Image acquisition device and image acquisition method

Abstract

The image acquisition device is an image acquisition device for acquiring a macro image of a sample, and includes a second irradiation unit that irradiates the sample obliquely with excitation light in at least one of a plurality of excitation wavelength ranges, a fluorescence filter unit that has a plurality of reflection wavelength ranges corresponding to the plurality of excitation wavelength ranges and a transmission wavelength range between each reflection wavelength range, an imaging lens that forms an image of fluorescence from the sample in response to the irradiated excitation light, and an image sensor that captures the light image formed by the imaging lens and outputs a fluorescence macro image of the sample, and a control device that sets acquisition conditions for the micro image of the sample based on the fluorescence macro image.

Description

[Technical field]

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

[0002] Patent Document 1 describes that a dark-field macro image is acquired by dark-field illumination, and micro image acquisition conditions are set based on the dark-field macro image. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2007-310231 A Summary of the Invention [Problem to be solved by the invention]

[0004] Conventionally, there has been a demand for acquiring clearer macro images and for setting more appropriate conditions for acquiring micro images.

[0005] The present disclosure has been made in consideration of the above-mentioned circumstances, and aims to set more appropriate micro image acquisition conditions. [Means for solving the problem]

[0006] An image acquisition device according to one embodiment of the present disclosure is an image acquisition device for acquiring a macro image of a sample, and includes: an excitation light irradiation unit that irradiates the sample obliquely with excitation light in at least one of a plurality of excitation wavelength ranges; a fluorescence filter unit that has a plurality of reflection wavelength ranges corresponding to the plurality of excitation wavelength ranges and a transmission wavelength range between each reflection wavelength range; an imaging lens that forms an image of fluorescence from the sample in response to the irradiated excitation light; and an image sensor that captures the light image formed by the imaging lens and outputs fluorescence macro image data of the sample; and a setting unit that sets acquisition conditions for the micro image of the sample based on the fluorescence macro image data.

[0007] An image acquisition method according to one aspect of the present disclosure is an image acquisition method for acquiring a macro-image of a sample, and includes an excitation light irradiation step of irradiating the sample obliquely with excitation light in at least one of a plurality of excitation wavelength ranges, an imaging step of imaging, using an image sensor, fluorescence that is generated from the sample in response to the irradiated excitation light and that has passed through a fluorescence filter section, each of which has a plurality of reflection wavelength ranges corresponding to the plurality of excitation wavelength ranges and a transmission wavelength range between each of the reflection wavelength ranges, and outputting fluorescence macro-image data of the sample, and a setting step of setting acquisition conditions for a micro-image of the sample based on the fluorescence macro-image data.

[0008] In the image acquisition device and image acquisition method according to one aspect of the present disclosure, excitation light is irradiated obliquely onto the sample, and an optical image of the fluorescence from the sample that has passed through a fluorescence filter unit and an imaging lens that reflect light in the excitation wavelength range is captured by an image sensor, and fluorescence macro image data of the sample is output. In this way, by irradiating the sample obliquely with excitation light, in a configuration in which a macro observation camera is provided directly above the sample, the fluorescence from the sample is appropriately captured by the image sensor. Furthermore, in the front stage of the image sensor, light in the excitation wavelength range is reflected by the fluorescence filter unit, so that only the fluorescence not including the excitation light is appropriately captured by the image sensor. The fluorescence macro image data captured in this way is output, and the acquisition conditions of the micro image of the sample are set, so that the acquisition conditions of the micro image can be appropriately set. That is, by capturing only the fluorescence by the image sensor through the fluorescence filter unit, the tissue in the sample is clearly detected based on the fluorescence macro image data, and the tissue area detection, the setting of the focus measurement position, and the like can be appropriately performed, and the acquisition conditions of the micro image can be appropriately set.

[0009] The setting unit may set the exposure time when the micro image is acquired based on the fluorescence macro image data. As described above, since the structure of the sample is clearly detected from the macro image data output from the macro observation camera, it is possible to set an appropriate exposure time when the micro image is acquired with high precision based on the macro image data.

[0010] The setting unit may extract a predetermined number of confirmation points that have a higher brightness than other regions in the fluorescence macro image data, and set the exposure time based on the brightness of the extracted confirmation points. In this way, the exposure time is set taking into account the brightness of the confirmation points, so that the exposure time can be set with higher accuracy.

[0011] The excitation light irradiating unit irradiates excitation light for each excitation wavelength range, the image sensor is a color image sensor that outputs fluorescence macro image data corresponding to each excitation wavelength range, and the setting unit may set the exposure time for each fluorescence macro image data corresponding to each excitation wavelength range. While an appropriate exposure time differs depending on the excitation wavelength range, by setting the exposure time for each fluorescence macro image data corresponding to each excitation wavelength range, the exposure time can be set with higher precision.

[0012] The excitation light irradiating unit may irradiate excitation light for each excitation wavelength range, the image sensor may be a color image sensor that outputs fluorescence macro image data corresponding to each excitation wavelength range, and the setting unit may perform tissue recognition to detect a tissue area contained in the sample for each fluorescence macro image data corresponding to each excitation wavelength range. By performing tissue recognition based on clear fluorescence macro image data that does not include excitation light, it is possible to detect a tissue area contained in the sample with high accuracy.

[0013] The excitation light irradiating unit irradiates excitation light for each excitation wavelength range, the image sensor is a color image sensor that outputs fluorescence macro image data corresponding to each excitation wavelength range, and the setting unit may set the focus measurement position for each fluorescence macro image data corresponding to each excitation wavelength range. Setting the focus measurement position based on clear fluorescence macro image data that does not contain excitation light allows the focus position to be set with high accuracy in accordance with the arrangement of the tissue.

[0014] The image acquisition device may further include a bright-field illumination unit that performs bright-field illumination on the sample. With this configuration, in addition to the above-mentioned fluorescence macro-image data, bright-field macro-image data can be acquired.

