism increases. This means that the subject is irradiated with collimated light beams from more directions, but the area of ​​the shooting area that is uniformly illuminated becomes smaller. When the number of sides of this polygon becomes infinite, the limit system becomes a cone, and this conical prism is called an axicon lens. When an axicon lens is used as the branching prism, the subject is irradiated with collimated light beams from all directions, but a Bessel beam is formed at the intersection area, and the area of ​​the shooting area that is uniformly illuminated becomes very small. From the above points, it is preferable to select a branching prism according to the size and properties of the phase object to be photographed.

[0024] It is preferable that the light from the branching prism is incident (deflected) at an equal angle toward the optical axis. To achieve this, it is preferable to use a regular polygonal pyramid prism or an axicon lens as the branching prism. It is also preferable that the irradiation angle of the light is approximately the NA (numerical aperture) of the imaging unit. Note that it is also effective to use an optical sheet or the like as the branching prism as long as it has the above-mentioned effect.

[0025] (light source) In this embodiment, an LED light source, a halogen light source, or the like can be used as the light source.

[0026] (Imaging unit) The imaging unit in this embodiment acquires optical information from the subject and images it. The imaging unit is preferably a digital camera equipped with a CCD or CMOS sensor as an imaging element. The larger the size of the imaging element included in the imaging device, the more preferable it is, and the more pixels it has. The more pixels there are, the higher the resolution of the subject by digital zoom, and the better the ability to depict the morphology of a minute subject such as a cell. An example of an imaging lens included in the imaging unit in this embodiment is a telecentric lens, and among telecentric lenses, it is more preferable to use a double-telecentric lens.

[0027] Although imaging lenses other than telecentric lenses are possible, telecentric lenses are preferable because they reduce unevenness in transmitted illumination.

[0028] In a lens with an angle of view, the longer the distance between the light irradiation unit and the lens, the smaller the area of ​​the light irradiation unit relative to the field of view becomes, and the smaller area of ​​the light irradiation unit captured becomes a bright image. In contrast, in the case of a telecentric lens with no angle of view, if the light irradiation unit is larger than the imaging area, an image with bright light can be obtained over the entire area regardless of the distance between the light irradiation unit and the lens.

[0029] If collimated light is irradiated from multiple directions, it will have the same effect as preparing multiple collimated light sources and irradiating the image from multiple directions at the same angle with respect to the optical axis of the imaging unit. However, there is an advantage to using a branching prism.

[0030] Figure 6 shows the state where collimated light is irradiated from multiple collimated light exit ports. Figure 6(a) shows the case where the distance between the collimated light exit ports is long, and Figure 6(b) shows the case where it is short. In this case, the distance to the position where the sample stage can be placed (005 range) is longer in (a).

[0031] When capturing a wide area, the illumination range is also wide and the width of the collimated light exit port must be made larger, which increases the distance from the exit end until the multiple illumination lights completely intersect.

[0032] In addition, a low-magnification lens for wide-area photography has a small NA, and the deflection angle of the illumination light required for visualizing a phase object is also small. When the deflection angle is small, the distance from the exit end until the intersection of the multiple illumination lights becomes long. Thus, in multi-directional oblique illumination for wide-area photography, the distance from the exit end to the subject tends to be long, and the imaging device also becomes large. For this reason, it is preferable that the interval between the multiple collimated light exit ports is as narrow as possible.

[0033] By using a branching prism, the distance between the collimated light exit ports can be made zero, making it possible to prevent the imaging device from becoming bulky.

[0034] (Installation part) In the present embodiment, the installation section for installing the phase object is provided in the region where the light beams emitted from the light irradiation section and coming from a plurality of different directions intersect for the first time as described above. With this configuration, the configuration of the imaging device can be made compact.

[0035] (Changes) The imaging device according to this embodiment may have an intersection area changer that changes the intersection area by displacing at least one of the light source, the collimator unit, and the axicon lens. That is, the changer in this embodiment can change the illumination position of the phase object.

[0036] By using the above imaging device, it is possible to provide an imaging device that can accurately obtain position information of cells, which are transparent phase objects, without mounting a phase plate on the lens.

[0037] [First embodiment] The configuration of an imaging device according to a first embodiment of the present invention will be described with reference to Fig. 2. In Fig. 2, the same components as those in Fig. 1 are in principle given the same reference numerals, and the description thereof will be omitted.

[0038] A typical observation target of the imaging device according to this embodiment is a cell. The subject is a 6-well polystyrene cell culture dish in which cells are adherently cultured. Note that the observation target may also be a floating cell.

