JP2012168088A - Fluorescent reaction detector - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescent reaction detector capable of stably detecting a microorganism or a virus included in a sample with high detection accuracy.SOLUTION: A fluorescent reaction detector 1 comprises a light source 2, an excitation filter, a mirror 43, a camera 4, and a computer 5. The excitation filter allows a plurality of kinds of excitation light X, different from one another in at least one of a wavelength and intensity, to simultaneously pass therethrough from the light source 2 to the mirror 43. The camera 4 captures a surface of a sample 8 irradiated with the excitation light X as a digital image. An image analysis part 52 for analyzing the digital image is constructed in the computer 5. The image analysis part 52 detects a specific fluorescent reaction in which a microorganism or a virus included in the sample 8 emits fluorescence when simultaneously irradiated with the plurality of kinds of excitation light X.

Description

  The present invention relates to a fluorescence reaction detection device that detects the presence or absence of a specific microorganism in a sample.

  In an insect test performed at an elementary school or the like, a sample collected by a subject applying an adhesive tape to the anus is inspected at an inspection institution, medical institution, or the like. In this case, the presence / absence of caterpillar eggs will be confirmed with a microscope, and the inspector will be skilled, and the inspection accuracy and efficiency will not be very good. For example, Patent Document 1 discloses an insect egg automatic inspection facility for observing an insect test paper with a microscope.

  On the other hand, for example, the foreign object detection method of Patent Document 2 discloses that when a parasite is excited by irradiation with visible light, the parasite fluoresces. And the presence or absence of the parasite which parasitizes meat is detected using this property. Patent Document 2 describes that a fluorescent image emitted from a subject is obtained by a digital camera, a CCD camera, or the like, image processing such as binarization is performed, and the presence or absence of a foreign object is determined by a computer. Yes.

Japanese Patent No. 3090492 JP 2007-286041 A

However, in the conventional examination of caterpillar eggs, such as Patent Document 2, there is only one kind of excitation light, such as UV excitation, B excitation, and G excitation, used when exciting the insect eggs.
Therefore, when trying to detect the presence or absence of a beetle egg using image processing by a computer, the detection accuracy is low and a stable detection result cannot be obtained.

  The present invention has been made in view of such conventional problems, and an object of the present invention is to provide a fluorescence reaction detection apparatus capable of stably detecting a microorganism or virus in a sample with high detection accuracy. .

The present invention is a fluorescence reaction detection device for detecting a specific fluorescence reaction of a microorganism or virus in a sample,
A light source that emits light including ultraviolet and visible light wavelengths;
An excitation filter that transmits only the wavelength component corresponding to the excitation light among the light emitted from the light source;
A mirror that reflects the excitation light transmitted through the excitation filter and irradiates the sample obliquely;
A camera for photographing the surface of the sample as a digital image;
A computer having an image analysis unit for analyzing the digital image,
The excitation filter is configured to simultaneously pass a plurality of types of excitation light having different wavelengths and intensities from the light source to the mirror,
The image analysis unit is in a fluorescence reaction detection apparatus that detects a specific fluorescence reaction in which a microorganism or virus contained in the sample fluoresces when the plurality of types of excitation light are simultaneously irradiated (claim) Item 1).

The fluorescence reaction detection apparatus of the present invention detects the presence or absence of specific microorganisms using a light source, an excitation filter, a mirror, a camera, and a computer.
The excitation filter of the present invention allows two types of excitation light having different wavelengths and intensities to simultaneously pass from the light source to the mirror. Then, the presence / absence of the specific microorganism is detected by analyzing the hue, luminance, and the like of the light that the specific microorganism contained in the sample fluoresces when the two types of excitation light are irradiated simultaneously.
Thereby, it is possible to obtain a fluorescence reaction of a microorganism that could not be obtained only by irradiating a specific type of excitation light to a specific microorganism, and the detection accuracy of the specific microorganism can be significantly improved.

  Therefore, according to the fluorescence reaction detection apparatus of the present invention, microorganisms or viruses in the sample can be stably detected with high detection accuracy.