[0015] The image acquisition device may further include a dark-field illumination unit that performs dark-field illumination on the sample. With this configuration, in addition to the above-mentioned fluorescence macro image data, dark-field macro image data can be acquired.

[0016] The image acquisition device may further include a switching unit configured to be able to move the fluorescence filter unit in and out of the optical axis of the fluorescence directed toward the imaging lens. With such a configuration, for example, it becomes possible to provide the fluorescence filter unit on the optical axis of the fluorescence only when acquiring the fluorescence macro image data, and to retract the fluorescence filter unit from the optical axis of the fluorescence in other cases (for example, when acquiring bright-field macro image data or dark-field macro image data), so that each image data can be appropriately acquired. Effect of the Invention

[0017] According to the present disclosure, the micro image acquisition conditions can be set more appropriately. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a diagram showing a schematic diagram of an image acquisition device according to the present embodiment. [Diagram 2]FIG. 2 shows bright-field, dark-field, and fluorescent macro images of the sample. [Diagram 3] FIG. 3 is a diagram showing a fluorescent macro image after preprocessing. [Figure 4] FIG. 4 is a diagram for explaining switching of macro image display. [Diagram 5] FIG. 5 is a diagram showing a display image during tissue recognition. [Figure 6] FIG. 6 is a diagram showing a display image of a fluorescence macro image and focus measurement points in full-color excitation. [Figure 7] FIG. 7 is a diagram for explaining the process of setting the focus measurement position. [Figure 8] FIG. 8 is a diagram showing a display image of a fluorescence macro image and a confirmation point in a certain excitation wavelength range. [Figure 9] FIG. 9 is a diagram illustrating the exposure time setting process. [Figure 10] FIG. 10 is a diagram for explaining switching of the fluorescence macro image display. [Figure 11] FIG. 11 is a flowchart showing the macro image capturing process. [Figure 12] FIG. 12 is a flowchart showing the tissue recognition process. [Figure 13] FIG. 13 is a flowchart showing the process of setting the focus measurement position. [Figure 14] FIG. 14 is a flowchart showing the exposure time setting process. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. In each drawing, the same or corresponding parts are denoted by the same reference numerals, and duplicated explanations will be omitted.

[0020] FIG. 1 is a diagram showing a schematic diagram of an image acquisition device 1 according to the present embodiment. The image acquisition device 1 is a device that irradiates light onto a glass slide 125 on which a sample S (specimen) is placed, scans the glass slide 125, and acquires an image of the sample S. The image acquisition device 1 is a device that acquires a macro image of the sample S in particular (details will be described later). As the sample S from which an image is acquired, for example, a biological sample such as a tissue section stained with a fluorescent dye is used. The glass slide 125 may be a preparation that seals the sample S. The image acquisition device 1 includes a microscope device 10, a control device 20 (setting unit) that controls image acquisition by the microscope device 10, a display device 30, and an input device 40.

[0021] The microscope device 10 includes a macro image acquisition section 110 , an excitation light source 130 , a first optical fiber 140 , a first irradiation section 150 , a fluorescent macro mirror 160 , a second optical fiber 170 , and a micro image acquisition section 190 .

[0022] The macro image acquisition unit 110 acquires a fluorescent macro image (fluorescent macro image data), a bright field macro image (bright field macro image data), and a dark field macro image (dark field macro image data) of the sample S. A macro image is a low-magnification image of the sample S (an image with a lower magnification than a micro image, and an image including the entire sample S). The macro image acquisition unit 110 acquires a low-resolution macro image corresponding to the entire image of the sample S. A macro image is an image for setting the imaging conditions for the sample S, specifically, the acquisition conditions for a micro image. A micro image is a high-magnification image of the sample S (an image with a higher magnification than a macro image, and an image obtained using a high-magnification lens such as 4x, 10x, 20x, or 40x).

[0023] A fluorescence macro image is a macro image of a sample S obtained by capturing the fluorescence generated by irradiating the sample S with excitation light. A bright-field macro image is a macro image of a sample S obtained using transmitted illumination. A dark-field macro image is a macro image of a sample S obtained by illuminating the sample S in a dark field using scattered light generated by irradiating the sample S with oblique illumination or the like, rather than directly using transmitted illumination.

[0024] The macro image acquisition unit 110 includes a sample holder 111, a scan stage 112, a bright field light source 113 (bright field illumination unit), a dark field light source 114 (dark field illumination unit), a dark field macro imaging shutter 115, a barcode imaging light source 116, a second irradiation unit 117 (excitation light irradiation unit), an imaging lens 118, a color image sensor (image sensor) 119, a fluorescent filter unit 120, and a switching unit 121. The imaging lens 118, the color image sensor 119, the fluorescent filter unit 120, and the switching unit 121 constitute a macro observation camera that captures a macro image.

[0025] The sample holder 111 is a holder for placing the transported slide glass 125. As described above, the sample S is placed on the slide glass 125. The scan stage 112 is an XY stage for moving the sample holder 111 in the XY directions (front-back and left-right directions), and after the sample holder 111 places the slide glass 125 on it, the scan stage 112 transports the sample holder 111 to a macro image acquisition position. The scan stage 112 may also transport the sample holder 111 to a micro image acquisition position by the micro image acquisition unit 190. In this case, the scan stage 112 may be a macro / micro switching stage capable of transporting the sample holder 111 to each of the macro image acquisition position and the micro image acquisition position.

[0026] The bright-field light source 113 is a bright-field illumination means used when acquiring a bright-field macro image, which is a macro image of the sample S. The bright-field light source 113 has an optical axis parallel to the optical axis of the imaging lens 118 of the macro observation camera. For example, the bright-field light source 113 is a white light source (for example, a white LED), and is provided directly below the slide glass 125 held by the sample holder 111. That is, the bright-field light source 113 is provided in a direction perpendicular to the placement surface of the slide glass 125 in the sample holder 111, and on the opposite side of the second irradiation unit 180 and the color image sensor 119 with respect to the sample S. The bright-field light source 113 performs bright-field illumination on the sample S from directly below, for example, via a diffusion plate (not shown).

