JP2012168086A - Fluorescent reaction detector - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescent reaction detector capable of stably detecting microorganisms or viruses in a sample with high detection accuracy.SOLUTION: A control part 51 changes at least one of the wave length and strength of excitation light X transmitted through a filter 3 and has a camera 4 to capture a plurality of kinds of digital images D1, D2 at excitation. An image processing part 52 obtains color space data for each pixel unit for the plurality of kinds of digital images D1, D2 at excitation, and performs collation calculation between pieces of color space data for the pixel units present at the same position in the plurality of kinds of digital images D1, D2 at excitation to obtain calculation processing data for each pixel unit. A determination part 53 compares the calculation processing data for each pixel unit with a specified range of calculation processing data that has been obtained in advance for a specific fluorescent reaction 81 to detect whether the specific fluorescent reaction 81 is present in a sample 8 or not.

Description

  The present invention relates to a fluorescence reaction detection apparatus that detects a specific fluorescence reaction caused by a specific microorganism or the like 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. .

1st invention is the fluorescence reaction detection apparatus which detects the specific fluorescence reaction of the microorganisms or virus in a sample,
A light source capable of irradiating the sample with excitation light having a predetermined wavelength through a filter;
A camera for photographing the surface of the sample as a digital image;
Photographed by the camera from the time point when at least one of the wavelength and intensity of the excitation light transmitted through the filter is changed, the time during which the excitation light is emitted from the light source, or when the excitation light is emitted from the light source A control unit that performs any one of the time changes to the time point to perform, and captures a plurality of types of excitation-time digital images with the camera;
For each of the plurality of types of excitation digital images, color space data for each pixel unit is obtained, and color space data for each pixel unit at the same position in each of the plurality of types of excitation digital images is collated with each other, An image processing unit for obtaining calculation processing data for each unit;
A determination unit for detecting the specific fluorescence reaction in the sample by comparing the calculation processing data for each pixel unit with a specified range of the calculation processing data obtained in advance for the specific fluorescence reaction. (1).

A second invention is a fluorescence reaction detection device for detecting a specific fluorescence reaction of a microorganism or virus in a sample,
A light source capable of irradiating the sample with excitation light having a predetermined wavelength through a filter;
A camera for photographing the surface of the sample as a digital image;
Either change at least one of the wavelength and intensity of the excitation light transmitted through the filter, or change the time from the start of irradiation of the excitation light from the light source to the time of shooting by the camera. A control unit that captures a plurality of types of excitation digital images with the camera;
For any of the digital images at the time of excitation, first color space data for each pixel unit is obtained, and among the first color space data for each pixel unit, a collection of pixel units within a predetermined specified range, Obtained as a temporary hit pixel, a pixel area having a size surrounding the temporary hit pixel is specified as a temporary hit area, and only the area corresponding to the temporary hit area for each of the other excitation digital images Second color space data for each pixel unit in the temporary hit area, and second color space data for each pixel unit in the area corresponding to the temporary hit area. In contrast, an image processing unit that performs a collation operation between pixel units at the same position and obtains calculation processing data for each of the pixel units in the temporary hit region,
A determination unit for detecting the specific fluorescence reaction in the sample by comparing the calculation processing data for each pixel unit in the temporary hit region with a predetermined range of the calculation processing data obtained in advance for the specific fluorescence reaction; The fluorescence reaction detection device is characterized by comprising: (Claim 2).

According to a first aspect of the present invention, there is provided a fluorescence reaction detection device, a light source capable of irradiating excitation light for exciting a specific fluorescence reaction through a filter, a camera for photographing a sample surface as a digital image, a control unit, an image A specific fluorescence reaction generated by a microorganism or virus is detected using a computer having a processing unit and a determination unit.
The control unit can irradiate the sample with a plurality of types of excitation light by operating the light source and the filter to change at least one of the wavelength and intensity of the excitation light transmitted through the filter. Then, the control unit operates the camera to capture a state in which the surface of the sample fluoresces as a digital image during excitation in each case where a plurality of types of excitation light is irradiated.

