Correction member and image acquisition device

Abstract

A correction member according to the present disclosure is for performing shading correction on a fluorescent image of a sample obtained by irradiation with excitation light. The correction member comprises a semiconductor substrate which includes: a front surface irradiated with excitation light; and a rear surface facing the reverse side from the front surface. The material of the semiconductor substrate includes an inorganic compound semiconductor that generates fluorescence by irradiation with excitation light.

Description

Correction component and image acquisition device

[0001] This disclosure relates to a correction member and an image acquisition device. This application claims priority under Japanese application No. 2025-025071, filed on 19 February 2025, and incorporates all the provisions of the said Japanese application.

[0002] As a correction member for performing shading correction on a fluorescence image (fluorescence image) generated from a sample in response to excitation light irradiation, for example, a fluorescence microscope slide described in Non-Patent Literature 1 is known. This fluorescence microscope slide is a fluorescence acrylic slide formed of fluorescence acrylic and has the characteristic of generating uniform fluorescence upon irradiation with excitation light. By utilizing this characteristic, shading correction is performed to correct for intensity unevenness and non-uniformity caused by the optical system of the image acquisition device, thereby improving the accuracy of the fluorescence image.

[0003] Thorlabs product information, microscope components, searched November 6, 2024, Internet (https: / / www.thorlabs.co.jp / newgrouppage9.cfm?objectgroup_ID=12142)

[0004] The fluorescent acrylic slides mentioned above are prone to fading (i.e., attenuation of fluorescence intensity) when exposed to excitation light. Therefore, if the same area of ​​the fluorescent acrylic slide is repeatedly irradiated with excitation light, uneven intensity may occur in the fluorescence emitted from the slide. Such uneven intensity can make it difficult to perform accurate shading correction on the fluorescence image of the actual sample, potentially making it difficult to obtain high-precision fluorescence images.

[0005] This disclosure provides a correction member and an image acquisition device capable of acquiring high-precision fluorescence images.

[0006] One aspect of the present disclosure is a corrective member, [1] a corrective member for performing shading correction on a fluorescence image of a sample obtained by irradiation with excitation light, comprising a semiconductor substrate having a surface to which the excitation light is irradiated and a back surface facing the opposite side of the surface, wherein the material of the semiconductor substrate includes an inorganic compound semiconductor that generates fluorescence upon irradiation with the excitation light.

[0007] The correction member described in [1] above comprises a semiconductor substrate having a surface to which excitation light is irradiated and a back surface facing the opposite side of the surface. The semiconductor substrate material contains an inorganic compound semiconductor. Compared to conventional fluorescent acrylic slides, inorganic compound semiconductors have the property of being less prone to discoloration even when exposed to excitation light. Therefore, even if the same area of ​​the semiconductor substrate is repeatedly irradiated with excitation light, it is less likely that intensity unevenness will occur in the fluorescence generated from the semiconductor substrate. In this case, shading correction can be performed accurately on the fluorescence image of the actual sample based on the intensity distribution of fluorescence from the semiconductor substrate, so that a high-precision fluorescence image can be obtained. Conventional fluorescent acrylic slides have an absorption band from the ultraviolet wavelength range to the visible wavelength range, but many have a low absorption rate in the red wavelength range to the near-infrared wavelength range, which is longer wavelength than those wavelength ranges. Therefore, even if excitation light in such long wavelength ranges is irradiated onto a fluorescent acrylic slide, it is not possible to stably obtain fluorescence of sufficient intensity from the fluorescent acrylic slide. On the other hand, in the correction member described in [1] above, the inorganic compound semiconductor can stably generate fluorescence of sufficient intensity even when excitation light in the long wavelength range is used, thus increasing the freedom of wavelength selection for the excitation light. Conventional fluorescent acrylic slides are transparent to excitation light, so when excitation light is irradiated onto a fluorescent acrylic slide, excitation occurs not only on the surface of the fluorescent acrylic slide but also at locations deep below the surface. As a result, fluorescence generated from the surface of the fluorescent acrylic slide and fluorescence generated at locations deep below the surface may overlap. In this case, it may be difficult to accurately correct the fluorescence image of the actual sample based on the intensity distribution of fluorescence generated from the fluorescent acrylic slide. On the other hand, in the correction member described in [1] above, the inorganic compound semiconductor has low transmittance to excitation light. Therefore, when excitation light is irradiated onto the semiconductor substrate, fluorescence is generated mainly only near the surface of the semiconductor substrate. In this case, the fluorescence image of the actual sample can be accurately corrected based on the intensity distribution of fluorescence from the semiconductor substrate, so a high-precision fluorescence image can be obtained.

[0008] The correction member according to one aspect of the present disclosure may be the one described in [2] "the correction member according to [1] above, further comprising a slide glass on which the back surface of the semiconductor substrate is placed". When the correction member includes the semiconductor substrate and the slide glass in this way, unlike the case where the correction member includes only the semiconductor substrate, the mechanical strength of the correction member can be increased by the slide glass, so that the semiconductor substrate does not need to be made unnecessarily large in order to ensure the mechanical strength of the correction member. As a result, the correction member can be formed at a low cost.

[0009] The correction member according to one aspect of the present disclosure may be the one described in [3] "the correction member according to [1] or [2] above, further comprising a cover glass covering the surface of the semiconductor substrate". In the correction member described in [3] above, the influence of aberration in the optical system for guiding the excitation light to the correction member can be made equivalent to the influence of aberration in the optical system for guiding the excitation light to the sample when actually acquiring the fluorescence image of the sample, so that a more accurate fluorescence image can be obtained.

[0010] The correction member according to one aspect of the present disclosure may be the one described in [4] "the correction member according to any one of [1] to [3] above, wherein the semiconductor substrate has a protective film formed on the surface of the semiconductor substrate and transmitting the excitation light and the fluorescence". In the correction member described in [4] above, by forming a protective film on the surface of the semiconductor substrate, the possibility of moisture in the air contacting the surface of the semiconductor substrate can be reduced. Thereby, it is possible to suppress a decrease in the emission intensity of the inorganic compound semiconductor caused by the surface of the semiconductor substrate being modified by moisture in the air. As a result, a more accurate fluorescence image can be obtained based on the fluorescence generated from the inorganic compound semiconductor.

[0011] The correction member according to one aspect of the present disclosure may be the one described in [5] "the correction member according to any one of [1] to [4] above, wherein the semiconductor substrate includes a pattern portion formed on the surface of the semiconductor substrate for focusing the imaging unit that images the fluorescence". In the correction member described in [5] above, based on the pattern portion formed on the surface of the semiconductor substrate, the focus of the imaging unit can be easily adjusted on the surface of the semiconductor substrate, so that a high-precision fluorescence image can be easily obtained.

[0012] The correction member according to one aspect of the present disclosure may be "[6] the correction member according to [5] above, wherein the pattern portion includes at least three portions arranged so as not to be on one straight line on the surface of the semiconductor substrate". In the correction member according to [6] above, when focusing the imaging unit on a pattern portion including three portions of the pattern portion arranged so as not to be on one straight line on the surface of the semiconductor substrate, even when the surface of the semiconductor substrate has an inclination, the focus surface can be surely aligned with the surface of the semiconductor substrate in consideration of the inclination, so that a fluorescence image with higher accuracy can be easily obtained.

[0013] The correction member according to one aspect of the present disclosure may be "[7] the correction member according to any one of [1] to [6] above, comprising a first semiconductor substrate and a second semiconductor substrate as the semiconductor substrates, wherein the bandgap wavelength of the inorganic compound semiconductor contained in the first semiconductor substrate is different from the bandgap wavelength of the inorganic compound semiconductor contained in the second semiconductor substrate". In the correction member according to [7] above, even when observing a plurality of samples that generate fluorescence with different wavelengths, the bandgap wavelengths of the first semiconductor substrate and the second semiconductor substrate can be appropriately set according to the wavelengths of the fluorescence from the respective samples, so that appropriate shading correction can be performed on the fluorescence images of the respective samples using the images of the fluorescence generated from the first semiconductor substrate and the second semiconductor substrate respectively by the irradiation of the excitation light.

[0014] An image acquisition apparatus in one aspect of the present disclosure is [8] "an image acquisition apparatus comprising: a correction member for performing shading correction on a fluorescence image of a sample obtained by irradiation with excitation light; a mounting section on which the correction member is placed; a light irradiation section for irradiating the correction member with the excitation light; and an imaging section for acquiring an image including fluorescence generated from the correction member by irradiation with the excitation light, wherein the correction member has a semiconductor substrate including a surface to which the excitation light is irradiated and a back surface facing the opposite side of the surface, and the material of the semiconductor substrate includes an inorganic compound semiconductor that generates fluorescence by irradiation with the excitation light." The image acquisition apparatus described in [8] above includes the correction member described in [1] above, and therefore the same effects as described in [1] above can be obtained.

