Image acquisition apparatus, image acquisition method, and program

Abstract

To execute characteristic evaluation on a cell that is a transparent phase object with a simpler configuration not having a ring slit and a phase plate.SOLUTION: An image acquisition apparatus is for a subject that is a phase object, and comprises: a first light irradiation unit that irradiates the subject with parallel light that is first light; a second light irradiation unit that irradiates the subject with second light at an incident angle different from the first light irradiation unit; an imaging lens that is formed by including a telecentric optical system; and an imaging unit that picks up an image of the subject irradiated with at least any one of the first light and second light through the imaging lens. An optical axis of the imaging lens is parallel to an optical path of the first light irradiation unit. The imaging unit is arranged, on the optical axis, on an opposite side of the first light irradiation unit with respect to the subject. An optical path length adjustment unit is arranged which controls a distance on the optical axis between the telecentric optical system and the subject.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to an image acquisition device, an image acquisition method, and a program. [Background technology]

[0002] To evaluate the characteristics of a cell, it is important to obtain the morphology of the cell and its chemical and physical properties (optical properties such as fluorescence) other than the morphology. The morphology of a cell, which is a transparent phase object, cannot be depicted by a normal bright-field optical system, and therefore a phase-contrast microscope is widely used to evaluate the characteristics of the cell. More specifically, a phase-contrast microscope is used in which a ring slit is installed in the transmitted illumination section and a phase plate is installed in the objective lens. Patent Document 1 discloses a system that can obtain morphological images and fluorescent properties by further combining this phase-contrast microscope with an excitation light irradiation section and a fluorescent filter for incident illumination. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 4936867 Summary of the Invention [Problem to be solved by the invention]

[0004] In the phase-contrast microscope disclosed in Patent Document 1, the same objective lens is used for phase-contrast observation and fluorescence observation, so that the images obtained by both observations can be superimposed with high accuracy. However, it requires the placement of a ring slit or a phase plate. In addition, there are restrictions on the lenses used to realize phase-contrast observation.

[0005] An embodiment of the present disclosure has been made in view of the above-mentioned problems. One of the objects of the present disclosure is to enable evaluation of characteristics of a phase object using a simpler configuration that does not include a ring slit or a phase plate. [Means for solving the problem]

[0006] In order to solve the above problem, an image acquisition device according to one aspect of the present disclosure includes: An image acquisition device for a subject that is a phase object, comprising: the imaging device comprises a first light irradiation unit which irradiates the subject with a first light, which is a parallel light; a second light irradiation unit which irradiates the subject with a second light at an incident angle different from that of the first light irradiation unit; an imaging lens which includes a telecentric optical system; and an imaging unit which images the subject irradiated with at least one of the first light and the second light, via the imaging lens, wherein the optical axis of the imaging lens is parallel to the optical path of the first light irradiation unit, and the imaging unit is disposed on the optical axis on the opposite side of the subject to the first light irradiation unit; and an optical path length adjustment unit which controls the distance on the optical axis between the telecentric optical system and the subject. Effect of the Invention

[0007] According to an embodiment of the present disclosure, it is possible to perform characteristic evaluation of a phase object with a simpler configuration that does not include a ring slit or a phase plate. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a diagram illustrating a schematic configuration of an image acquisition device according to an embodiment of the present disclosure. [Diagram 2] 13 is a diagram showing a schematic configuration of an imaging telecentric lens when the light from the second light irradiation unit is coaxially incident. FIG. [Diagram 3] FIG. 13 is a diagram showing a schematic configuration in which a second light irradiation unit is installed on the transmitted illumination side. [Figure 4] 13 shows an example of a defocused image in which the cell shape can be confirmed, acquired in the above embodiment. [Diagram 5] 1 is a diagram illustrating a schematic configuration of an image acquisition device according to a first embodiment of the present disclosure. [Figure 6] 10 is a flowchart showing a control flow of an image acquisition system according to a second embodiment. [Figure 7]FIG. 4 is a diagram showing an example of a second light irradiation unit in the embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Preferred embodiments of the present invention will be described below with reference to the drawings. However, the dimensions, materials, shapes, and relative positions of components exemplified in the following embodiments are arbitrary and can be changed according to the configuration of the device to which the present disclosure is applied or various conditions. In addition, the same reference symbols are used in the drawings to indicate elements that are identical or functionally similar. In the embodiments of the present invention, parallel light or parallel light may be approximately parallel light (approximately parallel light) or approximately parallel light within a range in which the effects of the present invention can be obtained.

[0010] Here, even in an optical system that does not use a ring slit and a phase plate, the presence of a transparent phase object can be confirmed by irradiating collimated light and shifting the focus from the focal position (defocusing). Therefore, even in a lens that does not contain a phase plate, for example, a lens for a commercially available digital camera, it is considered that the presence of a transparent phase object can be confirmed by preparing appropriate illumination light. The present disclosure described below is based on the knowledge that not only the presence of a minute transparent phase object such as a cell can be confirmed, but also its morphological information can be obtained by using a digital camera having a high-pixel image sensor in the imaging system and digitally zooming the captured image data.

[0011] In addition, by using a telecentric lens, for example a double-sided telecentric lens, as the imaging lens, it is possible to suppress the change in size due to defocus. Therefore, if a telecentric lens is used as an imaging lens for an image acquisition device exemplified by a digital camera using collimated light, the form of a transparent phase object can be visualized without a change in size. In Patent Document 1, the presence of a minute and transparent phase object such as a cell is confirmed by using a high-magnification objective lens such as a microscope. However, even when an image captured using a digital camera equipped with a high-pixel image sensor is digitally zoomed, the presence of a transparent phase object can be confirmed by using a telecentric lens. Based on the above knowledge, the embodiment of the present disclosure makes it possible to confirm a transparent phase object by irradiating a subject with collimated light, using a telecentric lens as an imaging lens, and obtaining an image in a defocused state.

[0012] However, in a collimated light irradiation system adjusted to a defocused position, only the presence of a transparent phase object or morphological information can be obtained, which is insufficient for evaluating cell characteristics. Therefore, in the present disclosure, excitation light for acquiring fluorescence characteristics is further irradiated onto the cell, and a fluorescence image is obtained in an infocus state. Then, by synthesizing an image obtained by changing the optical path length by moving on the optical axis of the imaging lens, etc., and the image obtained in a defocused state, it is possible to perform evaluation of the characteristics of a cell, which is a transparent phase object.

