JP2012168084A - Method for detecting specific microbial components - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting specific microbial components capable of stably detecting specific microorganisms or viruses in a sample with high detection accuracy.SOLUTION: A digital image at a first excitation time and a digital image at a second excitation time are obtained by irradiating a sample with a first excitation light and a second excitation light respectively and photographing a surface of the sample at the respective times (S1-S4). Color space data is obtained for each pixel unit in the digital image at the first excitation time and the digital image at the second excitation time, and collation calculation is performed on the color space data between the respective pixels that are at the same location to obtain calculation processing data (S5 and S6). The calculation processing data is collated with a specified range of calculation processing data that has been obtained in advance concerning a specific fluorescent reaction in which specific microorganisms or viruses develop at excitation times, to detect microorganisms or viruses in the sample (S7-S11).

Description

  The present invention relates to a method for detecting a specific biological component that detects a specific microorganism, virus, or the like in a sample.

  In the test for insects (eggs) carried out at schools and the like, for example, a test sample obtained by a subject applying cellophane to the anus is inspected at an inspection institution or medical institution. In this inspection, it is necessary to visually check with a microscope whether or not there is a caterpillar egg adhering to the cellophane. Therefore, the inspector requires skill, and it cannot be said that the inspection accuracy and the inspection efficiency are so excellent. 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, in the foreign substance detection method of Patent Document 2, it is shown 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 shows 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 / 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 a problem, and an object of the present invention is to provide a specific biological component detection method capable of stably detecting a specific microorganism or virus in a sample with high detection accuracy. is there.

1st invention is the specific biological component detection method which detects the said specific microorganisms or virus by detecting the specific fluorescence reaction which the specific microorganisms or virus in a sample generate | occur | produces at the time of excitation,
A first excitation light irradiation step of irradiating the sample with the first excitation light at a specific wavelength, intensity, and irradiation time;
A first digital image obtaining step of photographing the surface of the sample irradiated with the first excitation light and obtaining a first excitation digital image;
A second excitation light irradiation step of irradiating the sample with a second excitation light that is different from the first excitation light in at least one of wavelength, intensity, and irradiation time;
A second digital image acquisition step of photographing the surface of the sample irradiated with the second excitation light and acquiring a second excitation digital image;
For at least the first excitation digital image and the second excitation digital image, color space data for each pixel unit is obtained, collation is performed between pixel units at the same position, and calculation processing data is obtained for each pixel unit. The desired image processing process;
Detection for detecting microorganisms or viruses in the sample by comparing the calculation processing data for each pixel unit with a specified range of calculation processing data obtained in advance for the specific fluorescence reaction generated when the specific microorganism or virus is excited. And a method for detecting a specific biological component, characterized in that it comprises a step.

The second invention is a specific biological component detection method for detecting the specific microorganism or virus by detecting a specific fluorescence reaction generated when the specific microorganism or virus in the sample is excited.
A first excitation light irradiation step of irradiating the sample with the first excitation light at a specific wavelength, intensity, and irradiation time;
A first digital image obtaining step of photographing the surface of the sample irradiated with the first excitation light and obtaining a first excitation digital image;
A second excitation light irradiation step of irradiating the sample with a second excitation light that is different from the first excitation light in at least one of wavelength, intensity, and irradiation time;
A second digital image acquisition step of photographing the surface of the sample irradiated with the second excitation light and acquiring a second excitation digital image;
For either one of the first excitation time digital image or the second excitation time digital image, color space data for each pixel unit is obtained as reference color space data, and among the reference color space data, a predetermined color space data is determined. A group of pixel units within a specified range is 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 the first excitation digital image or the second excitation digital For only the region corresponding to the temporary hit region in the other of the images, color space data for each pixel unit is obtained as color space data for comparison, and the reference color space data for each pixel unit in the temporary hit region And the matching color space data for each pixel unit in the region corresponding to the temporary hit region, the pixel units at the same position And disjunction, and an image processing step of determining the operation processing data for each of the pixel unit of the temporary hit region,
The calculation processing data for each pixel unit in the temporary hit region is compared with a specified range of calculation processing data obtained in advance for the specific fluorescence reaction that occurs when the specific microorganism or virus is excited, in the sample. And a detection step of detecting a microorganism or a virus. (Claim 5)

In the specific biological component detection method of the first invention, the first irradiation step, the first digital image acquisition step, the second excitation light irradiation step, the second digital image acquisition step, and the image processing are performed. At least the process.
In the first irradiation step, the sample is irradiated with the first excitation light at a specific wavelength, intensity, and irradiation time. At this time, when a component that is excited by the first excitation light and emits fluorescence exists in the sample, these exhibit a fluorescence reaction.