[0015] An image acquisition apparatus in one aspect of the present disclosure may be [9] "an image acquisition apparatus according to [8] above, further comprising an image processing unit for processing the fluorescence image, wherein the image processing unit acquires the image including the fluorescence generated from the correcting member by irradiation of the correcting member with excitation light as shading correction data, and performs shading correction on the fluorescence image obtained by irradiation of the sample with excitation light using the shading correction data." In the image acquisition apparatus according to [9] above, as described above, inorganic compound semiconductors can stably generate fluorescence of sufficient intensity by irradiation with excitation light, so shading correction can be appropriately performed on the fluorescence image of the actual sample based on the intensity distribution of the fluorescence. This makes it possible to acquire a high-precision fluorescence image.

[0016] According to this disclosure, it is possible to provide a correction member and an image acquisition device that can acquire high-precision fluorescence images.

[0017] Figure 1 is a configuration diagram of an image acquisition device according to one embodiment. Figure 2 is a configuration diagram of a correction member provided in the image acquisition device shown in Figure 1. Figure 3 is a perspective view of the correction member shown in Figure 2. Figure 4 is a plan view of the correction member shown in Figure 3. Figure 5 is a cross-sectional view of the correction member along the line A1-A1 shown in Figure 4. Figure 6 is a plan view of the semiconductor substrate of the correction member shown in Figure 3. Figure 7 is a graph showing the time change of fluorescence intensity of the semiconductor substrate of Figure 3 and a conventional fluorescent acrylic slide, respectively. Figure 8 is a graph showing the relationship between the transmittance and excitation wavelength of a conventional fluorescent acrylic slide. Figure 9 is a graph showing the relationship between the fluorescence wavelength and excitation wavelength of the semiconductor substrate of Figure 3. Figure 10(a) is a diagram showing the properties of a III-V compound semiconductor, particularly a compound semiconductor containing As or P. Figure 10(b) is a diagram showing the properties of a III-V compound semiconductor, particularly a compound semiconductor containing Sb. Figure 11(a) is a diagram showing the properties of a II-VI compound semiconductor. Figure 11(b) shows the properties of a group I-III-VI2 compound semiconductor. Figures 12(a) and 12(b) are plan views showing the corrective member of Modification Example 1. Figure 13(a) is a plan view of the corrective member of Modification Example 2. Figure 13(b) is a cross-sectional view of the corrective member along the line A2-A2 in Figure 13(a). Figures 14(a), 14(b), and 14(c) are plan views showing the corrective member of Modification Example 3. Figures 15(a), 15(b), and 15(c) are plan views showing the corrective member of Modification Example 4. Figures 16(a), 16(b), and 16(c) are plan views showing the corrective member of Modification Example 5. Figures 17(a), 17(b), and 17(c) are plan views showing the corrective member of Modification Example 6.

[0018] Embodiments of this disclosure will be described in detail below with reference to the drawings. In each drawing, the same or corresponding parts are denoted by the same reference numerals, and redundant descriptions are omitted.

[0019] As shown in Figure 1, the image acquisition device 1 is a device that acquires a fluorescence image (hereinafter referred to as "fluorescence image G") of a sample S supported on a glass slide G1. The sample S is, for example, human or animal cells, tissues, organs, animals or plants themselves, plant cells, or tissues. As an example, the sample S is pathological cells stained with multiple fluorescent dyes. In the image acquisition device 1, the sample S is handled while it is held on the glass slide G1, and various images of the sample S are acquired.

[0020] The image acquisition device 1 comprises a slide glass G1 on which a sample S is placed, a cover glass G2 placed on the slide glass G1 so as to cover the sample S, a sample placement section 3 on which the slide glass G1 is placed, an imaging section 6 that acquires a fluorescence image G by irradiating the sample S with excitation light E, an excitation light source 7 (light irradiation section) that outputs excitation light E toward the sample S, and an image processing section 8 that processes the fluorescence image G acquired by the imaging section 6. Hereinafter, a predetermined horizontal direction will be referred to as the X-axis direction, a horizontal direction perpendicular to the X-axis direction will be referred to as the Y-axis direction, and the vertical direction will be referred to as the Z-axis direction.

[0021] The sample placement unit 3 includes a placement unit 31 and a moving stage 33. The placement unit 31 is mounted above the moving stage 33. A microscope slide G1 is placed on the placement unit 31. The moving stage 33 moves the placement unit 31 in the X-axis direction and the Y-axis direction, respectively. The moving stage 33 moves the field of view of the light detection unit 61, which will be described later, relative to the sample S.

[0022] The excitation light source 7 includes an optical output unit 71, an optical emission unit 72, and a light guide 73. The optical output unit 71 outputs excitation light E for each of a plurality of wavelength ranges. The wavelength range of the excitation light E includes wavelengths capable of exciting the fluorescent substance contained in the sample S. The wavelength range of the excitation light E may be, for example, 635 nm to 900 nm, or 650 nm to 800 nm. The optical emission unit 72 emits the excitation light E output from the optical output unit 71 and guided by the light guide 73 along the first optical path P1. The optical output unit 71 is a light source capable of switching and irradiating excitation light E of a plurality of wavelength ranges (wavelength distributions). The optical output unit 71 may be, for example, an LED (Light Emitting Diode) light source, a light source consisting of a plurality of monochromatic laser light sources, or a light source combining a white light source and a wavelength-selective optical element.

[0023] The imaging unit 6 includes an objective lens unit 55, a light detection unit 61, an optical path switching unit 62, a filter switching unit 63, a mirror 64, a mirror 65, an imaging lens 66, an optical filter unit 67, and a fluorescence filter unit 68.

[0024] The objective lens unit 55 includes a plurality of objective lenses 55a, a moving stage 55b, and a switching stage 55c. The plurality of objective lenses 55a are mounted below the moving stage 55b. The moving stage 55b is mounted below the switching stage 55c. In the imaging unit 6, the switching stage 55c rotates so that one of the objective lenses 55a is positioned on the optical axis of the photodetector 61 (the second optical path P2, described later). In the imaging unit 6, the objective lens 55a positioned on the optical axis of the photodetector 61 is moved in the Z-axis direction by the moving stage 55b so that the focus of the objective lens 55a positioned on the optical axis of the photodetector 61 is aligned with the sample S. The magnification of each objective lens 55a is, for example, 10x, 20x, or 40x. As an example, the switching stage 55c is a rotating type stage or a linear type stage.

[0025] The light-emitting section 72 of the excitation light source 7 is optically connected to the optical path switching section 62 by a first optical path P1. The mounting section 31 is optically connected to the optical path switching section 62 by a second optical path P2. The light-detecting section 61 is optically connected to the optical path switching section 62 by a third optical path P3. The first optical path P1 extends between the light-emitting section 72 and the optical path switching section 62. The second optical path P2 extends between the mounting section 31 and the optical path switching section 62. The third optical path P3 extends between the light-detecting section 61 and the optical path switching section 62.

[0026] As an example, in the imaging unit 6, the first optical path P1 extends along the Y-axis direction between the light emission unit 72 and the optical path switching unit 62. The second optical path P2 extends along the Z-axis direction between the optical path switching unit 62 and the mounting unit 31. The third optical path P3 extends along the Z-axis direction between the optical path switching unit 62 and the mirror 64. The third optical path P3 extends along the Y-axis direction between the mirror 64 and the mirror 65. The third optical path P3 extends along the Z-axis direction between the mirror 65 and the light detection unit 61.

[0027] The excitation light E emitted from the light emission unit 72 enters the optical path switching unit 62 along the first optical path P1. The excitation light E that enters the optical path switching unit 62 is reflected by the optical path switching unit 62 and travels along the second optical path P2, irradiating the sample S held on the slide glass G1. The excitation light E passes through the objective lens 55a of the objective lens unit 55 located on the second optical path P2. The fluorescence F1 generated by the irradiation of the sample S with excitation light E passes through the objective lens 55a of the objective lens unit 55 located on the second optical path P2. The fluorescence F1 that enters the optical path switching unit 62 passes through the optical path switching unit 62 and travels along the third optical path P3, where it is detected by the photodetector 61.