[0039] The installation stand is equipped with an attachment that can accommodate a general-purpose 6-well dish, and a leaf spring is provided to press the dish horizontally along two axes, and the dish is fixed to the installation stand when it is inserted. The installation stand is composed of two components, an upper and lower. The upper side, which the dish comes into contact with, has a wide opening that includes all six wells in the center of the dish, and the dish is designed so that only the periphery comes into contact with the component. The lower component has an opening that is only open in the observation area. The upper component is designed to move the dish together with a horizontal two-axis stage, while the lower component is fixed horizontally. The two-axis stage uses a manual stage with a rack and pinion configuration, but it can also be configured to use an automatic stage and be operated from a PC, etc. The well part to be observed is moved to the opening of the lower component using the two-axis stage, and then moved to the observation position.

[0040] In addition, even for dishes with different shapes, such as 35 mm dishes, it is possible to fix them to the installation stand by providing a special attachment.

[0041] The light source used is a high-brightness LED light source with a wavelength of 565 nm, a fiber bundle, and a telecentric lens. Uniform collimated light is achieved by inputting the LED light source into the fiber bundle and attaching a telecentric lens to the output end of the fiber bundle. The telecentric lens is installed so that it irradiates light vertically above the installation table. The telecentric lens used realizes uniform circular light with an output light of approximately φ50 mm.

[0042] The branching prism used was an acrylic regular pyramid with a square base measuring approximately 50 mm on each side. The angles of the four faces of the pyramid are approximately 10 degrees, and it is possible to deflect the collimated light incident perpendicularly by 5 degrees toward the optical axis. The material can be any material that can be used for prisms, such as transparent resin or glass.

[0043] The installation stand was positioned approximately 140 mm from the splitting prism. Figure 3(a) shows the illumination light distribution just before entering the splitting prism, and Figure 3(b) shows the illumination light distribution near the installation stand. The dotted lines in Figure 3(a) indicate the positions of the four sides of the splitting prism. After passing through the splitting prism, the illumination light is divided into four fan-shaped parts by the dotted lines in Figure 3(a), each of which moves at a 5° inclination toward the central optical axis. The illumination pattern near the installation stand is then as shown in Figure 3(b). The area where the illumination light from the four directions intersect at this time is a square with sides of approximately 18 mm.

[0044] The imaging section uses a commercially available mirrorless single-lens camera equipped with a full-size (36mm x 24mm) 8K pixel color CMOS sensor. The imaging telecentric lens uses a commercially available 2x telecentric lens that can be installed on the imaging section. If the mounts of the digital camera and the imaging telecentric lens are different, a conversion adapter can be used. The imaging telecentric lens faces vertically upwards, so that it can image the position of the opening in the lower part of the installation stand mentioned above. The imaging area in this case is 18mm x 12mm, and falls within the area (□18mm) where the lighting from four directions intersect, and the entire imaging area is within the range of the same multi-directional lighting.

[0045] The imaging unit and the telecentric lens are equipped with a one-axis stage 009 that moves vertically and is used for focus adjustment. There is also a monitor that displays images captured by the digital camera of the imaging unit in real time.

[0046] By using the imaging device described above, it is possible to provide a system that acquires correct positional information of cells, which are transparent phase objects present over a wide area within a dish.

[0047] [Second embodiment] The configuration of an image acquisition device according to a second embodiment of the present invention will be described with reference to Figures 4 and 5. In Figure 4, the same components as those in Figure 1 are in principle given the same reference numerals, and the description thereof will be omitted.

[0048] The device configuration is almost the same as that of the imaging device according to the first embodiment shown in Fig. 2, with the difference being that an axicon lens is used for the branching prism, and an illumination light scanning mechanism is attached to the illumination section to scan the illumination position in the horizontal direction. Only the differences from the imaging device according to the first embodiment will be explained below.

[0049] A glass axicon lens with a diameter of approximately 50 mm was used for the branching prism. The angle was 10 degrees, and the incident collimated light was tilted approximately 5 degrees toward the optical axis. Figure 5(a) shows the size and position of the illumination light in the imaging range. A Bessel beam area 010 is formed in the area where the light from both sides of the axicon lens intersect. This Bessel beam causes multiple collimated light beams from multiple directions to be irradiated onto only a portion of the center of the screen. Thus, only this portion is displayed as a cell image.

[0050] 011 is an illumination light scanning mechanism for moving the illumination position. As shown in Figure 5(b), the illumination light is scanned evenly vertically and horizontally on the subject in the imaging area. The shutter of the camera in the imaging unit is left open during this scan. By performing this process, it is possible to obtain images of cells that are depicted by irradiating the entire imaging area with collimated light from multiple directions. It is also effective to drive the scan function by moving the collimating means and axicon lens vertically and horizontally while keeping them horizontal, without tilting them. It is also effective to provide an image processing unit, obtain an image each time the illumination light is moved, and obtain an image of the cells in the entire imaging area by combining all the images.