[0013] That is, the image acquisition device of the present disclosure is irradiated with transmitted light by approximately collimated light from the vertical direction and light from other directions, and images a transparent phase object through a telecentric optical system. Then, using the telecentric optical system, cell characteristics are evaluated from a fluorescent image obtained in an in-focus state, and the presence of a transparent phase object is confirmed or morphological information is obtained from an image obtained in a defocused state. Hereinafter, an example of a specific image acquisition device of the present disclosure will be described with reference to FIG. 1 showing a schematic configuration thereof.

[0014] (Configuration of image acquisition device) The image acquisition device 100 according to an embodiment of the present disclosure includes an image acquisition unit 21 and an image processing unit 22. The image acquisition unit 21 includes a mounting stand 1, a first light irradiation unit 3, a second light irradiation unit 4, an imaging lens 5, an imaging unit 6, and an optical path length adjustment unit 7, which will be described later. The image processing unit 22 can be configured, for example, from a personal computer (PC) or a tablet terminal, and can be communicatively connected to the image acquisition unit 21 or a storage device (not shown) that stores images acquired in advance by the image acquisition unit 21. The image processing unit 22 can acquire images transmitted from the connected image acquisition unit 21 or storage device, and perform a synthesis process, etc., to be described later, on these images.

[0015] The installation table 1 is used when installing the subject 2 to be observed. The subject 2 is placed on the imaging optical axis OA of the imaging unit 6 between the imaging unit 6 (and the imaging lens 5) and the first light irradiation unit 3. The first light irradiation unit 3 irradiates the subject 2 with approximately collimated (parallel) light. The second light irradiation unit 4 irradiates the subject 2 with light at an incident angle different from that of the first light irradiation unit 3. The first light irradiation unit 3 irradiates the upper side (first side) of the subject 2 placed on the installation table 1, and the second light irradiation unit 4 irradiates the lower side (second side opposite to the first side) of the subject 2 placed on the installation table 1 through the installation table 1. The imaging lens 5 is configured to include a telecentric optical system. An example of a telecentric optical system is a telecentric lens. When a telecentric lens is used as the imaging lens, it may be referred to as an imaging telecentric lens hereinafter.

[0016] The imaging unit 6 acquires information on the subject 2 irradiated with light by the first light irradiation unit 3 and the second light irradiation unit 4. The imaging unit 6 receives the transmitted light irradiated from the first light irradiation unit 3 (transmission illumination) and transmitted through the subject 2. The imaging unit 6 also receives the incident light irradiated from the second light irradiation unit 4 (epi-illumination) and reflected by the subject 2. The optical path length adjustment unit 7 changes the optical path length between the subject 2 and the imaging lens 5. In the present disclosure, the imaging unit 6 also has an imaging control unit 6a. As described later, the imaging unit 6 can acquire, for example, a defocused image obtained by irradiation with light by the first light irradiation unit 3 and an infocus image obtained by irradiation with light by the second light irradiation unit 4, and these images can be transmitted to the image processing unit 22. The imaging control unit 6a operates, for example, the optical path length adjustment unit 7 based on the focus state obtained from the defocused image and the infocus image to adjust the optical path length described above.

[0017] It is preferable that the entire image acquisition unit 21 is covered with a housing so that no light enters from the outside. However, a door that can be opened and closed when placing the subject 2 on the placement stand 1 is necessary. In addition, since vibrations during imaging can cause fluctuations in the position of the subject 2 and image degradation, it is preferable to provide vibration countermeasures for the entire image acquisition unit 21. For example, it is preferable to install anti-vibration rubber or air springs under the entire image acquisition unit 21. 1 illustrates an inverted image acquisition device, but the form of the image acquisition device to which the present disclosure is applied is not limited to the example in FIG. 1, and the image acquisition device may also be used in an upright image acquisition device in which the vertical positional relationship is inverted. Also, the image acquisition unit 21 and the image processing unit 22 are configured as separate entities, but they may be integrated, and the imaging control unit 6a may be configured to be included in a PC or the like that includes the image processing unit 22 as necessary. The configuration of each of the above-mentioned image acquisition devices will be described below.

[0018] (subject) In the present disclosure, a transparent phase object is exemplified as the subject 2 to be observed. When the phase object is a cell, the subject 2 is a transparent container in which liquid and cells are sealed. In this case, a petri dish made of commercially available glass, polystyrene, or the like is used as the container. In addition, the liquid used may be a liquid for maintaining cells during cell culture, such as culture medium or PBS (Dulbecco's Phosphate Buffered Saline), or a liquid for preventing immobilized cells from drying. Note that the container needs to be optically transparent on the light irradiation side and the imaging unit side, but other sides, such as the side faces, do not need to be transparent.

[0019] The installation table 1 is arranged for installing the subject 2, and the imaging unit 6 side and the first light irradiation unit 3 side need to be optically transparent mechanisms. Specifically, for example, in the case of an inverted image acquisition device as shown in Fig. 1, it is preferable that the imaging area has a hole penetrating from the imaging unit 6 side to the first light irradiation unit 3 side. It is also preferable that a fixing jig is provided so that the above-mentioned container does not move from its placement position on the installation table 1 even with a small impact. In addition, since containers come in a variety of shapes, such as circular petri dishes and rectangular multi-well plates, it is preferable that the attachments and fixing jigs can be changed to match these sizes. Moreover, the imaging position can be changed by placing the installation table 1 on a two-axis XY stage. This allows imaging of different wells when the observation target is a multi-well plate. The imaging position may also be changed by moving the first light irradiation unit 3, the second light irradiation unit 4, the imaging lens 5, and the imaging unit 6 without moving the installation table 1. It is preferable that the scanning plane of the installation table 1 and the XY stage is perpendicular to the optical axis (OA) of the imaging unit 6 and the imaging lens 5 without any inclination.

[0020] (First light irradiation unit) The first light irradiating unit 3 irradiates the subject 2 with approximately collimated (parallel) light. More specifically, the first light irradiating unit 3 is composed of a radiation light source that is close to a point light source and a collimator that changes the light from the light source into parallel light. It is preferable that the light irradiated from the first light irradiating unit 3 to the subject is uniform, that is, that there is little variation in the intensity of the irradiated light. The optical path of the light irradiated from the first light irradiating unit is parallel to the optical axis of the imaging lens described below.

[0021] As a method for obtaining a point light source as a radiation light source, a pinhole may be installed in front of the light source. It is also effective to use the light emitted from the output end of a bundle fiber that takes in light from a light source as a point light source. A convex lens, a telecentric lens, or the like can be used as a collimator that converts light from a radiation light source into parallel light. The irradiation light used in the optical system exemplified here has a uniform light intensity distribution on the subject, and an image with suppressed intensity unevenness can be obtained. However, the application of the present disclosure is not limited to the example described here, and collimated light with non-uniform intensity may be irradiated, and visualization of a phase object can be realized even in this case. In addition, the first light irradiation unit 3 is not limited to the form exemplified here as long as it can form parallel light.