[0028] The filter switching unit 63 is located on the first optical path P1 between the light emission unit 72 and the optical path switching unit 62. The filter switching unit 63 is an optical device that incorporates two filters 631 and 632 and switches the two filters 631 and 632 in and out of the first optical path P1. For example, the two filters 631 and 632 are multibandpass filters that transmit light in multiple predetermined wavelength ranges that are different from each other. With a filter switching unit 63 configured in this way, excitation light E of two wavelength ranges can be switched and transmitted from the excitation light E of a predetermined wavelength range emitted from the light emission unit 72, and irradiated onto the sample S via the optical path switching unit 62. In the example in Figure 1, two filters 631 and 632 are incorporated, but three or more filters may be incorporated.

[0029] As described above, the photodetector 61 detects fluorescence F1 generated by irradiation with excitation light E. The photodetector 61 is, for example, a monochrome area image sensor. The photodetector 61 may also be a color area image sensor or a color separation sensor (for example, a multispectral sensor, a hyperspectral sensor, etc.). When a color separation sensor is used as the photodetector 61, it becomes easier to handle cases with a large number of dyes (for example, 10 or more colors). The photodetector 61 may also be a linear image sensor.

[0030] As described above, the optical path switching unit 62 switches the optical path of the excitation light E and the optical path of the fluorescence F1. The optical path switching unit 62 is configured to switch the optical paths of excitation light E in multiple different first wavelength ranges or multiple different second wavelength ranges from the first optical path P1 to the second optical path P2, and to switch the optical paths of fluorescence F1 in wavelength ranges shifted from the multiple first wavelength ranges or multiple second wavelength ranges from the second optical path P2 to the third optical path P3. Multiple different first wavelength ranges and multiple different second wavelength ranges mean multiple wavelength ranges that are far apart from each other. Wavelength ranges shifted from the multiple first wavelength ranges mean wavelength ranges that do not substantially overlap with the multiple first wavelength ranges. For example, the optical path switching unit 62 has a configuration in which a pair of triangular prism-shaped light-transmitting members are combined to form a cube, and a dichroic mirror, which is a dielectric multilayer film, is formed on the interface surface of the pair of light-transmitting members. The optical path switching unit 62 may be a fluorescence cube incorporating a plate-type dichroic mirror, an excitation bandpass filter, and a fluorescence bandpass filter.

[0031] The imaging unit 6 is equipped with a plurality of optical path switching units 62 having different specifications from each other (for example, the number of wavelength ranges, the upper limit of the wavelength ranges, and the lower limit of the wavelength ranges). The imaging unit 6 is configured to place one of the optical path switching units 62 at the position where the first optical path P1, the second optical path P2, and the third optical path P3 intersect. The imaging unit 6 is configured to allow each optical path switching unit 62 to be attached to and detached (replaced).

[0032] Mirror 64 is positioned on the third optical path P3. Mirror 65 is positioned on the third optical path P3 between mirror 64 and the photodetector 61. Mirror 64 reflects fluorescence F1 of multiple wavelength ranges emitted from the optical path switching unit 62 toward mirror 65. Mirror 65 reflects the fluorescence F1 of multiple wavelength ranges reflected by mirror 64 toward the photodetector 61, causing it to enter the photodetector 61. The imaging lens 66 is positioned on the third optical path P3 between mirror 64 and mirror 65. The imaging lens 66 images fluorescence F1 of multiple wavelength ranges onto the photodetector 61. In other words, the imaging lens 66 images the image of the field of view of the objective lens 55a positioned on the second optical path P2 onto the photodetector 61. The imaging lens 66 is, for example, a tube lens.

[0033] The optical filter section 67 includes an optical filter 67a. The optical filter 67a transmits fluorescence F1 in multiple wavelength ranges. The optical filter section 67 is configured to place the optical filter 67a on the third optical path P3 between the optical path switching section 62 and the imaging lens 66. The optical filter section 67 is configured to exclude the optical filter 67a from the third optical path P3 between the optical path switching section 62 and the imaging lens 66.

[0034] The optical filter 67a has transmission characteristics in which the transmittance changes in the fluorescence wavelength range, which includes multiple wavelength ranges. The optical filter 67a has transmission characteristics in which the wavelength and transmittance correspond one-to-one in the fluorescence wavelength range. The optical filter 67a has transmission characteristics in which the transmittance changes linearly in the fluorescence wavelength range.

[0035] The fluorescence filter section 68 includes a switching mechanism body 681, a plurality of single bandpass filters 682, and a light-transmitting section 683. Each single bandpass filter 682 is positioned in each of a plurality of apertures formed in the switching mechanism body 681. Each single bandpass filter 682 selectively transmits fluorescence F1 for each of a plurality of wavelength ranges. One single bandpass filter 682 substantially has the function of transmitting only fluorescence F1 of one wavelength range. The light-transmitting section 683 is an aperture formed in the switching mechanism body 681 and allows fluorescence F1 in the fluorescence wavelength range to pass through. The light-transmitting section 683 may be a light-transmitting member having transmission characteristics that allow fluorescence F1 in the fluorescence wavelength range to pass through. The light-transmitting section 683 may be positioned in an aperture formed in the switching mechanism body 681. The fluorescence filter section 68 is configured such that the light-transmitting section 683 and each of the plurality of single bandpass filters 682 are positioned on the third optical path P3 when the switching mechanism body 681 is switched. As an example, the switching mechanism body 681 is either a rotary type switching mechanism (e.g., a filter wheel) or a linear type switching mechanism (e.g., a slider).

[0036] The image processing unit 8 includes a processing unit 81 and a display unit 82. The processing unit 81 is a computer including a processor and memory. The image processing unit 8 is connected to the imaging unit 6, for example, via wired or wireless communication. The processing unit 81 acquires a fluorescence image G, which is image data showing the intensity distribution of fluorescence F1 in the sample S, from the photodetector 61 of the imaging unit 6, and processes the acquired fluorescence image G. The fluorescence image G to be processed by the processing unit 81 is an image of a region of the sample S corresponding to the field of view of the objective lens 55a (specifically, the field of view of the photodetector 61, which is an image sensor). The display unit 82 is a display that shows various images of the sample S.

[0037] The processing unit 81 has a function to perform shading correction on the fluorescence image G. Shading correction is a correction for intensity unevenness caused by the optical system including the imaging unit 6 and the excitation light source 7. In shading correction, shading correction data is acquired to correct for intensity unevenness that may occur in the fluorescence image G when the sample S is absent, and the intensity of the fluorescence image G is corrected using the shading correction data. More specifically, as shown in Figure 2, a correction member 2 is placed in place of the sample S and the slide glass G1 at the position where the sample S would be placed. In this state, excitation light E is irradiated onto the correction member 2 through the objective lens unit 55. Fluorescence F2 is generated from the correction member 2 upon irradiation with excitation light E. Fluorescence F2 is detected by the light detection unit 61 through the objective lens unit 55. The image of fluorescence F2 is acquired as shading correction data. The brightness value of each pixel in the fluorescence image G is divided by the shading correction data. This corrects the intensity unevenness caused by the optical system of the image acquisition device 1, improving the accuracy of the fluorescence image G. Fluorescence F2 is detected by the photodetector 61 in the image acquisition device 1, following the same path as fluorescence F1. In other words, the path of fluorescence F2 from the correction member 2 to detection by the photodetector 61 is described in the above-mentioned explanation of the path of fluorescence F1, but with fluorescence F2 replaced by fluorescence F1.

[0038] Furthermore, the configuration of the correction member 2 will be explained in more detail with reference to Figures 3 to 6.

[0039] As shown in Figures 3 to 5, the corrective member 2 includes, for example, a slide glass 21, a semiconductor substrate 23, and a cover glass 27. The slide glass 21 is a plate-shaped member with the Z-axis direction as the thickness direction. Specifically, the slide glass 21 is a glass plate formed from a glass material. The slide glass 21 is, for example, the same as the slide glass G1 described above, but may also be different from the slide glass G1. In this specification, light passing through a member means that at least a part of the light passes through the member, and it is not necessarily required that all of the light passes through the member.

[0040] As shown in Figure 4, the shape of the slide glass 21 as viewed along the Z-axis direction is, for example, a rectangle with the Y-axis direction as the longitudinal direction and the X-axis direction as the transverse direction. As shown in Figure 5, the slide glass 21 includes a front surface 21a and a back surface 21b facing the opposite side of the front surface 21a. The front surface 21a and the back surface 21b are, for example, planes perpendicular to the Z-axis direction and are arranged parallel to each other. The back surface 21b is placed on the mounting portion 31. The front surface 21a includes a barcode area 21c on which a barcode is attached. The barcode area 21c is, for example, located at the Y-axis end of the front surface 21a. The barcode information attached to the barcode area 21c is obtained by detecting the light reflected from the barcode.

