Image acquisition device and method for detecting target substance signal

Abstract

The present invention relates to an image acquisition device and method for detecting a target substance signal, which have improved sensitivity and uniformity compared to a conventional fluorescence microscope for acquiring a fluorescence image. The invention provides an image acquisition device for detecting a target substance signal. The image acquisition device comprises: (a) a camera for detecting a fluorescence signal; (b) light sources which are circularly arranged at the periphery of the camera at equal intervals; (c) a shielding light guide disposed above the light source and only allowing vertical light among the light emitted from the light source to pass through; and (d) a prism that refracts the light passing through the shielding light guide toward the sample.

Description

Technical Field

[0001] The present invention relates to an image acquisition device and method for detecting target substance signals, and more particularly, to an image acquisition device and method having improved sensitivity and uniformity compared to conventional fluorescence microscopes for acquiring fluorescence images. Background Art

[0002] In the past, various methods for detecting pathogens have been used. Existing methods collect samples from patients or infection sources, and then analyze the samples by optical or chemical means to determine the type of pathogen. However, this method not only requires a large number of samples, but also often relies on the skills of the tester to determine whether the test is successful, which has limitations in accurately diagnosing pathogens.

[0003] Especially in cases where symptoms are triggered by new pathogens or chemicals, a large number of samples may need to be obtained in order to analyze the symptoms, which can be a huge burden for patients.

[0004] To solve this problem, a method using PCR (polymerase chain reaction) to amplify pathogens is currently being used. PCR can amplify the DNA of pathogens and can obtain pathogens even from a small amount of samples, but the disadvantage is that it takes a long time to amplify DNA, so PCR may only be used to a limited extent in the initial diagnosis of pathogens.

[0005] Recently, due to bioterrorism or mass infection caused by pathogens, there is an urgent need for technology that can identify samples in a short time. For this reason, an immunoassay method that binds antibodies attached with fluorescent substances to pathogenic substances for detection is being widely introduced. Compared with the conventional PCR-based analysis method, this immunoassay method has the advantage of being able to confirm results quickly and only requires a small amount of sample to complete the analysis.

[0006] In this immunoassay method, since the luminescence of fluorescent substances is used to determine whether pathogens exist, a fluorescence microscope is used in most cases. For commonly used fluorescence microscopes, since the light path for providing excitation light and the light path for fluorescence generated by the sample are the same, a device is required to separate and observe these light paths. In order to separate these light paths, existing fluorescence microscopes generally use dichroic mirrors.

[0007] However, the dichroic mirror has the property of reflecting part of the fluorescence generated by the sample, which not only reduces the resolution but also may cause damage to the sample when strong excitation light is used to ensure the fluorescence intensity.

[0008] Therefore, it is necessary to develop a new type of fluorescence microscope that can obtain high-resolution fluorescence images while preventing damage to the sample. Summary of the invention

[0009] Technical issues

[0010] In order to solve the above problems, the present invention aims to provide an image acquisition device and method for detecting target substance signals, which have improved sensitivity and uniformity compared with conventional fluorescence microscopes for acquiring fluorescence images.

[0011] Technical Solution

[0012] In one aspect, an image acquisition device for detecting a target substance signal includes: (a) a camera for detecting a fluorescence signal; (b) a light source, which is arranged at equal intervals in a circle around the camera; (c) a shielding light guide, which is disposed above the light source and allows only vertically directed light emitted from the light source to pass through; and (d) a prism, which refracts light passing through the shielding light guide toward a sample.

[0013] In one embodiment, the shielding light guide may include: a central light guide portion, which is formed at the center of the shielding light guide and allows the fluorescent signal generated by the sample to pass through; and excitation light guide portions, which are installed at equal intervals on the periphery of the central light guide portion and allow the excitation light generated by the light source to pass through.

[0014] In one embodiment, a light absorbing coating may be formed on the inner side of the central light guiding portion or the excitation light guiding portion.

[0015] In one embodiment, when the inner diameter is R and the distance from the point of contact with the excitation light to the top is L, the R / L ratio of the excitation light guide is 0.05 to 0.5.

[0016] In one embodiment, the excitation light guide may have an inner diameter R of 2 mm to 5 mm and a length L of 10 mm to 50 mm.

[0017] In one embodiment, the shielding light guide includes 2 to 30 excitation light guide portions, and the light source is independently installed in each excitation light guide portion.

[0018] In one embodiment, an excitation light lens may be formed above the light source to focus the excitation light generated by the light source.

[0019] In one embodiment, the image acquisition device can acquire fluorescent images at a magnification of 0.5 times to 2 times.

[0020] In one embodiment, a prism may be mounted above the shielding light guide to refract the excitation light passing through the shielding light guide toward the sample.

[0021] In one embodiment, the light source may be a light emitting diode (LED) that emits excitation light having a wavelength of 400 nanometers to 700 nanometers.

[0022] The uniformity of the image acquisition device can reach 70% or higher, as shown in the following formula 1.

[0023] [Formula 1]

[0024] Uniformity = (minimum value of the fluorescence signal of each pixel area divided from the entire image analysis area / maximum value of the fluorescence signal of each pixel area divided from the entire image analysis area) × 100 (%)

[0025] The image acquisition device is five times more sensitive to fluorescence than conventional low-magnification fluorescence microscopes.

[0026] On the other hand, an image acquisition device for detecting a target substance signal includes: (i) a camera for detecting a fluorescent signal and an optical signal; (ii) a sample unit installed at a certain interval from the camera; (iii) a fluorescent unit for providing excitation light from the camera to the sample unit; and (iv) an optical component installed at a certain distance from the sample unit in a direction opposite to the camera and providing visible light to the camera, wherein the fluorescent unit includes: (1) a light source, which is arranged at equal intervals in a circle around the camera; (2) a shielding light guide, which is arranged above the light source and allows only vertical light emitted from the light source to pass through; and (3) a prism, which refracts light passing through the shielding light guide plate and the shielding light guide toward the sample.

[0027] In one embodiment, the sample may include magnetic particles.

[0028] In one embodiment, the magnetic particles may be formed into a microrod shape or a sphere shape.

[0029] In another aspect, an image analysis method for detecting a target material signal using an image acquisition device, wherein the image acquisition device includes: (i) a camera for detecting a fluorescent signal and an optical signal, (ii) a sample unit installed at a certain interval from the camera, (iii) a fluorescent unit for providing excitation light from the camera to the sample unit, and (iv) an optical component installed at a certain distance from the sample unit in a direction opposite to the camera and providing visible light to the camera. The image analysis method includes the following steps: ① injecting magnetic particles bound to the target material into each hole of a porous plate; ② fixing the porous plate to the sample unit; ③ powering the upper optical unit to acquire an optical image; and ④ powering the lower fluorescent unit to acquire a fluorescent image.

[0030] Beneficial Effects

[0031] According to the present invention, since the image acquisition device for detecting target substance signals can improve the uniformity and sensitivity of fluorescent signals by blocking the diffusion of excitation light when observing fluorescent signals, it is possible to acquire fluorescent images with higher resolution than existing fluorescent microscopes.

[0032] Furthermore, according to the present invention, since the image acquisition device for detecting a target substance signal can have high uniformity and sensitivity, a low-magnification fluorescent image can be acquired and a large-area sample can be analyzed simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram showing the structure of an image acquisition device for detecting target substance signals according to an embodiment of the present invention.

[0034] Figure 2 Schematic diagram showing the internal structure of an image acquisition device for detecting target substance signals according to an embodiment of the present invention.

[0035] Figure 3 FIG. 4 is a schematic diagram schematically illustrating generation of an optical image and a fluorescent image in an image acquisition device for detecting a target substance signal according to an embodiment of the present invention.