[0022] The optical axis of the parallel light emitted from the first light irradiating unit 3 is arranged to be parallel to the optical axis OA of the imaging unit 6 and the imaging lens 5. Therefore, in the case of the exemplified inverted imaging system, it is preferable that the first light irradiating unit 3 irradiates light vertically from above the subject 2. It is also preferable that the parallelism of both the optical axes of the imaging unit 6 and the imaging lens 5 is smaller than the maximum spread angle of the imaging telecentric lens used in the imaging lens 5. The imaging lens 5 may be a low-magnification lens.

[0023] In addition, in the first light irradiation unit 3 described above, a collimator is used to obtain substantially parallel light, but if substantially parallel light can be created, a collimator may not be used. For example, in the case of a radiation light source close to a point light source, if the distance from the light source to the subject 2 is increased and the imaging range of the subject 2 is narrowed, the light used for imaging approaches substantially parallel light. In reality, if the angle of incidence of light irradiated within the imaging range with respect to the imaging optical axis OA of the imaging unit 6 is less than the maximum spread angle of the imaging telecentric lens, an effect similar to that of using a collimator can be expected. That is, in this disclosure, substantially parallel light refers to light obtained by using a collimator and light that can be expected to have an effect similar to that of using a collimator. In addition, the light source wavelength of the first light irradiation unit may be white light, but an LED that emits a specific wavelength may be used to avoid blurring caused by chromatic aberration.

[0024] 1, the light from the light irradiation unit 3 is irradiated vertically, but it may be irradiated horizontally and reflected by a mirror tilted at 45 degrees so that the light is irradiated vertically onto the subject 2. In this way, the vertical size of the image acquisition unit 21 can be reduced. As a specific light source, a white light source such as an LED (Light Emitting Diode) light source or a halogen light source can be used. The wavelength band of the illumination light emitted from the light source causes blurring due to chromatic aberration of diffracted light. Therefore, a narrower band is more preferable to obtain a high-contrast image, so it is effective to use an LED with a narrow wavelength band width or to prepare various filters such as bandpass filters for an LED white light source with a wide wavelength band width.

[0025] (Imaging unit and telecentric lens for imaging) The imaging unit 6 acquires optical information from the subject 2, and the image processing unit 22 converts the optical information transmitted from the imaging unit 6 into an image. In the present disclosure, the imaging unit 6 may be, for example, a digital camera equipped with a CCD and a CMOS sensor in the imaging element. In this case, the imaging element preferably has a larger number of pixels, and the greater the number of pixels, the higher the resolution of the subject 2 by digital zoom, improving the ability to depict the morphology of a minute subject such as a cell.

[0026] It is more preferable to use a double-telecentric lens as the imaging telecentric lens used in the imaging lens 5. In this disclosure, a telecentric lens is used in the imaging lens 5, and because the chief ray of the telecentric lens is parallel to the optical axis, the following three effects can be obtained.

[0027] The first effect is that there is almost no change in the size of the subject when defocused. With a non-telecentric lens, the effect of defocusing not only causes blurring of the outline, but also causes a change in size equal to the angle of view. In contrast, with a telecentric lens, which has no angle of view, there is no change in size.

[0028] The second effect is that unevenness in transmitted illumination is suppressed. If a lens with a large angle of view is used instead of the imaging telecentric lens, the area of ​​the light irradiation unit within the angle of view becomes smaller as the distance between the first light irradiation unit 3 and the imaging lens 5 increases. Therefore, a larger light irradiation unit is required to obtain an image in which the entire imaging area is bright. In contrast, in the case of a telecentric lens, the area of ​​the light irradiation unit within the angle of view does not change even if the distance increases. Therefore, as long as the size of the first light irradiation unit 3 is equal to or larger than the imaging area, an image in which the entire area is bright can be obtained regardless of the distance between the light irradiation unit and the lens.

[0029] The third effect is that a uniform image can be obtained within the imaging plane. When a telecentric lens is used, the chief ray is parallel to the lens optical axis, so the light incident on the imaging element is limited to the components from the subject on the chief optical axis. Therefore, if uniform illumination light is incident on the imaging area, a relative evaluation of the subject within the plane can be realized using the output value of the imaging data.

[0030] It is preferable that the image generated by the image processing unit 22 can be immediately checked by an operator in order to check the imaging location and image quality. Therefore, it is preferable that the image processing unit 22 is connected to a monitor (not shown) that displays the generated image information. It is also preferable that the imaging section and the imaging telecentric lens are interchangeable as appropriate depending on the type of subject 2 and the contents of measurement.

[0031] (Second light irradiation unit) The second light irradiation unit irradiates the subject 2 with light from an angle different from that of the first light irradiation unit, and can be used to acquire the fluorescence characteristics of the subject 2 by irradiating it with excitation light. When acquiring the fluorescence characteristics of the subject 2, the excitation light is irradiated by epi-illumination to acquire fluorescence information in order to improve the signal-to-noise ratio of the image. In this case, it is preferable to irradiate the subject 2 with excitation light obliquely from the imaging unit 6 side, as shown in FIG. 1, for example. It is preferable that the light irradiated from the second light irradiation unit to the subject is uniform, that is, that there is little variation in the intensity of the irradiated light.

[0032] In addition, when the light irradiated is uniform, approximately collimated light, the amount of light on the irradiated surface irradiated from an angle becomes uniform, which is effective for homogenizing the image and relative evaluation within the screen. To obtain such uniform approximately collimated light, it can be achieved by irradiating a collimator with uniform light. A convex lens or a telecentric lens can be used for the collimator. The uniform incident light can be achieved by passing the light from the light source through a diffusion plate or a bundle fiber. In addition, a rod lens that emits uniform light by repeatedly reflecting the incident light inside a polygonal prism like a kaleidoscope is also effective. It is also effective to use these in combination. Figure 7 shows an example of a second light irradiation unit that combines a light guide 710, a rod lens 711, and a telecentric lens 712. It is preferable to use a bundle fiber for the light guide 710. It is necessary to adjust the focus position so that it is on the subject when a rod lens is used. In addition, the uniform light means that the light amount distribution in the observation area is gentle and there is no partial change up and down. When performing fluorescence imaging, it is necessary to install a filter (for fluorescence) between the subject 2 and the imaging section 6 that removes excitation light and transmits only fluorescence (not shown).