[0027] The dark-field light source 114 is a dark-field illumination means (oblique illumination means) used when acquiring a dark-field macro image, which is a macro image of the sample S. The dark-field light source 114 has an optical axis inclined with respect to the optical axis of the imaging lens 118 of the macro observation camera. For example, the dark-field light source 114 is a white light source (for example, a white LED) and is provided obliquely below the slide glass 125 held by the sample holder 111. That is, the dark-field light source 114 is provided in a direction perpendicular to the mounting surface of the slide glass 125 in the sample holder 111 and on the opposite side of the second irradiation unit 180 and the color image sensor 119 with respect to the sample S. The dark-field light source 114 performs dark-field illumination on the sample S from an obliquely downward direction. The dark-field macro imaging shutter 115 blocks the diffusion plate (not shown) of the bright-field light source 113 when capturing the dark-field macro image. This makes it possible to perform appropriate dark-field illumination even in a configuration in which the bright-field light source 113 and the dark-field light source 114 coexist.

[0028] The barcode imaging light source 116 is a light source that emits light to read a barcode attached to the glass slide 125 when acquiring a bright-field macro image, etc. By reading the barcode, the glass slide 125 (i.e., the sample S) can be uniquely identified. That is, the control device 20 described later may uniquely identify the glass slide 125 (i.e., the sample S) from the information of the barcode.

[0029] The second irradiation unit 117 is a fluorescence macro excitation light irradiation unit used when acquiring a fluorescence macro image, which is a macro image of the sample S. The second irradiation unit 117 irradiates the excitation light output from the excitation light source 130 obliquely from above the sample S. The excitation light here is light having at least one of a plurality of excitation wavelength ranges. In this way, by irradiating the sample S with the excitation light from obliquely above, in a configuration in which a macro observation camera (fluorescence filter unit 120, imaging lens 118, and color image sensor 119) is provided directly above the sample S, it is possible to appropriately image the fluorescence from the sample S without interference between the configuration for irradiating the excitation light and the macro observation camera.

[0030] The components relating to the excitation light emitted from the second irradiating section 117, that is, the excitation light source 130, the first guiding optical fiber 140, the first irradiating section 150, the fluorescent macro mirror 160, and the second guiding optical fiber 170, will be described.

[0031] The excitation light source 130 is a light source (e.g., an LED) that generates and outputs excitation light for acquiring a fluorescence macro-image in the macro image acquiring section 110, and excitation light for acquiring a fluorescence micro-image in the micro image acquiring section 190. In this manner, in the image acquiring device 1, the same excitation light source 130 is used as the light source for acquiring a fluorescence macro-image and a fluorescence micro-image. Note that, in this embodiment, the same excitation light source 130 is described as being used as the light source for acquiring a fluorescence macro-image and a fluorescence micro-image, but this is not limiting, and different light sources may be used as the light sources for acquiring a fluorescence macro-image and a fluorescence micro-image.

[0032] The excitation light source 130 is configured to be capable of emitting excitation light in a plurality of excitation wavelength ranges. The excitation light source 130 can emit the excitation light while switching the excitation wavelength range. The plurality of excitation wavelength ranges correspond to the respective reflection wavelength ranges of the fluorescence filter unit 120, which is, for example, a multi-bandpass filter (details will be described later).

[0033] The first optical fiber 140 is an optical fiber that guides the excitation light output from the excitation light source 130 to the first irradiation unit 150. The first irradiation unit 150 irradiates the excitation light guided from the excitation light source 130 through the first optical fiber 140 toward the fluorescent macro mirror 160. The fluorescent macro mirror 160 is disposed on the optical path of the excitation light irradiated from the first irradiation unit 150, and reflects the excitation light and guides it to the second optical fiber 170. Note that, when acquiring a fluorescent micro image, the angle of the mirror of the fluorescent macro mirror 160 is changed, and the excitation light is guided toward the micro image acquisition unit 190. The second optical fiber 170 guides the excitation light input through the fluorescent macro mirror 160 to the second irradiation unit 117 of the macro image acquisition unit 110.

[0034] The fluorescence filter section 120 is provided in a region directly above the sample S between the sample S and the color image sensor 119, and has a plurality of reflection wavelength ranges corresponding to a plurality of excitation wavelength ranges, and a transmission wavelength range between each of the reflection wavelength ranges. According to such a fluorescence filter section 120, light in the excitation wavelength range is reflected, and therefore, the light in the excitation wavelength range is prevented from being imaged by the color image sensor 119.

[0035] The fluorescence filter section 120 may be, for example, a filter used in a Pinkel arrangement including a multi-bandpass filter. In a configuration in which a multi-bandpass filter is used, it is not necessary to switch filters in response to switching of the excitation wavelength range, and one scan of the stage operation is sufficient.

[0036] The switching unit 121 is a filter wheel that holds one or more multi-bandpass filters of the fluorescence filter unit 120, and is configured to switch between a state in which a multi-bandpass filter is arranged on the optical axis of the fluorescence and a state in which a multi-bandpass filter is not arranged (a state without a filter) by rotating. That is, the switching unit 121 is a member configured to be able to insert and remove the fluorescence filter unit 120 onto the optical axis of the fluorescence toward the imaging lens 118. For example, the switching unit 121 may provide the fluorescence filter unit 120 on the optical axis of the fluorescence only when a fluorescence macro image is acquired, and may retract the fluorescence filter unit 120 from the optical path of the fluorescence in other cases (for example, when a bright-field macro image or a dark-field macro image is acquired). When a bright-field macro image or a dark-field macro image is acquired, the switching unit 121 may switch to a multi-bandpass filter different from that used when the fluorescence macro image was acquired. Note that the switching unit 121 is not limited to a filter wheel, and may be a slide mechanism that slides one or more multi-bandpass filters of the fluorescence filter unit 120 to arrange them on the optical axis. Furthermore, even if the multi-bandpass filter is placed on the optical axis, it is possible to acquire bright-field macro images and dark-field macro images, so the switching unit 121 does not necessarily have to be provided.