In addition to this, the control unit can change the time from the start of irradiation of excitation light from the light source to the time of shooting by the camera, and can take a plurality of types of excitation-time digital images by the camera.
At these times, when microorganisms or viruses are present in the sample, the microorganisms are excited by the excitation light and emit predetermined fluorescence. Further, the state of fluorescence emitted from the sample varies depending on the wavelength, intensity, or imaging time of the excitation light.

  The image processing unit obtains color space data for each pixel unit for a plurality of types of excitation digital images. The image processing unit collates the color space data in pixel units at the same position in the plurality of types of excitation digital images, and obtains calculation processing data for each pixel unit. For example, the calculation processing data can collate color space data of two pixels at the same position for two excitation digital images, and the same for three excitation digital images. The color space data of the three pixels at the position can be collated.

  The determination unit collates the calculation processing data for each pixel unit with a specified range of calculation processing data obtained in advance for the specific fluorescence reaction. Here, the specified range of the calculation processing data obtained in advance for the specific fluorescence reaction is set by analyzing the calculation processing data of the pixel showing the specific fluorescence reaction in a plurality of types of excitation digital images obtained for microorganisms or viruses. It is.

The conditions of the excitation light used when detecting the specific fluorescence reaction are the same as the conditions of the excitation light used when setting the reference for setting the specified range. Then, the calculation processing data at the time of detection and the specified range of the calculation processing data at the time of setting the reference are compared when the conditions of the excitation light are the same.
In this way, when the calculation processing data for each pixel unit is within the specified range of the calculation processing data obtained in advance for the specific fluorescence reaction, the determination unit determines that there is a specific fluorescence reaction that is caused by microorganisms or viruses in the sample. Can be determined.

By the way, the sample also contains dust (foreign matter not detected) that shows a fluorescence reaction other than the specific fluorescence reaction, and this dust also emits a predetermined fluorescence reaction. However, the fluorescence property differs between the specific fluorescence reaction and the fluorescence reaction with dust depending on the conditions of the wavelength, intensity, and irradiation time (imaging time) of the excitation light.
So, when multiple types of excitation digital images are taken, for example, the fluorescence reaction due to dust is captured in all the excitation digital images, while the specific fluorescence reaction is clearly in any of the excitation digital images. The case where it is photographed is assumed. In this case, by performing the above collation operation, the influence of the fluorescent reaction due to dust can be removed, and the specific fluorescent reaction can be distinguished and detected. The fluorescence reaction caused by microorganisms or viruses can be clearly distinguished from the fluorescence reaction caused by other dust (foreign matter not detected).

In the fluorescence reaction detection device of the second invention, the processing performed by the image processing unit is different from the case of the first invention.
In the image processing unit of the second invention, it is determined whether or not the first color space data for each pixel unit of any one of the excitation-time digital images is within a predetermined specified range. Then, only one or a plurality of specific pixel areas within the specified range among the entire pixel areas of the digital image are set as temporary hit areas used when calculating subsequent calculation processing data.

Then, second color space data for each pixel unit is obtained for only the region corresponding to the temporary hit region for the other digital images at the time of excitation. Thereby, the process for obtaining the second color space data can be reduced.
In this way, the arithmetic processing data is obtained only for a specific limited pixel region in the entire pixel region of the digital image. Thereby, the process which calculates | requires arithmetic processing data can be decreased.
In addition, in the second invention, the same function and effect as in the first invention can be obtained.

  In the image processing unit, the color space data to be collated with the first color space data can be color space data obtained for each pixel unit in three or more types of excitation digital images. For example, for three types of excitation digital images, the first color space data for each pixel unit in the temporary hit area, the second color space data for each pixel unit in the area corresponding to the temporary hit area, and the first color space data The three color space data can be collated for each pixel unit at the same position.

  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.