[0033] The excitation light is selected to have a wavelength suited to the excitation characteristics of the fluorescent label, etc., and the type of light source is not limited as long as it irradiates light of that wavelength. A light source that emits white light, for example, a light source that combines a mercury lamp with an excitation filter (a filter that transmits light of a specific wavelength), can also be used. The filter (for fluorescence) must also be selected taking into account the tradeoff between the fluorescence wavelength from the subject 2 and the excitation light wavelength. It is preferable that the excitation light and the filter (for fluorescence) are easily replaceable.

[0034] When the subject 2, such as a cell, is contained in a container, such as a petri dish, the oblique incident light may irradiate the container wall and cause stray light. In order to reduce this stray light, it is preferable to realize coaxial incident illumination by making the excitation light enter the telecentric lens from the side and passing it through a light path changing member, such as a dichroic mirror, installed on the telecentric lens.

[0035] FIG. 2 shows a schematic configuration of an example of an optical system that realizes such incidence of excitation light. In the example shown in FIG. 2, the second light irradiation unit 4 is fixed to the side of the telecentric lens, which is the imaging lens 5, and is configured to directly input the excitation light into the telecentric lens. A dichroic mirror 9 is disposed inside the telecentric lens. The excitation light is reflected by this dichroic mirror 9 and is irradiated onto the subject 2 from the telecentric lens along an optical axis parallel to the imaging optical axis OA of the imaging unit 6. In this configuration, in order to remove reflected light originating from the excitation light from the subject 2, it is necessary to install a filter 8 that does not pass the excitation light but passes only the fluorescence just before imaging.

[0036] Furthermore, by using the second light irradiating unit 4, it is possible to obtain characteristics of the subject 2 other than the fluorescence characteristics. An example of an image acquisition device for obtaining characteristics other than the fluorescence characteristics will be described with reference to the schematic diagram of FIG. 3. In the example shown in FIG. 3, the second light irradiating unit 4 is configured to irradiate the imaging unit 6 with light from the back side of the subject 2. In this manner, in a configuration in which light is irradiated from the back side of the subject 2, when light is irradiated at an angle equal to or greater than the maximum spread angle with respect to the optical axis of the imaging telecentric lens, which is the imaging lens 5, a dark-field illumination image is obtained, and when light is irradiated at an angle equal to or less than the maximum spread angle, an oblique illumination image is obtained.

[0037] In this example, a telecentric lens is used as the imaging lens 5. However, an optical system that can achieve the same effect as the telecentric lens can be provided by using a plurality of optical members and placed at the position of the imaging lens. Although the configuration may become complicated, the design freedom regarding the placement of the dichroic mirror and the like is increased by using such a configuration. In this disclosure, the telecentric optical system includes each of the aspects of these optical systems and the aspect of the telecentric lens.

[0038] (Optical path length adjustment section) Even if nearly collimated light from the first light irradiation unit 3 is incident parallel to the optical axis of the imaging telecentric lens (imaging lens 5) and the subject 2 is imaged at the focus position of the imaging lens 5, the cells, which are transparent phase objects, are not visualized. However, by slightly defocusing from the focus position, the transparent cells are depicted. FIG. 4 shows examples of images obtained by imaging a transparent phase object by changing the defocus amount (Z) from the focus position. As can be seen from FIG. 4, by changing the defocus amount, it is possible to grasp the transparent cells.

[0039] On the other hand, since a fluorescent image is light emitted from a cell, high-quality information can be obtained by capturing the image at the focal position. Therefore, when using the second light irradiating unit 4 to image the subject 2 using the light, it is preferable to capture the image in focus. Therefore, it is preferable to obtain a defocused image when using the first light irradiating unit 3 and an infocus image when using the second light irradiating unit 4, and it is necessary to change the optical path length when acquiring both images.

[0040] In the present disclosure, an optical path length adjustment unit 7 is provided as a means for changing the optical path length. As the optical path length adjustment unit 7, there is a method of changing the distance between the imaging lens 5 and the subject 2. In this case, a one-axis stage for moving the installation table 1 or a one-axis stage for moving the imaging unit 6 and the imaging lens 5 can be used. In addition, when the second light irradiation unit 4 irradiates the subject 2 with light from an oblique angle as in the configuration exemplified in FIG. 1, the irradiation surface will shift if the installation table 1 is moved. Therefore, in the case of a configuration in which the installation table 1 is moved, it is preferable to move the second light irradiation unit 4 in the same direction in synchronization with the installation table 1 in order to maintain the light irradiation conditions. With such a configuration, the light irradiation conditions in the imaging area of ​​the subject do not change even if the optical path length is changed. In addition, a manual stage or an electric stage can be used for the one-axis stage described here.

[0041] It is also effective to insert a member having a different refractive index between the imaging lens 5 and the subject 2 as the optical path length adjustment unit 7. FIG. 5 shows an example of such a configuration in which a turret-type filter replacement unit 10 is provided for the fluorescence filter 8, and multiple fluorescence filters can be selected using the filter replacement unit 10. In the configuration illustrated in FIG. 5, when excitation light is used by the second light irradiation unit 4, the focus is set to be adjusted on the subject 2 with the corresponding fluorescence filter 8 installed. It is preferable to use the one-axis stage described above for focus adjustment in this case.

[0042] In this configuration, when the second light irradiation unit 4 is used, the subject 2 is imaged in-focus using the fluorescent filter 8. When the first light irradiation unit 3 is used, the filter replacement unit 10 is driven to create a defocused state by switching to a filter that does not have a fluorescent filter or has a different optical path length from the fluorescent filter 8 used when the second light irradiation unit 4 is used. This makes it possible to switch between in-focus and defocus by simply driving the filter replacement unit 10 without the need to move the subject 2 or the imaging unit 6, thereby reducing the risk of the position of the observation target shifting when switching.

[0043] (Image processing unit) When the optical path length is changed in parallel to the optical axis of the imaging lens 5 by the optical path length adjustment unit 7, the size and position of the image do not change because a telecentric lens is used. Therefore, the positions of the defocused image and the infocus image correspond to each other, and superimposition of the two images can be easily performed. For example, a cell can be evaluated by superimposing a defocused image (morphological image) obtained using the first light irradiation unit 3 and an infocus image whose fluorescent characteristics are obtained using the excitation light from the second light irradiation unit 4. The image processing unit 22 performs this superimposition process. In addition to the superimposition, the image processing unit 22 may count the number of cells from the defocused image and count the number of cells that show fluorescent characteristics from the infocus image to calculate the ratio of cells having the characteristics. The processing contents performed by the image processing unit 22 are not limited to these, and any analysis using the infocus image and the defocused image can be performed. In the above system, the light sources of both light irradiation units may be controlled in conjunction with the image capture unit 6. In addition, when an electric stage is used for the optical path length adjustment unit 7 or the XY stage attached to the installation table 1, these may also be controlled in conjunction with each other. In addition, it is also effective to simplify the system by consolidating the light source control units of both light irradiation units, the imaging control unit, and the controller of the motorized stage into one. In this case, it is preferable to provide some measure to ensure sufficient heat dissipation.