[0037] The fluorescence filter unit 120 may be, for example, a filter used in a Sedat arrangement including a plurality of switchable single-band filters. In this case, the switching unit 121 may switch the single-band filters according to each excitation wavelength. In such a configuration, a scanning operation is performed for each excitation wavelength range.

[0038] The imaging lens 118 is a lens that forms an image of the light transmitted through the fluorescence filter unit 120, that is, the fluorescence from the sample S corresponding to the irradiated excitation light. The imaging lens 118 forms an image of the entire slide glass 125 on the color image sensor 119.

[0039] The color image sensor 119 is an image sensor that captures the light image formed by the imaging lens 118 and outputs a fluorescence macro image of the sample S. The color image sensor 119 captures an image of the entire slide glass 125 formed by the imaging lens 118. Note that, although the present embodiment will be described assuming that the color image sensor 119 is used, a monochrome image sensor that is an image sensor may be used instead of the color image sensor 119.

[0040] The control device 20 is a control means for controlling the image acquisition operation in the microscope device 10, setting image acquisition conditions, and processing image data of the acquired sample S. The control device 20 is configured by a computer including, for example, a CPU and necessary memory, and storage devices such as a hard disk. A display device 30 and an input device 40 are connected to the control device 20. The display device 30 is, for example, an LED display, an OLED display, or a liquid crystal display, and is used to display an operation screen required for the operation of the image acquisition device 1, or to display an image of the acquired sample S. The input device 40 is, for example, a keyboard or a mouse, and is used to input information required for image acquisition, input instructions regarding the image acquisition operation, and the like.

[0041] When acquiring a macro image, the control device 20 controls the scan stage 112 so that the sample S is transported to a macro image acquisition position. When acquiring a fluorescent macro image, the control device 20 controls the excitation light source 130 so that excitation light in a predetermined excitation wavelength range is irradiated, and controls the switching unit 121 so that a multi-bandpass filter of the fluorescence filter unit 120 corresponding to the excitation wavelength range is arranged. When acquiring a bright-field macro image, the control device 20 controls the bright-field light source 113 so that bright-field illumination is performed. When acquiring a dark-field macro image, the control device 20 controls the dark-field light source 114 so that dark-field illumination is performed, and controls the dark-field macro imaging shutter 115 so that a diffusion plate (not shown) of the bright-field light source 113 is blocked. Moreover, when acquiring a micro image, the control device 20 controls the scan stage 112 so that the sample S is transported to a micro image acquisition position. Then, the control device 20 controls the micro image acquisition unit 190 so that a micro image is acquired under the set micro image acquisition conditions described later.

[0042] The control device 20 sets the acquisition conditions of the micro image of the sample S based on the fluorescence macro image captured by the color image sensor 119. The acquisition conditions of the micro image are, for example, the position of the focus measurement point (focus measurement position) and the exposure time when the micro image is acquired. That is, the control device 20 sets the position of the focus measurement point (focus measurement position) and the exposure time when the micro image is acquired based on the fluorescence macro image. Furthermore, the control device 20 performs tissue recognition to detect the area of ​​the tissue contained in the sample S based on the fluorescence macro image, as a prerequisite for setting the position of the focus measurement point and the exposure time described above. The control device 20 sets the micro scan area when the micro image is acquired based on the area of ​​the tissue contained in the sample S detected by the tissue recognition. The focus measurement point may be set to, for example, a point (confirmation point) having a higher brightness than other areas. Furthermore, the control device 20 may perform tissue recognition for each fluorescence macro image of excitation light having a different excitation wavelength range, for example, when a filter used in the above-mentioned Sedat arrangement is used as the fluorescence filter unit 120. Furthermore, the control device 20 may set the micro scan area for each fluorescence macro image of excitation light having a different excitation wavelength range.

[0043] FIG. 2 is a diagram showing a bright-field macro image, a dark-field macro image, and a fluorescent macro image of a prepared sample S. Here, an image of sample S of sample I is shown as the image of sample S. In FIG. 2, the tissue-recognized region is shown as a rectangular tissue recognition region A1. Also, in FIG. 2, a plurality of set focus measurement points are shown by cross marks. As is clear from FIG. 2, in the bright-field macro image and the dark-field macro image, the tissue recognition cannot be performed with high accuracy for sample I. For example, in the bright-field macro image of sample I, the tissue recognition region A1 is extremely small, and in the dark-field macro image of sample I, the tissue recognition region A1 is larger than the actual tissue region. Since the tissue recognition is not performed appropriately, it is natural that the focus measurement point cannot be set appropriately in the bright-field macro image and the dark-field macro image of sample I. Specifically, the focus measurement point is set on air bubbles, dust, etc. other than the tissue.

[0044] In contrast, in the fluorescence macro image of sample I, the tissue recognition area A1 generally coincides with the area of ​​the actual tissue S10, and tissue recognition can be performed with high accuracy. As described above, by using a fluorescence macro image, the accuracy of tissue recognition can be improved.

[0045] On the other hand, the focus measurement points are set so as to overlap the tissue S10 in the fluorescence macro image of sample I, but there are some parts that are set in an area (an inappropriate area) that does not overlap the tissue S10 in the fluorescence macro image of sample I. In this regard, for example, by performing preprocessing to invert the negative and positive of the fluorescence macro image before recognizing the tissue and setting the focus measurement points, as shown in FIG. 3, the accuracy of tissue recognition can be further improved and the accuracy of setting the focus measurement points can be improved. FIG. 3 shows the setting of the tissue recognition and the focus measurement points in the fluorescence macro image after the preprocessing of sample I. In the example shown in FIG. 3, the tissue recognition area A1 coincides with the area of ​​the actual tissue S10, and all the focus measurement points are set so as to overlap the tissue S10 in the fluorescence macro image.