[0036] Figure 4 Schematic diagram showing a shielding light guide and a lens structure according to an embodiment of the present invention.

[0037] Figure 5 is a schematic diagram schematically illustrating alignment of excitation light in an excitation light guide according to an embodiment of the present invention.

[0038] Figure 6 Schematic diagram showing the effect of the length of the excitation light guide on the focusing degree of the excitation light according to an embodiment of the present invention.

[0039] Figure 7 is a schematic diagram showing the shape of a prism according to an embodiment of the present invention.

[0040] Figures 8 to 12 is a schematic diagram showing uniformity experimental results according to an embodiment of the present invention.

[0041] Fig.13 is a schematic diagram showing the results of a resolution experiment according to an embodiment of the present invention.

[0042] Fig.14 is a schematic diagram showing the experimental results of resolution and sensitivity according to an embodiment of the present invention.

[0043] Fig.15 is a chart showing sensitivity experiment results according to an embodiment of the present invention.

[0044] Fig.16 are photographs showing sensitivity experiment results according to an embodiment of the present invention. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention will be described in detail below. If it is believed that the detailed description of any prior art related to the present invention may obscure the core content of the present invention, the detailed description will be omitted. It should be understood that, unless the context clearly states otherwise, the singular form also includes the plural form, and it is further understood that the use of the term "including" or "having" in this specification specifies the existence of the stated features, steps, operations, components, parts, but does not exclude the existence or addition of one or more other features, numbers, steps, operations, components, parts. In addition, when performing a method or a manufacturing method, unless a specific order is clearly specified in the context, the various processes constituting the method may occur in a manner different from the specified order. That is, the respective steps can be performed in the order described, almost simultaneously, or in an order opposite to the order described.

[0046] The technology disclosed in this specification is not limited to the embodiments, but can be implemented in other forms. However, the exemplary embodiments introduced here are to make the disclosed content thorough and complete, and fully convey the spirit of the present invention to those skilled in the art. In the figure, in order to clearly represent the components of each device, the component sizes such as width or thickness are slightly enlarged. When the figure is described as a whole, it is from the observer's point of view, and when it is mentioned that one element is located on another element, this includes all possible meanings, that is, one element can be directly located on another element, or other elements can be interspersed between them. In addition, those skilled in the art can implement the spirit of the present invention in various other forms without departing from the technical spirit of the present invention. In addition, the same reference numerals in multiple figures represent elements that are substantially the same as each other.

[0047] In this specification, the term "and / or" includes any combination of a plurality of listed items or any one of them. In this specification, "A or B" may include "A", "B", or "A and B".

[0048] The present invention relates to an image acquisition device for detecting target substance signals, comprising: (a) a camera, the camera is used to detect fluorescence signals; (b) a light source, the light sources are arranged at equal intervals in a circle on the periphery of the camera; (c) a shielding light guide, the shielding light guide is arranged above the light source and only allows vertical light emitted from the light source to pass through; and (d) a prism, the prism refracts the light passing through the shielding light guide toward a sample.

[0049] The following will refer to Figure 1 and Figure 2 To describe the present invention. Figure 1 The internal structure of the image acquisition device of the present invention is shown. Figure 2 FIG. 4 shows the internal structure of the image acquisition device viewed from the side.

[0050] The camera 100 is used to detect the fluorescence generated by the excitation light, and a common optical camera can be used. Generally speaking, the fluorescent substance used in immunoassay may have an emission peak of 400 nanometers to 800 nanometers. That is, the fluorescence generated by the excitation light in the sample is generally within the visible light range, so a common optical camera can be used as the camera 100.

[0051] However, when the fluorescence generated by the sample has multiple colors, a camera can be installed for each color for observation. For example, three types of cameras can be installed to detect the three colors of red, blue, and yellow, and a dichroic mirror that can selectively reflect each color can be installed in front of the camera to separate the light path according to each color. Specifically, certain wavelengths of color can be separated by the first dichroic mirror so that the fluorescence generated by the sample can be detected by the first camera; the light that passes through the first dichroic mirror can be separated into certain wavelengths again through the same process in the second dichroic mirror. The remaining wavelengths of light can be detected by the third camera. That is, through two dichroic mirrors, the fluorescence generated by the sample can be divided into three parts according to the wavelength and incident on each camera, and each camera can measure the maximum peak wavelength of each fluorescence by calculating the difference in the intensity of the incident light. Therefore, due to the different types of fluorescent substances attached to each sample, the peak wavelength of the generated fluorescence will also be different, so that the type of each sample can be determined.

[0052] The light source 310 is installed to provide excitation light to the sample 200, and can be used to provide light for exciting the fluorescent substance included in the sample 200. Generally, in a fluorescence microscope, a short-wave laser is often used as the excitation light. However, generating laser light requires large equipment or a large amount of electricity. Therefore, recently, a fluorescence microscope using a light emitting diode (LED) as a light source has been widely used to reduce the size of the overall device. Light emitting diodes not only require a small amount of energy due to high power conversion efficiency, but also contribute to the miniaturization of the device due to their very small size.

[0053] In this case, the light emitting diode can provide light that can excite the fluorescent substance contained in the sample 200. Specifically, it is preferable to generate excitation light with a wavelength of 400 nanometers to 700 nanometers. In addition, the light emitting diode can use a light emitting diode that generates short-wave light, or a light emitting diode that can emit light of 400 nanometers to 700 nanometers in a continuous spectrum, and can form excitation light of a desired wavelength by using an appropriate excitation light filter for each fluorescent substance.

[0054] Specifically, light-emitting diodes generally generate light of a single color. That is, light-emitting diodes can emit red light, green light, or blue light, and some diodes can also emit ultraviolet light. In the case of the present invention, a light-emitting diode that emits ultraviolet light is used, but a light-emitting lens containing a fluorescent substance excited by infrared rays is installed above the light emitter, so a light-emitting diode that can form the entire visible light wavelength can be used. That is, the light-emitting semiconductor in the light-emitting diode can generate infrared light, but because the fluorescent substance contained in the light-emitting lens is excited by infrared light and emits light, light of the desired spectrum can be formed. In addition, in this application, light of various wavelengths can be provided by replacing only the light-emitting lens without replacing the entire light-emitting diode.

[0055] In addition, the light emitting diode can be powered by a voltage of 1 volt to 5 volts and a current of 100 milliamperes to 500 milliamperes to adjust the amount of light to 20 lumens to 200 lumens. It is known that the amount of light is proportional to the voltage and current provided, and it is known that a voltage and current at or below a certain level are provided to a general light emitting diode to generate light. When the voltage and current are provided within the above range, the light emitting diode can have an appropriate amount of light, minimize heat generation, and have a long life. However, if the voltage and current are lower than the above range, the amount of light may be reduced, and thus it may be difficult to excite light; and when the voltage and current exceed the above range, heat generation may become severe, thereby shortening the life.

[0056] In addition, the light source 310 may include an excitation filter that transmits wavelengths from 466 nm to 526 nm with a bandwidth of 60 nm (see Figure 3 ). As described above, the light source can generate light with a wavelength of 400 nanometers to 700 nanometers. However, the commonly used excitation light peak wavelength is 466 nanometers to 526 nanometers. When excitation light of a wavelength other than the excitation light is provided, it may be reflected from the sample and observed as a false positive. Therefore, by using an excitation filter, the wavelength portion other than the excitation light can be blocked.