[0044] (Automatic Processing Unit) In many cell characteristic evaluations, the measurement procedure is often predefined, and after the imaging location is determined, the predefined processing is often performed. In this case, it is effective for the efficiency of data acquisition work to automatically perform defocused image acquisition, infocus image acquisition, and image processing using a predefined algorithm. In the present disclosure, for example, the imaging control unit 6a executes this series of processing.

[0045] In the present disclosure, for example, after placing the subject 2 on the installation stand 1 and determining the imaging location, automatic processing is started by an imaging start switch (not shown). After starting the automatic processing, the first light irradiation unit 3 is turned on, the optical path length adjustment unit 7 adjusts the imaging unit 6 to a defocused position, and a defocused image is acquired. Thereafter, the first light irradiation unit 3 is turned off, and the second light irradiation unit 4 is turned on. At the same time, the optical path length adjustment unit 7 adjusts the imaging unit 6 to an infocus position, and an infocus image is acquired. Thereafter, the second light irradiation unit 4 is turned off, and the image processing unit 22 performs superimposition processing of the acquired defocused image and infocus image, etc. After the processing, it is also effective to display the processing result on a monitor (not shown). This operation is an example of automatic processing, and the order of the above-mentioned processing does not matter. It is preferable that the series of operations executed by the imaging control unit 6a be stored in the imaging control unit 6a, for example, so that they are executed automatically.

[0046] By using the image acquisition device 100 as described above, it is possible to evaluate the characteristics of cells, which are transparent phase objects, without using the conventional configuration in which a phase plate is mounted on a lens.

[0047] [First Example] Next, the configuration of an image acquisition device according to a first embodiment of the present disclosure will be described with reference to the schematic diagram of Fig. 5. In the drawing, the same components as those in the configuration previously described as an embodiment of the present disclosure are generally designated by the same reference numerals, and detailed description thereof will be omitted below.

[0048] A typical observation subject of this image acquisition device is a cell, which is an example of the above-mentioned transparent phase object, and this cell is the subject 2. In this embodiment, the subject 2 exists as cells that are adherently cultured in a 6-well plate for cell culture made of polystyrene, and the cells are further immobilized and then stained with a specific fluorescent label. Note that TBS (Tris-buffered saline) is injected into the 6-well plate as a liquid to prevent drying, but the observation subject may be a floating cell.

[0049] In this embodiment, the installation table 1 is provided with an attachment on which a general-purpose 6-well plate (a container having six recesses, hereinafter referred to as a container) can be installed. The attachment is provided with a fixing means (e.g., a spring means such as a leaf spring) for pressing the container in two horizontal directions, and when the container is inserted, it is fixed to the installation table 1. The installation table 1 is composed of two members, an upper and lower. The upper member with which the container comes into contact is composed of an opening that is open over a wide area including all six recesses in the center of the container, and the container comes into contact with the member only at the periphery. The lower member has an opening that is open only in the observation area. The upper member can be moved together with the container on a horizontal two-axis stage, while the lower member is fixed in the horizontal direction.

[0050] The two-axis stage described here employs a manual stage with a rack-and-pinion configuration, but it is not necessary to be limited to this. In the case of coarse movement, linear guides can be installed in two axial directions, and the installation table 1 itself can be moved manually, and in the case of fine movement, a micro stage or the like can be used. In addition, it may be possible to switch between coarse and fine movement. An automatic stage may be adopted and operated from, for example, an external control device such as a PC. During observation, the recessed portion to be observed is moved to the opening of the lower member by the two-axis stage and positioned at the observation position. In addition, the installation table 1 in this embodiment is designed to be able to fix containers of different shapes, such as a 35 mm dish, by providing a dedicated attachment.

[0051] The first light irradiation unit 3 uses an illumination system that combines a high-brightness LED light source that emits light with a wavelength of 535 nm, a bundle fiber, and a telecentric lens. In this embodiment, the light emitted from the LED light source is made incident on the bundle fiber, and the light is emitted through a telecentric lens attached to the output end of the bundle fiber, so that uniform collimated light can be irradiated onto the subject 2. The telecentric lens used in the imaging lens 5 is installed so as to irradiate light in a vertical direction above the installation table 1. In this embodiment, the telecentric lens used realizes uniform circular light with an output light of about φ50 mm.

[0052] The imaging unit 6 employs a commercially available mirrorless single-lens digital camera equipped with a full-size (36 mm x 24 mm) 8K pixel color CMOS sensor. The imaging lens 5 employs a commercially available 2x telecentric lens that can be installed in the imaging unit 6 as an imaging telecentric lens. 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 upward so that it can image the position of the opening in the lower member of the installation stand 1 described above. The imaging unit 6 and imaging lens 5 are mounted on an electric single-axis stage that moves vertically. In addition, a monitor (not shown) is also provided to display images captured by the digital camera constituting the imaging unit 6 in real time.

[0053] The cells in the 6-well plate (container) are irradiated with uniform collimated light of 535 nm from the first light irradiation unit 3 in the vertical direction, and the motorized one-axis stage is moved. At that time, the image acquired by the imaging unit 6 is displayed on the monitor in real time, and the part where the cells are located is enlarged and displayed by digital zoom. The image of the cells displayed on the monitor at that time is exemplified in FIG. 4. As shown in the figure, the image of the cells is not displayed at the focus position, and the image of the cells is displayed when the focus position is shifted forward or backward. According to FIG. 4, it can be understood that in this embodiment, it is preferable to move the focus position by about 50 μm. In addition, the brightness and darkness of the displayed image are inverted depending on the direction of focus shift, but which one to select may be appropriately selected depending on, for example, the display state of the observation target.

[0054] The second light irradiation unit 4 may be provided with a plurality of light sources, or may be configured to switch between the light sources and emit light. Light from a plurality of light sources may be multiplexed. In this embodiment, an illumination system is used that combines a high-intensity LED light source that emits light of a plurality of wavelengths including light of a wavelength of 365 nm, a quartz bundle fiber, and a telecentric lens. The input end of the quartz bundle fiber is branched, and LEDs of the respective wavelengths are attached to each input end. The switching and multiplexing of the light sources are performed by turning on and off each LED and adjusting the light amount. In this embodiment, the input end of the quartz bundle fiber is replaceable with a plurality of LEDs. A telecentric lens is attached to the output end of the quartz bundle fiber. With this configuration, even when the output light is irradiated obliquely to the subject 2, the light amount distribution on the irradiation surface becomes constant. It is also effective to insert a rod lens between the bundle fiber and the telecentric lens to improve the uniformity of the light amount. In this embodiment, a uniform light amount is obtained in a range of about 15 mm x 15 mm by inserting a rod lens.