[0051] By using the imaging device according to the present embodiment described above, it is possible to provide an imaging device that acquires positional information of cells, which are transparent phase objects present over a wide area within a dish, at correct positions.

[0052] [Third embodiment] The configuration of an imaging device according to a third embodiment of the present invention will be described with reference to Fig. 7. In the figure, the same components are generally given the same reference numerals and the description thereof will be omitted.

[0053] The configuration of the imaging device of this embodiment can be explained with reference to Fig. 7. The imaging device configuration is almost the same as that of the first embodiment shown in Fig. 2, with the difference being that a fluorescent illumination unit 012 is attached to the subject to obtain a fluorescent image. Only the differences will be explained below.

[0054] The fluorescent illumination unit irradiates the subject with light from an angle different from that of the light irradiation unit, and can be used to obtain the fluorescent characteristics of the subject by irradiating it with excitation light. When obtaining the fluorescent characteristics of the subject, the excitation light is irradiated by epi-illumination to improve the signal-to-noise ratio of the image to obtain the fluorescent information. For example, as shown in Figure 7, it is preferable to irradiate the subject with excitation light obliquely from the imaging unit side. In addition, when the irradiated light is uniform, the amount of light on the irradiated surface irradiated obliquely becomes uniform, which is effective for homogenizing the image and relative evaluation within the screen. This uniform approximately collimated light can be realized by making the light enter a pinhole or bundle fiber from the light source and installing a collimator such as a convex lens or a telecentric lens, as in the above-mentioned light irradiation unit. By using an imaging device that irradiates obliquely in this way, a commercially available telecentric lens can be used for the lens. It is necessary to install a filter (for fluorescence) between the subject and the imaging unit to remove the excitation light and transmit only the fluorescence.

[0055] The excitation light is selected to have a wavelength that matches the excitation characteristics of the fluorescent label, etc., and there is no limitation on the type of light source as long as it irradiates the wavelength. A combination of a wide-band xenon lamp and an excitation filter can also be used. The filter (for fluorescence) must also be selected based on the balance between the fluorescence wavelength from the subject and the excitation light wavelength. It is preferable that the excitation light and the filter (for fluorescence) are easily replaceable.

[0056] In addition, when the subject is in a container such as a cell, the oblique incident light may be irradiated onto the container wall, causing stray light. In order to reduce this stray light, it is preferable to make the second light irradiation unit enter the telecentric lens from the side as shown in FIG. 7(b), and to realize coaxial incident illumination via a dichroic mirror 014 installed on the telecentric lens. It is preferable that this dichroic mirror has wavelength characteristics that reflect the excitation light and pass the fluorescence generated by the subject and the transmitted illumination light. It is also preferable to install a filter (for fluorescence) 013.

[0057] By using the imaging device described above, it is possible to provide an imaging device that can obtain positional information of cells, which are transparent phase objects present over a wide area within a dish, at the correct positions and obtain fluorescent staining results for each cell. [Explanation of symbols]

[0058] 001 Light source 002 Collimation part 003 Branching Prism 004 Installation part 005 First Intersection Area 006 Subject (phase object) 007 Imaging unit 008 Imaging lens 009 1-axis stage 010 Bessel beam region 011 Intersection area change section 012 Fluorescent lighting section 013 Filter (for fluorescence) 014 Dichroic mirror 020 Optical axis of imaging lens 030 Light irradiation unit 100 Imaging device

Claims

1. An imaging device for an image of a phase object, comprising an imaging unit that images the phase object via an imaging lens, a light irradiation unit including a collimator unit that converts light emitted from a light source into substantially parallel light, and a branching prism that branches and emits the substantially parallel light so that the substantially parallel light is non-parallel to the optical axis of the imaging lens and is irradiated onto the phase object from a plurality of directions different from each other; an installation unit for installing the phase object in a region where light beams emitted from the light irradiation unit and coming from a plurality of different directions intersect for the first time; An imaging device having the above configuration.

2. The imaging device according to claim 1 , wherein the branching prism is a polygonal pyramid prism.

3. The imaging device according to claim 1 , wherein the branching prism is an axicon lens.

4. The imaging device according to claim 3 , further comprising an intersection area changing unit that changes the intersection area by displacing at least one of the light source, the collimator unit, and the axicon lens.

5. The imaging device of claim 1 , wherein the phase object comprises a cell.

6. The imaging device according to claim 1 , wherein the collimating portion includes a convex lens or a telecentric lens.

Images