[0057] In addition, the light source 310 may include a heat dissipation unit for discharging the heat generated by the light source. The function of the heat dissipation unit is to discharge the heat generated by the light source, which can be achieved by simply arranging heat sinks around the light source, or by using a thermal medium to transfer the heat generated by the light source to other parts and process it. In detail, one end of the heat pipe including a heat source inside can be installed in the heating unit of the light source, and a heat sink or a cooling unit can be installed at the other end of the heat pipe. In this way, the heat generated by the light source can be transferred to the heat sink or the cooling unit through the heat pipe and processed. If the heat generated by the light source cannot be quickly discharged to the outside, it may not only cause damage to the light source unit, but also the heat may be transferred to the generated excitation light, causing atomization. Therefore, by installing the heat dissipation unit in the above manner, the heat generated by the light source can be quickly discharged.

[0058] The light sources 310 may be arranged in a circular pattern outside the camera 100 (see Figure 3 ). In general, since the excitation light generated by the light source has linearity, when the light source is placed only on one side of the camera, a shadow may appear depending on the direction. In addition, since the amount of light increases only in the direction in which the light source is arranged, there is a disadvantage that the intensity of the generated fluorescence is inconsistent (asymmetric). Therefore, in the present invention, in order to eliminate the asymmetry of the excitation light, a plurality of light sources are installed, and they are arranged in a circle outside the camera, thereby minimizing the asymmetric supply of the excitation light.

[0059] In addition, as a method of solving the asymmetry of the prior art, a method of installing a strong light source below the camera and refracting the light generated by the light source to supply it to the periphery of the camera has been adopted. However, in this case, the camera may malfunction due to the heat generated by the strong light source, and the fluorescent image supplied to the camera may be distorted due to the heat generated by the light source. In addition, a method of providing excitation light using a dichroic mirror is also widely used. However, since not only the quality of the fluorescent image obtained by the dichroic mirror is reduced, but also the amount of light is reduced when the dichroic mirror is used for reflection, a strong light source is required to obtain the desired fluorescent image.

[0060] However, in the case of the present invention, multiple small light sources (light emitting diodes 310) are arranged in a circle on the main surface of the camera 100, and then these small light sources are used to provide excitation light, which not only achieves energy saving and device miniaturization, but also minimizes the asymmetry of the excitation light.

[0061] In this case, 2 to 30 light sources 310 may be installed at even intervals. As described above, when only one light source is used, asymmetry of the excitation light may occur; and when the number of light sources exceeds 30, the increase in the number of light sources may make the device too large in size.

[0062] Since the light emitting diode of the present invention emits hemispherically diffused excitation light, it is preferred to use only light irradiated at a specific angle as the excitation light by appropriately controlling the emission. If hemispherically diffused light is used, the excitation light may irradiate unnecessary parts to form fluorescence, and it is also difficult to provide uniform light. The specific method and apparatus will be described later.

[0063] An excitation light lens 340 may be installed above the light source to focus the excitation light generated by the light source. As described above, a common light emitting diode emits light in a hemispherical direction. When such a light emitting diode is used directly and the remaining excitation light except for the excitation light having a specific direction is blocked, a large amount of light loss may occur. Therefore, it is preferred to install an excitation light lens above the light source to mainly focus the excitation light generated by the light source.

[0064] In this case, the excitation light lens 340 can be independently mounted on each light source, or can be manufactured in a ring shape so that one lens covers the entire light source (see FIG. Figure 4 B). In other words, the excitation light lens can be independently mounted on each light source, or can be made into a ring shape by processing a single lens, and the entire excitation light lens can be manufactured in one piece.

[0065] When the excitation light lens 340 is manufactured in one piece, it is preferred to form a protrusion ( Figure 4 B). In this case, the protrusion acts as a convex lens, which can focus the light generated by the light source in one direction. For existing annular lenses, the cross-section of the annular lens is manufactured into a semicircular shape to focus the light generated by the light source in a specific direction. However, although such existing lenses can focus light in the direction of the lens cross-section, they have limitations in focusing light in the tangent direction of the lens. Therefore, in the case of the present invention, the excitation light lens 340 is manufactured into a ring shape, and a protrusion is formed on each light source, which can achieve the same effect as installing a convex lens on each light source. The advantage of the integrated excitation lens 340 is that the installation cost is lower than installing an excitation lens for each light-emitting diode separately, and the entire lens can be replaced at one time when the focal length needs to be changed or the color of the excitation lens needs to be changed.

[0066] As described above, the excitation light focused by the excitation lens 340 can pass through the shielding light guide 320 (see Figure 3 ). The shielding light guide 320 can serve as a filter that only allows vertical light from the light source to pass through. As described above, the excitation light generated by the light source 310 can pass through the excitation lens 340 and be focused at a specific angle. However, even if the excitation light passes through the excitation lens 340, it still propagates in a straight line at a specific angle. In addition, the excitation light that does not pass through the excitation lens may diffuse to the surroundings and irradiate upward (ambient light). Therefore, it is best to use the shielding light guide 320 to remove ambient light and excitation light emitted at a certain angle or more.

[0067] To this end, the shielding light guide 320 may include a central light guide portion 321 located at the center of the shielding light guide and allowing a fluorescent signal generated from a sample to pass therethrough, and excitation light guide portions 322 installed at equal intervals outside the central light guide portion and allowing excitation light generated by a light source to pass therethrough.

[0068] In the present invention, the camera 100 is mounted in the center and the light sources 310 are arranged in a circle around the camera (see FIG. Figure 3 ). Therefore, the shielding light guide member 320 should also be manufactured into a corresponding shape accordingly.

[0069] In detail, a central light guide portion 321 that allows fluorescence generated by the sample to pass through may be formed at the center of the shielding light guide. The central light guide portion 321 is located between the sample and the camera so that the fluorescence generated by the sample can pass through. The camera may be installed below the central light guide portion 321, and the sample may be located above the central light guide portion 321. In other words, the fluorescence generated by the sample and excited by the light source may be observed by the camera through the central light guide portion (see FIG. 1 ). Figure 3 and 4 A).

[0070] In this case, it is preferred that the diameter of the lower end of the central light guide portion is similar to the diameter of the camera's outermost lens 110. If the diameter of the central light guide portion is much larger than the diameter of the camera's outermost lens 110, light may enter between the camera lens and the central light guide portion 321, which may reduce the resolution during observation. On the other hand, if the diameter of the central light guide portion is much smaller than the diameter of the camera's outermost lens 110, the camera's field of view may become narrow.

[0071] In the present invention, the term "camera outermost lens" refers to the outermost lens in the camera lens arrangement, usually the lens that the external light first contacts. Generally speaking, the diameter of this camera outermost lens can be expressed by the lens aperture.

[0072] In addition, the central light guide portion 321 may be manufactured to have the same diameter from the top to the bottom, but if the size of the sample and the size of the camera outermost lens 110 are different, it may be manufactured in a tapered shape to match the respective sizes. In other words, when the diameter of the camera outermost lens is much larger than the size of the sample unit on which the sample is mounted, the central guide portion 321 may be manufactured in a form in which the diameter of the sample unit side is reduced and the diameter of the camera outermost lens portion is increased, and vice versa. This allows the fluorescence generated by the sample to be effectively transmitted to the camera.

[0073] The excitation light guide portion 322 may be arranged at the periphery of the central light guide portion 321 (see Figure 4 A) The excitation light guide portion 322 is a portion through which the excitation light generated from the light source passes and provides the excitation light to the sample, and is formed along the same direction as the central light guide portion, but the direction of the light passing therethrough is opposite.

[0074] The excitation light guide portion 322 can absorb and remove the remaining excitation light except the excitation light with a specific angle (see Figure 5). Specifically, in order to produce uniform fluorescence, it is preferred that the excitation light is provided at a narrower angle. If the excitation light is provided at a wide angle, the excitation light may irradiate an unnecessary portion, and light other than the sample fluorescence may be observed. In addition, in order to prevent this, when the irradiation range of the excitation light is reduced, the excitation light may fail to irradiate the peripheral portion. Therefore, in the present invention, the excitation light having a certain angle or a larger angle is removed by the excitation light guide portion 322, so that higher resolution sample fluorescence can be obtained.