[0041] As shown in Figure 3, the semiconductor substrate 23 is a plate-shaped member with the Z-axis direction as the thickness direction. Specifically, the semiconductor substrate 23 is a semiconductor substrate formed from an inorganic compound semiconductor. In other words, the material of the semiconductor substrate 23 includes an inorganic compound semiconductor. An inorganic compound semiconductor is a compound semiconductor composed of two or more elements, including an inorganic element, that exhibits semiconductor properties. Inorganic compound semiconductors do not include inorganic semiconductors composed of a single element such as Si (silicon) or Ge (germanium). Inorganic compound semiconductors generate PL (photoluminescence) emission as fluorescence F2 when irradiated with excitation light E. PL emission is light generated when a substance is irradiated with light and excited electrons return to the ground state. The wavelength of fluorescence F2 is longer than the wavelength of excitation light E. For example, if the wavelength of excitation light E is 600 nm or more and 650 nm or less, the wavelength of fluorescence F2 is 650 nm or more and 800 nm or less. The wavelength of fluorescence F2 may be the same as fluorescence F1, or it may be different from fluorescence F1. When compound semiconductors, which are direct bandgap semiconductors, are used, it is possible to obtain sufficient fluorescence intensity F2. When indirect bandgap semiconductors such as Si (silicon) or Ge (germanium) are used, it is difficult to obtain sufficient fluorescence intensity F2, so indirect bandgap semiconductors are not suitable for shading correction.

[0042] The image of fluorescence F2 generated from the inorganic compound semiconductor contained in the semiconductor substrate 23 is used as shading correction data. The semiconductor substrate 23, as a correction member, may be housed in a slide rack in the same way as the slide glass 21, or it may be removed from the slide rack in the same way as the slide glass 21 and placed on the mounting section 31 (see Figure 1). Alternatively, the semiconductor substrate 23 may be mounted on the mounting section 31, and the semiconductor substrate 23 may be placed on the optical path of the excitation light E when performing shading correction.

[0043] Inorganic compound semiconductors include, for example, III-V compound semiconductors, II-VI compound semiconductors, I-III-VI2 compound semiconductors, mixed crystal semiconductors using these semiconductors, or heterostructures of these semiconductors. Examples of III-V compound semiconductors include As-based compound semiconductors containing As (arsenic), P-based compound semiconductors containing P (phosphorus), and Sb-based compound semiconductors containing Sb (antimony).

[0044] Examples of As-based compound semiconductors include GaAs (gallium arsenide), InAs (indium arsenide), and AlAs (aluminum arsenide). Examples of mixed crystal semiconductors using As-based compound semiconductors include, for example, Ga １－ｘ , ｙ Al １－ｘ As (gallium aluminum arsenide), In １－ｘ Ga ｘ As (indium gallium arsenide), In １－ｘ Ga ｘ As ｙ P １－ｙ (indium gallium arsenide phosphorus), or In １－ｘ Ga ｘ As ｙ N １－ｙ (indium gallium arsenide nitrogen). The inorganic compound semiconductor may be one formed by forming Ga ｘ Al １－ｘ As or In １－ｘ Ga ｘ As ｙ N <000001⑧> on a GaAs substrate, or it may be one formed by forming In １－ｘ Ga ｘ As ｙ P １－ｙ on an InP substrate.

[0045] Examples of P-based compound semiconductors include GaP (gallium phosphorus) and InP (indium phosphorus). Examples of mixed crystal semiconductors using P-based compound semiconductors include, for example, In １－ｘ Ga ｘ P (indium gallium phosphorus) and In １－ｘ Ga ｘ As ｙ P １－ｙ (indium gallium arsenide phosphorus).

[0046] Examples of Sb-based compound semiconductors include GaSb (gallium antimony), InSb (indium antimony), and AlSb (aluminum antimony). An example of a mixed crystal semiconductor using an Sb-based compound semiconductor is GaAs. １－ｘ Sb ｘ (Gallium arsenide antimony), InAs １－ｘ Sb ｘ (Indium arsenide antimony), AlAs １－ｘ Sb ｘ (Aluminum arsenide antimony), Al ｘ Ga １－ｘ Sb (aluminum gallium antimony), and Ga ｘ In １－ｘ As ｙ Sb １－ｙ (Gallium indium arsenide antimony) is one example.

[0047] II-VI compound semiconductors are compound semiconductors composed of a Group II element of the periodic table (e.g., Zn (zinc), Cd (cadmium), Be (beryllium), or Mg (magnesium)) and a Group VI element (e.g., O (oxygen), S (sulfur), Se (selenium), or Te (tellurium)). Examples of II-VI compound semiconductors include ZnO (zinc oxide), CdO (cadmium oxide), ZnS (zinc sulfide), CdS (cadmium sulfide), ZnSe (zinc selenide), CdSe (cadmium selenide), ZnTe (zinc telluride), CdTe (cadmium telluride), BeTe (beryllium telluride), MgTe (magnesium telluride), ZnMgSe (zinc magnesium selenium), and ZnMgTe (zinc magnesium telluride).

[0048] Group I-III-VI2 compound semiconductors are compound semiconductors composed of Group I elements (e.g., Cu (copper), Ag (silver)), Group III elements (e.g., Ga (gallium), In (indium), Al (aluminum)), and Group VI elements (e.g., S (sulfur), Se (selenium), Te (tellurium)). Examples of Group I-III-VI2 compound semiconductors include CuGaS2 (copper gallium sulfide), CuGaSe2 (copper gallium selenide), CuInS2 (copper indium sulfide), CuInSe2 (copper indium selenide), AgGaS2 (silver gallium sulfide), AgGaSe2 (silver gallium selenide), AgInS2 (silver indium sulfide), AgInSe2 (silver indium selenide), CuAlTe2 (copper aluminum telluride), and AgAlTe2 (silver aluminum telluride).

[0049] As shown in Figure 4, the shape of the semiconductor substrate 23 as viewed along the Z-axis is, for example, a square with sides along the Y-axis and sides along the X-axis. The semiconductor substrate 23 is placed on a glass slide 21. When viewed along the Z-axis, the area of ​​the semiconductor substrate 23 is smaller than the area of ​​the glass slide 21. The semiconductor substrate 23 is positioned, for example, to overlap the center of the glass slide 21. The length of each side of the semiconductor substrate 23 as viewed along the Z-axis is, for example, 10 mm. The thickness of the semiconductor substrate 23 along the Z-axis is, for example, thinner than the thickness of the glass slide 21 along the Z-axis. The thickness of the semiconductor substrate 23 along the Z-axis is, for example, 0.2 mm.

[0050] As shown in Figure 5, the semiconductor substrate 23 includes a surface 23a and a back surface 23b facing the opposite side of the surface 23a. The surface 23a and the back surface 23b are, for example, planes perpendicular to the Z-axis direction and are arranged parallel to each other. The back surface 23b is placed on the surface 21a of the slide glass 21.

[0051] As shown in Figures 3 and 4, a pattern portion 24 for focusing the imaging unit 6 is formed on the surface 23a. Focusing the imaging unit 6 specifically means aligning the focus of the objective lens 55a with the surface 23a.

[0052] As shown in Figure 6, the pattern portion 24 includes a first pattern 24a and a second pattern 24b. Each of the first pattern 24a and the second pattern 24b is, for example, a metal reflective film composed of a plurality of shaded portions, and is formed on the surface 23a by vapor deposition. The plurality of shaded portions forming each of the first pattern 24a and the second pattern 24b extend along directions inclined with respect to the X-axis and Y-axis directions. Each of the first pattern 24a and the second pattern 24b may be, for example, a metal reflective film composed of a plurality of vertical lines parallel to the X-axis direction or horizontal lines parallel to the Y-axis direction.

[0053] The materials used for the first pattern 24a and the second pattern 24b are, for example, Ti (titanium) or Cr (chromium), which do not easily emit light even when irradiated with excitation light E. The shapes of the first pattern 24a and the second pattern 24b are, for example, rectangles with the Y-axis direction as the longitudinal direction and the X-axis direction as the transverse direction. The first pattern 24a and the second pattern 24b extend parallel to each other at positions separated in the X-axis direction. The first pattern 24a and the second pattern 24b are arranged, for example, in a pair of positions that sandwich the center C of the surface 23a in the X-axis direction.

