Inspection device

Abstract

To provide a method enabling measurement of the intensity of a spot image signal by easily and directly specifying a position and shape of a spot without performing the gridding operation and increasing the manufacturing cost of a biochip.SOLUTION: An inspection device (1) is provided, comprising: a light and dark switching mechanisms (13-16) which can switch between a light field mode in a state where a biochip (3) is irradiated with light and a dark field mode in a state where the light is shielded with respect to the biochip (3); and an imaging apparatus (11) in which the light and dark switching mechanisms (13,14,16) image a light field image in the light field mode, and the dark switching mechanisms (13,14,16) image a dark field image in the dark field mode.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an inspection apparatus for inspecting biological substances using a biomolecule microarray (hereinafter referred to as a biochip or a chip), and more particularly to an inspection apparatus for accurately and simply performing position information operations on an image for spot image analysis.

Background Art

[0002] In the present specification and claims, the "spot image" refers to an image obtained by imaging the position and shape of a spot, which is a minute site on the substrate of a biochip where a substance to be immobilized is immobilized.

[0003] A measuring apparatus for a biochip that arranges a substance for detection in a large number of minute spots and performs multi-item simultaneous inspection of a single specimen needs to recognize the position of each spot, observe the chemiluminescence or fluorescence emitted from the spot as an image, and digitize the signal intensity for each spot.

[0004] The measuring method of a biochip includes an image acquisition method of collectively measuring with a camera and a scanning method using a confocal laser. In any case, the obtained spot image signal must be digitized. That is, an operation of converting into information such as the peak intensity and average intensity of each of the numerous spot images is required.

[0005] Since the image of a biochip cannot usually digitize the signal intensity etc. for each spot as it is, an operation called "gridding" is performed by analysis software. Gridding refers to an operation of inputting the number of spots in the vertical and horizontal directions on the chip, the distance (interval) between each spot, and the diameter of the spot, and surrounding each spot with a square frame or a circle.

[0006] The purpose of processing assuming the frame surrounding this spot is to cope with the fact that there are spots in the biochip image that do not react, that is, do not emit light.

[0007] By the gridding operation, the maximum value, average value, or median value of each spot image is calculated, that is, quantified. By using these as the signal values of the spot images, quantitative analysis of the biochip becomes possible.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] The gridding of spot images cannot be done uniquely. This is because spot coordinates cannot be uniformly specified for two-dimensional coordinates on the image. This is because the spot positions on the biochip on the image may actually deviate from the designed grid point positions, and the positions where the biochip is mounted on the measuring device may deviate slightly for each biochip. Therefore, it can be considered that the spot positions usually have a deviation of ±100 to 300 μm. In contrast, the size of the spots is generally 20 to 400 μm in diameter.

[0010] In biochip images, even if the same chip is used, it is necessary to perform gridding for each spot image. That is, because there is no guarantee that the spot positions are uniformly determined for each chip. Also, dealing with variations in spot size and differences in shape is an issue in biochip measurement. High reliability and simplification of such gridding operations are issues in biochip measurement.

[0011] One solution is to refine the spot position. That is, the position setting is refined so that the displacement of the spot position on the image is always constant. For this purpose, it is necessary to minimize errors in the shape of the biochip itself, errors in the spot position within the chip, mounting errors of the biochip with respect to the measuring device, etc., and keep them within ±20 μm or less. Although it is not impossible to achieve, it increases costs and is not suitable for applications such as inspections.

[0012] As one method for simplifying the gridding operation, as described in Patent Document 1, there is a method of providing a position indicator in the biochip, first recognizing this position indicator, using it as coordinate information, and performing gridding from the separately provided spot coordinate information. In this case, since the recognition process of the position indicator and the high-precision improvement of the position accuracy of the spot are required, it becomes a factor in cost increase.

[0013] The technical problem of the present invention is to simply and directly identify the spot position and shape without performing the gridding operation and without incurring the cost of manufacturing the biochip, and enable measurement of the spot image signal intensity.

Means for Solving the Problem

[0014] In order to solve the above technical problem, the inspection device of the invention described in claim 1 is a biochip on which a plurality of beads on which a fixed substance that emits light by the reaction of a biological substance and a reagent are fixed in a spot shape, a light and dark switching mechanism capable of switching between a bright field mode in which the biochip is irradiated with light and a dark field mode in which the biochip is shielded from light, an imaging device that images the spot image of the fixed substance on the biochip, wherein the light and dark switching mechanism images a bright field image in the bright field mode and images a dark field image in the dark field mode, Means for specifying the spot position and shape by applying image processing technology to the spot image obtained under bright field in a state where the reaction solution that reacts with the biological substance and the reagent are injected, and digitizing the signal intensity of the same spot image under dark field; and includes 、 The light switching mechanism includes a hood that surrounds and shields the biochip from light, and an illumination device disposed inside the hood that can irradiate light onto the biochip, wherein the illumination device is lit in the bright field mode and turned off in the dark field mode. The bright field image and the dark field image are captured by the same imaging device at the same position. It is characterized by the following.