[0075] Explaining the working principle of the excitation light guide 322 in more detail, the interior of the excitation light guide 322 is manufactured into a cylindrical shape, and the excitation light can pass through the interior. In this case, since the excitation light passes through the long cylinder, only the excitation light with a specific angle can pass directly without being reflected by the interior of the excitation light guide, while the excitation light with a certain angle or greater will contact the inner wall of the excitation light guide. In other words, when a light absorbing coating is formed on the inner wall of the excitation light guide, the excitation light with a certain angle or greater that contacts the wall of the excitation light guide can be removed (see Figure 5 ). Existing excitation light filters usually use filters with simple slits or dot-shaped openings, but in this case, it may be difficult to remove the excitation light that deviates from the angle, and the amount of excitation light passing through will also be reduced. Therefore, in the present invention, by optimizing the length of the excitation light guide, it is possible to provide only excitation light less than a specific angle while maintaining a high amount of excitation light.

[0076] To this end, when the inner diameter is R and the distance from the excitation light contact point to the top is L, the R / L ratio of the excitation light guide portion 322 may be between 0.05 and 0.5. As described above, the excitation light guide portion has a certain length and can remove excitation light with an angle greater than a certain value. However, if the length of the excitation light guide portion exceeds a certain length, the size of the entire system may increase, and the amount of excitation light provided to the sample may decrease ( Figure 6 In addition, if the length of the excitation light guide is shorter than a certain length, the irradiation range may be too wide, resulting in a decrease in the effect of the excitation light guide ( Figure 6 However, when the diameter of the excitation light guide is larger, the angle of the excitation light passing through will be larger and the amount of light will increase, so the length of the excitation light guide must be longer. Therefore, the inner diameter and length of the excitation light guide should maintain a certain ratio. If R / L is less than 0.05, the length of the excitation light guide will be too long and the amount of excitation light supplied may be reduced; and if R / L exceeds 0.5, the angle of the excitation light supplied will become larger, resulting in a decrease in the resolution of the fluorescence image.

[0077] Regardless of the ratio, the inner diameter of the excitation light guide portion 322 is preferably 2 mm to 5 mm. The size of the light-emitting diode used for the light source is usually not more than 5 mm, and in the present invention, in order to miniaturize the device, it is preferred to use a light-emitting diode with a size of 2 mm to 5 mm as the light source 310. Therefore, the inner diameter of the excitation light guide portion is also preferably 2 mm to 5 mm to match the size of the light-emitting unit of the light-emitting diode. If the inner diameter of the excitation light guide portion 322 is less than 2 mm, the amount of excitation light passing through will be reduced, and the amount of fluorescent light of the sample may be insufficient; if the inner diameter of the excitation light guide portion 322 exceeds 5 mm, the size of the device will increase and the image resolution will also decrease. In addition, the length of the excitation light guide portion can be 10 mm to 50 mm to maintain an appropriate ratio with the inner diameter of the excitation light guide portion. In addition, the length of the excitation light guide portion can refer to the length of the entire excitation light guide portion or the distance from the excitation light contact point to the top.

[0078] The light absorbing coating can be formed inside the central light guide 321 or the excitation light guide 322. As described above, a coating capable of absorbing light is required inside the excitation light guide. In addition, a light absorbing coating can also be formed on the central light guide to remove the image reflected by the wall of the central light guide. That is, the present invention obtains a fluorescent image while supplying excitation light through a shielding light guide, but the resolution and accuracy of the image can be improved by minimizing the light reflection of the wall of the shielding light guide. In this case, the light absorbing coating can be any coating that can absorb excitation light and fluorescence, but preferably includes carbon black, carbon nanotubes or graphene. Since carbon black, carbon nanotubes or graphene themselves are black and easy to absorb light, in particular, carbon nanotubes can make the light incident on one side of the tube diffusely reflected and absorbed inside the tube structure. In addition, since graphene has a two-dimensional structure, the incident light can be reflected and absorbed between each graphene layer. In other words, when carbon nanotubes and graphene are included in the light absorbing layer, a higher light absorption effect can be achieved.

[0079] In addition, the light source 310 can be independently installed in each excitation light guide portion 322 (see Figure 3 ). That is, a light guide portion may be installed above each light source, so the excitation light generated by each light source is preferably provided to the sample through the excitation light guide portion 322. As described above, since it is preferred to install 2 to 30 light sources, it is also preferred to install 2 to 30 excitation light guide portions.

[0080] In addition, the light source 310 can be installed outside the excitation light guide 322, but part or all of the light source 310 can also be installed inside the excitation light guide 322. When the light source is installed outside, it may have the advantages of easy heat dissipation, convenient installation and maintenance, but if the alignment with the excitation light guide is inaccurate, the amount of excitation light provided to the sample may be reduced. In addition, when the light source is installed inside the excitation light guide, the overall size of the device increases, and it may make it difficult to dissipate the heat generated by the light source, but once the light source is aligned, it can prevent the light source from being misaligned even if it is impacted.

[0081] A prism 330 is installed above the shielding light guide member, which can refract the light (excitation light) passing through the excitation light guide portion 320 toward the sample (see Figure 3 ). In this case, it is best to use prisms arranged in a ring above the shielding light guide or manufactured in a ring shape to avoid interfering with the observation of sample fluorescence.

[0082] Figure 7 A shows the prism of the present invention, and Figure 7 B shows a cross-sectional view of the prism.

[0083] The prism is used to focus the excitation light to the center of the sample and Figure 7 As shown in A, the prism can be installed in a ring shape above the excitation light guide, and the inner portion thereof can be made higher than the outer portion. That is, in the case of the prism of the present invention, the upper portion can form an oblique line that gradually decreases outward. In addition, the upper portion of the prism can have an angle of 30 to 40 degrees relative to the horizontal plane (i.e., the surface in contact with the excitation light guide), preferably an angle of 31 to 38 degrees, and more preferably an angle of 35.5 degrees. If the upper angle is less than 30 degrees, the excitation light will not be fully focused to the center, resulting in a decrease in the uniformity of the excitation light. If the angle exceeds 40 degrees, the amount of light in the peripheral portion may be reduced, also resulting in a decrease in uniformity.

[0084] Furthermore, in the case of a prism, in order to cover the entire excitation light guide, the height of the inner portion may be 3 mm to 8 mm, preferably 6 mm to 7 mm, and more preferably 6.4 mm. If the height of the inner portion is less than 3 mm, all the excitation light generated by the excitation light guide may not be completely refracted; if the height exceeds 8 mm, the size of the prism will be too large, and external light other than the excitation light may be introduced, thereby reducing the signal-to-noise ratio.

[0085] The image acquisition device can acquire fluorescent images at a magnification of 0.5 to 2 times. For existing fluorescence microscopes, due to the low resolution, images with a magnification of 2 to 100 times are usually acquired. This is because, as mentioned above, due to the use of a large number of dichroic mirrors, the resolution of the image may be reduced, and due to the imbalance of the excitation light, the uniformity and clarity are also reduced. Therefore, most fluorescence microscopes only observe a portion of the particles attached to a hole or the entire target material. However, in the case of the present invention, since uniform excitation light can be provided without the use of a dichroic mirror, a high-resolution image can be obtained. Therefore, unlike the existing fluorescence microscope, according to the present invention, an image with a magnification of 0.5 to 2 times, preferably 1 time, can be acquired, which means that the image of the entire sample unit can be acquired and analyzed at the same time.

[0086] The image acquisition device may have a uniformity of 70% or more, as expressed in Equation 1 below.