BRIEF DESCRIPTION OF THE DRAWINGS Explanatory drawing which shows the structure of the fluorescence reaction detection apparatus concerning Example 1. FIG. FIG. 3 is an explanatory diagram schematically illustrating a first excitation digital image according to the first embodiment. FIG. 3 is an explanatory diagram schematically illustrating a second excitation digital image according to the first embodiment. FIG. 3 is an explanatory diagram schematically showing a result of performing a collation operation on a first excitation digital image and a second excitation digital image according to the first embodiment and schematically showing the collation image; FIG. 3 is an explanatory diagram showing a state in which the first color space data and the second color space data according to the first embodiment are collated in a pixel unit for each dot. Binarization processing of whether or not the arithmetic processing data obtained by collating the first color space data and the second color space data in the pixel unit for each dot is within the specified range according to the first embodiment Explanatory drawing which visualizes and shows the result of having performed. FIG. 3 is an explanatory diagram illustrating a hit area surrounding a hit pixel according to the first embodiment. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory diagram schematically illustrating a state in which a pixel area corresponding to a hit area is enlarged and displayed on a monitor for an elementary digital image according to Example 1; 2 is a flowchart showing a procedure for detecting a specific fluorescence reaction of a microorganism or the like according to the first embodiment. FIG. 9 is an explanatory diagram schematically showing a state in which a temporary hit pixel and a temporary hit area are specified in a first excitation digital image according to the second embodiment. FIG. 7 is an explanatory diagram schematically showing a region corresponding to a temporary hit region in a second excitation digital image according to the second embodiment. FIG. 6 schematically shows a result of performing a collation operation on the first color space data in the temporary hit area and the second color space data in the area corresponding to the temporary hit area according to the second embodiment as a visualized image. Illustration.

A preferred embodiment of the fluorescence reaction detection apparatus according to the first and second inventions described above will be described.
In the first and second inventions, 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 first invention, the determination unit obtains, as a hit pixel, a pixel unit within the specified range of the calculation processing data obtained in advance for the specific fluorescence reaction from the calculation processing data for each pixel unit. It is preferable to detect the presence of microorganisms or viruses in the sample when the shape and area in which the hit pixels are formed side by side are within the prescribed range of the shape and area determined in advance for the specific fluorescence reaction (claim) Item 3).
In the second invention, the determination unit hits a pixel unit within the specified range of the calculation processing data obtained in advance for the specific fluorescence reaction among the calculation processing data for each pixel unit in the temporary hit region. Detected as microorganisms or viruses in the sample when the shape and area where the hit pixels are formed are within the specified range of the shape and area determined in advance for the specific fluorescence reaction. (Claim 4).

  In these cases, the determination unit can detect the microorganisms or viruses by confirming the shape and size of the specific fluorescence reaction for the sample exhibiting the specific fluorescence reaction. Therefore, the detection accuracy of microorganisms or viruses can be improved.

In the first and second inventions, the determination unit specifies a pixel area having a size surrounding the shape formed by the hit pixels detected as having the specific fluorescence reaction as a hit area, It is preferable that the control unit captures an elementary digital image when the excitation light is not irradiated by the camera, and enlarges and displays a pixel area corresponding to the hit area in the elementary digital image on the monitor (claim). Item 5).
In this case, the pixel area detected as having the specific fluorescence reaction is enlarged and displayed on the monitor, so that the person who inspects actually confirms the portion of the elementary digital image where the microorganism or virus has been detected by visually checking. be able to.

The color space data is at least one of red, green, blue hue, and luminance. In the image processing unit, the collation operation performed between the pixel units at the same position is performed by the hue. And an error calculation or arithmetic calculation performed on at least one of the luminances.
In this case, the image processing unit can appropriately obtain the arithmetic processing data for each pixel unit in the excitation digital image.

The wavelength of the excitation light is preferably a wavelength corresponding to any of UV excitation, B excitation, and G excitation.
In this case, excitation light that irradiates the sample from the light source through the filter is appropriate.
Here, UV excitation (near ultraviolet rays) can be excitation light having a wavelength in the range of 320 to 380 nm. B excitation (blue light) can be excitation light having a wavelength in the range of 380 to 495 nm. G excitation (green light) can be excitation light having a wavelength in the range of 495 to 570 nm.