Explanatory drawing which shows the structure of the fluorescence reaction detection apparatus concerning an Example. Explanatory drawing which shows the fluorescence reaction detection apparatus concerning an Example in the state seen from upper direction. Explanatory drawing which shows the 1st excitation filter concerning an Example in the state seen from the AA line in FIG. Explanatory drawing which shows the 2nd excitation filter concerning an Example in the state seen from the BB line in FIG. Explanatory drawing which shows typically the state which irradiates excitation light with respect to a sample from the diagonal concerning an Example. Explanatory drawing which shows the digital image at the time of the 1st excitation concerning an Example schematically. Explanatory drawing which shows the digital image at the time of the 2nd excitation concerning an Example schematically. Explanatory drawing which visualizes the result of having performed collation calculation with respect to the digital image at the time of 1st excitation, and the digital image at the time of 2nd excitation concerning an Example, and shows schematically as a collation image. Explanatory drawing which visualizes the state which collates 1st color space data and 2nd color space data concerning the Example in the pixel unit for every dot. A binarization process for determining whether or not arithmetic processing data obtained by collating the first color space data and the second color space data in a pixel unit for each dot is within a specified range according to the embodiment. Explanatory drawing which visualizes and shows the result performed. Explanatory drawing which shows the hit area | region surrounding a hit pixel concerning an Example. Explanatory drawing which shows schematically the state which expanded and displayed on the monitor the pixel area | region corresponded to a hit area | region about the elementary digital image concerning an Example.

A preferred embodiment of the above-described fluorescence reaction detection apparatus of the present invention will be described.
In the present invention, the microorganism may be various parasites, parasite eggs, bacteria, and the like. Examples of the parasites include worms, roundworms, whipworms, flukes, worms, reduced tapeworms, broad-headed crested worms, oriental hairy nematodes, and striped worms. Parasite eggs can be those eggs.
Examples of bacteria include cocci, bacilli, and spiral bacteria.
The virus can be various viruses composed of nucleic acids and proteins.

  The sample can be stool, blood, or the like. The microorganisms and the like can be present in stool, blood and the like. Parasites lurking in the blood include, for example, Ustermann lung fluke, Miyazaki lung fluke, schistospores, malaria pathogens, Japanese schistosomes, filamentous insects, filariae, and cantonese schistosomiasis.

In the present invention, the excitation filter comprises two rotatable switching filters arranged in parallel in close proximity to each other, and the switching filter emits light having different wavelengths in the circumferential direction of the rotating disk that does not transmit light. A plurality of kinds of filter parts to be transmitted are arranged side by side, and both of the two switching filters are emitted from the light source among the plurality of kinds of filter parts by rotating around the rotation center of the rotating disk. The filter unit disposed at the light passing position through which the transmitted light passes can be selected, and the excitation light having different wavelengths can be selected by the excitation filter from the filter unit disposed at the light passing position in the two switching filters. It is preferable to transmit the light simultaneously to the mirror (claim 2).
In this case, it is possible to irradiate the sample with excitation light of two types of wavelengths via the mirror by a transmission filter having a simple structure using two switching filters.

  Further, the plurality of types of filter units transmit a UV excitation filter unit that transmits light having a wavelength of UV excitation from the light emitted from the light source, and transmit light having a wavelength of B excitation from the light emitted from the light source. It is preferable that a B excitation filter unit to be transmitted and a G excitation filter unit to transmit light having a wavelength of G excitation from the light emitted from the light source are included.

In this case, any two excitation lights of UV excitation, B excitation, and G excitation can be irradiated to the sample simultaneously.
The plurality of types of filter units may include those that absorb ultraviolet rays and transmit visible light.
Further, only a notch or a hole can be formed at a position where the filter portion is formed in the circumferential direction of the rotating disk, and a portion that transmits light emitted from the light source as it is can be formed.

  In addition, two of the excitation filters including the two switching filters are arranged in series at the light passage position, and one of the excitation filters is UV light from the light emitted from the light source to the plurality of types of filter units. A UV excitation filter that transmits light of the excitation wavelength, a B excitation filter that transmits light of the B excitation wavelength from the light emitted from the light source, and a wavelength of G excitation from the light emitted from the light source. A G excitation filter that transmits light, and the other excitation filter includes a plurality of types of filters that reduce the intensity of light emitted from the light source in a plurality of stages. It is also possible to include a neutral density filter section (claim 4).

In this case, the intensity of any two excitation lights of UV excitation, B excitation, and G excitation can be adjusted by the neutral density filter unit, and the sample can be irradiated simultaneously.
Moreover, any one of UV excitation, B excitation, and G excitation can be simultaneously irradiated to the sample as two types of excitation light having different intensities.
In the two switching filters constituting one excitation filter, the plural types of filter units may include those that absorb ultraviolet rays and transmit visible rays. In addition, in the two switching filters constituting either one of the excitation filters and the other excitation filter, only a notch or a hole is formed at the position where the filter portion is formed in the circumferential direction of the turntable and emitted from the light source. It is also possible to form a part that transmits the light as it is.