[0087] [Equation 1]

[0088] Uniformity = (minimum value of the fluorescence signal of each pixel area divided from the entire image analysis area / maximum value of the fluorescence signal of each pixel area divided from the entire image analysis area) × 100 (%)

[0089] Uniformity represents the difference between the maximum and minimum values ​​of the fluorescence signal in each pixel area divided from the entire image analysis area, and it is generally believed that the higher the uniformity, the more uniform the light provided. In particular, in fluorescence microscopy, fluorescent substances are excited by excitation light. In this case, the analysis is performed by observing the fluorescence intensity and quantity, so it is very important to provide uniform excitation light in the observable area. In the case of the present invention, the uniformity expressed by the above equation 1 can reach 70% or higher, preferably 80% to 100%, and more preferably 85% to 96%. When the uniformity is lower than 80%, it may be difficult to provide uniform excitation light, so additional correction may be required or incomplete observation may result. In addition, since 100% means that the same light is provided to the entire area, it is impossible to have a value higher than this.

[0090] In addition, uniformity can be divided into uniformity of the overall area and uniformity of the observable area (see Figure 8 ), although high uniformity of the entire area is ideal, it is more important that the uniformity of the observable area is high. In particular, the area observed by the fluorescence microscope corresponds to the pixels of the observable area, not the pixels of the entire area, so the uniformity of the observable area should be 80% or higher.

[0091] The image acquisition device can be five times more sensitive to fluorescence than existing fluorescence microscopes. In this case, the sensitivity can be expressed by the mean fluorescence intensity (MFI), which is the average value of the fluorescence signal intensity.

[0092] It is generally believed that the higher the fluorescence value observed under the same conditions, the higher the sensitivity to fluorescence. In the case of the image acquisition device of the present invention, when using a lens of the same magnification, its sensitivity can be 5 times or more than that of an existing low-magnification fluorescence microscope, preferably 5 times to 20 times, and more preferably 8 times to 12 times. That is, when using a lens under the same conditions, the image acquisition device of the present invention can obtain a fluorescence value that is 5 times or more higher than that of an existing low-magnification fluorescence microscope. In order to obtain the same fluorescence value as the present invention, a high-magnification fluorescence microscope with a high-magnification lens should be used when using an existing fluorescence microscope, or the exposure time of the excitation light should be increased. However, as described above, when a high-magnification lens is used, the area that can be observed at one time may be reduced, which may increase the overall analysis time, and when exposed to excitation light for a long time, the signal-to-noise ratio may be reduced, and photobleaching may occur.

[0093] The present invention relates to an image acquisition device for detecting a target material signal, comprising: (i) a camera for detecting a fluorescent signal and an optical signal; (ii) a sample unit installed at a certain distance from the camera; (iii) a fluorescent unit for providing excitation light from the camera to the sample unit; and (iv) an optical component installed at a certain distance from the sample unit in a direction opposite to the camera and providing visible light to the camera, wherein the fluorescent unit comprises: (1) a light source, which is arranged at equal intervals in a circle on the periphery of the camera; (2) a shielding light guide, which is arranged above the light source and allows only vertical light from the light emitted from the light source to pass through; and (3) a prism, which refracts light passing through the shielding light guide plate to the sample.

[0094] For general fluorescence microscopes, only the fluorescence generated by exciting the fluorescent substances in the sample can be observed. However, in this case, non-luminescent samples cannot be observed, so only the presence or absence of fluorescence can be confirmed, and additional tests are required to determine the proportion and type of the sample. However, when optical observation is performed at the same time, even non-luminescent samples can be observed, making it easier to judge whether there is fluorescence in the sample. However, due to the different formation mechanisms of fluorescence signals and optical signals, different light sources must be used. Especially in the case of generating fluorescence signals, in order to maximize the fluorescence of the sample, the excitation light should be provided in the same direction as the camera, so it is necessary to optimize the position of the excitation light that generates the fluorescence signal and the position of the observation light that generates the optical signal.

[0095] In the case of the present invention, since the position and amount of the sample are specified using an optical signal, it is preferable to provide observation light of the optical signal from the opposite side of the sample (see Figure 3 ). In the existing optical microscope, the light source is provided from the objective lens part so that the shape of the surface can be easily observed, but in the present invention, since only the appearance of the sample, such as size, position and quantity, needs to be observed, the observation light can be installed as a transmission type. That is, in the case of the present invention, the excitation light of the fluorescent image is provided from the same direction as the camera, and the observation light of the optical image is provided from the opposite direction to the camera, so that the optical image and the fluorescent image can be selectively or simultaneously acquired.

[0096] The configuration of the fluorescent unit 300 and the configuration of the camera 100 used above are the same as those described above, and thus will not be described in detail.

[0097] The sample unit 200 is a part that fixes and holds the sample, and may be provided with a microplate on which the sample is directly placed or includes the sample.

[0098] In chemical, biochemical and / or biological analysis, multiwell plates are standard tools for processing and analyzing multiple samples. Multiwell plates can have various shapes, sizes and forms, but are usually manufactured in standard sizes and shapes and have standard well arrangements. Standard well arrangements include 96-well plates (12×8 well arrangements), 384-well plates (24×16 well arrangements) and 1536-well plates (48×32 well arrangements), and various other types of commercial multiwell plates can be used. In the present invention, a microwell plate containing a sample is used as a sample unit, but since the microwell plate has similar specifications to a commercial multiwell plate, it can be easily compatible with various traditional bioanalysis techniques.

[0099] The sample may include magnetic particles. In a general immunoassay method, a fluorescent substance is attached to an antibody, and then the sample is provided in a solution containing the antibody, and the antigen is bound by an antigen-antibody reaction to perform a test. However, this method has a disadvantage that it is not easy to separate non-competitive antibodies. In the present invention, the antibody is attached to magnetic particles, and then the antibody is used to perform an antigen-antibody reaction, thereby simplifying the operation during immunodiagnosis.

[0100] To this end, the magnetic particles may be formed into a microrod or spherical shape, preferably, the magnetic particles may be in a microrod shape.

[0101] The magnetic particles of the present invention will be described below based on the microrod shape.

[0102] The magnetic particle of the present invention may include: a core containing a magnetically responsive metal; a shell layer surrounding the core and having a uniform thickness; and a capture probe introduced onto the shell layer for capturing a biological substance.

[0103] The core part may include a magnetically reactive metal, preferably a magnetically reactive metal or a metal alloyed with a magnetic metal. The magnetic metal may be an alloy containing iron (Fe), nickel (Ni), cobalt (Co) and manganese (Mn). The magnetically reactive metal may contain transition metals such as iron, nickel, cobalt and manganese as main components, as well as rare earth metals such as gadolinium (Gd), terbium (Tb) and samarium (Sm), and may contain boron (B), silicon (Si), carbon (C) and the like. Representative examples of magnetically reactive metals may be iron alloys or cobalt alloys. A specific example of an iron alloy may be Fe. 70 B 15 Si 10 C5, an example of a cobalt alloy may be Co 68 Mn7Si 10 B 15 . In addition, the magnetic metal can be mixed or alloyed with copper (Cu), gold (Au), silver (Ag), iron (Fe) or platinum (Pt), and more preferably, a superparamagnetic metal can be included. Although the core part itself does not directly have magnetism, it can be magnetized when a magnet approaches, and there is an attraction between it and the magnet, so it can be effectively used to use a magnet for movement and mixing. However, in the present invention, it is preferred to use paramagnetic substances rather than ferromagnetic substances, in order to reduce the aggregation phenomenon caused by the magnetic attraction between the magnetic particles after the magnet is removed. In this case, if a ferromagnetic substance is used, due to the aggregation phenomenon, the individual fluorescence observation of each particle may become difficult. Therefore, the core part of the present invention preferably uses paramagnetic particles. In addition, the magnetic particles should have a size and specific gravity that will not float in water so that they can be quickly collected or separated by an external magnet such as a permanent magnet or an electromagnet.