[0054] The surface 23a on which the first pattern 24a and the second pattern 24b are formed includes four sides S1, S2, S3, and S4. Sides S1 and S2 are located opposite each other in the X-axis direction and extend along the Y-axis direction. Sides S3 and S4 are located opposite each other in the Y-axis direction and extend along the X-axis direction. The first pattern 24a is positioned between side S1 and the center C in the X-axis direction. The second pattern 24b is positioned between side S2 and the center C in the X-axis direction.

[0055] The first pattern 24a includes a first corner P11 and a second corner P12 located on opposite sides in the Y-axis direction. The first corner P11 and the second corner P12 are positioned closest to side S1 in the X-axis direction and on opposite sides in the Y-axis direction. The second pattern 24b includes a side P13 extending along the Y-axis direction. Side P13 is positioned closest to side S2 in the X-axis direction. The focus plane of the imaging unit 6 is aligned with the pattern portion 24, which includes the three parts: the first corner P11, the second corner P12, and side P13.

[0056] The first corner P11, the second corner P12, and the side P13 are positioned on the surface 23a at locations offset from the same straight line, that is, at locations that are not aligned on the same straight line. In other words, the first corner P11, the second corner P12, and the side P13 are positioned at locations that are non-collinear on the surface 23a. For example, the first corner P11, the second corner P12, and the side P13 are positioned at locations that form the three vertices of a triangle on the surface 23a.

[0057] If a pattern portion 24 including at least three points is formed on the surface 23a, the imaging unit 6 can be easily focused on the surface 23a. These three points do not necessarily have to be the first corner P11, the second corner P12, and the edge P13. At least one of the three points may be a part of the pattern portion 24 other than the first corner P11, the second corner P12, and the edge P13. In order to focus the imaging unit 6 on the surface 23a, a pattern portion 24 including four or more points may be formed on the surface 23a.

[0058] As shown in Figure 5, a protective film 25 may be formed on the surface 23a. The protective film 25 covers the entire surface 23a, including the pattern portion 24. The area of ​​the protective film 25 viewed along the Z-axis is, for example, the same as the area of ​​the surface 23a viewed along the Z-axis. The protective film 25 is an insulating dielectric film. The protective film 25 is, for example, SiO ２This is a silicon oxide film formed from silicon oxide. The protective film 25 is used to protect the semiconductor substrate 23 from moisture contained in the air. Moisture contained in the air can modify the semiconductor substrate 23 by coming into contact with it, potentially degrading its luminescence. By forming the protective film 25 on the surface 23a, it is possible to prevent the degradation of the luminescence of the semiconductor substrate 23.

[0059] The protective film 25 has light transmittance that allows excitation light E and fluorescence F2 to pass through. Therefore, when excitation light E is irradiated toward the correction member 2, the excitation light E passes through the protective film 25 and irradiates the surface 23a of the semiconductor substrate 23. Fluorescence F2 generated from the semiconductor substrate 23 by the irradiation of excitation light E is detected by the photodetector 61 through the protective film 25. In this way, the protective film 25 does not affect the irradiation of excitation light E or the detection of fluorescence F2. As the material for the protective film 25, SiO2 is used, which has little effect on the irradiation of excitation light E and the detection of fluorescence F2. ２ (Silicon oxide) and MgF ２ (Magnesium fluoride) is one example.

[0060] The cover glass 27 is a plate-shaped member with the Z-axis direction as its thickness. For example, the cover glass 27 is a glass plate formed of a glass material that transmits excitation light E and fluorescence F2. The cover glass 27 is, for example, the same as the cover glass G2 described above, but may also be different from the cover glass G2. The cover glass 27 covers the entire surface 23a of the semiconductor substrate 23. The area of ​​the cover glass 27 viewed along the Z-axis direction is, for example, the same as the area of ​​the surface 23a viewed along the Z-axis direction. Since the cover glass 27 also transmits excitation light E and fluorescence F2, it does not affect the irradiation of excitation light E and the detection of fluorescence F2, similar to the protective film 25.

[0061] When the image acquisition device 1 acquires a fluorescence image G of the sample S, first, as shown in Figure 2, with the sample S absent, i.e., with the correction member 2 placed in the position where the sample S would be placed, the correction member 2 is irradiated with excitation light E. The excitation light E passes through the cover glass 27 and protective film 25 and irradiates the surface 23a of the semiconductor substrate 23. Fluorescence F2 is generated from the semiconductor substrate 23 upon irradiation with excitation light E. The image of fluorescence F2 is acquired as shading correction data.

[0062] Next, as shown in Figure 1, with the sample S in place, i.e., with the sample S placed in the position where the correction member 2 was placed, the sample S is irradiated with excitation light E. The excitation light E passes through the cover glass G2 and irradiates the sample S. Fluorescence F1 is generated from the sample S upon irradiation with excitation light E. The image of fluorescence F1 is acquired as the fluorescence image G of the sample S. Shading correction is performed on the fluorescence image G using shading correction data. This results in a highly accurate fluorescence image G.

[0063] Next, with reference to Figures 7 to 11, the advantages of using the semiconductor substrate 23 for the correction member 2 will be explained.

[0064] Conventionally, fluorescent acrylic slides have been known as correction components used in image acquisition devices that acquire images of fluorescence generated from a sample in response to excitation light irradiation. Fluorescent acrylic slides have the characteristic of generating uniform fluorescence upon irradiation with excitation light. This characteristic can be used to perform shading correction to compensate for intensity unevenness caused by the optical system of the image acquisition device. However, these fluorescent acrylic slides have the following three problems.

[0065] The first challenge is that fluorescent acrylic slides are prone to fading (i.e., a decrease in fluorescence intensity) upon exposure to excitation light. Graph G100, shown in Figure 7, illustrates the time evolution of fluorescence intensity (fluorescence intensity) emitted from the fluorescent acrylic slide upon irradiation with excitation light. As shown in Figure 7, the fluorescence intensity of the fluorescent acrylic slide decreases over time. In graph G100, a 635 nm LED is used as the excitation light irradiated onto the fluorescent acrylic slide, and the excitation light density is 12 mW / mm². ２ That is the case.

[0066] Therefore, if the same area of ​​a fluorescent acrylic slide is repeatedly irradiated with excitation light, the fluorescence intensity at that area decreases. As a result, unevenness in intensity occurs in the fluorescence image generated from the fluorescent acrylic slide. In other words, when observing areas of a fluorescent acrylic slide that have been irradiated with excitation light and areas that have not been irradiated with excitation light in the same field of view, the difference in fluorescence intensity between these areas appears as a step in brightness value. When such unevenness in intensity occurs, it becomes difficult to perform accurate shading correction on the fluorescence image of the actual sample, and it may become difficult to obtain a high-precision fluorescence image.

[0067] The second challenge is that most fluorescent acrylic slides do not have sufficient emission intensity for excitation light in the wavelength range from red to near-infrared wavelengths. Figure 8 shows a graph illustrating the relationship between the wavelength of excitation light (excitation wavelength) and the transmittance of the fluorescent acrylic through which the excitation light is transmitted. In Figure 8, graph G101 shows the transmission spectrum of red fluorescent acrylic. Graph G102 shows the transmission spectrum of orange fluorescent acrylic. Graph G103 shows the transmission spectrum of yellow fluorescent acrylic. Graph G104 shows the transmission spectrum of green fluorescent acrylic. Graph G105 shows the transmission spectrum of blue fluorescent acrylic.

[0068] In recent years, image acquisition devices that obtain fluorescence images of samples have increasingly used excitation light in the long-wavelength range, such as red light around 635 nm or near-infrared light around 740 nm. However, as shown in graphs G101 to G105 in Figure 8, all colored fluorescent acrylic slides have an absorption band in the wavelength range from ultraviolet to visible wavelengths, but no absorption band exists in the long-wavelength range beyond that range (for example, above 635 nm). In other words, all colored fluorescent acrylic slides have weak absorption to long-wavelength excitation light such as red light or near-infrared light, so even if a fluorescent acrylic slide is irradiated with such long-wavelength excitation light, almost no fluorescence is generated from the fluorescent acrylic slide. Therefore, it is difficult to perform shading correction on the fluorescence image of a sample using such long-wavelength excitation light.