  Next, in the first digital image acquisition step, the surface of the sample irradiated with the first excitation light is photographed to acquire a first excitation digital image. That is, by photographing the surface of the sample with a digital camera or the like, for example, the fluorescence state emitted by the component contained in the sample can be acquired as a digital image. As a component that emits fluorescence, foreign substances such as dust (foreign substances that are not to be detected) are assumed in addition to microorganisms or viruses.

  On the other hand, in the second irradiation step, the sample is irradiated with second excitation light composed of excitation light that differs from the first excitation light in at least one of wavelength, intensity, and irradiation time. At this time, when a component that is excited by the second excitation light and emits fluorescence exists in the sample, these exhibit a fluorescence reaction. Since the second excitation light is different from the first excitation light in at least one of wavelength, intensity, and irradiation time, the sample generates fluorescence in a pattern different from that in the case of the first excitation light.

Next, in the second digital image acquisition step, the surface of the sample irradiated with the second excitation light is photographed to acquire a second excitation digital image. Similarly to the first digital image acquisition step, the fluorescence state emitted by the component contained in the sample can be acquired as a digital image. As a component that emits fluorescence, foreign substances such as dust (foreign substances that are not to be detected) are assumed in addition to microorganisms or viruses.
The first excitation-time digital image and the second excitation-time digital image show different fluorescence states because the excitation light is different.

  Next, in the image processing step, color space data for each pixel unit is obtained for at least the first excitation digital image and the second excitation digital image. Then, the color space data for each pixel unit in the first excitation digital image and the color space data for each pixel unit in the second excitation digital image are collated with pixel units at the same position. The calculation processing data is obtained for each pixel unit. By performing the above collation calculation, detection in a detection process performed in a subsequent process becomes easy. That is, when the sample contains microorganisms or viruses, the fluorescence generated by these can be made more prominent. Further, when the sample contains foreign matter such as dust, the fluorescence emitted by the foreign matter can be made inconspicuous.

  Next, in the detection step, the processing data for each pixel unit is collated with a specified range of the processing data obtained in advance for the specific fluorescence reaction that occurs when the specific microorganism or virus is excited, and the sample Detects microorganisms or viruses in it. Here, the specified range of the calculation processing data obtained in advance for the specific fluorescence reaction is obtained by analyzing the calculation processing data of the pixels that reflected the specific fluorescence reaction in a plurality of excitation-time digital images obtained for the specific microorganism or virus. It is set.

The conditions of the excitation light (first excitation light and second excitation light) used at the time of detecting the specific fluorescence reaction are the same as the conditions of the excitation light used at the time of 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.
Thus, in the detection step, 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 specific microorganism or There is a specific fluorescent reaction emitted by the virus, and the presence of the specific microorganism or virus can be estimated.

By the way, in general, a sample contains foreign matters other than detection targets such as dust in addition to microorganisms or viruses, and the foreign matters also generate fluorescence by excitation light. However, the fluorescence properties of the specific fluorescence reaction that occurs when a microorganism or virus is excited and the fluorescence reaction that generates foreign matter such as dust differ depending on the wavelength, intensity, and irradiation time (imaging time) of the excitation light. .
Therefore, as in the first aspect of the invention, at least two types of excitation light, the first excitation light and the second excitation light, are irradiated, and at least two of the first excitation digital image and the second excitation digital image. When a type of excitation digital image is taken, for example, a specific fluorescence reaction is taken in any excitation digital image, while in any excitation digital image and other excitation digital images, It is assumed that the shooting method differs depending on the strength. 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 foreign matters such as dust.
When at least two types of excitation digital images, i.e., the first excitation digital image and the second excitation digital image, are captured, for example, fluorescence due to foreign matter is captured in all the excitation digital images, It is assumed that the specific fluorescence reaction is clearly captured in any digital image upon excitation. In this case, by performing the above collation operation, it is possible to remove the influence of the fluorescent reaction due to the foreign matter, and to make the specific fluorescent reaction due to the microorganism or virus stand out and be detected. The fluorescence reaction caused by microorganisms or viruses can be clearly distinguished from the fluorescence reaction caused by foreign matters such as dust.

Next, in the second invention, as in the first invention, the first excitation light irradiation step, the first digital image acquisition step, the second excitation light irradiation step, and the second The specific biological component is detected by performing a digital image acquisition step, the image processing step, and the detection step, but the image processing step is different from that of the first invention.
That is, in the image processing step of the second invention, the reference color space data for each pixel unit of either the first excitation digital image or the second excitation digital image is predetermined. It is determined whether it is within the 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, for the other excitation digital image, the color space data for collation for each pixel unit is obtained only for the region corresponding to the temporary hit region. As a result, it is possible to reduce the processing for obtaining the matching color space data.
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.