[0104] The core may occupy 60% or more of the total volume of the magnetic particles. For example, the core may occupy 60% to 99%, preferably 75% to 99%, of the total volume of the magnetic particles. In addition, the size (such as diameter or length) of the magnetic particles may be tens of microns to hundreds of microns, and the specific gravity is preferably 5 or higher, such as 5 or higher and not more than 30. When the magnetic particles 130 are nanoparticles less than 1 micron, even if the magnetic particles 130 have a high specific gravity (such as the specific gravity of iron is 7.876), they may float in water. In this case, in the bioassay process of using magnetic particles to detect biological substances in the hole, due to the low magnetic force of the fine particles, the magnetic control of the magnetic particles when applying the magnetic field may not be smooth enough, which may cause separation difficulties. When the magnetic bar moves up and down in the hole to promote immune response, the fine particles will break away from and attach. In the process of the magnetic bar moving up and down, due to the light weight, the magnetic particles once attached to the magnetic bar may not fall back to the bottom of the hole even after removing the magnetic field, but remain on the magnetic bar due to non-specific binding. In this case, when the biological substances in the hole are detected, the reproducibility of the quantitative analysis may decrease.

[0105] At the same time, the magnetic particles form a core-shell structure by having a magnetically reactive metal in the center and a shell layer surrounding it. The shell layer can be made of organic or inorganic material, preferably glass.

[0106] The glass may be a compound selected from soda lime, borosilicate, aluminosilicate, silicon dioxide, alkali silicate, Pyrex and quartz as its main component. It is preferably used for experiments requiring heat resistance, acid resistance and water resistance, and may be borosilicate, which can minimize nonspecific binding with antibodies.

[0107] The shell surface may contain hydrophilic functional groups.

[0108] The shell surface can be coupled to the capture probe described below, including various functional groups, the functional groups are hydrophilic functional groups, the hydrophilic functional groups can be silanol groups or carboxyl groups, amino groups, hydroxyl groups, thiol groups or aldehyde groups derived from silanol groups. More preferably, the functional groups can be silanol groups.

[0109] In addition, the hydrophilic functional group can be directly bound to the capture probe, but it is also preferred to use a linker to bind the hydrophilic functional group and the capture probe. The linker is located between the hydrophilic functional group and the capture probe so that the capture probe can be easily attached to the shell layer, preferably a silane compound can be used, more preferably aminosilane can be used, and most preferably 3-aminopropyltrimethoxysilane (APTES) can be used.

[0110] The magnetic particles may introduce capture probes on the shell layer for capturing biological materials (antigens), and the capture probes may be antibodies with biomarkers as antigens.

[0111] 1 to 80 / nm can be introduced on the shell surface 2 hydrophilic functional groups.

[0112] Since the linker or capture probe is attached to the magnetic particle by binding to the hydrophilic functional group of the shell layer, the number of linkers or capture probes that can be bound can be determined according to the number of hydrophilic functional groups. Therefore, when a plurality of hydrophilic functional groups are formed on the surface of the magnetic particle, a large number of linkers or capture probes can be attached to the surface of the magnetic particle. In this case, when the density of the hydrophilic functional groups is lower than 1 / nm 2 When the fluorescence is low, it may be difficult to measure. When the density of hydrophilic functional groups exceeds 80 / nm 2 When the fluorescence is increased, there may be no further increase due to interference during probe binding.

[0113] The optical unit 400 is a part that provides visible light to the sample unit for optical observation, and may include a light source using a light emitting diode (see Figure 3). In this case, the light source used may be a light source using a light emitting diode (LED), and unlike the fluorescent unit, since the light source emits visible light that propagates in a straight line, an optical lens may be formed at the front end of the light emitting diode. That is, the fluorescent unit has a shielding light guide structure to avoid interference with the camera, but the optical unit may directly use LED lighting because it is not affected by such interference.

[0114] In this case, the light source of the optical unit 400 can be supplied with a power supply of 1V to 5V and 1mA to 50mA, and the light intensity can be adjusted to 1 lumen to 100 lumens. It is easy to perform optical observation of the sample within the above range, but if it exceeds the above range, it may be difficult to accurately observe the sample due to the excessive light intensity. At the same time, if it is lower than the above range, it may be difficult to observe the sample on the sample unit due to the excessive light intensity.

[0115] The present invention relates to an image analysis method, which uses an image acquisition device to detect a target material signal, wherein the image acquisition device includes: (i) a camera for detecting a fluorescent signal and an optical signal, (ii) a sample unit installed at a certain interval from the camera, (iii) a fluorescent unit that provides excitation light from the camera to the sample unit, and (iv) an optical component installed at a certain distance from the sample unit in a direction opposite to the camera and providing visible light to the camera. The image analysis method includes the following steps: ① injecting magnetic particles bound to a target material into each hole of a porous plate; ② fixing the porous plate to the sample unit; ③ supplying power to the upper optical unit to obtain an optical image; and ④ supplying power to the lower fluorescent unit to obtain a fluorescent image.

[0116] The above step ① is a process of binding the target material to the surface of magnetic particles and then injecting it into the wheel plate. In this case, as mentioned above, a capture probe is formed on the surface of the magnetic particles, and in this case, the capture probe can bind to the target material (antigen). However, in this case, since it is impossible to observe using fluorescence, a second antibody that can bind to another part of the target material is mixed, and the second antibody can contain a fluorescent substance.

[0117] In the case of using the above-mentioned second antibody, when the antigen is attached to the surface of the magnetic particles, the second antibody will also be attached to the antigen, so the magnetic particles will emit fluorescence; when there is no antigen attached, the second antibody will not be attached, so the magnetic particles will not emit fluorescence. In this way, when each capture probe is bound to a magnetic particle with a different length or size, it can compete for different antigens according to each size, which means that the antigen type can be read by simply observing the fluorescence. In conventional fluorescence analysis, the fluorescence spectrum is separated to confirm the peak wavelength of each fluorescence, and a complicated method of reading the antigen type is used by this method. However, in the case of the present invention, the antigen type can be immediately determined by simply comparing the size of the magnetic particles attached with the antigen. In addition, since the fluorescent substance that is not attached to the antigen can be easily removed from the magnetic particles by simple washing, false positive fluorescence emission can be minimized.

[0118] As described above, magnetic particles bound to a target material may be injected into each well of a multi-well plate.

[0119] After the multi-well plate is prepared as above, the multi-well plate can be fixed to the sample unit. For the sample unit, since a fixing device capable of binding the multi-well plate is formed, it is preferred to use a fixing device to fix the multi-well plate.

[0120] When the multiwell plate is attached as above, the upper optical unit can be powered to acquire an optical image. In this case, the light source of the optical unit provides visible light from the opposite side of the camera based on the sample unit, so an image showing the size and number of magnetic particles can be acquired instead of directly observing the surface.

[0121] After the optical image acquisition as described above is completed, the fluorescent unit is powered to acquire a fluorescent image. As described above, the fluorescent image of the present invention is an image with a magnification of 0.5 to 2 times, so the image of the entire sample unit can be acquired at the same time, which can generate a large area image compared to a conventional fluorescence microscope, and the large area image can be analyzed at a high speed.

[0122] Next, exemplary embodiments of the present invention will be described with reference to the accompanying drawings so that those skilled in the art can easily practice the present invention. In addition, when describing the present invention, if it is considered that a detailed description of a related known configuration or function may unnecessarily obscure the core content of the present invention, the detailed description will be omitted. Some of the features shown in the figures have been enlarged, reduced or simplified for ease of description, and these figures and their components are not necessarily drawn to scale. However, those skilled in the art will easily understand these details.