[0046] The control device 20 may perform tissue recognition and position setting of the focus measurement point (setting of the focus measurement position) based on a fluorescence macro image (pseudo bright-field fluorescence macro image) that has been pre-processed to invert the captured fluorescence macro image from negative to positive, as described above. Note that the control device 20 may control the display device 30 to switch between displaying a bright-field macro image, a dark-field macro image, a fluorescence macro image, and a pre-processed fluorescence macro image (pseudo bright-field fluorescence macro image) for the same sample S, as shown in Fig. 4. In this case, as shown in Fig. 4, a display icon for each image may be displayed on the display device 30, and the displayed macro image may be switched by clicking the display icon.

[0047] The processing of the control device 20 and the display on the display device 30 when sequentially performing tissue recognition, position setting of the focus measurement point, and exposure time setting will be described below with reference to Figs. 5 to 10. Fig. 5 is a diagram showing a display image on the display device 30 during tissue recognition. As shown in Fig. 5, the control device 20 controls the display device 30 so that the tissue S10 of the sample S that has undergone tissue recognition, the tissue recognition area A1, and the estimated size Si of the tissue S10 of the sample S are displayed together.

[0048] Next, the control device 20 sets focus conditions for the sample S that has undergone tissue recognition, and controls the display device 30 so that an image in which, for example, five focus measurement points are superimposed on the tissue S10 of the sample S is displayed as shown in FIG. 6. FIG. 6 is a diagram showing a display image of a fluorescence macro image and focus measurement points in full-color excitation. The fluorescence macro image in full-color excitation is a fluorescence macro image detected in a state in which the sample S is irradiated with excitation light in all excitation wavelength ranges. FIG. 7 is a diagram for explaining the focus condition setting process. For example, suppose that the fluorescence macro image shown in FIG. 7(a) is acquired by irradiating excitation light in a certain excitation wavelength range (for example, Violet (wavelength range of 360 nm to 400 nm)) and setting the fluorescence filter unit 120 according to the excitation light. Then, as shown in FIG. 7(b), the control device 20 extracts a predetermined number (for example, five) of confirmation points that are brighter than other regions from the fluorescence macro image, and sets each of the extracted five confirmation points as focus measurement points. Then, the control device 20 controls the display device 30 so that the set focus measurement point is superimposed on the fluorescence macro image in full-color excitation, as shown in FIG. 7(c). In this way, the control device 20 may extract a focus measurement point from a fluorescence macro image detected according to excitation light in a certain excitation wavelength range (e.g., violet), and control the display device 30 so that the extracted focus measurement point is superimposed on the fluorescence macro image in full-color excitation. For example, when a filter used in the Pinkel arrangement described above is used as the fluorescence filter unit 120, the control device 20 may set a focus measurement point only from a fluorescence macro image corresponding to excitation light in a single excitation wavelength range, as described above. Furthermore, when a filter used in the Sedat arrangement described above is used as the fluorescence filter unit 120, the control device 20 may set a focus measurement point for each fluorescence macro image of excitation light with different excitation wavelength ranges.

[0049] Next, the control device 20 sets an exposure time for each fluorescence macro image corresponding to the excitation wavelength range. That is, the control device 20 controls the excitation light source 130 so that excitation light is sequentially irradiated for each excitation wavelength range, and the color image sensor 119 outputs a fluorescence macro image corresponding to each excitation wavelength range. The control device 20 extracts a predetermined number (e.g., three) of confirmation points that are brighter than other regions in the fluorescence macro image, and controls the display device 30 so that an image is displayed in which, for example, three confirmation points indicated by crosses are superimposed on the tissue S10 of the sample S, as shown in Fig. 8. Fig. 8 is a diagram showing a display image of a fluorescence macro image and confirmation points in a certain excitation wavelength range.

[0050] FIG. 9 is a diagram for explaining the exposure time setting process. As described above, the control device 20 extracts three points (see FIG. 9(a)) that are independent of each other and have higher intensity (brightness) than other regions as confirmation points in descending order from a fluorescence macro image (see FIG. 9(a)) corresponding to excitation light of a certain excitation wavelength range (for example, cyan (wavelength range of 460 nm to 490 nm)). The control device 20 then sets the exposure time based on the brightness of the extracted confirmation points (three points). Since the position of maximum brightness is known, the exposure time can be set with high accuracy. As described above, the control device 20 performs such exposure time setting process for each fluorescence macro image corresponding to the excitation wavelength range. The control device 20 may control the display device 30 so that the fluorescence macro image display is appropriately switched by switching the excitation wavelength range of the excitation light, as shown in FIG. 10. In the example shown in FIG. 10, a fluorescence macro image of all-color excitation (Common (for example, a wavelength range combining a plurality of different monochromatic lights)), a fluorescence macro image of violet, and a fluorescence macro image of cyan are switched.

[0051] Next, the processing executed by the image acquisition device 1 will be described with reference to Figs. 11 to 14. Fig. 11 is a flowchart showing a macro image capturing processing. Fig. 12 is a flowchart showing a tissue recognition processing. Fig. 13 is a flowchart showing a focus measurement position setting processing. Fig. 14 is a flowchart showing an exposure time setting processing. These processings are, for example, processings relating to setting of acquisition conditions for micro images.

[0052] The macro image capturing process will be described. As shown in Fig. 11, first, a bright-field macro image is captured (step S1). Specifically, the control device 20 controls the scan stage 112 so that the sample S is transported to a macro image capturing position, and controls the bright-field light source 113 so that bright-field illumination is performed. As a result, the bright-field macro image is captured by the color image sensor 119.