  As described above, according to the specific biological component detection method of the present invention, the presence or absence of microorganisms or viruses in a 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. The result of having performed collation operation on the reference color space data in the temporary hit area and the color space data for collation in the area corresponding to the temporary hit area according to the second embodiment is schematically shown as a visualized image. Illustration.

Next, a preferred embodiment of the specific biological component detection method of the first and second inventions will be described.
In the specific biological component detection method, a specific microorganism or virus in the sample can be detected.
Examples of the microorganism include a parasite, a parasitic egg, and a bacterium. Examples of parasites include worms, roundworms, whipworms, flukes, worms, contracted tapeworms, broad-lined headworms, oriental hairy nematodes, and striped worms. Parasite eggs are those eggs.
Examples of bacteria include cocci, bacilli, and spiral bacteria.
In addition, as viruses, there are various viruses composed of nucleic acids and proteins.

  The sample can be stool, blood, or the like. The microorganism or virus may be present in stool, blood and the like. Parasites lurking in blood insects include, for example, Westermann lung fluke, Miyazaki lung fluke, schistospores, malaria pathogens, Japanese schistosomiasis, filamentous insects, filariae, and Guangdong schistosomes.

  In the first aspect of the invention, the first excitation light and the second excitation light may be further irradiated with excitation light while changing at least one of wavelength, intensity, and irradiation time. The surface of the sample irradiated with light is photographed, and the excitation light irradiation-digital image acquisition step of acquiring a digital image at excitation is performed at least once. In the image processing step, the first excitation digital image and the first excitation image are acquired. It is preferable to obtain the calculation processing data for at least one or more excitation digital images acquired in the excitation light irradiation-digital image acquisition step together with the two excitation excitation digital images.

  In the second aspect of the invention, the first excitation light and the second excitation light are further irradiated with excitation light by changing at least one of wavelength, intensity, and irradiation time, and the excitation light is irradiated. The surface of the sample is photographed and the excitation light irradiation-digital image acquisition process for acquiring the digital image at the excitation is performed at least once. In the image processing process, the digital image at the first excitation or the digital at the second excitation is performed. An area corresponding to the temporary hit area in the other one of the first excitation digital image and the second excitation digital image for the temporary hit pixel and the temporary hit area for any one of the images. And at least one or more of the excitation-time digital images acquired in the excitation light irradiation-digital image acquisition step. For each region, color space data for each pixel unit is obtained as matching color space data, the reference color space data for each pixel unit in the temporary hit region, and the above in the region corresponding to the temporary hit region. It is preferable that the pixel data in the same position is collated with the matching color space data for each pixel unit to obtain calculation processing data for each pixel unit in the temporary hit region. 6).

  As described above, by performing the excitation light irradiation-digital image acquisition step and performing the collation operation, it is possible to further eliminate the influence of the fluorescence reaction due to the foreign matter. That is, in this case, since the above collation operation can be performed on three or more types of excitation digital images respectively obtained by irradiating three or more types of excitation light, the influence of the fluorescence reaction due to foreign substances can be further removed. The specific fluorescence reaction caused by the microorganisms or viruses can be detected more clearly. Therefore, in this case, the specific fluorescence reaction caused by microorganisms or viruses can be more clearly distinguished from the fluorescence reaction caused by foreign substances.

Next, for the first invention, in the detection step, out of the calculation processing data for each pixel unit, calculation processing data obtained in advance for a specific fluorescence reaction that occurs when the specific microorganism or virus is excited. A pixel unit within a specified range is obtained as a hit pixel, and when the shape and area of the hit pixels formed side by side are within the specified range of the shape and area determined in advance for the specific fluorescence reaction, It is preferable to detect the presence of the specific microorganism or virus (claim 3).
In the second aspect of the invention, in the detection step, a specific fluorescence reaction that occurs during excitation of the specific microorganism or virus is calculated in advance in the calculation processing data for each pixel unit in the temporary hit region. When the pixel unit within the specified range of the arithmetic processing data is determined as a hit pixel, and the shape and area formed by the hit pixels are within the specified range of the shape and area determined in advance for the specific fluorescence reaction It is preferable to detect the presence of the specific microorganism or virus in the sample (claim 7).

  In these cases, in the detection step, for the sample in which the specific fluorescence reaction generated when the microorganism or virus is excited is detected, the shape and size of the specific fluorescence reaction is confirmed to detect the microorganism or virus. Can do. Therefore, the detection accuracy of microorganisms or viruses can be improved.