[0123] Example 1

[0124] like Figure 1 and Figure 2As shown, an image acquisition device for detecting target material signals is prepared. The equipment used is as follows:

[0125] Camera: ac1920-40um (Basler)

[0126] Fluorescent unit light source: 505nm LED (OSRAM, LV CRBP.01)

[0127] Optical unit light source: White LED (Seoul Semiconductor, LW514)

[0128] Shielding light guide: A central light guide portion with an inner diameter of 30 mm is formed in the center, and 21 excitation light guide portions with an inner diameter of 4 mm are formed around the central light guide portion. The shielding light guide is 25 mm long and has a total outer diameter of 50 mm.

[0129] Excitation light lens: Figure 4 As shown in B, a bulge is formed on the upper part of the light source.

[0130] Example 2

[0131] Example 2 is the same as Example 1 above, except that only four excitation light guide portions are formed in the shielding guide light guide, and only four fluorescent unit light sources are used.

[0132] Example 3

[0133] Example 3 is the same as the above-mentioned Example 1, except that only one excitation light guide portion is formed in the shielding light guide member, and only one fluorescent unit light source is used.

[0134] Example 4

[0135] Example 4 is the same as Example 1 described above, except that 25 excitation light guide portions are formed in the shielding light guide member, and 25 fluorescent unit light sources are used.

[0136] Example 5

[0137] Example 5 is the same as Example 1 above, except that the length of the shielding light guide is 10 mm.

[0138] Example 6

[0139] Example 6 is the same as Example 1 above, except that the length of the shielding light guide is 5 mm.

[0140] Example 7

[0141] Example 7 is the same as Example 1 above, except that the length of the shielding light guide is 50 mm.

[0142] Example 8

[0143] Example 8 is the same as Example 1 above, except that the length of the shielding light guide is 60 mm.

[0144] Example 9

[0145] Example 9 is the same as Example 1 above, except that the inner diameter of the excitation light guide is 2 mm.

[0146] Example 10

[0147] Example 10 is the same as Example 1 above, except that the inner diameter of the excitation light guide is 1 mm.

[0148] Example 11

[0149] Example 11 is the same as Example 1 above, except that the inner diameter of the excitation light guide is 5 mm.

[0150] Example 12

[0151] Example 12 is the same as Example 1 above, except that the inner diameter of the excitation light guide is 8 mm.

[0152] Example 13

[0153] Example 13 is the same as Example 1 above, except that no light absorbing paint is used.

[0154] Comparative Example 1

[0155] Comparative Example 1 is the same as Example 1 described above, except that no shielding light guide is used.

[0156] Comparative Example 2

[0157] The process is the same except that the shielding light guide is replaced with a slit-type light shield used in the past.

[0158] Experimental Example 1

[0159] In order to analyze the uniformity of the image acquisition device of the present invention, cylindrical biotin-conjugated magnetic particles with a size of 300 microns were prepared and treated with streptavidin-conjugated fluorescent substances.

[0160] The prepared cylindrical magnetic microparticles bound to the fluorescent substance were placed in the reaction well part, and the 852 (horizontal) × 852 (vertical) pixel area appearing in the fluorescence measurement mode of the image acquisition device was divided into 6 (horizontal) × 6 (vertical) equal parts, and the magnetic microparticles bound to the fluorescent substance were moved to obtain the fluorescent signal. The uniformity was analyzed using the minimum and maximum values ​​of the fluorescent signal in a total of 36 areas.

[0161] [Formula 1]

[0162] Uniformity=(minimum value of the fluorescence signal of each pixel area divided from the entire image analysis area / maximum value of the fluorescence signal of each pixel area divided from the entire image analysis area)×100(%).

[0163] [Table 1]

[0164] Uniformity (%) - Total Uniformity (%) - Observable area Example 1 89.87 95.40 Example 2 77.87 86.83 Example 3 61.14 76.98 Example 4 78.49 90.44 Example 5 77.31 86.69 Example 6 63.57 71.58 Example 7 76.32 85.26 Example 8 64.18 72.45 Example 9 78.11 81.15 Example 10 63.16 71.29 Example 11 74.73 85.28 Example 12 69.45 72.58 Example 13 81.59 80.19 Comparative Example 1 65.73 70.75 Comparative Example 2 57.72 63.85

[0165] As shown in Table 1, in Example 1 of the present invention ( Figure 8 A), the uniformity (which indicates the ratio of the minimum light amount to the maximum light amount) is high, so there is almost no difference in the light amount. However, when the number of fluorescent unit light sources generating excitation light is reduced (Example 2, Figure 8 B) or when only one light source is used (Example 3), it is confirmed that the uniformity decreases. In particular, when only one light source is used (Example 3), as Fig. 9 As shown in A, it was observed that the light was concentrated on one side of the observable area. In contrast, when 25 light sources were used (Example 4), no further increase in uniformity was found.

[0166] In addition, as the length of the shielding light guide is shortened (Example 5 ( Fig. 9 B) and Example 6( Fig.10 A)), the uniformity is reduced, especially for the sample 6 with a thickness of 5 mm, the uniformity is significantly reduced and the light quantity difference is large (see Fig.10 A). On the other hand, when the length of the shielding light guide increases, that is, the length of the excitation light guide portion is longer, if the length exceeds a certain length (Example 8, length 60 mm), the intensity of the guided excitation light is weakened, resulting in a difference in light quantity.

[0167] When the inner diameter of the excitation light guide is reduced (Examples 9 and 10), similar situations can be seen. In particular, in Example 10 where the inner diameter is excessively reduced, the overall light amount is reduced and the light in the peripheral portion is weakened, so that the uniformity is significantly reduced (see Fig.11 A).

[0168] On the contrary, when the inner diameter increases (Example 11, Example 12), it can be seen that the amount of light decreases, especially in Example 11, although the uniformity is maintained to a certain extent, the overall amount of light decreases ( Fig.11 B). Therefore, it can be seen that the inner diameter and length ratio of the excitation light guide should be kept within a certain range.

[0169] In Example 13 where the light-absorbing paint is not used, since the excitation light is scattered and reflected and propagates through the inside, the change in uniformity is small.

[0170] In Comparative Example 1 ( Fig.12 A), the uniformity is very low and the excitation light leaks, so it can be seen that the light amount in the center of the identifiable light area is low. Therefore, it can be seen that the use of the shielding light guide of the present invention can provide uniform excitation light to the entire sample. In addition, when the existing slit-type light shielding plate (Comparative Example 2, Fig.12 B), the uniformity is greatly reduced, which means that the existing slit-type light shield alone cannot provide uniform light to the sample.

[0171] Experimental Example 2

[0172] The prepared fluorescent substance-bound cylindrical magnetic particles were placed in the reaction well portion and measured with an exposure time of 50 milliseconds in the fluorescence measurement mode of the image acquisition device, and the measurement results using 1x, 4x, and 10x lenses were compared to measure the resolution of the image acquisition device.

[0173] like Fig.13 As shown, in Examples 2 and 4 including Example 1 of the present invention, high-resolution photos can be obtained even with a 1x lens. In other words, in the case of the present invention, not only can large-area photography be achieved with a 1x lens, but also normal analysis resolution can be achieved when enlarging photos for analysis.