It is preferable that the camera is capable of photographing a surface of 1 (μm ² ) or less in the sample as a subject for one pixel of the digital image to be photographed by combining a CMOS camera and an optical lens. 5).
In this case, in the fluorescence reaction detection apparatus using a camera, the surface of the sample can be magnified and observed as in the case of observing with a microscope.

It is preferable that the excitation light irradiated from the mirror to the sample is irradiated at an incident angle of 27 to 33 ° with respect to the plane direction of the holding body that holds the sample.
In this case, when the surface of the sample when irradiated with the excitation light is photographed by the camera, a shadow portion can be formed in a state where the microorganisms contained in the sample fluoresce. The shadowed portion is formed such that the microbe or the like has a three-dimensional shape, and light is applied obliquely so that the width on one side where the light strikes the microbe or the like is small and the width on the other side is large.

Hereinafter, embodiments of the fluorescence reaction detection apparatus of the present invention will be described with reference to the drawings.
As shown in FIG. 1, the fluorescence reaction detection device 1 of this example detects the presence or absence of a specific fluorescence reaction 81 of a microorganism or virus in a sample 8. The fluorescence reaction detection apparatus 1 includes the following light source 2, excitation filter 3, mirror 43, camera 4, and computer 5.
The light source 2 is configured to emit light including ultraviolet and visible light wavelengths. The excitation filter 3 transmits only the wavelength component corresponding to the excitation light X among the light emitted from the light source 2. The mirror 43 is arranged to reflect the excitation light X transmitted through the excitation filter 3 and irradiate the sample 8 obliquely. The camera 4 is configured to capture the surface of the sample 8 as a digital image.

  The excitation filter 3 is configured to simultaneously transmit a plurality of types of excitation light X having different wavelengths and intensities from the light source 2 to the mirror 43. The computer 5 is constructed by constructing an image analysis unit 52 for analyzing a digital image. The image analysis unit 52 is configured to detect a specific fluorescence reaction 81 in which a microorganism or virus contained in the sample 8 fluoresces when a plurality of types of excitation light X are irradiated simultaneously. In FIG. 1, the fluorescence emitted from the sample 8 is indicated by an arrow X1.

Below, it demonstrates in detail with reference to FIGS. 1-12 about the fluorescence reaction detection apparatus 1 of this example.
The fluorescence reaction detection apparatus 1 detects a specific fluorescence reaction that occurs when the microorganisms or viruses in the sample 8 are irradiated with the excitation light X.
FIG. 1 is a diagram schematically showing the configuration of the fluorescence reaction detection apparatus 1. As shown in the figure, the light source 2 of this example is constituted by an ultraviolet lamp 2 that emits light having wavelengths of ultraviolet light and visible light.

The excitation filter 3 of this example includes a first excitation filter 3A for switching the wavelength of transmitted light and a second excitation filter 3B for switching the intensity of transmitted light. The first excitation filter 3A and the second excitation filter 3B are arranged in series at the light passage position E through which the light emitted from the light source 2 passes.
The first excitation filter 3 </ b> A and the second excitation filter 3 </ b> B are each formed by arranging two rotatable switching filters 3 in close proximity to each other.
Here, FIG. 2 is a diagram showing the fluorescence reaction detection device 1 as seen from above (the direction in which imaging is performed by the camera 4). 3 is a diagram showing the first excitation filter 3A as viewed from the front (in the direction of arrows AA in FIG. 2). FIG. 4 is a diagram showing the second excitation filter 3B in the front (B- in FIG. 2). It is a figure shown in the state seen from B line arrow direction.

As shown in FIG. 3, the two switching filters 3 in the first excitation filter 3 </ b> A are provided with a plurality of types of filter portions 33 that transmit light of different wavelengths in parallel in the circumferential direction of the rotating disk 32 that does not transmit light. Become.
The plurality of types of filter portions 33 are provided at predetermined intervals in the circumferential direction on the outer peripheral portion of the turntable 32. In the switching filter 3 of this example, filter portions 33 are formed at three positions at 90 ° intervals with respect to the rotating disk 32. The three filter parts 33 are provided with filter members that allow only light of a predetermined wavelength to pass through the notches 321 formed in the rotary disk 32. At the position where the filter unit 33 is formed in the circumferential direction of the turntable 32, only the notch 321 is formed with a space in the circumferential direction of 90 ° from the filter unit 33, and the light emitted from the light source 2 passes therethrough as it is. A portion 33D is formed.

  As shown in FIG. 2, both of the two switching filters 31 rotate around the rotation center O of the turntable 32, so that light emitted from the light source 2 among the plurality of types of filter units 33 passes. The filter part 33 arrange | positioned in the passage position E can be selected. Further, the passage portion 33 </ b> D can be arranged at the light passage position E by rotating the switching filter 31.