In the first and second aspects of the invention, the pixel region having a size surrounding the shape in which the hit pixels are formed side by side in the detection step is specified as a hit region, and the excitation light is not irradiated. It is preferable to perform a display step of acquiring an elementary digital image and enlarging and displaying on the monitor a pixel region corresponding to the hit region in the elementary digital image.
In this case, by enlarging and displaying on the monitor the pixel area where the presence of the specific fluorescence reaction is detected, it is possible to visually confirm the area where the microorganism or virus is detected in the elementary digital image.

The color space data is at least one of red, green, and blue hues and luminance, and the collation operation in the image processing step is performed on at least one of the hue and luminance. It is preferable that it is an error calculation or an arithmetic operation to be performed.
In this case, the arithmetic processing data for each image unit can be appropriately obtained in the image processing step.

Moreover, it is preferable that the wavelength of said 1st excitation light and said 2nd excitation light is a wavelength corresponding to either of UV excitation, B excitation, or G excitation, respectively (Claim 10).
In this case, microorganisms or viruses can be excited appropriately.
Here, UV excitation (near ultraviolet rays) can be excitation light having a wavelength of 320 to 380 nm. The B excitation (blue light) can be excitation light having a wavelength of 380 to 495 nm. The G excitation (green light) can be excitation light having a wavelength of 495 to 570 nm.
In addition, also when performing the said excitation light irradiation-digital image acquisition process, it is preferable that the wavelength of excitation light is a wavelength corresponded in any one of UV excitation, B excitation, or G excitation, respectively.

In addition, it is preferable that at least one of the first excitation light and the second excitation light is composed of a plurality of types of excitation light in which at least one of wavelength and intensity is different.
That is, it is preferable to simultaneously irradiate a plurality of types of excitation light having different wavelengths and / or intensities as at least one of the first excitation light and the second excitation light.
In this case, as at least one of the first excitation time digital image and the second excitation time digital image, a digital image when a plurality of types of excitation light having different wavelengths and intensities are simultaneously irradiated. Can be obtained. In this case, there may be a case where it is possible to obtain a specific fluorescence reaction that cannot be obtained simply by irradiating a microorganism or virus with excitation light having one wavelength and intensity. In some cases, the detection accuracy of microorganisms or viruses can be significantly improved. For example, in the case of a caterpillar (egg), the detection accuracy can be particularly improved.

Example 1
Next, examples according to the specific biological component detection method of the present invention will be described.
In the specific biological component detection method of this example, the first excitation light irradiation step, the first excitation digital image acquisition step, the second excitation light irradiation step, the second excitation digital image acquisition step, the image processing step, and the detection step, To detect specific microorganisms or viruses in the sample.

  In the first excitation light irradiation step, the sample is irradiated with the first excitation light at a specific wavelength, intensity, and irradiation time. In the first excitation digital image acquisition step, the surface of the sample irradiated with the first excitation light is photographed to acquire a first excitation digital image.

  In the second excitation light irradiation step, the sample is irradiated with second excitation light that differs from the first excitation light in at least one of wavelength, intensity, and irradiation time. In the second excitation digital image acquisition step, the surface of the sample irradiated with the second excitation light is photographed to acquire a second excitation digital image.

Further, in the image processing step, color space data for each pixel unit is obtained for at least the first excitation digital image and the second excitation digital image, and collation is performed between the pixel units at the same position. The calculation processing data is obtained every time.
In the detection process, the processing data for each pixel unit is checked against the specified range of the processing data obtained in advance for the specific fluorescence reaction that occurs when the specific microorganism or virus is excited, and the microorganism or virus in the sample is detected. To do.

  In this example, as shown in FIG. 1, using the fluorescence detection apparatus 1 including the light source 2, the camera 4, the control unit 51, the image processing unit 52, and the determination unit 53, A first excitation digital image acquisition step, a second excitation light irradiation step, a second excitation digital image acquisition step, an image processing step, and a detection step are performed.

In the fluorescence detection device 1, the light source 2 can irradiate the sample 8 with excitation light X having a specific 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 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 generated when the specific microorganism or virus is excited at the time of excitation light irradiation. It is configured to detect the presence or absence of a specific fluorescence reaction.

  The light source 2 and the control unit 51 perform the first excitation light irradiation process and the second excitation light irradiation process, and the camera 4 and the control unit 51 perform the first digital image acquisition process and the second digital image acquisition process. It can be carried out. Further, the image processing unit 52 and the determination unit 53 can perform the above-described image processing step and detection step.