In addition, at least one of the plurality of types of excitation-time digital images is obtained when the sample is simultaneously irradiated with a plurality of types of the excitation light in which at least one of wavelength and intensity is different. A digital image is preferable.
In this case, it may be possible to obtain a specific fluorescence reaction that could not be obtained simply by irradiating a microorganism or virus with one kind of wavelength and intensity of excitation light. In some cases, the detection accuracy of microorganisms or viruses can be significantly improved.

Hereinafter, embodiments of the fluorescence reaction detection apparatus of the present invention will be described with reference to the drawings.
Example 1
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 device 1 includes the following light source 2, camera 4, control unit 51, image processing unit 52, and determination unit 53.
The light source 2 can irradiate the sample 8 with excitation light X having a predetermined wavelength through the filter 3. The camera 4 is configured to capture the surface of the sample 8 as a digital image.

The control unit 51, the image processing unit 52, and the determination unit 53 are constructed in the computer 5, and the light source 2 and the camera 4 are connected to the computer 5.
The control unit 51 changes at least one of the wavelength and intensity of the excitation light X transmitted through the filter 3 or changes the time from when the irradiation of the excitation light X from the light source 2 is started to when the camera 4 takes an image. Any one of them is performed, and a plurality of types of excitation-time digital images D1 and D2 are captured by the camera 4. The image processing unit 52 obtains color space data for each pixel unit for the plurality of types of excitation digital images D1 and D2, and each pixel unit at the same position in the plurality of types of excitation digital images D1 and D2. The color space data is collated with each other to obtain operation processing data for each pixel unit. The determination unit 53 is configured to detect the presence / absence of the specific fluorescence reaction 81 in the sample 8 by comparing the calculation process data for each pixel unit with a specified range of the calculation process data obtained in advance for the specific fluorescence reaction 81. It is.

Below, it demonstrates in detail with reference to FIGS. 1-9 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. A filter 3 that transmits light of a specific wavelength is disposed at the light passage position of the ultraviolet lamp 2, and the sample 8 is irradiated with excitation light X having a desired wavelength and intensity by appropriately replacing the filter 3. be able to.

The wavelength of the excitation light X that passes through the filter 3 and irradiates the sample 8 corresponds to one of UV excitation (320 to 380 nm), B excitation (380 to 495 nm), or G excitation (495 to 570 nm). It is. The filter 3 includes a UV excitation filter 3 that transmits light having a wavelength of UV excitation from light emitted from the ultraviolet lamp 2, and a B excitation filter 3 that transmits light having a wavelength of B excitation from light emitted from the ultraviolet lamp 2. And a G excitation filter 3 that transmits light having a wavelength of G excitation from the light emitted from the ultraviolet lamp 2.
Further, the light source 2 is configured to change the intensity of the excitation light X by passing through the neutral density filter 30. The neutral density filter 30 is disposed at a position facing the filter 3. And the wavelength and intensity | strength of the excitation light X can be changed by changing the filter 3 and the neutral density filter 30 to be used.
Further, the excitation light X can be irradiated from the light source 2 to the sample 8 by being reflected by the mirror 43.

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 the 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. 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 control unit 51 of this example transmits a control signal to the light source 2, emits light from the light source 2, transmits a control signal to the camera 4, and images the surface of the sample 8 by the camera 4. Is configured to do.

  In the fluorescence reaction detection apparatus 1 of this example, two types of excitation-time digital images D1 and D2 in which the wavelengths of the excitation light X emitted from the light source 2 and the filter 3 are different from each other are taken. 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).

In addition, at least one of the two types of excitation digital images D1 and D2 may be a digital image when the sample 8 is simultaneously irradiated with excitation light X having different wavelengths from the light source 2 and the filter 3. it can. Further, the excitation light X irradiated at the same time can be irradiated with different wavelengths and different intensities. Further, the excitation light X irradiated at the same time can be irradiated with only different intensities.
Each of the excitation-time digital pixels D1 and D2 can be photographed while the sample 8 is irradiated with the excitation light X, or can be photographed immediately after the sample 8 is irradiated with the excitation light X.

  FIG. 2 schematically shows the first excitation digital image D1, and FIG. 3 schematically shows the second excitation digital image D2. In FIG. 4, 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. 2 and 3, 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. 4, when the 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.