  In the first excitation filter 3A, the three filter units 33 of the two switching filters 31 include a UV excitation filter unit 33A that transmits light having a wavelength of UV excitation (320 to 380 nm) from the light emitted from the light source 2, and a light source. The B excitation filter 33B that transmits light having a wavelength of B excitation (380 to 495 nm) from the light emitted from 2 and the light having a wavelength of G excitation (495 to 570 nm) from the light emitted from the light source 2 are transmitted. The G excitation filter unit 33C. Each of the two switching filters 31 is formed by forming a UV excitation filter portion 33A, a B excitation filter portion 33B, a G excitation filter portion 33C, and a passage portion 33D with respect to the rotating plate 32.

  Thus, the excitation light X having different wavelengths can be simultaneously transmitted to the mirror 43 by the first excitation filter 3 </ b> A configured by the two switching filters 31. Note that when the person to be inspected visually confirms a specific part of the sample 8, the passage 33D is disposed at the light passage position E, and the light emitted from the light source 2 is passed through the mirror 43.

  As shown in FIG. 4, the two switching filters 31 in the second excitation filter 3 </ b> B have a plurality of kinds of different light intensities to be transmitted in the circumferential direction of the rotating disk 32 that does not transmit light (different amounts of light are different from each other). The neutral density filter 34 is provided in parallel. Similarly to the case of the first excitation filter 3A, the second excitation filter 3B is also provided with the neutral density filter portions 34 at three positions with a 90 ° interval in the circumferential direction with respect to the rotating disk 32. The three types of neutral density filter sections 34 are configured by disposing filter members that perform a predetermined amount of dimming with respect to the notches 321 formed in the rotating disk 32. Only a notch 321 is formed at a position where the neutral density filter portion 34 is formed in the circumferential direction of the turntable 32 and spaced from the neutral density filter portion 34 in the circumferential direction by 90 °, and the light emitted from the light source 2 is emitted. A passing portion 33D is formed to pass through as it is.

The three types of neutral density filter sections 34 of this example are those with a light reduction amount of 75%, those with a light reduction amount of 50%, and those with a light reduction amount of 25%. In addition, each switching filter 31 in the second excitation filter 3B is formed with a passing portion 33D that is 90% apart from the neutral density filter portion 34 and has a reduced light amount of 0%.
By using the second excitation filter 3B, the intensity of light irradiated from the light source 2 to the mirror 43 can be changed to 100%, 75%, 50%, and 25%.

  For example, when simultaneously irradiating the sample 8 with two types of excitation light X having different wavelengths, in the first excitation filter 3A, the filter unit 33 arranged at the light passing position E is switched between one switching filter 31 and the other. The filters 31 are different from each other. For example, when the sample 8 is irradiated with two types of excitation light X having different intensities at the same time, the filter unit 34 disposed at the light passage position E in the second excitation filter 3B is replaced with one switching filter 31 and the other. Are different from each other.

  As shown in FIGS. 2 to 4, the first excitation filter 3 </ b> A and the second excitation filter 3 </ b> B are provided from the periphery of each filter unit 33 and each neutral density filter unit 34 arranged at the light passing position E in each switching filter 31. A blocking member 35 for blocking leaking light is provided. The blocking member 35 is formed with a transmission hole 351 for transmitting light, and the light emitted from the light source 2 is transmitted through the first excitation filter 3A, the second excitation filter 3B, and the transmission hole 351, and the mirror 43 Is irradiated.

As shown in FIG. 1, the camera 4 of this example is configured by combining an optical lens 42 having a predetermined magnification with a CMOS camera (digital camera) 41 that can be digitally processed by a computer 5. The camera 4 selects an appropriate number of pixels according to each detection target of microorganisms or viruses, and enlarges the digital image to 1000 to 10000 times by the magnification of the optical lens 42 and the magnification of the CMOS camera 41 by digital zoom. be able to. A CMOS camera 41 having a resolution of 20 million pixels or more can be used. The camera 4 can shoot a surface of 1 (μm ² ) or less in the sample 8 as a subject with respect to one pixel of a digital image to be photographed by combining the CMOS camera 41 and the optical lens 42.

The camera 4 is arranged in a state where the surface of the sample 8 is photographed from above with the lens portion of the CMOS camera 41 facing downward. The light source (ultraviolet lamp) 2 is arranged in a horizontal direction perpendicular to the camera 4, and the mirror 43 is arranged in a direction perpendicular to the camera 4 and facing the light source 2. is there. Further, a filter 44 for blocking the ultraviolet rays and protecting the camera 4 is disposed in front of the lens portion of the camera 4 (in front of the optical lens 42 in this example).
The CMOS camera 41 can replace a plurality of types of optical lenses 42 having different magnifications, and can slide up and down.