[0053] Next, it is determined whether the sample S is a fluorescent sample (step S2), and if it is not a fluorescent sample, the process ends. On the other hand, if the sample S is a fluorescent sample, a dark-field macro image is captured (step S3). Specifically, the control device 20 controls the dark-field light source 114 to perform dark-field illumination, and controls the dark-field macro imaging shutter 115 to block the diffusion plate (not shown) of the bright-field light source 113. As a result, a dark-field macro image is captured by the color image sensor 119.

[0054] Next, a fluorescence macro image is captured (step S4). Specifically, the control device 20 controls the excitation light source 130 so as to irradiate excitation light in a predetermined excitation wavelength range (excitation light irradiation step), and controls the switching unit 121 so as to arrange the multi-bandpass filter of the fluorescence filter unit 120 according to the excitation wavelength range. This causes the color image sensor 119 to capture a fluorescence macro image (imaging step).

[0055] The tissue recognition process (setting step) will be described. As shown in FIG. 12, first, it is determined whether the sample S is a fluorescent sample (step S11), and if it is not a fluorescent sample, a bright-field macro image is analyzed (step S12). Specifically, the control device 20 performs tissue recognition based on the bright-field macro image. On the other hand, if the sample S is a fluorescent sample, first, a dark-field macro image is analyzed (step S13). Specifically, the control device 20 performs tissue recognition based on the dark-field macro image.

[0056] Next, pre-processing of the fluorescent macro image is performed (step S14). Specifically, the control device 20 performs an inversion process to invert the negative / positive of the fluorescent macro image. This improves the accuracy of tissue recognition. Finally, the fluorescent macro image after the inversion process is analyzed (step S15). Specifically, the control device 20 performs tissue recognition based on the fluorescent macro image after the inversion process.

[0057] The setting process (setting step) of the focus measurement position will be described below. As shown in Fig. 13, first, it is determined whether the sample S is a fluorescent sample (step S21), and if it is not a fluorescent sample, the focus measurement position is set using a bright field macro image (step S22).

[0058] On the other hand, when the sample S is a fluorescent sample, first, it is determined whether or not a filter used in the Pinkel arrangement is used as the fluorescent filter unit 120 (step S23). When a filter used in the Pinkel arrangement is used, a focus measurement position is set using a fluorescence macro image corresponding to excitation light of a single excitation wavelength range (step S24). Specifically, the control device 20 extracts, for example, five confirmation points having higher brightness than other regions from a fluorescence macro image corresponding to excitation light of a certain excitation wavelength range (e.g., violet), and sets each of the extracted five confirmation points as a focus measurement point. On the other hand, when a filter used in the Sedat arrangement rather than the Pinkel arrangement is used, a focus measurement point is set for each fluorescence macro image of excitation light of different excitation wavelength ranges (step S25).

[0059] The exposure time setting process will be described below. As shown in Fig. 14, first, it is determined whether the sample S is a fluorescent sample (step S31), and if it is not a fluorescent sample, the exposure time is set using a bright field macro image (step S32).

[0060] On the other hand, if the sample S is a fluorescent sample, first, the exposure time is set using the fluorescent macro image (step S33). Specifically, the control device 20 sets the exposure time for each fluorescent macro image according to the excitation wavelength range. The control device 20 extracts, for example, three confirmation points that are brighter than other regions in the fluorescent macro image, and sets an initial value of the exposure time based on the confirmation points.

[0061] Next, the effects of the image acquisition device 1 according to this embodiment will be described.

[0062] The image acquisition device 1 of this embodiment is an image acquisition device that acquires a macro image of a sample S, and is equipped with a second irradiation unit 117 that irradiates the sample S obliquely with excitation light in at least one of a plurality of excitation wavelength ranges, a fluorescence filter unit 120 that has a plurality of reflection wavelength ranges corresponding to the plurality of excitation wavelength ranges and a transmission wavelength range between each reflection wavelength range, an imaging lens 118 that forms an image of fluorescence from the sample S in accordance with the irradiated excitation light, and a color image sensor 119 that captures the light image formed by the imaging lens 118 and outputs a fluorescence macro image of the sample S, and a control device 20 that sets acquisition conditions for a micro image of the sample S based on the fluorescence macro image.

[0063] In the image acquisition device 1, excitation light is irradiated obliquely onto the sample S, and an optical image of the fluorescence from the sample S that has passed through the fluorescence filter section 120, which reflects light in the excitation wavelength range, and the imaging lens 118 is captured by the color image sensor 119, and a fluorescence macro image of the sample S is output. In this way, by irradiating the sample S obliquely with excitation light, in a configuration in which a macro observation camera is provided directly above the sample S, the fluorescence from the sample S is appropriately captured by the color image sensor 119. Furthermore, because light in the excitation wavelength range is reflected by the fluorescence filter section 120 upstream of the color image sensor 119, only the fluorescence not including the excitation light is appropriately captured by the color image sensor 119. The fluorescence macro image captured in this way is output, and the acquisition conditions for the micro image of the sample S are set, so that the acquisition conditions for the micro image can be appropriately set. In other words, since only the fluorescence is captured by the color image sensor 119 through the fluorescent filter section 120, the tissue in the sample S can be clearly detected based on the fluorescent macro image, and the tissue area detection, setting of the focus measurement position, etc. can be appropriately performed, and the conditions for acquiring the micro image can be appropriately set.

[0064] The control device 20 may set the exposure time when acquiring the micro image based on the fluorescent macro image. As described above, since the texture of the sample S is clearly detected from the macro image output from the macro observation camera, it is possible to set an appropriate exposure time when acquiring the micro image with high accuracy based on the macro image.

[0065] The control device 20 may extract a predetermined number of confirmation points that are brighter than other regions in the fluorescence macro image, and set the exposure time based on the brightness of the extracted confirmation points. In this way, the exposure time is set taking into account the brightness of the confirmation points, so that the exposure time can be set with higher accuracy.