[0055] By irradiating the subject 2 with the emitted light from the second light irradiation unit 4 from a position tilted by approximately 30 degrees or more from the optical axis of the imaging lens 5, the subject 2 is irradiated with light having a uniform light amount over an area of ​​approximately 18 mm x approximately 15 mm. This is a condition that allows the imaging range of 18 mm x 12 mm captured by the imaging lens 5 (2x telecentric lens) and full-size CMOS sensor to be almost irradiated, and light with a uniform light amount can be irradiated over the imaging range.

[0056] The filter installed in front of the imaging unit is preferably configured to change the wavelength of light transmitted by switching the filter, and may be automatic or manual. In the case of manual operation, an operation unit for switching may be provided outside the housing. The position after switching may be fixed. In this embodiment, a turret-type filter exchange unit 10 is provided between the subject 2 and the imaging lens 5, and multiple optical filters can be attached. Specifically, when the first light irradiation unit 3 is turned on, a filter that can pass light in the 535 nm band is selected, and when the second light irradiation unit 4 is turned on, a filter with band characteristics corresponding to the fluorescence characteristics to be obtained is selected. In this embodiment, for example, when irradiating light of 365 nm, a 400 nm long-pass filter is used. Although FIG. 5 shows a configuration in which four types of optical filters can be changed, it is not necessary to limit the number to four, and it is effective to increase the number of types as necessary.

[0057] In this embodiment, the optical path length adjustment unit 7 uses a one-axis stage attached to the imaging unit 6. By driving this one-axis stage, the in-focus and defocus of the imaging lens 5 are adjusted. As described above, when filters with different optical path lengths are installed in the filter replacement unit 10, the automatic stage that drives the filter replacement unit can also be used as the optical path length adjustment unit 7.

[0058] As described above, according to the image acquisition device of this embodiment, physical information such as morphological information and fluorescent information of cells, which are transparent phase objects, can be acquired without mounting a phase plate on the lens, and cell characteristics can be evaluated.

[0059] [Second Example] Next, automatic imaging of an image acquisition device according to a second embodiment of the present disclosure will be described. As in the first embodiment, the same components as those described in the first embodiment of the present disclosure are generally designated by the same reference numerals, and detailed description thereof will be omitted.

[0060] In this embodiment, a process of acquiring physical information of a cell, which is a transparent phase object, by automatic imaging processing using the image acquisition device shown in FIG. 5 described as the first embodiment will be described with reference to FIG. 6. FIG. 6 is a flow chart showing a system flow related to the physical information acquisition processing according to this embodiment. In this embodiment, steps S601 to S606 in FIG. 6 are processes performed manually, and steps S607 to S618 are processes automatically performed by the imaging control unit 6a. These automatically performed processes are controlled by an algorithm recorded in advance in the imaging control unit 6a.

[0061] (Manual process) In the process of acquiring physical information of a transparent cell, first, in step S601, an operator places a stained cell that has been adherently cultured on a 6-well polystyrene cell culture plate on the setting table 1 as the subject 2. After the placement, in step S602, the operator turns on the first light irradiating unit 3 via an input device such as a control panel (not shown) provided on the image acquisition device, and irradiates the subject 2 with collimated light having a wavelength of 535 nm. Then, in step S603, the operator operates the turret-type filter replacement unit 10 to place a filter (for transmitted light) that transmits 535 nm light at a predetermined position on the imaging optical axis OA of the imaging unit 6.

[0062] In the next step S604, the image acquired by the imaging unit 6 is displayed on a monitor (not shown) by the image processing unit 22. In step S605, the operator operates the one-axis stage, which is the optical path length adjustment unit 7, to a position (defocus position) where the cell morphology can be seen while viewing the image of the subject 2 displayed on the monitor. After a suitable defocus position is obtained, in step S606, the operator operates the two-axis XY stage associated with the installation table 1 to move the subject 2 on the installation table 1, and selects the imaging location while viewing the cell image displayed on the monitor.

[0063] (automatic process) After the defocus position is determined and the imaging location is selected, the operator causes the imaging control unit 6a to execute the imaging process start process in step S607 via the above-mentioned input device. More specifically, in this embodiment, the subsequent automatic process is started by selecting a start button on the imaging operation screen displayed on the monitor. When the automatic process is started, the imaging control unit 6a shifts the flow to step S608.

[0064] In step S608, the imaging control unit 6a first starts controlling each component according to the transmitted light imaging conditions set by default. If the flow is followed, the first light irradiating unit 3 is turned on to emit light with a wavelength of 535 nm, and the filter exchange unit 10 places a filter for transmitted light on the imaging optical axis OA of the imaging unit 6. In step S608, the imaging control unit 6a automatically checks whether the image acquisition device corresponds to this condition, and if it is different from the setting, drives these components so that it is set to this condition. In the following step S609, the imaging control unit 6a drives the one-axis stage, which is the optical path length adjustment unit 7, to set the defocus position. If the flow is followed, the defocus position is adjusted to the visually set position at this stage, but in this embodiment, automatic adjustment is performed to the pre-set defocus position.

[0065] Here, a method for setting the defocus position will be described. First, the imaging lens 5 is moved back and forth in the optical axis direction near the focus position on the optical axis defined by the optical conditions of the imaging lens 5, and an image of the subject 2 is acquired at each point. Next, the contrast of the image obtained by, for example, the image processing unit 22 is measured, and the imaging position of the imaging lens 5 when the image with the lowest contrast is obtained is set as the focus position, and the coordinates on the optical axis at that time are recorded. After that, the one-axis stage is moved by a predefined amount. In this embodiment, the defined amount is 50 μm, and the position where the imaging lens 5 is brought closer to the subject 2 by 50 μm from the focus position is set as the defocus position.

[0066] After the imaging control unit 6a moves the imaging lens 5 to the defocus position, in step S610, the imaging control unit 6a acquires a defocused image of the subject 2 by the digital camera of the imaging unit 6. After acquiring the defocused image, the imaging control unit 6a moves the flow to step S611. Next, in step S611, the imaging control unit 6a drives the one-axis stage to move the imaging lens 5 to the focus position recorded in step S609.

[0067] In step S612, the imaging control unit 6a turns off the first light emitting unit 3, and in step S613, operates the filter replacing unit 10 to place the fluorescence filter 8 corresponding to the second light emitting unit 4 on the imaging optical axis OA of the imaging unit 6. In this embodiment, the fluorescence filter 8 and the transmitted light filter have the same thickness of 2 mm and are made of the same glass material. Therefore, even if the filter is replaced, the optical path length does not change.