Advantages of the Invention

[0018] According to the invention described in claim 1, it is possible to switch between a bright-field mode and a dark-field mode by a light and dark switching mechanism for imaging, and without performing a gridding operation and without incurring the cost of fabricating a biochip, simply and directly identify the spot position and shape, and enable measurement of the spot image signal intensity. Also, Claim 1 According to the invention described in [claim number], by turning on the lighting device in the bright-field mode, it is possible to capture a bright-field spot image in a state where the light quantity is stable. Furthermore, Claim 1 The invention described in [claim number] can identify the spot position and shape in the bright-field mode and capture the light emission at each spot in the dark-field mode, and can digitize the spot image signal intensity.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0020] Next, specific examples of embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to the following embodiments.

[0021] FIG. 1 is an explanatory diagram of the inspection apparatus of the present invention. FIG. 1A is an explanatory diagram of a state where the hood has moved upward, and FIG. 1B is an explanatory diagram of a state where the hood has moved downward. The inspection apparatus 1 according to an embodiment of the present invention has a chip tray 2. A biochip 3 is supported on the upper surface of the chip tray 2. Above the biochip 3, a camera 11 as an example of an imaging device is arranged. On the biochip 3 side of the camera 11, a lens 12 as an example of an optical system is arranged.

[0022] A hood 13 is arranged so as to surround the camera 11. The hood 13 is made of a light-shieldable material and is configured in a hollow cylindrical shape (dark cylindrical shape). The hood 13 is supported by a moving device 14 so as to be movable in the vertical direction. The moving device 14 moves the hood 13 between the upper bright-field position shown in FIG. 1A and the lower dark-field position shown in FIG. 1B. In the bright-field position, external light can be irradiated onto the biochip 3, and in the dark-field position, the biochip 3 is surrounded by the hood 13 and is in a light-shielded state. Also, in the dark-field position, the biochip can be irradiated by an illumination device to enter the bright-field mode.

[0023] An illumination device 16 is arranged at a position inside the hood 13 when the hood 13 has moved to the dark-field position above the biochip 3. The illumination device 16 can be anything as long as white light can be irradiated onto the biochip. As an example, white LEDs can be used. The light and dark switching mechanism 13 to 16 of the embodiment is constituted by the hood 13, the moving device 14, the illumination device 16, a control circuit for controlling the moving device 14 and the illumination device 16, etc. Note that the light and dark switching mechanism 13 to 16 of the embodiment has been exemplified as having the illumination device 16, but is not limited thereto. For example, when only external light is used for imaging in the bright field, it is also possible to adopt a configuration without the illumination device 16.

[0024] The biochip 3 can adopt a known biochip, and the configuration of the biochip 3 will be outlined. Generally speaking, it consists of a substrate for immobilizing biological substances, etc. and a storage part for containing reaction solutions, reagents, etc. A layer for holding an immobilization carrier (hereinafter referred to as beads) for immobilizing an immobilized substance composed of a biological substance is provided on the substrate, and the beads are spot-fixed on the layer. As the beads, organic microparticles, inorganic microparticles, or magnetic microparticles can be used. Note that it is also possible to directly immobilize the immobilized substance on the biochip 3 without using an immobilization carrier.

[0025] Next, the specific usage method of the biochip 3 will be outlined. As the immobilized substance, an antigen is previously immobilized on the beads and spotted on the substrate. An antibody, which is a biological substance, is injected into the storage part, and after adding a reaction solution, etc. and reacting for a certain period of time, a luminescent reagent is injected and a luminescent spot image is captured. As the luminescent reagent, either a chemiluminescent reagent or a fluorescent reagent can be used.

[0026] There are generally two types of devices corresponding to the above usage method. One is a mass production device that separates a reaction part with a long reaction time and an imaging part with a short processing time. After the reaction is completed, the chip is mounted on the imaging part to capture a luminescent spot image. The other is a POCT (Point Of Care Test) device in which the reaction part and the imaging part are integrated. In any application, the biochip 3 and the camera 11 can be used.