Hereinafter, the fluorescence reaction detection device 1 of this example and the specific biological component detection method using the same will be described in detail with reference to FIGS.
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 by the digital zoom of the CMOS camera 41. be able to. In this example, a filter 44 for blocking the ultraviolet rays and protecting the camera 4 is disposed in front of the optical lens 42 of the camera 4.
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, the wavelengths of the excitation light X emitted from the light source 2 and the filter 3 are made different from each other, and two types of excitation light are respectively irradiated to the sample 8 (the first excitation light irradiation step and the first excitation light irradiation step and the first excitation light irradiation step). (2 excitation light irradiation process). And the digital image at the time of excitation (1st digital image at the time of excitation and 2nd digital image at the time of excitation) D1, D2 is image | photographed with the camera 4 (a 1st digital image acquisition process and a 2nd digital image acquisition process).
The two types of digital images D1 and D2 at the time of excitation differ in the wavelength of the excitation light X in the first excitation light irradiation step and the second excitation light irradiation step, and the intensity or irradiation time (imaging time) of the excitation light X is also mutually different. You can shoot differently. The two types of excitation digital images D1 and D2 can also be photographed with different intensities or irradiation times (imaging time) of the excitation light X in the first excitation light irradiation step and the second excitation light irradiation step. .

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 caused by foreign matter not detected such as dust and a specific fluorescence reaction 81 caused by microorganisms are photographed in both of the two types of excitation digital images D1 and D2. However, at least for the specific fluorescence reaction 81, it is assumed that the one of the excitation digital image D1 and the other excitation digital image D2 are photographed differently depending on the intensity of fluorescence. At this time, as shown in FIG. 4, when the color space data for each pixel unit for the two types of digital images D1 and D2 at the time of excitation is obtained and the collation operation is performed, the specific fluorescence reaction 81 caused by microorganisms or viruses is changed to dust or the like. It can be clearly distinguished from the fluorescence reaction 82 caused by foreign matter. In addition, the specific fluorescence reaction 81 shown with the dotted line in FIG. 2 means that the specific fluorescence reaction 81 shown with the continuous line shown in FIG. 3 is weaker.

  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 FIG. 2 and FIG. 3, the foreign substance exhibits a fluorescence reaction 82 even when irradiated with the excitation light X having a weak intensity or the excitation light X for a short period of time. Are assumed to show a clear specific fluorescent reaction 81 only after irradiation with excitation light X with high 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 for the two types of excitation digital images D1 and D2 is obtained and the collation operation is performed, the influence of the fluorescence reaction 82 due to the foreign matter is canceled, 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 a microorganism or the like exhibits a fluorescent reaction is later than that of a foreign substance.

The image processing unit 52 obtains color space data for each pixel unit at least for each of the first excitation digital image and the second excitation digital image, and performs a collation operation between the pixel units at the same position. An image processing step for obtaining calculation processing data for each unit can be performed. The color space data obtained here indicates the hue data of red (R), green (G), and blue (B), which are the three primary colors, as 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.
Note that depending on the detection target of the microorganism or virus, 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, and B1 + B2, and at least one of R1-R2, G1-G2, and B1-B2, at least one of R1 × R2, G1 × G2, and 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 be performed as any one of Y1 + Y2, Y1-Y2, Y1 × Y2, Y1 / Y2, Y2-Y1, and 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.

In the determination unit 53, the calculation processing data for each pixel unit is checked against the specified range of the calculation processing data obtained in advance for the specific fluorescence reaction that occurs when the specific microorganism or virus is excited, A detection step of detecting a virus can be performed.
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 whether the arithmetic processing data obtained by collating the first color space data d1 and the second color space data d2 (see FIG. 5) in the pixel unit for each dot is within the specified range. The result of the binarization process is visualized and shown.
The determination unit 53 of this example hits a pixel unit within the specified range of the calculation processing data obtained in advance for the specific fluorescence reaction 81 generated when the specific microorganism or virus is excited among the calculation processing data for each pixel unit. Obtained as pixel H. Then, when the shape and area in which the hit pixels H are formed side by side are within the specified 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.

  In addition, for each pixel in the image processing unit 52, when | R1-R2 |, | G1-G2 |, | B1-B2 | 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 perform a display process of enlarging display.
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, it is possible to actually confirm visually the region 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, that is, a method for detecting a specific biological component will be described with reference to the 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 (first excitation light irradiation step; step S1 in the figure). And after predetermined time passes, the surface of the sample 8 is image | photographed with the camera 4 (1st digital image acquisition process; 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 digital image D1 at the time of excitation that has been taken is captured and stored in the image processing unit 52 (see FIG. 1) of the computer 5.