The mirror 43 has its reflection surface arranged obliquely toward the sample 8, and reflects light emitted from the light source 2 in the horizontal direction obliquely toward the sample 8. The excitation light X irradiated to the sample 8 from the mirror 43 is irradiated at an incident angle θ of 27 to 33 ° with respect to the plane direction of the holding body 7 holding the sample 8.
FIG. 5 is a diagram schematically showing a state in which the excitation light X is irradiated to the sample 8 from an oblique direction. As shown in the figure, by irradiating the excitation light X at an incident angle θ of 27 to 33 ° with respect to the plane direction of the holding body 7, the identification of microorganisms or the like that can be contained in the sample 8 is emitted. The shadow reaction K can be appropriately generated in the fluorescence reaction 81. Thereby, in the image analysis part 52, the shape of the specific fluorescence reaction 81 can be easily detected. In the figure, reference numeral 80 denotes a microorganism or the like in the sample 8.
In addition, the portion of the shadow K is formed such that the width of one side where the light strikes the microorganism or the like is small and the width of the other side is large when the microorganism or the like has a three-dimensional shape. .

In the fluorescence reaction detection apparatus 1 of this example, two types of excitation digital images D1 and D2 when the sample 8 is irradiated with excitation light X that is transmitted through the two excitation filters 3A and 3B and have different wavelengths. Is photographed by the camera 4. These two types of digital images D1 and D2 at the time of excitation can be photographed while varying the wavelength of the excitation light X and also varying the intensity or irradiation time (imaging time) of the excitation light X. Further, the two types of excitation digital images D1 and D2 can be photographed with different excitation light X intensity or irradiation time (imaging time).
When at least one of the two types of excitation digital images D1 and D2 is irradiated simultaneously with the excitation light X from the two excitation filters 3 to the sample 8 with at least one of the wavelength and the intensity being different from each other. Digital image.

  The image analysis unit 52 of this example obtains color space data for each pixel unit for each of the two types of excitation digital images D1 and D2, and color space data for each pixel unit in the first excitation digital image D1. And the color space data for each pixel unit in the second excitation-time digital image D2 are collated between pixel units at the same position, and calculation processing data is obtained for each pixel unit. Then, the image analysis unit 52 compares the calculation processing data for each pixel unit with the specified range of the calculation processing data obtained in advance for the specific fluorescence reaction 81 to detect the presence or absence of the specific fluorescence reaction 81 in the sample 8. .

  FIG. 6 schematically shows the first excitation digital image D1, and FIG. 7 schematically shows the second excitation digital image D2. Further, in FIG. 8, the result of the collation operation performed on the first excitation digital image D1 and the second excitation digital image D2 is visualized and schematically shown as a collation image D3.

  The wavelength of the excitation light X can be made different from each other in the two types of excitation-time digital images D1 and D2 as the wavelength of UV excitation, B excitation, or G excitation. In this case, for example, as shown in FIGS. 6 and 7, a fluorescence reaction 82 due to dust (foreign matter other than the detection target) appears in both of the two types of digital images D1 and D2 at the time of excitation, while as shown in FIG. Assume that the specific fluorescence reaction 81 due to microorganisms or the like clearly appears in the second excitation digital image D2. At this time, as shown in FIG. 8, when color space data for each pixel unit is obtained for the two types of digital images D1 and D2 at the time of excitation, the influence of the fluorescence reaction 82 due to dust is canceled out. The specific fluorescence reaction 81 caused by microorganisms or the like can be clearly detected. In this case, it is effective when there is a wavelength of the excitation light X in which microorganisms or the like have a particularly strong fluorescence reaction.

  In addition, when excitation light X having a wavelength of UV excitation, B excitation, or G excitation is used, two types of excitation digital images D1 and D2 photographed with different intensities of the excitation light X should be used. You can also. In this case, for example, as shown in FIGS. 6 and 7, dust (foreign matter other than the detection target) clearly shows the fluorescence reaction 82 even when irradiated with the weak excitation light X or the excitation light X for a short time. On the other hand, as shown in FIG. 7, it is assumed that microorganisms or the like clearly show a specific fluorescence reaction 81 only after irradiation with strong excitation light X or irradiation with excitation light X for a long time. At this time, as shown in FIG. 8, when color space data for each pixel unit is obtained for the two types of digital images D1 and D2 at the time of excitation, the influence of the fluorescence reaction 82 due to dust is canceled out. The specific fluorescence reaction 81 caused by microorganisms or the like can be clearly detected. In this case, it is effective when the time when microorganisms or the like show a fluorescence reaction is late compared to dust.