[0027] Figure 2 is an explanatory diagram of the imaging process of the inspection device, Figure 2A is an explanatory diagram of the conventional imaging process, and Figure 2B is an explanatory diagram of the imaging process of the embodiment. In Figure 2A, in the conventional method, first, the position information (number, arrangement, size) of the spots is input by dedicated software, and then a spot image is acquired under dark field. Then, gridding is performed for each chip, for example, adjustment of the displacement of the spot position that should be at the center of the frame, etc. Next, the spot image signal intensity is digitized by dedicated software.

[0028] In FIG. 2B, in the inspection apparatus 1 of the embodiment, first, a spot image is acquired under bright field. That is, with the biochip 3 being irradiated with light, the camera 11 captures a light scattering image due to the irradiated light. The state in which the biochip 3 is irradiated with light is a state where the light and dark switching mechanisms 13 to 16 are in the bright field mode, (i) moving the hood 13 to the bright field position and turning off the illumination device 16, (ii) moving the hood 13 to the bright field position and turning on the illumination device 16, (iii) moving the hood 13 to the dark field position and turning on the illumination device 16, Among these three patterns, the user can select and it is possible to capture an image under bright field. In the pattern of (iii), instead of natural external light that is easily affected by the installation environment of the inspection apparatus 1, using the illumination device 16, with the hood 13 lowered and shielded from light, it is possible to capture images under both bright field (and dark field). Therefore, in the patterns of (ii) and (iii) that use illumination, especially in the pattern of (iii), it is less affected by external light, and it is possible to capture a bright field spot image with a stable light amount.

[0029] In summary, for a spot image, for one chip, it is possible to obtain a light-emitting spot image captured in dark field and a light scattering spot image captured in bright field at the same position and by the same camera.

[0030] And by extracting the edge of the spot image in bright field, the position and shape of the spot are directly identified. That is, without inputting prior information on the spot position for each biochip as in the conventional method, the position and shape of the spot can be identified. In principle, information on the number of spots is not required in this way, but it is always beneficial from the perspective of fail-safe to constantly confirm that it is consistent using prior information on the number of spots. The identification of the position and shape of the spot is not limited to the edge extraction method. For example, it is also possible to identify the position and shape of the spot by any image processing technique such as feature extraction using AI (artificial intelligence) or a method of comparing with a reference spot image prepared in advance. In addition, as an example of the bright-field image, not only the scattered light of the beads in the reaction solution but also the spots of a normal biochip on which a biological substance without beads is stamped (fixed) can always obtain scattered light from the spots because salts, which are the composition of the stamp solution, precipitate in the dry state without the reaction solution.

[0031] Next, an image of the same spot is acquired under dark field. That is, in a state where the biochip 3 is not irradiated with light (shielded state), chemiluminescence, fluorescence of a biological substance, etc., or scattered light of these lights from beads is imaged by the camera 11. In the embodiment, the state where the biochip 3 is shielded can be realized when the light and dark switching mechanisms 13 to 16 are in the dark field mode, and the hood 13 is moved to the dark field position and the illumination device 16 is turned off. Then, based on the captured image under dark field, the spot image signal intensity is digitized by dedicated software.

[0032] Therefore, the spot image in the embodiment becomes an image in a luminescent state when the immobilized substance, biological sample, and reagent react and emit light, and becomes an image in a non-luminescent state when they do not emit light. Also, in a state with illumination light or external light (bright field), the image includes the light reflected and scattered at the spot position of the biochip by the illumination light, etc. That is, the spot image refers to an image in which the reaction between the biological substance fixed in a spot shape on the substrate and the reagent appears as a spot-like luminescence, non-luminescence, or light scattering phenomenon.

[0033] Note that the camera 11 in the embodiment uses a fixed-focus lens 12 without a zoom function and the angle of view is set to be constant, so the distance between the lens 12 and the spot of the biochip 3 is unchanged. Therefore, the positions of the respective spots in the bright-field image and the dark-field image do not change. That is, if the bright-field image and the dark-field image are superimposed, the spot images of both will match.

[0034] In the inspection apparatus 1 of the embodiment having the above configuration, by performing edge extraction of the spot image acquired under bright field, the spot position and shape can be directly specified, and the signal intensity of the same spot image can be quantified under dark field. The method for determining the position and shape of the spot image of the biochip 3 according to this embodiment can accurately and simply determine the position and shape within the range of normal machining accuracy without specially specifying the spot position accuracy during the manufacture of the biochip 3 or the mounting position accuracy of the biochip 3 in the inspection apparatus 1 during measurement.