  Further, when the excitation light X having any wavelength of UV excitation, B excitation, or G excitation is used, the intensity of the excitation light X or the imaging time (from the time when the irradiation of the excitation light X from the light source 2 is started) It is also possible to use two types of digital images D1 and D2 at the time of excitation, which are photographed with different times). In this case, for example, as shown in FIGS. 2 and 3, dust (foreign matter other than the detection target) clearly shows the fluorescence reaction 82 even when irradiated with the excitation light X having a weak intensity or the excitation light X for a short time. On the other hand, as shown in FIG. 3, it is assumed that microorganisms or the like clearly show a specific fluorescence reaction 81 only after irradiation with excitation light X having a strong intensity or irradiation with excitation light X for a long time. At this time, as shown in FIG. 4, when the 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 in the image processing unit 52 of this example indicates 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 processing unit 52, the collation calculation performed between the pixel units at the same position in the digital image indicated by a large number of pixels can be the following various calculations.
The collation operation of this example is an error operation or an arithmetic operation performed on the red, green, and blue hues in the color space data d1, d2 of the two types of excitation digital images D1, D2.

FIG. 5 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.

As arithmetic operations, at least one of R1 + R2, G1 + G2, B1 + B2, at least one of R1-R2, G1-G2, B1-B2, and at least one of R1 × R2, G1 × G2, B1 × B2 As at least one of R1 / R2, G1 / G2, B1 / B2, as at least one of R2-R1, G2-G1, B2-B1, or at least of R2 / R1, G2 / G1, B2 / B1 It can be done as one.
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.
In this way, the arithmetic processing data obtained by performing the collation operation in the image processing 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 determination unit 53, is a plurality (2) when the specific microorganisms or the like to be detected are irradiated with excitation light X having at least one of wavelength and intensity different from each other. Is obtained as a result of performing a collation operation on a pixel in which the specific fluorescence reaction 81 is reflected in the excitation digital image.
In addition, the prescribed range of the arithmetic processing data which is a determination criterion in the determination unit 53 is different in time from the time when the irradiation of the excitation light X is started to a specific microorganism or the like to be detected to the time when the camera 4 takes an image. In a plurality of (two) excitation-time digital images when the images are taken, it is also possible to obtain 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. 6 shows binarization as to whether or not the arithmetic processing data obtained by collating the first color space data d1 and the second color space data d2 in a pixel unit for each dot is within a specified range. The result of processing is visualized and shown.
The determination unit 53 of this example obtains, as hit pixels H, pixel units that are within the specified range of the calculation processing data obtained in advance for the specific fluorescence reaction 81 among the calculation 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 processing 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 same applies to the case where the image processing unit 52 performs an arithmetic operation.

The determination unit 53 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.
4 and 7 show a hit area A surrounding the hit pixel H. FIG.
In the determination unit 53 of this example, the binarized arithmetic processing data obtained by extracting the hit pixels H is matched with the maximum contour of the hit pixel shape formed by arranging the hit pixels H in accordance with the X of the digital image. Surrounded by a rectangle with sides parallel to the axis and the Y axis. 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 determination unit 53 can detect the area (number of hit pixels H) of the hit pixel shape surrounded by the 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 determination unit 53 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. When the ratio is equal to or greater than the ratio, it can be determined that the specific fluorescent reaction 81 is present in the sample 8.
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. 8 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.

Moreover, in the determination part 53, the prescription | regulation range of the shape and area about the specific fluorescence reaction 81 can be preset about several types of microorganisms. Then, the determination unit 53 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, etc. 8 can be used to estimate the types of microorganisms and the like present in 8. In this case, not only the presence or absence of microorganisms but also the type can be detected.
The determination unit 53 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. It can also be estimated.

Next, a procedure for detecting a specific fluorescence reaction 81 such as a microorganism using the fluorescence reaction detection apparatus 1 will be described with reference to a flowchart of FIG.
First, the sample 8 is irradiated with the first excitation light X having a predetermined wavelength and intensity from the light source 2 (step S1 in the figure). Then, after a predetermined time has elapsed, the surface of the sample 8 is photographed by the camera 4 (S2). At this time, the sample 8 is excited by the first excitation light X to cause various fluorescence reactions, and the camera 4 captures fluorescence of predetermined visible light. In addition, the first excitation digital image D <b> 1 that has been shot is captured and stored in the image processing unit 52 of the computer 5.