The color space data obtained by the image analysis unit 52 of this example indicates the hue data of red (R), green (G), and blue (B), which are the three primary colors, by numerical values in a predetermined number of gradations. . The color space data in this example is obtained by decomposing light of a specific wavelength detected by the camera 4 into red, green, and blue data. Each of the red, green, and blue data is assigned 256 gradations, and each data (each primary color) is indicated by a numerical value of 0-255.
Further, the color space data can include luminance (brightness) expressed using the three primary colors. The luminance Y can be obtained from Y = a1 * R + a2 * G + a3 * B, where a1 to a3 are coefficients, R is red, G is green, and B is blue. The coefficients a1 to a3 are the values of red, green, and blue for a plurality of digital images when light of a plurality of wavelengths is captured by the camera 4, and X1 to Y = (a1 × X1 + a2 × X2 + a3 × X3) / 3 It can be obtained by substituting for X3.
Depending on the detection target of microorganisms or viruses, the color space data may be only luminance data.

In the image analysis unit 52, a collation operation performed between pixel units at the same position in a digital image indicated by a large number of pixels can be the following error calculation.
FIG. 9 shows a state in which the first color space data d1 and the second color space data d2 are collated in pixel units for each dot. As shown in the figure, the numerical values represented by 0 to 255 of red, green, and blue in the first color space data d1 for the first excitation digital image D1 are R1, G1, and B1, and the second excitation digital image. The numerical values represented by 0 to 255 of red, green, and blue in the second color space data d2 for D2 are R2, G2, and B2.
The error calculation includes at least one of | R1-R2 | (absolute value of R1-R2), | G1-G2 | (absolute value of G1-G2), | B1-B2 | (absolute value of B1-B2). As can be done.

  In addition, the error calculation is performed by setting the luminance value in the first color space data d1 for the first excitation digital image D1 to Y1, and the luminance value in the second color space data d2 for the second excitation digital image D2 to Y2. As | Y1-Y2 |. The arithmetic operation can also be performed as any one of Y1 + Y2, Y1-Y2, Y1 * Y2, Y1 / Y2, Y2-Y1, Y2 / Y1.

In these calculations, R1 to R3, G1 to G3, and B1 to B3 are set to 0 when the numerical value is less than 0, and to 255 when the numerical value exceeds 255. Further, when the numerical value that becomes the denominator by division is 0, it is set to 255, and when the numerical value that becomes the numerator by division is 0, it is set to 0.
Thus, the arithmetic processing data obtained by performing the collation operation in the image analysis unit 52 is represented by a numerical value in the range of 0 to 255.

The specified range of the calculation processing data, which is a determination criterion in the image analysis unit 52, is a plurality of (when a specific microorganism or the like to be detected is irradiated with excitation light X having at least one of wavelength and intensity different from each other ( Two digital images at the time of excitation are taken and obtained as a result of the collation operation performed on the pixels in which the specific fluorescence reaction 81 is reflected in the digital image at the time of excitation.
In addition, the specified range of the arithmetic processing data that is a determination criterion in the image analysis unit 52 is the time from the start of irradiation of the excitation light X to the specific microorganisms or the like to be detected to the time of shooting by the camera 4. In a plurality (two) of excitation-time digital images taken with different images, it can be obtained as a result of performing a collation operation on pixels in which the specific fluorescence reaction 81 is reflected in the excitation-time digital image.

The specified range of the arithmetic processing data can be set with a suitable margin with respect to the color space data of hue and luminance when the specific fluorescence reaction 81 is actually photographed for a specific microorganism or the like. At this time, the specific fluorescent reaction 81 can be photographed a plurality of times to determine the width of the specified range.
When the specific fluorescent reaction 81 is actually inspected for the sample 8, the excitation light X under the same conditions as the excitation light X used when determining the prescribed range of the calculation processing data that is the determination criterion is used. 8 is irradiated. In addition, when the wavelength or intensity of the excitation light X is different, the type of excitation light X used at the time of setting reference and at the time of detection is made the same.