[0069] The third challenge is that when a fluorescent acrylic slide is irradiated with excitation light, excitation occurs not only on the surface of the slide but also deep within it. In this case, when excitation occurs deep within the surface of the fluorescent acrylic slide, the fluorescence generated from the surface and the fluorescence generated deep within the surface may overlap. In this case, it may not be possible to correctly perform shading correction on the fluorescence image of the actual sample based on the intensity distribution of fluorescence generated from the fluorescent acrylic slide.

[0070] In this embodiment, the three problems described above are solved by providing the corrective member 2 with a semiconductor substrate 23 containing an inorganic compound semiconductor.

[0071] Regarding the first issue, inorganic compound semiconductors have the property of being resistant to fading even when exposed to excitation light. Graph G10 in Figure 7 shows the time change in fluorescence intensity (fluorescence intensity) generated from the inorganic compound semiconductor upon irradiation with excitation light. As is clear from graphs G10 and G100, the fluorescence intensity of the inorganic compound semiconductor in this embodiment does not decrease as easily over time compared to conventional fluorescent acrylic slides. In graph G10, a 635 nm LED is used as the excitation light irradiated onto the inorganic compound semiconductor, and the density of the excitation light is 40 mW / mm². ２ That is the case.

[0072] Therefore, in this embodiment, even if the same location on the semiconductor substrate 23 is repeatedly irradiated with excitation light E over a long period of time, the fluorescence intensity at that location hardly decreases, so unevenness in intensity is less likely to occur in the image of fluorescence F2 generated from the semiconductor substrate 23. As a result, in this embodiment, it becomes possible to perform accurate shading correction on the fluorescence image G based on the image of fluorescence F2. This makes it possible to obtain a highly accurate fluorescence image G.

[0073] Regarding the second issue, inorganic compound semiconductors have emission properties in response to excitation light in the wavelength range from red wavelengths to near-infrared wavelengths. By changing the elemental composition ratio of the inorganic compound semiconductor, the wavelength of the excitation light irradiated onto the inorganic compound semiconductor and the wavelength of fluorescence generated from the inorganic compound semiconductor upon irradiation with excitation light can be shifted toward longer wavelengths. For example, it is possible to obtain an inorganic compound semiconductor that generates fluorescence at 700 nm upon irradiation with 635 nm excitation light, and it is also possible to obtain an inorganic compound semiconductor that generates fluorescence at 800 nm upon irradiation with 740 nm excitation light. The relationship between the wavelength of the excitation light irradiated onto the inorganic compound semiconductor and the wavelength of fluorescence generated from the inorganic compound semiconductor upon irradiation with that excitation light can be adjusted by changing the elemental composition ratio of the inorganic compound semiconductor forming the inorganic compound semiconductor.

[0074] Figure 9 shows graphs illustrating the relationship between the wavelength of excitation light irradiated onto an inorganic compound semiconductor (excitation wavelength) and the wavelength of fluorescence emitted from the inorganic compound semiconductor as a result of that irradiation (fluorescence wavelength). In Figure 9, graph G11 shows the wavelength and intensity of fluorescence emitted from the inorganic compound semiconductor by excitation light at 440 nm. Graph G12 shows the wavelength and intensity of fluorescence emitted from the inorganic compound semiconductor by excitation light at 460 nm. Graph G13 shows the wavelength and intensity of fluorescence emitted from the inorganic compound semiconductor by excitation light at 480 nm. Graph G14 shows the wavelength and intensity of fluorescence emitted from the inorganic compound semiconductor by excitation light at 500 nm. Graph G15 shows the wavelength and intensity of fluorescence emitted from the inorganic compound semiconductor by excitation light at 520 nm. Graph G16 shows the wavelength and intensity of fluorescence emitted from the inorganic compound semiconductor by excitation light at 540 nm. Graph G17 shows the wavelength and intensity of fluorescence emitted from the inorganic compound semiconductor by excitation light at 560 nm. Graph G18 shows the wavelength and intensity of fluorescence emitted from an inorganic compound semiconductor by excitation light at 580 nm. Graph G19 shows the wavelength and intensity of fluorescence emitted from an inorganic compound semiconductor by excitation light at 600 nm.

[0075] Figure 9 shows that the inorganic compound semiconductor used has fluorescence emission peaks around 680 nm and 850 nm. These emission peaks are highest when excitation light in the 580 nm to 600 nm range (see graphs G18 and G19). Figure 9 uses an inorganic compound semiconductor that generates fluorescence at 680 nm or 850 nm when irradiated with excitation light in the 580 nm to 600 nm range.

[0076] As mentioned above, fluorescence emitted from inorganic compound semiconductors is PL emission. The wavelength of this PL emission changes depending on the band gap of the inorganic compound semiconductor. The band gap of an inorganic compound semiconductor changes depending on the elemental composition ratio of the inorganic compound semiconductor. Therefore, by changing the composition ratio of the inorganic compound semiconductor, it is possible to change the wavelength of fluorescence emitted from the inorganic compound semiconductor. Accordingly, it is also possible to change the wavelength of the excitation light used to generate fluorescence. By changing the elemental composition ratio of the inorganic compound semiconductor, it is possible to obtain an inorganic compound semiconductor that has a fluorescence emission peak at other wavelengths, such as around 780 nm. The band gap wavelength of an inorganic compound semiconductor can be considered to be the same as the wavelength of fluorescence emitted from the inorganic compound semiconductor. By adjusting the elemental composition ratio of the inorganic compound semiconductor so that the band gap wavelength of the inorganic compound semiconductor, i.e., the wavelength of fluorescence emitted from the inorganic compound semiconductor, is between 650 nm and 850 nm, the wavelength of the excitation light irradiated onto the inorganic compound semiconductor can be adjusted to between 500 nm and 600 nm.

[0077] Figures 10 and 11 illustrate how the bandgap wavelength of an inorganic compound semiconductor can be controlled by the elemental composition ratio of the inorganic compound semiconductor. Figure 10(a) shows the properties of a group III-V compound semiconductor, particularly a compound semiconductor containing As or P. Figure 10(b) shows the properties of a group III-V compound semiconductor, particularly a compound semiconductor containing Sb. Figure 11(a) shows the properties of a group II-VI compound semiconductor. Figure 11(b) shows the properties of a group I-III-VI2 compound semiconductor. In each figure, the left vertical axis represents the bandgap energy, the right vertical axis represents the bandgap wavelength corresponding to the bandgap energy, and the horizontal axis represents the lattice constant.

[0078] In Figure 10(a), for example, Ga on a GaAs substrate ｘ Al １－ｘ When using As as an inorganic compound semiconductor, ｘ Al １－ｘBy adjusting x, which represents the composition ratio of As, the bandgap wavelength can be changed along the dashed vertical line representing the lattice constant of the GaAs substrate. １－ｘ Ga ｘ As ｙ N １－ｙ Similarly, when the formed material is used as an inorganic compound semiconductor, In １－ｘ Ga ｘ As ｙ N １－ｙ By adjusting x and y, which represent the composition ratio, the bandgap wavelength can be changed along the dashed vertical line that represents the lattice constant of the GaAs substrate. １－ｘ Ga ｘ As ｙ P １－ｙ Similarly, when the formed material is used as an inorganic compound semiconductor, In １－ｘ Ga ｘ As ｙ P １－ｙ By adjusting x and y, which represent the composition ratio, the bandgap wavelength can be changed along the dashed vertical line that shows the lattice constant of the InP substrate.

[0079] In this way, by adjusting the elemental composition ratio of the inorganic compound semiconductor, the band gap wavelength, i.e., the fluorescence wavelength, can be controlled. For example, to obtain a band gap wavelength of 650 nm to 850 nm, the composition ratio should be adjusted so that it falls within the hatched region in Figure 10(a). In Figure 10(b), for example, Al ｘ Ga １－ｘ As, Al ｘ Ga １－ｘ Sb, and Ga ｘ In １－ｘ As ｙ Sb １－ｙ By adjusting x and y, which represent the respective composition ratios, to fit within the hatched region, a band gap wavelength of, for example, 650 nm to 850 nm can be obtained. In Figures 11(a) and 11(b), by adjusting x and y, which represent the respective elemental composition ratios of the II-VI compound semiconductor and the I-III-VI2 compound semiconductor, to fit within the hatched region, a band gap wavelength of, for example, 650 nm to 850 nm can be obtained.