  Next, the sample 8 is irradiated with the second excitation light X having at least one of the wavelength and intensity different from the first excitation light X from the light source 2 (second excitation light irradiation step; S3). And after predetermined time passes, the surface of the sample 8 is image | photographed with the camera 4 (2nd digital image acquisition process; 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, the image processing steps (S5 and S6) are performed as follows.
Specifically, first, 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 operation or arithmetic operation 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 to obtain operation processing data for each pixel unit (S6). ).

Next, a detection process (S7-S11) is performed as follows.
Specifically, first, calculation processing data for each pixel unit is within a specified range of calculation processing data obtained in advance for fluorescence (specific fluorescence reaction 81; see FIG. 3) generated by a specific microorganism or virus to be detected. (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 specific biological component detection method 1 of this example, as described above, the first irradiation step (S1), the first digital image acquisition step (S2), the second excitation light irradiation step (S3), and the second At least a digital image acquisition step (S4), an image processing step (S5 and S6), and a detection step (S7 to S11) are performed (see FIG. 9).
In the first irradiation step, the sample 8 is irradiated with the first excitation light X at a specific wavelength, intensity, and irradiation time (see FIG. 1). Next, in the first digital image acquisition step, the surface of the sample 8 irradiated with the first excitation light X is photographed by the camera 4 to obtain a first excitation digital image D1 (see FIG. 2). In this example, foreign matter such as dust, which is a component that emits fluorescence when excited by the first excitation light, is present in the sample 8, and thus the foreign matter shows a fluorescence reaction 82 in the first excitation digital image D <b> 1 (FIG. 2). In the first excitation digital image D1, microorganisms or viruses are excited by the first excitation light, and these show a weak specific fluorescence reaction 81.

On the other hand, in the second irradiation step, the sample 8 is irradiated with the second excitation light X composed of excitation light different from the first excitation light in at least one of wavelength, intensity, and irradiation time. Next, in the second digital image acquisition step, the surface of the sample 8 irradiated with the second excitation light X is photographed by the camera 4, and a second excitation digital image D2 is acquired. Similar to the first digital image acquisition step, the fluorescence state of the sample 8 can be acquired as a digital image by photographing the surface of the sample 8. Since at least one of the wavelength, intensity, and irradiation time of the second excitation light X is different from that of the first excitation light, the sample 8 emits fluorescence in a pattern different from that of the first excitation light. In this example, since there is a foreign substance that emits fluorescence not only by the first excitation light but also by the second excitation light X in the sample 8, these foreign substances also show a fluorescence reaction 82 in the second excitation digital image D2. (See FIG. 3). Further, in the second excitation digital image D2, microorganisms or viruses are excited by the second excitation light, and these show a strong specific fluorescence reaction 81.
Thus, the first excitation digital image D1 and the second excitation digital image D2 show different fluorescence states because the excitation light is different (see FIGS. 2 and 3).

  In the image processing step, color space data d1 and d2 for each pixel unit are obtained for the first excitation digital image D1 and the second excitation digital image D2. Then, the color space data d1 for each pixel unit in the first excitation digital image D1 and the color space data d2 for each pixel unit in the second excitation digital image D2 are collated with each other at the same position. Calculation is performed to obtain calculation processing data obtained by performing error calculation or arithmetic calculation for each pixel unit.

  Next, in the detection step, the calculation processing data for each pixel unit is collated with a specified range of calculation processing data obtained in advance for the specific fluorescence reaction 81 generated when a specific microorganism or virus is excited. If the calculation processing data for each pixel unit is within the specified range of the calculation processing data obtained for the specific fluorescence reaction 81 in advance, it is determined that there is a specific fluorescence reaction 81 generated by microorganisms or the like in the sample 8; Microorganisms can be detected.

Incidentally, in general, the sample 8 contains foreign matters such as dust in addition to microorganisms or viruses, and the foreign matter also generates a fluorescent reaction 82 by the excitation light X (see FIGS. 2 and 3). 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, as in this example, when two types of excitation digital images D1 and D2 of a first excitation digital image and a second excitation digital image are taken, for example, the specific fluorescence reaction 81 is generated in any excitation digital image. D1 and D2 are also photographed, while either excitation digital image D1 (D2) and other excitation digital image D2 (D1) are photographed depending on the intensity of fluorescence (specific fluorescence reaction 81) Are assumed to be different (see FIGS. 2 and 3). 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 foreign matters such as dust.
Therefore, according to the specific biological component detection method of this example, the presence or absence of microorganisms or viruses in the sample can be stably detected with high detection accuracy.