  Next, the sample 8 is irradiated with the second excitation light X having at least one of the wavelength and the intensity different from the first excitation light X from the light source 2 (S3). And after predetermined time passes, the surface of the sample 8 is image | photographed with the camera 4 (S4). At this time, the sample 8 is excited by the second excitation light X to cause various fluorescent reactions, and the camera 4 captures fluorescence of predetermined visible light. The second excitation digital image D <b> 2 that has been shot is captured and stored in the image processing unit 52 of the computer 5.

Next, first color space data d1 for each pixel unit in the first excitation digital image D1 and second color space data d2 for each pixel unit in the second excitation digital image D2 are obtained (S5).
Next, an error calculation or an arithmetic calculation collation operation is performed on the first color space data d1 for each pixel unit and the second color space data d2 for each pixel unit, and calculation processing data for each pixel unit is obtained. Obtain (S6).

Next, it is determined whether or not the calculation processing data for each pixel unit is within the specified range of the calculation processing data obtained in advance for the specific fluorescence reaction 81 (S7). Then, the pixels within the specified range are extracted as hit pixels H (S8). On the other hand, if all the pixels are not within the specified range, it is determined that the specific fluorescence reaction 81 has not been detected in the sample 8 (S11).
The hit pixel H is extracted, and it is determined whether or not the hit pixel shape formed by the gathering of the hit pixels H is within the prescribed range of the shape and area obtained in advance for the specific fluorescence reaction 81 (S9). If the hit pixel shape is within the specified range, it is detected that there is a specific fluorescence reaction 81 caused by microorganisms or the like (S10). On the other hand, if the hit pixel shape is not within the specified range, the process is terminated assuming that the specific fluorescence reaction 81 has not been detected in the sample 8 (S11).

In the fluorescence reaction detection apparatus 1 of this example, the control unit 51 operates the light source 2 and the filter 3 to change at least one of the wavelength and intensity of the excitation light X, and uses the two types of excitation light X as the sample 8. Can be irradiated. Then, the control unit 51 operates the camera 4 to photograph the state in which the surface of the sample 8 fluoresces as the excitation digital images D1 and D2 in each case where the two types of excitation light X are irradiated.
At this time, if a microorganism or the like is present in the sample 8, the microorganism or the like is excited by the excitation light X and emits a predetermined fluorescence. The state of fluorescence emitted from the sample 8 varies depending on the wavelength and intensity of the excitation light X.

  The image processing unit 52 obtains color space data d1 and d2 for each pixel unit for the two types of excitation-time digital images D1 and D2. Then, the image processing unit 52 is in the same position with respect to the color space data d1 of each pixel unit in the first excitation digital image D1 and the color space data d2 of each pixel unit in the second excitation digital image D2. A collation operation is performed between certain pixel units to obtain operation processing data obtained by performing error operation or arithmetic operation for each pixel unit.

  The determination unit 53 collates 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. Then, when the calculation processing data for each pixel unit is within the specified range of the calculation processing data obtained in advance for the specific fluorescence reaction 81, the determination unit 53 determines the specific fluorescence reaction 81 that is emitted by microorganisms or the like in the sample 8. It can be determined that there is.

By the way, as shown in FIGS. 2 and 3, the sample 8 includes 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. 4, by performing the above-described collation operation, the influence of the fluorescent reaction 82 caused by dust can be removed, and the specific fluorescent reaction 81 can be distinguished 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 this example, microorganisms and the like in the sample 8 can be stably detected with high detection accuracy.

(Example 2)
In this example, the processes in the image processing unit 52 and the determination unit 53 are different from those in the first embodiment.
The image processing unit 52 of the present example can reduce the amount of image processing by specifying a pixel area having a predetermined fluorescence reaction in the first excitation digital image D1 and performing a collation operation only on this pixel area. Is.
The controller 51 of this example also takes two types of excitation-time digital images D <b> 1 and D <b> 2 when the two types of excitation light X are irradiated by the camera 4.