[0080] As described above, conventional fluorescent acrylic slides do not have a strong absorption band in the long wavelength range, such as 635 nm or 740 nm, but this long wavelength range is a wavelength range that is commonly used in image acquisition devices such as fluorescence microscopes. In contrast, in this embodiment, as described above, the wavelength of fluorescence F2 generated from the inorganic compound semiconductor can be changed by adjusting the elemental composition ratio of the inorganic compound semiconductor, and the wavelength of the excitation light E corresponding to that fluorescence wavelength can also be changed. Therefore, by setting the wavelength of fluorescence F2 generated from the inorganic compound semiconductor and the wavelength of the excitation light E for exciting fluorescence F2 to the long wavelength range as described above, even when using excitation light E in the long wavelength range, fluorescence F2 of sufficient intensity can be generated from the inorganic compound semiconductor, and shading correction can be performed on the fluorescence image G of the sample S. Thus, in this embodiment, shading correction to the fluorescence image G can be performed by appropriately matching the wavelength range of the image acquisition device 1 actually used. Therefore, the degree of freedom in selecting the wavelength of the excitation light E used in the image acquisition device 1 can be increased.

[0081] Regarding the third issue, inorganic compound semiconductors have low transmittance to excitation light. In this embodiment, when excitation light E is irradiated onto the semiconductor substrate 23, fluorescence F2 is generated only near the surface 23a of the semiconductor substrate 23. In this case, shading correction can be appropriately performed on the fluorescence image G of the actual sample S based on the intensity distribution of fluorescence F2 generated from the semiconductor substrate 23, thereby enabling the acquisition of a highly accurate fluorescence image G.

[0082] As described above, this embodiment can solve the first, second, and third problems.

[0083] As in this embodiment, the corrective member 2 may include a slide glass 21 on which the back surface 23b of the semiconductor substrate 23 is placed. When the corrective member 2 includes both the semiconductor substrate 23 and the slide glass 21, unlike when the corrective member 2 includes only the semiconductor substrate 23, the slide glass 21 can increase the mechanical strength of the corrective member 2. Therefore, the semiconductor substrate 23 does not need to be made unnecessarily large to ensure the mechanical strength of the corrective member 2. As a result, the corrective member 2 can be manufactured at a low cost.

[0084] As in this embodiment, the correction member 2 may include a cover glass 27 that covers the surface 23a of the semiconductor substrate 23. In this embodiment, the effect of aberrations in the optical system that guides excitation light E to the correction member 2 can be made equivalent to the effect of aberrations in the optical system that guides excitation light E to the sample S when actually acquiring a fluorescence image G of the sample S, thereby enabling the acquisition of a more accurate fluorescence image G.

[0085] As in this embodiment, the semiconductor substrate 23 may have a protective film 25 formed on its surface 23a that transmits excitation light E and fluorescence F2. By forming the protective film 25 on the surface 23a of the semiconductor substrate 23, the possibility of moisture in the air coming into contact with the surface 23a of the semiconductor substrate 23 can be reduced. This makes it possible to suppress the decrease in the luminescence intensity of the inorganic compound semiconductor caused by the modification of the surface 23a of the semiconductor substrate 23 by moisture in the air. As a result, a more accurate fluorescence image G can be obtained based on the fluorescence F2 generated from the inorganic compound semiconductor.

[0086] As in this embodiment, the semiconductor substrate 23 may include a pattern portion 24 formed on the surface 23a of the semiconductor substrate 23 for focusing the imaging unit 6 that images the fluorescence F2. In this case, the imaging unit 6 can be easily focused on the surface 23a of the semiconductor substrate 23 using the pattern portion 24 formed on the surface 23a of the semiconductor substrate 23 as a reference, so that a high-precision fluorescence image G can be easily obtained.

[0087] As in this embodiment, the pattern portion 24 may include at least three portions (i.e., a first corner portion P11, a second corner portion P12, and a side portion P13) that are not aligned on a single straight line on the surface 23a of the semiconductor substrate 23. In this case, when the imaging unit 6 focuses on the pattern portion 24 including the three portions of the pattern portion 24 that are not aligned on a single straight line on the surface 23a of the semiconductor substrate 23, even if there is a tilt on the surface 23a of the semiconductor substrate 23, the focus plane can be reliably aligned with the surface 23a of the semiconductor substrate 23, taking that tilt into consideration, so that a more accurate fluorescence image G can be easily obtained.

[0088] As in this embodiment, the image processing unit 8 may acquire an image of fluorescence F2 generated from the semiconductor substrate 23 by irradiation of the correction member 2 with excitation light E as shading correction data. In this embodiment, as described above, the inorganic compound semiconductor can stably generate fluorescence F2 of sufficient intensity by irradiation with excitation light E, so the image processing unit 8 can perform appropriate shading correction on the fluorescence image G of the actual sample S based on the intensity distribution of fluorescence F2. This makes it possible to obtain a highly accurate fluorescence image G.

[0089] Several modifications of this embodiment will be described below. In each of the following modifications, the cover glass 27 and protective film 25 will be omitted from the description, but the cover glass 27 and protective film 25 may be provided.

[0090] <Modification 1> As shown in Figures 12(a) and 12(b), the number of semiconductor substrates does not necessarily have to be one, but may be multiple. The number of semiconductor substrates may be a number other than those shown in Figures 12(a) and 12(b) (for example, four or more).

[0091] The correction member 2A shown in Figure 12(a) comprises a first semiconductor substrate 231, a second semiconductor substrate 232, and a third semiconductor substrate 233. The first semiconductor substrate 231, the second semiconductor substrate 232, and the third semiconductor substrate 233 have the same configuration as, for example, the semiconductor substrate 23 described above. The first semiconductor substrate 231, the second semiconductor substrate 232, and the third semiconductor substrate 233 are arranged in a line along the Y-axis direction on the surface 21a of the slide glass 21.

[0092] The first semiconductor substrate 231, the second semiconductor substrate 232, and the third semiconductor substrate 233 have different bandgap wavelengths. The respective bandgap wavelengths of the first semiconductor substrate 231, the second semiconductor substrate 232, and the third semiconductor substrate 233 are determined according to the fluorescence wavelength of the sample to be observed. Therefore, the fluorescence wavelength of the sample placed on the first semiconductor substrate 231, the fluorescence wavelength of the sample placed on the second semiconductor substrate 232, and the fluorescence wavelength of the sample placed on the third semiconductor substrate 233 are different from each other. The bandgap wavelength of the first semiconductor substrate 231 is, for example, 400 nm to 500 nm. The bandgap wavelength of the second semiconductor substrate 232 is, for example, 500 nm to 600 nm. The bandgap wavelength of the third semiconductor substrate 233 is, for example, 600 nm to 700 nm.

[0093] In the correction member 2A shown in Figure 12(a), even when observing multiple samples with different fluorescence wavelengths, the bandgap wavelengths of the first semiconductor substrate 231, the second semiconductor substrate 232, and the third semiconductor substrate 233 can be appropriately set according to the fluorescence wavelength of each sample. Therefore, appropriate shading correction can be performed on the fluorescence image of each sample using the fluorescence images generated from each of the first semiconductor substrate 231, the second semiconductor substrate 232, and the third semiconductor substrate 233 by irradiation with excitation light. Accordingly, the correction member 2A makes it possible to accurately and efficiently acquire fluorescence images of multiple samples placed on multiple semiconductor substrates (i.e., the first semiconductor substrate 231, the second semiconductor substrate 232, and the third semiconductor substrate 233) when the multiple semiconductor substrates are placed on a single slide glass 21.

[0094] The correction member 2B shown in Figure 12(b) comprises a first semiconductor substrate 234 and a second semiconductor substrate 235. The first semiconductor substrate 234 and the second semiconductor substrate 235 have the same configuration as, for example, the semiconductor substrate 23 described above. The first semiconductor substrate 234 and the second semiconductor substrate 235 are arranged in a line along the X-axis direction on the surface 21a of the slide glass 21.

[0095] The first semiconductor substrate 234 and the second semiconductor substrate 235 have different band gap wavelengths. The respective band gap wavelengths of the first semiconductor substrate 234 and the second semiconductor substrate 235 are determined according to the fluorescence wavelength of the sample to be observed. Therefore, the fluorescence wavelength of the sample placed on the first semiconductor substrate 234 and the fluorescence wavelength of the sample placed on the second semiconductor substrate 235 are different from each other. The respective band gap wavelengths of the first semiconductor substrate 234 and the second semiconductor substrate 235 may be the same as, for example, the respective band gap wavelengths of the first semiconductor substrate 231 and the second semiconductor substrate 232. The same effect as the correction member 2A described above can be obtained even with the correction member 2B.