(Example 2)
This example is an example in which the processes in the image processing step and the detection step are different from those in the first embodiment.
The image processing process of this 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 matching operation only on this pixel area. It is.

  Also in this example, similarly to Example 1, the fluorescence detection apparatus 1 is used to perform the first excitation light irradiation step, the first digital image acquisition step, the second excitation light irradiation step, and the second digital image acquisition step. Thus, two types of excitation digital images (first excitation digital image D1 and second excitation digital image D2) when two types of excitation light X are irradiated are taken by the camera 4 (FIGS. 1, 10, and 10). And FIG. 11).

  In the image processing step of this example, the image processing unit 52 of the fluorescence detection device 1 converts the first color space data (reference color space data) d1 for each pixel unit of the first excitation digital image D1. Ask. Then, among the reference color space data d1 for each pixel unit, a group of pixel units within a predetermined specified range is obtained as a temporary hit pixel H ′, and a pixel area having a size surrounding the temporary hit pixel H ′ is temporarily determined. The hit area A ′ is specified.

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 specified range can be the specified range of the reference color space data d1 obtained for the specific fluorescence reaction 81 in advance.

In the image processing step, second color space data (collation color space data) for each pixel unit of only the area A ′ corresponding to the temporary hit area A ′ in the second excitation digital image D2. d2 is obtained.
FIG. 11 schematically shows a region A ′ corresponding to the temporary hit region A ′ in the second excitation digital image D2. In the image processing step, the reference color space data d1 for each pixel unit in the temporary hit area A ′ and the matching color space data d2 for each pixel unit in the area corresponding to the temporary hit area A ′ are used. On the other hand, comparison processing is performed between pixel units 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 reference color space data d1 in the temporary hit area A ′ and the color space data d2 for collation 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 reference color space data d1 and the collation color space data d2 can be an error operation or an arithmetic operation as in the first embodiment.
In the detection process of this example, the determination unit 53 of the fluorescence detection device 1 is 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 in the temporary hit area A ′. Is determined as a hit pixel H. 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 the microorganism or virus can be clearly distinguished from the fluorescence reaction caused by other dust (foreign matter not detected).

In this example, for the second excitation-time digital image D2, the color space data for comparison d2 for each pixel unit is obtained for only the area corresponding to the temporary hit area A ′. As a result, it is possible to reduce the processing for obtaining the matching color space data d2.
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 (11)