FIG. 10 shows the binarization of whether or not the arithmetic processing data obtained by collating the first color space data d1 and the second color space data d2 in pixel units for each dot is within a specified range. The result of processing is visualized and shown.
The image analysis unit 52 of this example obtains, as hit pixels H, pixel units that are within the specified range of the computation processing data obtained in advance for the specific fluorescence reaction 81 from the computation processing data for each pixel unit. Then, when the shape and area in which the hit pixels H are formed side by side are within the prescribed range of the shape and area obtained in advance for the specific fluorescence reaction 81, it is detected that microorganisms or viruses are present in the sample 8. The hit pixel H is obtained when the binarization processing is performed with 1 when the calculation processing data of each pixel unit is within the specified range of the calculation processing data and 0 when the calculation processing data is not within the specified range of the calculation processing data. It is assumed that the pixel is 1.
The specified range of the shape and area for the specific fluorescence reaction 81 can be set with a moderate margin to the value obtained by actually measuring the shape and area of a specific microorganism or the like to be detected.

  Further, for each pixel in the image analysis unit 52, for example, when | R1-R2 |, | G1-G2 |, | B1-B2 | are performed as an error calculation, all of these values are within a specified range. When it becomes, it can be set as the hit pixel H. In addition, when one or two of these values are within the specified range, the hit pixel H can be determined.

The image analysis unit 52 specifies a pixel region having a size surrounding a shape in which the hit pixels H detected as having the specific fluorescence reaction 81 are formed side by side as the hit region A.
8 and 11 show a hit area A surrounding the hit pixel H. FIG.
In the image analysis unit 52 of this example, the binarized arithmetic processing data obtained by extracting the hit pixels H is adapted to the maximum contour of the hit pixel shape in which the hit pixels H are formed side by side. Surrounded by a rectangular shape with sides parallel to the X and Y axes. Then, the pixel region surrounded by the rectangular shape is specified as the hit region A, and it is determined whether or not the hit pixel shape approximates the shape of the specific fluorescence reaction 81 such as a microorganism to be detected. .

  The image analysis unit 52 can detect the area (number of hit pixels H) of the hit pixel shape surrounded by a rectangular shape, the center of gravity position, and the like. When the hit pixel shape is within the specified range of the shape and area of the specific fluorescence reaction 81 for microorganisms or the like, the position, number, etc., of the hit pixel shape in the entire digital image at the time of excitation are specified. The specified range of the area of the specific fluorescence reaction 81 has a predetermined size range that is not less than a predetermined area and not more than a predetermined area. If the hit pixel shape is too large or too small, it does not fall within the specified range of the area of the specific fluorescence reaction 81.

In addition to detecting the hit pixel shape, the image analysis unit 52 has a predetermined ratio of the area (number of pixels) occupied by the hit pixel H in the total area (number of pixels) constituting the excitation digital image. It is also possible to determine that the specific fluorescent reaction 81 is present in the sample 8 when the ratio is equal to or greater than the ratio.
The control unit 51 of this example captures the elementary digital image D4 when the excitation light X is not irradiated by the camera 4, and a monitor in which the pixel region corresponding to the hit region A in the elementary digital image D4 is connected to the computer 5. 6 is configured to be enlarged and displayed.
FIG. 12 schematically shows a state in which the pixel area B corresponding to the hit area A is enlarged and displayed on the monitor 6 for the elementary digital image D4. In this example, the person who inspects can actually visually confirm the part detected in the elementary digital image D4 that microorganisms or the like are present.

Further, in the image analysis unit 52, the prescribed range of the shape and area for the specific fluorescence reaction 81 can be set in advance for a plurality of types of microorganisms and the like. Then, the image analysis unit 52 determines whether the shape and area formed by arranging the hit pixel shapes are included in a predetermined range of the shape and area obtained in advance for each of a plurality of types of microorganisms, The type of microorganisms or the like present in the sample 8 can be estimated. In this case, not only the presence or absence of microorganisms but also the type can be detected.
Further, the image analysis unit 52 determines the type of microorganism or the like according to the ratio of the area (number of pixels) of the hit pixel H having a predetermined shape in the hit area A to the area (number of pixels) of the rectangular hit area A. Can also be estimated.

In the fluorescence reaction detection device 1 of this example, the excitation filter 3 allows two types of excitation light X having different wavelengths and intensities to pass simultaneously from the light source 2 to the mirror 43. Then, the presence or absence of the specific microorganism is detected by analyzing the hue, luminance, and the like of the light emitted by the specific microorganism contained in the sample 8 when the two types of excitation light X are simultaneously irradiated by the image analysis unit 52. To do.
Thereby, it is possible to obtain a fluorescence reaction 81 of a microorganism that could not be obtained only by irradiating a specific type of excitation light X to a specific microorganism, and the detection accuracy of the specific microorganism can be remarkably improved.