[0096] <Modification 2> The corrective member 2C shown in Figure 13(a) and Figure 13(b) comprises a slide glass 21A having a surface 21a on which a recess 21d is formed. The shape of the recess 21d as viewed along the Z-axis direction corresponds to the shape of the semiconductor substrate 23, and is, for example, a square. The semiconductor substrate 23 is placed in the recess 21d. The semiconductor substrate 23 is, for example, fitted into the recess 21d. The depth of the recess 21d from the surface 21a in the Z-axis direction is, for example, deeper than the thickness of the semiconductor substrate 23 in the Z-axis direction. Even with the corrective member 2C, the same effect as the corrective member 2 described above can be obtained.

[0097] <Modification 3> As shown in Figures 14(a), 14(b), and 14(c), the shape of the semiconductor substrate does not necessarily have to be a square, and may be other shapes. The shape of the semiconductor substrate may be other shapes than those shown in Figures 14(a), 14(b), and 14(c).

[0098] The semiconductor substrate 23A of the corrective member 2D shown in Figure 14(a) is a rectangle extending along the X-axis when viewed along the Z-axis. The semiconductor substrate 23B of the corrective member 2E shown in Figure 14(b) is circular when viewed along the Z-axis. The semiconductor substrate 23C of the corrective member 2F shown in Figure 14(c) is a pentagon when viewed along the Z-axis. The same effect as the corrective member 2 described above can be obtained with corrective members 2D, 2E, and 2F.

[0099] <Modification 4> As shown in Figures 15(a), 15(b), and 15(c), the pattern formed on the surface of the semiconductor substrate does not necessarily have to be along the Y-axis direction, and may extend along other directions. The pattern on the semiconductor substrate may extend along directions other than those shown in Figures 15(a), 15(b), and 15(c).

[0100] The first pattern 24a and the second pattern 24b formed on the surface 23a of the semiconductor substrate 23D of the corrective member 2G shown in Figure 15(a) extend along directions that are inclined in both the X-axis and Y-axis directions. The first pattern 24a and the second pattern 24b formed on the surface 23a of the semiconductor substrate 23E of the corrective member 2H shown in Figure 15(b) extend along the X-axis direction. The semiconductor substrate 23F of the corrective member 2J shown in Figure 15(c) is arranged to be inclined in both the X-axis and Y-axis directions. The first pattern 24a and the second pattern 24b formed on the surface 23a of the semiconductor substrate 23F also extend along directions that are inclined in both the X-axis and Y-axis directions. The same effects as the corrective member 2 described above can be obtained with corrective members 2G, 2H, and 2J.

[0101] <Modification 5> As shown in Figures 16(a), 16(b), and 16(c), the patterned portion formed on the surface of the semiconductor substrate does not necessarily have to include two patterns, but may include three or more patterns. The patterned portion may include a number of patterns other than those shown in Figures 16(a), 16(b), and 16(c).

[0102] The pattern portion 24A of the semiconductor substrate 23G provided by the corrective member 2K shown in Figure 16(a) includes four patterns 24c, 24d, 24e, and 24f. Patterns 24c, 24d, 24e, and 24f are arranged, for example, at the vertices of a square on the surface 23a of the semiconductor substrate 23G. The pattern portion 24B of the semiconductor substrate 23H provided by the corrective member 2L shown in Figure 16(b) includes three patterns 24c, 24d, and 24f. The pattern portion 24C of the semiconductor substrate 23J provided by the corrective member 2M shown in Figure 16(c) includes five patterns 24c, 24d, 24e, 24f, and 24g. Pattern 24g is arranged, for example, at the center of a square on the surface 23a of the semiconductor substrate 23J. The same effects as the corrective member 2 described above can be obtained with the corrective members 2K, 2L, and 2M.

[0103] <Modification 6> As shown in Figures 17(a), 17(b), and 17(c), the pattern formed on the surface of the semiconductor substrate does not necessarily have to include two rectangular patterns, but may include patterns of other shapes. The pattern may include patterns of shapes other than those shown in Figures 17(a), 17(b), and 17(c).

[0104] The pattern portion 24D of the semiconductor substrate 23K of the corrective member 2N shown in Figure 17(a) is composed of a single annular pattern. The pattern portion 24E of the semiconductor substrate 23L of the corrective member 2P shown in Figure 17(b) includes a rectangular pattern 24h extending along the X-axis and a rectangular pattern 24i extending along the Y-axis. Patterns 24h and 24i are arranged to intersect each other at the center of the surface 23a of the semiconductor substrate 23L, forming a cross shape. The pattern portion 24F of the semiconductor substrate 23M of the corrective member 2Q shown in Figure 17(c) includes rectangular patterns 24j and 24k arranged parallel to each other and extending along the Y-axis. Patterns 24j and 24k are metallic reflective films composed of multiple dot-like portions. The same effects as the corrective member 2 described above can be obtained with corrective members 2N, 2P, and 2Q.

[0105] This disclosure is not limited to the embodiments and modifications described above, and various other modifications are possible. For example, the embodiments and modifications described above may be combined with each other to the extent that they do not contradict each other, depending on the required purpose and effect. In the embodiments and modifications described above, the case in which the “semiconductor substrate” of this disclosure is a plate-shaped member is illustrated. The “semiconductor substrate” of this disclosure may be a member other than a plate-shaped member. The “correction member” of this disclosure may not have a slide glass. In this case, the “semiconductor substrate” of this disclosure may have the same size as a slide glass and may function as a slide glass. In other words, the “semiconductor substrate” of this disclosure itself may be used in place of a slide glass.

[0106] 1...Image acquisition device, 2, 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2J, 2K, 2L, 2M, 2N, 2P, 2Q...Correction members, 6...Imaging unit, 7...Excitation light source (light irradiation unit), 8...Image processing unit, G1, 21, 21A...Slide glass, 21a...Surface, 23, 23A, 23B, 23C, 23D, 23E, 23F, 23G, 23H ,23J,23K,23L,23M...semiconductor substrate, 23a...surface, 24,24A,24B,24C,24D,24E,24F...patterned area, 25...protective film, G2,27...cover glass, 31...mounting area, 231,234...first semiconductor substrate, 232,235...second semiconductor substrate, E...excitation light, F1,F2...fluorescence, G...fluorescence image, S...sample.

Claims

1. A correction member for performing shading correction on a fluorescence image of a sample obtained by irradiation with excitation light, comprising a semiconductor substrate having a surface to which the excitation light is irradiated and a back surface facing the opposite side of the surface, wherein the material of the semiconductor substrate includes an inorganic compound semiconductor that generates fluorescence upon irradiation with the excitation light.

2. The corrective member according to claim 1, further comprising a slide glass on which the back surface of the semiconductor substrate is placed.

3. The corrective member according to claim 1 or 2, further comprising a cover glass that covers the surface of the semiconductor substrate.

4. The corrective member according to any one of claims 1 to 3, wherein the semiconductor substrate has a protective film formed on the surface of the semiconductor substrate that transmits the excitation light and the fluorescence.

5. The corrective member according to any one of claims 1 to 4, wherein the semiconductor substrate includes a pattern formed on the surface of the semiconductor substrate for focusing the imaging unit that images the fluorescence.

6. The corrective member according to claim 5, wherein the pattern portion includes at least three portions arranged on the surface of the semiconductor substrate so as not to be aligned on a single straight line.

7. A corrective member according to any one of claims 1 to 6, comprising a first semiconductor substrate and a second semiconductor substrate, wherein the band gap wavelength of the inorganic compound semiconductor contained in the first semiconductor substrate is different from the band gap wavelength of the inorganic compound semiconductor contained in the second semiconductor substrate.

8. An image acquisition device comprising: a correction member for performing shading correction on a fluorescence image of a sample obtained by irradiation with excitation light; a mounting section on which the correction member is placed; a light irradiation section for irradiating the correction member with the excitation light; and an imaging section for acquiring an image including fluorescence generated from the correction member by irradiation with the excitation light, wherein the correction member has a semiconductor substrate including a surface to which the excitation light is irradiated and a back surface facing the opposite side of the surface, and the material of the semiconductor substrate includes an inorganic compound semiconductor that generates fluorescence by irradiation with the excitation light.

9. The image acquisition apparatus according to claim 8, further comprising an image processing unit for processing the fluorescence image, wherein the image processing unit acquires the image including the fluorescence generated from the correcting member by irradiation of the correcting member with excitation light as shading correction data, and performs shading correction on the fluorescence image obtained by irradiation of the sample with excitation light using the shading correction data.

Images