[0174] In addition, a comparative experiment with existing fluorescence microscopes was conducted. Fig.14 As shown, the prepared fluorescent substance combined with cylindrical magnetic particles is placed in the reaction hole part, and the image acquisition device is used in the fluorescence measurement mode to measure the exposure time of 50 milliseconds, 200 milliseconds and 500 milliseconds; for Comparative Example 3, a fluorescence microscope is used to measure the same magnetic particles under the same exposure time conditions with 1x, 4x, 10x and 20x lenses, and the measurement results are compared. Fig.14As shown, Example 1 of the present invention can normally acquire images under all conditions, but in Comparative Example 3, in order to obtain the same image as the present invention, a lens with at least 4 times magnification is required, and preferably a 10 times lens is used to obtain a normal image. That is, in the case of the present invention, a high-resolution image of a large area can be acquired with a 1 times lens, while in the case of the existing fluorescence microscope, only a small area image can be acquired due to the low resolution and light quantity, so the present invention can analyze a large area in a short time.

[0175] Experimental Example 3

[0176] The magnetic microrod manufactured by the present invention is a glass-coated microwire made according to the content described in Korean Patent Application No. 10-2018-0130436. In addition, the diagnostic system for performing sensitivity analysis on the image acquisition device of the present invention using the manufactured magnetic microrod is configured to perform the diagnostic system according to the content described in Korean Patent Application No. 10-2020-0123989 by the following steps:

[0177] Step 1: Fabrication and surface treatment of magnetic microrods.

[0178] Step 2: Fabrication of giant magnetic microrod conjugates with immobilized primary antibodies.

[0179] Step 3: Quality Control (QC) of giant magnetic microrods with immobilized primary antibody.

[0180] Step 4: Preparation of immunoreactive materials.

[0181] Step 5: Sample addition step.

[0182] Step 6: Mixing / heating step.

[0183] Step 7: Washing step.

[0184] Step 8: Detection signal measurement step.

[0185] [GFAP and UCHL1 detection]

[0186] RSMP-coupled capture antibodies (GFAP_cAb_09 and UCHL1_cAb_02) and biotin-coupled detection antibodies (GFAP_dAb_03 and UCHL1_dAb_07) manufactured as described above were used. Antigen GFAP from Hytest and antigen UCHL1 from Origene were added to PBS or serum at concentrations of 0, 3.125 pg / ml, 6.25 pg / ml, 12.5 pg / ml, 25 pg / ml, 50 pg / ml, 100 pg / ml, 200 pg / ml, 400 pg / ml, 800 pg / ml, 1600 pg / ml and 3200 pg / ml, respectively, and then the experiment was carried out according to the method of Experimental Example 1 described above.

[0187] In addition, a comparative sensitivity analysis was performed with the GFAP and UCHL1 DuoSet ELISA kit products from R&D Systems.

[0188] By applying the image acquisition device of the present invention to the diagnostic system, the test results of GFAP and UCHL1 concentrations in the range of 0 to 3200 pg / ml were compared and analyzed, confirming that its sensitivity is 40 times higher than that of ELISA (see Fig.15 and Fig.16 ).

[0189] Experimental Example 4

[0190] An experiment was conducted to measure the sensitivity of the image acquisition device of the present invention to fluorescence. The fluorescence image of the present invention and the fluorescence image of the existing fluorescence microscope were obtained, and then the respective fluorescence values ​​(MFI) were compared. In this case, the experimental method was the same as that of Experimental Example 2 above.

[0191] [Table 2]

[0192]

[0193] As shown in Table 2 and Fig.14 As shown, Example 1 of the present invention has a sensitivity to fluorescence that is approximately 10 times greater than that of an existing fluorescence microscope when using a 1x lens. In order for an existing fluorescence microscope to achieve fluorescence sensitivity similar to that of the present invention, it is confirmed that a lens with a magnification of 10x or more is required, and an exposure time of 4x or more is required.

[0194] The above has described in detail certain parts of the present invention. For those skilled in the art, this detailed description is only an exemplary embodiment, and the scope of the present invention is not limited by this detailed description. Therefore, the actual scope of the present invention will be defined by the claims and their equivalents.

Claims

1. An image acquisition device for detecting a target substance signal, comprising: (a) a camera, wherein the camera is used to detect fluorescent signals; (b) light sources, which are arranged in a circular shape with equal intervals around the camera; (c) a shielding light guide member, which is disposed above the light source and allows only vertically directed light from the light source to pass therethrough; and (d) A prism that refracts light that has passed through the shielding light guide toward a sample.

2. The image acquisition device according to claim 1, wherein: The shielding light guide member comprises: a central light guide portion formed at the center of the shielding light guide member and through which a fluorescent signal generated by a sample passes; and The excitation light guide portions are installed at equal intervals on the periphery of the central light guide portion and allow the excitation light generated by the light source to pass through.

3. The image acquisition device according to claim 2, wherein: A light absorbing coating is formed on the inner side of the central light guiding portion or the excitation light guiding portion.

4. The image acquisition device according to claim 2, wherein: When the inner diameter is R and the distance from the point of contact with the excitation light to the top is L, the R / L ratio of the excitation light guide portion is 0.05 to 0.

5.

5. The image acquisition device according to claim 4, wherein: The excitation light guide has an inner diameter R of 2 mm to 5 mm and a length L of 10 mm to 50 mm.

6. The image acquisition device according to claim 2, wherein: The shielding light guide member includes 2 to 30 excitation light guide portions, and the light source is independently installed in each excitation light guide portion.

7. The image acquisition device according to claim 1, wherein: An excitation light lens is formed above the light source, and the excitation light lens is used to focus the excitation light generated by the light source.

8. The image acquisition device according to claim 1, wherein: The image acquisition device acquires a fluorescent image at a magnification of 0.5 to 2 times.

9. The image acquisition device according to claim 1, wherein: The prism is installed above the shielding light guide to refract the excitation light passing through the shielding light guide toward the sample.

10. The image acquisition device according to claim 1, wherein: The light source is a light emitting diode (LED) which emits excitation light with a wavelength of 400 nanometers to 700 nanometers.

11. The image acquisition device according to claim 1, wherein: The uniformity of the image acquisition device is 70% or higher, as shown in Formula 1 below. [Formula 1] Uniformity = (minimum value of the fluorescence signal in the pixel area / maximum value of the fluorescence signal in the pixel area) × 100 (%) 12. The image acquisition device according to claim 1, wherein: The image acquisition device has a fluorescence sensitivity five times that of a conventional low-magnification fluorescence microscope.

13. An image acquisition device for detecting a target substance signal, comprising: (i) a camera for detecting fluorescent signals and optical signals; (ii) a sample unit mounted at a certain distance from the camera; (iii) a fluorescence unit providing excitation light from the camera to the sample unit; as well as (iv) an optical component installed at a distance from the sample unit in a direction opposite to the camera and providing visible light to the camera, Wherein, the fluorescent unit comprises: (1) light sources, which are arranged in a circular shape with equal intervals around the camera; (2) a shielding light guide member, which is disposed above the light source and allows only vertically directed light from the light source to pass therethrough; and (3) A prism that refracts light that has passed through the light-shielding plate or light-shielding guide member toward the sample specimen.

14. The image acquisition device according to claim 13, wherein: The sample includes magnetic particles.

15. The image acquisition device according to claim 14, wherein: The magnetic particles are formed into a microrod shape or a sphere shape.

16. An image analysis method for detecting a target substance signal using an image acquisition device, the image acquisition device comprising: (i) a camera for detecting a fluorescent signal and an optical signal, (ii) a sample unit installed at a certain distance from the camera, (iii) a fluorescent unit for providing excitation light from the camera to the sample unit, and (iv) an optical component installed at a certain distance from the sample unit in a direction opposite to the camera and providing visible light to the camera, the image analysis method comprising: ①Inject magnetic particles bound to the target substance into each well of the multi-well plate; ② Fixing the multi-well plate to the sample unit; ③ Supply power to the upper optical unit to acquire optical images; and ④ Supply power to the lower fluorescent unit to acquire fluorescent images.

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