[0068] Immediately after turning on the second light irradiating unit 4 in step S614, the imaging control unit 6a acquires a fluorescent image of the subject 2 with the digital camera of the imaging unit 6 in step S615. Thereafter, the imaging control unit 6a turns off the second light irradiating unit 4 in step S616. In this manner, by shortening the lighting time of the second light irradiating unit 4, optical damage to the subject 2 can be reduced as much as possible.

[0069] After turning off the second light irradiation unit 4, the imaging control unit 6a shifts the flow to step S617. In step S617, the image processing unit 22 executes a process of superimposing the defocused image acquired in step S610 and the infocus image acquired in step S615. In step S618, the image generated by the superimposition process is displayed on a monitor (not shown), and the imaging control unit 6a ends a series of imaging processes.

[0070] As described above, in this embodiment, the operator manually sets the imaging location. Then, the imaging control unit 6a appropriately turns on the first light irradiating unit 3 and the second light irradiating unit 4 to automatically obtain a defocused image and an infocus image of the subject 2, and automatically generates a superimposed image of these and displays it on the monitor. This makes it possible to easily obtain physical information such as morphological information and fluorescent information of a cell, which is a transparent phase object, without mounting a phase plate on the lens, and to evaluate the cell characteristics.

[0071] This embodiment describes an example of a method for setting or acquiring a defocused image and an infocus image acquired by automatic processing. That is, the order of acquiring and processing both images, the filter (for fluorescence) conditions and the number of images acquired when capturing an infocus image, and the image processing conditions can be appropriately changed according to the protocol to be implemented.

[0072] As described above, the image acquisition device according to one embodiment of the present disclosure acquires an image of the subject 2, which is a transparent phase object. The image acquisition device includes a first light irradiation unit 3, a second light irradiation unit 4, an imaging unit 6, and an optical path length adjustment unit 7. The first light irradiation unit 3 irradiates the subject 2 with parallel light (approximately parallel light) as the first light, for example, by using a collimator. The second light irradiation unit 4 irradiates the subject 2 with second light (for example, excitation light) at an incident angle different from that of the first light irradiation unit 3. The imaging unit 6 is configured to include a telecentric lens (imaging lens) 5 as a telecentric optical system, and images the subject 2 irradiated with the first light and the second light via the telecentric lens 5. The imaging unit 6 has an imaging optical axis OA parallel to the optical path of the first light irradiation unit 3, and is disposed on the imaging optical axis OA on the opposite side of the subject 2 from the first light irradiation unit 3. The optical path length adjustment unit 7 can control the position of the imaging lens 5 on the imaging optical axis OA to control the focus state of the subject 2 to be in-focus or out-of-focus. With the above configuration, even when a digital camera using a high-pixel imaging element is used, it is possible to obtain morphological information and information about physical properties of a minute and transparent phase object by digitally zooming the obtained image.

[0073] The image acquisition device described above may further include an image processor 22 that synthesizes a defocused image acquired when the first light is irradiated and an infocus image acquired when the second light is irradiated. By acquiring a defocused image and an infocus image using a telecentric optical system, these images can be easily synthesized. In addition, the second light may be, for example, an excitation light in order to obtain physical characteristics such as the fluorescent characteristics of transparent cells, and the image acquisition unit 6 may capture a fluorescent image of the subject 2.

[0074] In the above-mentioned image acquisition device, the telecentric optical system (imaging lens 5) can have an optical path changing unit that changes the optical path of the second light so that the optical path is parallel to the imaging optical axis OA of the imaging unit 6. In the above-mentioned embodiment, the dichroic mirror 9 is used as the optical path changing unit, but the aspect of the optical path changing unit is not limited to this as long as a similar effect is obtained. In such an aspect, the second light irradiating unit 4 may irradiate the dichroic mirror 9 with the second light. In addition, in the case where such an optical path changing unit is not provided, the second light irradiating unit 4 may be disposed on the same side as the imaging unit 6 with respect to the subject 2, and may irradiate the second light from the opposite side to the first light.

[0075] Furthermore, in the above-mentioned image acquisition device, the optical path length adjustment unit 7 may control the position of the imaging lens 5 so that the irradiation range of the second light on the subject 2 does not deviate from the imaging area. In the case of an aspect in which the second light is obliquely irradiated on the subject 2 as in the example shown in FIG. 1, for example, if only the installation table 1 is moved for focusing, the irradiation surface will shift, and a fluorescent image at a desired position will not be obtained. By performing such control, a fluorescent image at a desired position can always be obtained. Note that in the present disclosure, as a specific control, the optical path length adjustment unit 7 controls the position of the imaging lens 5 or the distance between the imaging lens 5 and the subject 2 while keeping the positional relationship between the subject 2 (installation table 1) and the second light emission part of the second light irradiation unit 4 constant. However, as long as the above-mentioned irradiation range can be kept within the imaging area, for example, the aspect is not limited to the disclosed example. Also, the subject 2 such as a transparent cell can be accommodated in a container whose upper surface and lower surface, which are arranged approximately perpendicular to the imaging optical axis OA of the imaging unit 6, are transparent to the first light and the second light.

[0076] The present disclosure can also configure an image acquisition method in which the subject 2 is irradiated with the first light from the above-mentioned first light irradiation unit 3, the subject 2 is irradiated with the second light from the second light irradiation unit 4, and an image of the subject 2 is acquired via a telecentric optical system. In the image acquisition method, a defocused image of the subject 2 is acquired in a defocused state using the first light, and an infocus image of the subject 2 is acquired in an infocus state using the second light. Then, in the image processing unit 22, a composite image is generated using the defocused image and the infocus image. By performing the above method, it is possible to evaluate the characteristics of a minute cell, which is a transparent phase object, with a simpler configuration that does not have a ring slit or a phase plate.

[0077] (Other Examples) The present invention can also be realized by providing a program for implementing one or more functions of the above-described embodiments to a system or device via a network or a storage medium, and having a computer in the system or device read and execute the program. The computer may have one or more processors or circuits, and may include multiple separate computers or a network of multiple separate processors or circuits so that the computer can read and execute executable instructions.