The image processing unit 52 of the present example obtains first color space data d1 for each pixel unit for the first excitation digital image D1. Then, the image processing unit 52 obtains, as the temporary hit pixel H ′, a group of pixel units within the predetermined specified range from the first color space data d1 for each pixel unit, and surrounds the temporary hit pixel H ′. A pixel area having a size is specified as a temporary hit area A ′.
FIG. 10 schematically shows a state in which the temporary hit pixel H ′ and the temporary hit area A ′ are identified in the first excitation digital image D1. The temporary hit area A ′ is formed so as to be surrounded by a rectangular shape having sides parallel to the X axis and the Y axis of the digital image in accordance with the maximum outline of the hit pixel shape formed by arranging the temporary hit pixels H ′. .
The predetermined prescribed range can be a prescribed range of the first color space data d1 obtained in advance for the specific fluorescence reaction 81.

In addition, the image processing unit 52 obtains second color space data d2 for each pixel unit for only the area A ′ corresponding to the temporary hit area A ′ for the second excitation digital image D2.
FIG. 11 schematically shows a region A ′ corresponding to the temporary hit region A ′ in the second excitation digital image D2. Then, the image processing unit 52 performs first color space data d1 for each pixel unit in the temporary hit area A ′ and second color space data d2 for each pixel unit in the area corresponding to the temporary hit area A ′. Are compared with each other at the same position, and calculation processing data is obtained for each pixel unit in the temporary hit area A ′.
FIG. 12 visualizes the result of the collation operation performed on the first color space data d1 in the temporary hit area A ′ and the second color space data d2 in the area A ′ corresponding to the temporary hit area A ′. This is schematically shown as an image D3.

The collation operation performed on the first color space data d1 and the second color space data d2 can be an error operation or an arithmetic operation as in the first embodiment.
The determination unit 53 of this example selects the pixel units within the specified range of the calculation processing data obtained for the specific fluorescence reaction 81 in advance among the calculation processing data for each pixel unit in the provisional hit area A ′. Asking. The determination unit 53 determines that microorganisms or the like are present in the sample 8 when the shape and area formed by the hit pixels H are within the specified range of the shape and area obtained in advance for the specific fluorescence reaction 81. Can be detected.

  In this example, when two types of excitation digital images D1 and D2 are photographed, for example, the specific fluorescence reaction 81 is photographed in any excitation digital image, while the first excitation digital image D1 and The second excitation digital image D2 is assumed to have a different shooting method. In this case, by performing the above collation operation, the fluorescence reaction caused by microorganisms or viruses can be clearly distinguished from the fluorescence reaction caused by other dust (foreign matter not detected).

In this example, the second color space data d2 for each pixel unit is obtained for only the region corresponding to the temporary hit region A ′ for the second excitation digital image D2. Thereby, the process for obtaining the second color space data d2 can be reduced.
In this way, it is possible to reduce the processing for obtaining the arithmetic processing data by obtaining the arithmetic processing data only for a specific limited pixel region in the entire pixel region of the digital image.
Also in this example, other configurations are the same as those of the first embodiment, and the same effects as those of the first embodiment can be obtained.

DESCRIPTION OF SYMBOLS 1 Fluorescence reaction detection apparatus 2 Light source 3 Filter 4 Camera 5 Computer 51 Control part 52 Image processing part 53 Judgment part 6 Monitor 8 Sample 81 Specific fluorescence reaction X Excitation light D1, D2 Excitation digital image D4 Elementary digital image d1, d2 Color space Data H Hit pixel A Hit area

Claims (8)