[0066] The control device 20 may set an exposure time for each fluorescence macro image corresponding to each excitation wavelength range. Although an appropriate exposure time differs depending on the excitation wavelength range, by setting an exposure time for each fluorescence macro image corresponding to each excitation wavelength range, the exposure time can be set with higher accuracy.

[0067] Based on the fluorescence macro image, the control device 20 may perform tissue recognition to detect a tissue area contained in the sample S or set a microscan area. Furthermore, the control device 20 may perform tissue recognition to detect a tissue area contained in the sample S or set a microscan area for each fluorescence macro image corresponding to each excitation wavelength range. By performing tissue recognition based on a clear fluorescence macro image that does not include excitation light, the tissue area contained in the sample S can be detected with high accuracy.

[0068] The control device 20 may set the focus measurement position based on the fluorescence macro image. Furthermore, the control device 20 may set the focus measurement position for each fluorescence macro image corresponding to each excitation wavelength range. By setting the focus measurement position based on a clear fluorescence macro image that does not contain excitation light, the focus measurement position can be set with high accuracy according to the arrangement of the tissue.

[0069] The image acquisition device 1 may further include a bright-field light source 113 that performs bright-field illumination on the sample S. With this configuration, in addition to the above-mentioned fluorescence macro image, a bright-field macro image can be acquired.

[0070] The image acquisition device 1 may further include a dark-field light source 114 that performs dark-field illumination on the sample S. With this configuration, in addition to the above-mentioned fluorescence macro image, a dark-field macro image can be acquired.

[0071] The image acquisition device 1 may further include a switching unit 121 configured to enable the fluorescence filter unit 120 to be inserted or removed from the optical axis of the fluorescence directed toward the imaging lens 118. With such a configuration, for example, it becomes possible to provide the fluorescence filter unit 120 on the optical axis of the fluorescence only when a fluorescence macro image is acquired, and to retract the fluorescence filter unit 120 from the optical axis of the fluorescence in other cases (for example, when a bright-field macro image or a dark-field macro image is acquired), making it possible to appropriately acquire each image data.

[0072] Finally, various exemplary embodiments included in the present disclosure are described below in [E1] to [E9] and [E10] to [E16].

[0073] [E1] An image acquisition device for acquiring a macro image of a sample, comprising: an excitation light irradiation unit that obliquely irradiates the sample with excitation light in at least one of a plurality of excitation wavelength ranges; a macro observation camera having a fluorescence filter unit having a plurality of reflection wavelength ranges corresponding to the plurality of excitation wavelength ranges and a transmission wavelength range between the reflection wavelength ranges, an imaging lens that forms an image of the fluorescence from the sample corresponding to the irradiated excitation light, and an image sensor that captures the light image formed by the imaging lens and outputs fluorescence macro image data of the sample; A setting unit that sets acquisition conditions for a micro image of the sample based on the fluorescence macro image data.

[0074] [E2] The image acquisition device according to [E1], wherein the setting unit sets an exposure time when acquiring the micro image based on the fluorescence macro image data.

[0075] [E3] The image acquisition device described in [E2], wherein the setting unit extracts a predetermined number of confirmation points that are brighter than other areas in the fluorescence macro image data, and sets the exposure time based on the brightness of the extracted confirmation points.

[0076] [E4] The excitation light irradiating unit irradiates the excitation light for each of the excitation wavelength ranges, the image sensor is a color image sensor, and the color image sensor outputs the fluorescence macro image data corresponding to each of the excitation wavelength ranges, The image acquisition device according to [E2] or [E3], wherein the setting unit sets the exposure time for each of the fluorescence macro image data corresponding to each of the excitation wavelength ranges.

[0077] [E5] The excitation light irradiating unit irradiates the excitation light for each of the excitation wavelength ranges, the image sensor is a color image sensor, and the color image sensor outputs the fluorescence macro image data corresponding to each of the excitation wavelength ranges, The image acquisition device described in any one of [E1] to [E4], wherein the setting unit performs tissue recognition to detect a tissue area contained in the sample for each of the fluorescence macro image data corresponding to each of the excitation wavelength ranges.

[0078] [E6] The excitation light irradiating unit irradiates the excitation light for each of the excitation wavelength ranges, the image sensor is a color image sensor, and the color image sensor outputs the fluorescence macro image data corresponding to each of the excitation wavelength ranges, The image acquisition device according to any one of [E1] to [E5], wherein the setting unit sets a focus measurement position when acquiring a micro image for each of the fluorescence macro image data corresponding to each of the excitation wavelength ranges.

[0079] [E7] The image acquisition device according to any one of [E1] to [E6], further comprising a bright-field illumination unit that performs bright-field illumination on the sample.

[0080] [E8] The image acquisition device according to any one of [E1] to [E7], further comprising a dark-field illumination unit that performs dark-field illumination on the sample.

[0081] [E9] The image acquisition device according to any one of [E1] to [E8], further comprising a switching unit configured to enable the fluorescence filter unit to be inserted or removed onto the optical axis of the fluorescence directed toward the imaging lens.

[0082] [E10] 1. A method for acquiring a macro image of a sample, comprising: an excitation light irradiation step of obliquely irradiating the sample with excitation light having at least one of a plurality of excitation wavelength ranges; an imaging step of imaging, using an image sensor, fluorescence that is generated from the sample in response to the irradiated excitation light and that has been transmitted through a fluorescence filter unit having a plurality of reflection wavelength ranges corresponding to the plurality of excitation wavelength ranges and a transmission wavelength range between each of the reflection wavelength ranges, and outputting fluorescence macro image data of the sample; and setting conditions for acquiring a micro-image of the sample based on the fluorescence macro-image data.

[0083] [E11] The image acquisition method according to [E10], in the setting step, an exposure time for acquiring the micro image is set based on the fluorescence macro image data.