[0078] The above disclosure includes the following configurations, methods, and programs. (Configuration 1) An image acquisition device for a subject that is a phase object, comprising: A first light irradiation unit that irradiates the subject with parallel light that is a first light; a second light irradiation unit that irradiates the subject with a second light at an incident angle different from that of the first light irradiation unit; An imaging lens including a telecentric optical system; an imaging unit that captures an image of the subject illuminated with at least one of the first light and the second light via the imaging lens; Equipped with an optical axis of the imaging lens is parallel to an optical path of the first light irradiation unit; the imaging unit is disposed on the optical axis on an opposite side of the subject to the first light irradiation unit, An image acquisition device comprising an optical path length adjustment unit that controls a distance on the optical axis between the telecentric optical system and the subject. (Configuration 2) 2. The image acquisition device according to claim 1, further comprising an image processing unit that combines a defocused image of the subject acquired when irradiating the subject with the first light and an infocus image of the subject acquired when irradiating the subject with the second light. (Configuration 3) 3. The image acquisition device according to claim 1, wherein the second light is used to acquire a fluorescent image from the subject. (Configuration 4) 4. The image acquisition device according to any one of configurations 1 to 3, wherein the second light irradiation unit is disposed on the same side as the imaging unit with respect to the subject. (Configuration 5) an optical path changing unit that changes an optical path of the second light so as to be parallel to the optical axis; 5. The image acquisition device according to configuration 4, wherein the second light irradiating unit emits the second light to the optical path changing unit. (Configuration 6) An image acquisition device described in any one of configurations 1 to 5, wherein the optical path length adjustment unit controls the position of the telecentric optical system while maintaining a constant positional relationship between the subject and the second light emission unit of the second light irradiation unit. (Configuration 7) The image acquisition device according to any one of configurations 1 to 6, wherein the subject is contained in a container whose upper and lower surfaces, which are arranged approximately perpendicular to the optical axis, are transparent to the first light and the second light. (Method 1) An image acquisition method using an image acquisition device including: a first light irradiating unit that irradiates a subject that is a phase object with parallel light that is a first light; a second light irradiating unit that irradiates the subject with a second light at an incident angle different from that of the first light irradiating unit; an imaging lens configured to include a telecentric optical system; and an imaging unit that images the subject irradiated with at least one of the first light and the second light via the imaging lens, acquiring a defocused image of the object in a defocused state using the first light; acquiring an in-focus image of the object in an in-focus state using the second light; generating a composite image using the defocused image and the infocus image. (Program 1) A program which, when executed by a computer, causes the computer to execute each step of the image acquisition method described in Method 1. [Explanation of symbols]

[0079] 1: installation stand, 2: subject, 3: first light irradiation unit, 4: second light irradiation unit, 5: imaging lens, 6: imaging unit, 6a: imaging control unit, 7: optical path length adjustment unit, 8: filter, 9 dichroic mirror, 21: image acquisition unit, 22: image processing unit

Claims

1. An image acquisition device for a subject that is a phase object, a first light irradiation unit that irradiates the subject with collimated light that is a first light; a second light irradiating unit that irradiates the subject with second light at an incident angle different from that of the first light irradiating unit; an imaging lens including a telecentric optical system; an imaging unit that captures an image of the subject illuminated with at least one of the first light and the second light via the imaging lens; Equipped with an optical axis of the imaging lens is parallel to an optical path of the first light irradiation unit; the imaging unit is disposed on the optical axis on an opposite side of the subject from the first light irradiation unit, An image acquisition device comprising an optical path length adjustment unit that controls the distance on the optical axis between the telecentric optical system and the subject.

2. The image acquisition device according to claim 1 , further comprising an image processing unit that combines a defocused image of the subject acquired when irradiated with the first light and an infocus image of the subject acquired when irradiated with the second light.

3. The image acquisition device according to claim 2 , wherein the second light is emitted from the second light emitting unit as excitation light for generating fluorescence in the subject.

4. The image acquisition device according to claim 1 , wherein the second light irradiation unit is disposed on the same side as the imaging unit with respect to the subject.

5. an optical path changing unit that changes the optical path of the second light so that the optical path is parallel to the optical axis; The image acquisition device according to claim 4 , wherein the second light irradiating unit emits the second light toward the optical path changing unit.

6. The image acquisition device according to claim 1 , wherein the optical path length adjustment unit controls the position of the telecentric optical system while maintaining a constant positional relationship between the subject and the second light emission unit of the second light irradiation unit.

7. The image acquisition device according to claim 1 , wherein the subject is accommodated in a container whose upper and lower surfaces, which are arranged substantially perpendicular to the optical axis, are transparent to the first light and the second light.

8. An image acquisition device as described in Claim 7, wherein the subject is contained in a container that is transparent to not only the first light and the second light, but also to fluorescence emitted from the subject.

9. An image acquisition device as described in claim 1, wherein the first light irradiation unit includes a first light source and a first collimator that converts light emitted from the first light source into parallel light, which is the first light.

10. An image acquisition device as described in claim 9, wherein the first collimator is a telecentric lens.

11. An image acquisition device as described in claim 1, wherein the second light is collimated light.

12. An image acquisition device as described in Claim 11, wherein the second light irradiation unit includes a second light source and a second collimator that converts light emitted from the second light source into collimated light, which is the second light.

13. An image acquisition device as described in claim 1, wherein the telecentric optical system is a telecentric lens.

14. An image acquisition device as described in claim 1, wherein the phase object is a cell.

15. The optical path length adjustment unit changes a position on the optical axis of the telecentric optical system from an in-focus position of the subject to a predetermined defocus position, The image acquisition device according to claim 1 , wherein the imaging unit captures an image of the subject at the defocus position.

16. An image acquisition device for a subject that is a phase object, comprising: a first light irradiation unit that irradiates the subject with collimated light, which is a first light, and that includes a light source and a collimator that converts the light emitted from the light source into the collimated light, which is the first light; an imaging lens including a telecentric optical system; an imaging unit that captures an image of the subject illuminated with the first light via the imaging lens, an optical axis of the imaging lens is parallel to an optical path of the first light irradiation unit; the imaging unit is disposed on the optical axis on an opposite side of the subject from the first light irradiation unit, an optical path length adjustment unit changes a position on the optical axis of the telecentric optical system from an in-focus position of the object to a predetermined defocus position; The imaging unit is an image acquisition device that captures an image of the subject at the defocus position.

17. a first light irradiation unit that irradiates a subject that is a phase object with parallel light that is a first light; a second light irradiating unit that irradiates the subject with second light at an incident angle different from that of the first light irradiating unit; an imaging lens including a telecentric optical system; an imaging unit that captures an image of the subject irradiated with at least one of the first light and the second light via the imaging lens, acquiring a defocused image of the object in a defocused state using the first light; acquiring an in-focus image of the object in an in-focus state using the second light; generating a composite image using the defocused image and the in-focus image.

18. A program that, when executed by a computer, causes the computer to execute each step of the image acquisition method according to claim 17.

Images