[0035] Specifically, by capturing a bright field image, the spot position can be accurately acquired, and the signal intensity can be accurately calculated even for weak emission and non-emission (accurately, emission cannot be detected) spots in the dark field image.

[0036] In addition, since the position and shape of the spot image are determined for each biochip 3, it is not affected by the shape accuracy of the biochip 3 itself or the variation in the spot position accuracy within the biochip 3.

[0037] As a result, the accuracy of quantifying the spot image signal intensity is increased, and for example, the expectations of the patient and doctor, who are users of the antigen-antibody reaction result, can be met.

Example

[0038] FIG. 3 is an explanatory diagram of a photographed image of the biochip of Example 1. FIG. 3A is a diagram of an example of a bright field spot image, and FIG. 3B is a diagram obtained by edge extraction of the spot image of FIG. 3A.

[0039] In FIG. 3A, beads to which a biological substance was bound and a reaction solution were spotted 63 times on a biochip substrate. In addition, a protein without beads and a reaction solution were spotted 6 times. Further, 12 spots in the area where there were no spots were prepared. As can be seen from the figure, the bead spots could be clearly imaged as a bright field image due to the scattered light from the beads. However, the protein spots and the areas without spots were not imaged as bright field images.

[0040] Figure 3B enhanced the spot contour by using edge extraction, which is one of the known image processing techniques, for the spot image clearly captured as a bright-field image. The 63 bead spots were clearly represented, and the purpose of the bright-field image was achieved.

[0041] Figure 4 is an explanatory diagram of the dark-field image of the biochip in Figure 3. Figure 4A is a diagram of the image obtained by performing digitization processing on the dark-field spot image, and Figure 4B is a diagram of the image obtained by enhancing the contrast of the image in Figure 4A. As can be seen from Figure 4A, although there is light and dark in each bead spot, digitization processing was performed based on the signal intensity of each acquired spot image. In Figure 4B, by enhancing the contrast of the dark-field image in Figure 4A, a clearer spot image can be obtained, and digitization processing can also be easily performed. The description of actual numerical data is omitted.

[0042] (Modification example) In the above embodiment, the form in which the hood 13 and the camera 11 are arranged above the biochip 3 is exemplified, but it is not limited thereto. For example, the hood 13 and the camera 11 can be arranged below or on the side of the biochip 3 and can be moved in a direction approaching or separating from the biochip 3. When the camera 11 or the like is arranged below or on the side of the biochip 3, it is possible to take a photograph in a dry state inside the biochip 3, or to seal the liquid inside the biochip 3 with a cover or the like and turn the biochip 3 upside down or sideways to take a photograph. Also, for example, it is possible to form the bottom plate of the biochip 3 with a transparent material such as acrylic and configure it to be imaged with a camera or the like from below the bottom plate. At this time, in order to enable the taking of a dark-field image, it is desirable to arrange a light-shielding member (lid or cover) on the upper surface or side surface of the biochip 3. Furthermore, in the embodiment, a camera is exemplified as an example of the imaging device, but it is not limited thereto. For example, it is also possible to use a video camera capable of taking still images or moving images, an infrared camera capable of imaging light in the non-visible region, a photometer (for example, a confocal scanning meter) capable of acquiring the emission position and its intensity, etc.

Explanation of Symbols

[0043] 1…Inspection device, 3…Biochip, 11…Imaging device, 13…Hood, 13~16…Brightness and darkness switching mechanism, 14…Moving device, 16…Illumination device.

Claims

【Claim 1】 A biochip on which a plurality of beads fixed with a substance to be immobilized that emits light by the reaction of a biological substance and a reagent are fixed in a spot shape, A light and dark switching mechanism capable of switching between a bright field mode in which light is irradiated onto the biochip and a dark field mode in which the biochip is shielded from light, An imaging device that images a spot image of the substance to be immobilized on the biochip, wherein the light and dark switching mechanism images a bright field image in the bright field mode and images a dark field image in the dark field mode, Means for specifying the spot position and shape by applying an image processing technique to the spot image obtained under bright field in a state where a reaction solution that reacts with the biological substance and the reagent are injected, and quantifying the signal intensity of the same spot image under dark field, comprising The light and dark switching mechanism includes a hood that shields and blocks light around the biochip, and an illumination device disposed inside the hood that can irradiate light onto the biochip, the illumination device being lit in the bright field mode and turned off in the dark field mode, The bright field image and the dark field image are imaged at the same position by the same imaging device An inspection device characterized by this.

Images