A fluorescence reaction detection device for detecting a specific fluorescence reaction of a microorganism or virus in a sample,
A light source capable of irradiating the sample with excitation light having a predetermined wavelength through a filter;
A camera for photographing the surface of the sample as a digital image;
Either change at least one of the wavelength and intensity of the excitation light transmitted through the filter, or change the time from the start of irradiation of the excitation light from the light source to the time of shooting by the camera. A control unit that captures a plurality of types of excitation digital images with the camera;
For each of the plurality of types of excitation digital images, color space data for each pixel unit is obtained, and color space data for each pixel unit at the same position in each of the plurality of types of excitation digital images is collated with each other, An image processing unit for obtaining calculation processing data for each unit;
A determination unit for detecting the specific fluorescence reaction in the sample by comparing the calculation processing data for each pixel unit with a specified range of the calculation processing data obtained in advance for the specific fluorescence reaction. A fluorescence reaction detection device characterized by the above. A fluorescence reaction detection device for detecting a specific fluorescence reaction of a microorganism or virus in a sample,
A light source capable of irradiating the sample with excitation light having a predetermined wavelength through a filter;
A camera for photographing the surface of the sample as a digital image;
Either change at least one of the wavelength and intensity of the excitation light transmitted through the filter, or change the time from the start of irradiation of the excitation light from the light source to the time of shooting by the camera. A control unit that captures a plurality of types of excitation digital images with the camera;
For any of the digital images at the time of excitation, first color space data for each pixel unit is obtained, and among the first color space data for each pixel unit, a collection of pixel units within a predetermined specified range, Obtained as a temporary hit pixel, a pixel area having a size surrounding the temporary hit pixel is specified as a temporary hit area, and only the area corresponding to the temporary hit area for each of the other excitation digital images Second color space data for each pixel unit in the temporary hit area, and second color space data for each pixel unit in the area corresponding to the temporary hit area. In contrast, an image processing unit that performs a collation operation between pixel units at the same position and obtains calculation processing data for each of the pixel units in the temporary hit region,
A determination unit for detecting the specific fluorescence reaction in the sample by comparing the calculation processing data for each pixel unit in the temporary hit region with a predetermined range of the calculation processing data obtained in advance for the specific fluorescence reaction; And a fluorescence reaction detecting device.   In the fluorescence reaction detection device according to claim 1, the determination unit, among the calculation processing data for each pixel unit, a pixel unit within a specified range of the calculation processing data obtained in advance for the specific fluorescence reaction, Detected as microorganisms or viruses in the sample when the shape and area formed as a hit pixel are within the specified range of the shape and area determined in advance for the specific fluorescence reaction. A fluorescence reaction detection apparatus characterized by:   3. The fluorescence reaction detection device according to claim 2, wherein the determination unit is within a specified range of calculation processing data obtained in advance for the specific fluorescence reaction among calculation processing data for each pixel unit in the temporary hit region. When a certain pixel unit is obtained as a hit pixel and the shape and area formed by arranging the hit pixels are within the prescribed range of the shape and area obtained in advance for the specific fluorescence reaction, microorganisms or A fluorescence reaction detection apparatus for detecting the presence of a virus. 5. The fluorescence reaction detection device according to claim 3, wherein the determination unit uses, as a hit region, a pixel region having a size surrounding a shape in which the hit pixels detected when the specific fluorescence reaction is present are formed side by side. Identify,
The control unit is configured to take an elementary digital image when the excitation light is not irradiated with the camera, and to enlarge and display a pixel area corresponding to the hit area in the elementary digital image on a monitor. Fluorescence reaction detector. In the fluorescence reaction detection device according to any one of claims 1 to 5, the color space data is at least one of red, green, blue hue, and luminance,
In the image processing section, the collation operation performed between the pixel units at the same position is an error operation or an arithmetic operation performed on at least one of the hue and luminance, and a fluorescence reaction detection apparatus.   The fluorescence reaction detection apparatus according to any one of claims 1 to 6, wherein the wavelength of the excitation light is a wavelength corresponding to any of UV excitation, B excitation, and G excitation. Fluorescence reaction detector.   The fluorescence reaction detection device according to any one of claims 1 to 7, wherein at least one of the plurality of types of excitation digital images is a plurality of types of the above-described plurality of types in which at least one of wavelength and intensity is different. A fluorescence reaction detection apparatus, which is a digital image when excitation light is simultaneously applied to the sample from the light source.

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