As shown in FIGS. 6 and 7, the sample 8 also contains dust (foreign matter not detected) other than the specific fluorescence reaction 81, and this dust is also a predetermined fluorescence reaction 82. Will be issued. However, the fluorescence property of the specific fluorescence reaction 81 and the fluorescence reaction 82 caused by dust differ depending on conditions such as the wavelength and intensity of the excitation light X. Therefore, when two types of excitation digital images D1 and D2 are photographed, for example, the fluorescence reaction 82 due to dust is photographed in two types of excitation digital images D1 and D2, while the specific fluorescence reaction 81 It is assumed that the digital image D2 is clearly captured at the time of double excitation. In this case, as shown in FIG. 8, by performing the above collation operation, the influence of the fluorescent reaction 82 caused by dust can be removed, and the specific fluorescent reaction 81 can be made to stand out and detected. The specific fluorescence reaction 81 caused by microorganisms or the like can be clearly distinguished from the fluorescence reaction 82 caused by other dust (foreign matter not detected).
Therefore, according to the fluorescence reaction detection device 1 of the present example, a specific microorganism or the like in the sample 8 can be stably detected with high detection accuracy.

DESCRIPTION OF SYMBOLS 1 Fluorescence reaction detection apparatus 2 Light source 3 Filter 3A, 3B Excitation filter 31 Switching filter 32 Turntable 33A, 33B, 33C Filter part 34A, 34B, 34C Attenuation filter part 4 Camera 43 Mirror 5 Computer 51 Control part 52 Image analysis part 6 Monitor 8 Sample 81 Specific fluorescence reaction X Excitation light D1, D2 Excitation digital image

Claims (6)

A fluorescence reaction detection device for detecting a specific fluorescence reaction of a microorganism or virus in a sample,
A light source that emits light including ultraviolet and visible light wavelengths;
An excitation filter that transmits only the wavelength component corresponding to the excitation light among the light emitted from the light source;
A mirror that reflects the excitation light transmitted through the excitation filter and irradiates the sample obliquely;
A camera for photographing the surface of the sample as a digital image;
A computer having an image analysis unit for analyzing the digital image,
The excitation filter is configured to simultaneously pass a plurality of types of excitation light having different wavelengths and intensities from the light source to the mirror,
The fluorescence analysis detection apparatus, wherein the image analysis unit detects a specific fluorescence reaction in which a microorganism or virus contained in the sample fluoresces when the plurality of types of excitation light are simultaneously irradiated. 2. The fluorescence reaction detection device according to claim 1, wherein the excitation filter includes two rotatable switching filters arranged in parallel in proximity to each other,
The switching filter includes a plurality of types of filter units that transmit light of different wavelengths in parallel in the circumferential direction of the rotating disk that does not transmit light,
Both of the two switching filters rotate around the rotation center of the rotating disk, and are arranged at a light passing position through which light emitted from the light source passes among the plurality of types of filter units. Is selectable,
A fluorescence reaction detection apparatus characterized in that excitation light having mutually different wavelengths is simultaneously transmitted to the mirror from the filter unit disposed at the light passage position in the two switching filters by the excitation filter.   3. The fluorescence reaction detection apparatus according to claim 2, wherein the plurality of types of filter units are emitted from the light source and a UV excitation filter unit that transmits light having a wavelength of UV excitation from the light emitted from the light source. A B excitation filter that transmits light having a wavelength of B excitation from light and a G excitation filter that transmits light having a wavelength of G excitation from light emitted from the light source are included. Fluorescence reaction detector. The fluorescence reaction detection device according to claim 2, wherein the two excitation filters including the two switching filters are arranged in series at the light passage position,
On the other hand, the excitation filter includes a UV excitation filter unit that transmits light of a wavelength of UV excitation from the light emitted from the light source to the plurality of types of filter units, and a wavelength of B excitation from the light emitted from the light source. A B excitation filter that transmits the light of G, and a G excitation filter that transmits light of the wavelength of G excitation from the light emitted from the light source,
The other excitation filter includes a plurality of types of neutral density filter units that reduce the intensity of light emitted from the light source in a plurality of stages in the plurality of types of filter units. Detection device. 5. The fluorescence reaction detection device according to claim 1, wherein the camera is a combination of a CMOS camera and an optical lens, and the one sample of the digital image to be photographed in the sample as a subject. A fluorescence reaction detection device capable of photographing a surface of 1 (μm ² ) or less.   6. The fluorescence reaction detection apparatus according to claim 1, wherein the excitation light applied to the sample from the mirror is 27 to 33 ° with respect to a planar direction of the holding body that holds the sample. A fluorescence reaction detection device configured to irradiate at an incident angle.

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