A specific biological component detection method for detecting the specific microorganism or virus by detecting a specific fluorescence reaction generated when a specific microorganism or virus in a sample is excited,
A first excitation light irradiation step of irradiating the sample with the first excitation light at a specific wavelength, intensity, and irradiation time;
A first digital image obtaining step of photographing the surface of the sample irradiated with the first excitation light and obtaining a first excitation digital image;
A second excitation light irradiation step of irradiating the sample with a second excitation light that is different from the first excitation light in at least one of wavelength, intensity, and irradiation time;
A second digital image acquisition step of photographing the surface of the sample irradiated with the second excitation light and acquiring a second excitation digital image;
For at least the first excitation digital image and the second excitation digital image, color space data for each pixel unit is obtained, collation is performed between pixel units at the same position, and calculation processing data is obtained for each pixel unit. The desired image processing process;
Detection for detecting microorganisms or viruses in the sample by comparing the calculation processing data for each pixel unit with a specified range of calculation processing data obtained in advance for the specific fluorescence reaction generated when the specific microorganism or virus is excited. And a specific biological component detection method comprising the steps of:   2. The specific biological component detection method according to claim 1, wherein the first excitation light and the second excitation light are further subjected to excitation light by changing at least one of a wavelength, intensity, and irradiation time with respect to the sample. Irradiation, imaging of the surface of the sample irradiated with the excitation light, and at least one excitation light irradiation-digital image acquisition step of acquiring a digital image at the time of excitation are performed. In the image processing step, at the time of the first excitation The specific biological component detection characterized in that the arithmetic processing data is obtained for at least one or more excitation digital images acquired in the excitation light irradiation-digital image acquisition step together with the digital image and the second excitation digital image. Method.   3. The specific biological component detection method according to claim 1, wherein in the detection step, the specific fluorescence reaction that occurs when the specific microorganism or virus is excited is calculated in advance in the calculation processing data for each pixel unit. When the pixel unit within the specified range of the arithmetic processing data is determined as a hit pixel, and the shape and area formed by the hit pixels are within the specified range of the shape and area determined in advance for the specific fluorescence reaction Detecting the presence of the specific microorganism or virus in the sample. The specific biological component detection method according to claim 3, wherein a pixel region having a size surrounding a shape in which the hit pixels are formed side by side in the detection step is specified as a hit region,
A specific biological component detection method comprising: performing a display step of acquiring an elementary digital image when the excitation light is not irradiated and enlarging and displaying a pixel region corresponding to the hit region in the elementary digital image on a monitor . A specific biological component detection method for detecting the specific microorganism or virus by detecting a specific fluorescence reaction generated when a specific microorganism or virus in a sample is excited,
A first excitation light irradiation step of irradiating the sample with the first excitation light at a specific wavelength, intensity, and irradiation time;
A first digital image obtaining step of photographing the surface of the sample irradiated with the first excitation light and obtaining a first excitation digital image;
A second excitation light irradiation step of irradiating the sample with a second excitation light that is different from the first excitation light in at least one of wavelength, intensity, and irradiation time;
A second digital image acquisition step of photographing the surface of the sample irradiated with the second excitation light and acquiring a second excitation digital image;
For either one of the first excitation time digital image or the second excitation time digital image, color space data for each pixel unit is obtained as reference color space data, and among the reference color space data, a predetermined color space data is determined. A group of pixel units within a specified range is 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 the first excitation digital image or the second excitation digital For only the region corresponding to the temporary hit region in the other of the images, color space data for each pixel unit is obtained as color space data for comparison, and the reference color space data for each pixel unit in the temporary hit region And the matching color space data for each pixel unit in the region corresponding to the temporary hit region, the pixel units at the same position And disjunction, and an image processing step of determining the operation processing data for each of the pixel unit of the temporary hit region,
The calculation processing data for each pixel unit in the temporary hit region is compared with a specified range of calculation processing data obtained in advance for the specific fluorescence reaction that occurs when the specific microorganism or virus is excited, in the sample. And a specific biological component detection method comprising a detection step of detecting a microorganism or a virus.   The specific biological component detection method according to claim 5, wherein the first excitation light and the second excitation light are further irradiated with excitation light by changing at least one of wavelength, intensity, and irradiation time, and the excitation light The excitation light irradiation-digital image acquisition process of acquiring the digital image at the time of excitation is performed at least once by photographing the surface of the sample irradiated with the above, and in the image processing process, the digital image at the time of the first excitation or the second image is acquired. The temporary hit pixel and the temporary hit area are obtained for any one of the excitation-time digital images, and the temporary hit area in the other of the first excitation-time digital image or the second excitation-time digital image is determined. The temporary hit in the corresponding region and at least one or more excitation-time digital images acquired in the excitation light irradiation-digital image acquisition step. For the area corresponding to the area, the color space data for each pixel unit is obtained as the color space data for comparison, and the reference color space data for each pixel unit in the temporary hit area and the temporary hit area are equivalent. The collation color space data for each pixel unit in the region is collated with pixel units at the same position, and calculation processing data is obtained for each pixel unit in the temporary hit region. A specific biological component detection method as a feature.   7. The specific biological component detection method according to claim 5, wherein in the detection step, the specific microorganism or virus is generated at the time of excitation among the arithmetic processing data for each pixel unit in the temporary hit region. The pixel unit within the specified range of the calculation processing data obtained in advance for the specific fluorescence reaction is obtained as a hit pixel, and the shape and area formed by arranging the hit pixels in advance are defined in advance for the specific fluorescence reaction. A method for detecting a specific organism, characterized in that when it is within a range, it is detected that the specific microorganism or virus is present in the sample. The specific biological component detection method according to claim 7, wherein a pixel region having a size surrounding a shape in which the hit pixels are formed side by side in the detection step is specified as a hit region,
A specific biological component detection method comprising: performing a display step of acquiring an elementary digital image when the excitation light is not irradiated and enlarging and displaying a pixel region corresponding to the hit region in the elementary digital image on a monitor . The specific biological component detection method according to any one of claims 1 to 8, wherein the color space data is at least one of red, green, blue hue, and luminance,
The specific biological component detection method, wherein the collation operation in the image processing step is an error operation or an arithmetic operation performed on at least one of the hue and luminance.   10. The specific biological component detection method according to claim 1, wherein the wavelengths of the first excitation light and the second excitation light are any of UV excitation, B excitation, and G excitation, respectively. The specific biological component detection method, wherein the wavelength corresponds to   The specific biological component detection method according to any one of claims 1 to 10, wherein at least one of the first excitation light and the second excitation light is a plurality in which at least one of wavelength and intensity is different. A method for detecting a specific biological component, comprising a kind of excitation light.

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