[0084] [E12] An image acquisition method as described in [E11], in which, in the setting step, a predetermined number of confirmation points which are brighter than other areas in the fluorescence macro image data are extracted, and the exposure time is set based on the brightness of the extracted confirmation points.

[0085] [E13] In the excitation light irradiation step, the excitation light is irradiated for each of the excitation wavelength ranges, In the imaging step, the fluorescence macro image data corresponding to each of the excitation wavelength ranges is output, The image acquisition method according to [E11] or [E12], wherein in the setting step, the exposure time is set for each of the fluorescence macro image data corresponding to each of the excitation wavelength ranges.

[0086] [E14] In the excitation light irradiation step, the excitation light is irradiated for each of the excitation wavelength ranges, In the imaging step, the fluorescence macro image data corresponding to each of the excitation wavelength ranges is output, The image acquisition method described in any one of [E11] to [E13], in which the setting step performs tissue recognition to detect an area of ​​tissue contained in the sample for each of the fluorescence macro image data corresponding to each of the excitation wavelength ranges.

[0087] [E15] In the excitation light irradiation step, the excitation light is irradiated for each of the excitation wavelength ranges, In the imaging step, the fluorescence macro image data corresponding to each of the excitation wavelength ranges is output, The image acquisition method according to any one of [E11] to [E14], in which the setting step sets a focus measurement position when acquiring a micro image for each of the fluorescence macro image data corresponding to each of the excitation wavelength ranges.

[0088] [E16] The image acquisition method according to any one of [E10] to [E15], further comprising a switching step of moving the fluorescent filter unit in and out onto the optical axis of the fluorescent light directed toward the imaging lens. [Explanation of symbols]

[0089] 1...image acquisition device, 20...control device (setting section), 113...bright field light source (bright field illumination section), 114...dark field light source (dark field illumination section), 117...second irradiation section (excitation light illumination section), 118...imaging lens, 119...color image sensor (image sensor), 120...fluorescence filter section, 121...switching section, S...sample (specimen).

Claims

1. An image acquisition device for acquiring a macro image of a sample, comprising: an excitation light irradiation unit that obliquely irradiates the sample with excitation light having at least one of a plurality of excitation wavelength ranges; a macro observation camera having a fluorescence filter unit having a plurality of reflection wavelength ranges corresponding to the plurality of excitation wavelength ranges and a transmission wavelength range between the reflection wavelength ranges, an imaging lens that forms an image of the fluorescence from the sample corresponding to the irradiated excitation light, and an image sensor that captures the light image formed by the imaging lens and outputs fluorescence macro image data of the sample; a setting unit that sets acquisition conditions for the fluorescence micro-image of the sample based on the fluorescence macro-image data, the setting unit sets an exposure time when the fluorescence micro-image is acquired based on the fluorescence macro-image data; the excitation light irradiation unit has a same excitation light source as a light source for acquiring the fluorescence macro-image and the fluorescence micro-image, An image acquisition device, wherein the fluorescence macro image is a low-resolution image corresponding to an entire image of the sample.

2. The image acquisition device according to claim 1 , wherein the setting unit extracts a predetermined number of confirmation points that are brighter than other areas in the fluorescence macro image data, and sets the exposure time based on the brightness of the extracted confirmation points.

3. The excitation light irradiating unit irradiates the excitation light for each of the excitation wavelength ranges, the image sensor is a color image sensor, and the color image sensor outputs the fluorescence macro image data corresponding to each of the excitation wavelength ranges, The image acquisition device according to claim 1 , wherein the setting section sets the exposure time for each of the fluorescence macro image data corresponding to each of the excitation wavelength ranges.

4. The excitation light irradiating unit irradiates the excitation light for each of the excitation wavelength ranges, the image sensor is a color image sensor, and the color image sensor outputs the fluorescence macro image data corresponding to each of the excitation wavelength ranges, The image acquisition device according to claim 1 , wherein the setting unit performs tissue recognition to detect a tissue region contained in the sample for each of the fluorescence macro image data corresponding to each of the excitation wavelength ranges.

5. The excitation light irradiating unit irradiates the excitation light for each of the excitation wavelength ranges, the image sensor is a color image sensor, and the color image sensor outputs the fluorescence macro image data corresponding to each of the excitation wavelength ranges, 3. The image acquiring device according to claim 1, wherein the setting section sets a focus measurement position when acquiring the fluorescence micro image for each of the fluorescence macro image data corresponding to each of the excitation wavelength ranges.

6. The image acquisition device according to claim 1 , further comprising a bright-field illumination unit that performs bright-field illumination on the sample.

7. The image acquisition device according to claim 1 , further comprising a dark-field illumination unit that provides dark-field illumination to the sample.

8. 8. The image acquisition device according to claim 6, further comprising a switching unit configured to be able to move the fluorescence filter unit in and out onto an optical axis of the fluorescence directed toward the imaging lens.

9. 1. A method for acquiring a macro image of a sample, comprising: an excitation light irradiation step of obliquely irradiating the sample with excitation light having at least one of a plurality of excitation wavelength ranges; an imaging step of imaging, using an image sensor, fluorescence that is generated from the sample in response to the irradiated excitation light and that has been transmitted through a fluorescence filter unit having a plurality of reflection wavelength ranges corresponding to the plurality of excitation wavelength ranges and a transmission wavelength range between each of the reflection wavelength ranges, and outputting fluorescence macro image data of the sample; and setting conditions for acquiring a fluorescence micro-image of the sample based on the fluorescence macro-image data, In the setting step, an exposure time for acquiring the fluorescence micro-image is set based on the fluorescence macro-image data; the excitation light irradiation step uses the same excitation light source as a light source for acquiring the fluorescence macro-image and the fluorescence micro-image; An image acquisition method, wherein the fluorescence macro image is a low-resolution image corresponding to an entire image of the sample.

Images