WO2014156797A1 - Detection device and detection method - Google Patents

Abstract

A detection device (100) is provided with: a securing unit (20) for securing a collection substrate (200) that can collect on the surface thereof particles in a sample; an excitation light source (30) for irradiating the surface of the secured collection substrate with excitation light; an imaging unit (40) that includes a plurality of sensors, arranged in two dimensions, for imaging at least part of the area of the surface of the secured collection substrate for an imaging range; a heating unit (50) for heating the collection substrate; and a calculation unit (101) for calculating the amount of particles of biological origin out of the particles collected on the surface of the collection substrate using an image that is imaged after heating.

Description

Detection apparatus and detection method

The present invention relates to a detection apparatus and a detection method, and more particularly to a detection apparatus and a detection method for detecting biological particles in a specimen.

As an apparatus for counting microorganisms in a sample such as the atmosphere, there is an apparatus for measuring fluorescence derived from microorganisms (very weak autofluorescence emitted by microorganisms itself or fluorescence by a fluorescent reagent that acts specifically on microorganisms).

For example, Japanese Patent Application Laid-Open No. 2008-187935 (hereinafter referred to as Patent Document 1) collects microorganisms, stains them with a fluorescent staining reagent, obtains a fluorescent image, and determines the microorganisms (viable bacteria / Disclosed is a microbe counting apparatus for discriminating between dead cells) and contaminants.

JP 2008-187935 A Japanese Patent Laid-Open No. 3-43069 JP 2003-38163 A

However, when the number of microorganisms is accurately counted one by one, or when the number of microorganisms is small and the concentration is low, the amount of fluorescence becomes weak, making detection difficult. Therefore, an expensive detector such as a photomultiplier tube, a sensor with an electron multiplying function, or a cooled CCD (Charge Coupled Device Image Sensor) is required. In addition, because it is necessary to separate biological particles from substances other than biological particles that emit fluorescence such as chemical fibers that exist in the air, it is possible to detect scattered light (size information) from microorganisms at the same time. An apparatus for separation is required. Therefore, there is a problem that the configuration of the apparatus becomes complicated and the cost becomes high.

In order to cope with the above-mentioned problems, a technique using a fluorescent reagent which is disclosed in the above-mentioned Patent Document 1 and acts specifically on microorganisms can be employed. However, the technique of Patent Document 1 requires a fluorescent reagent to act on a specimen before measurement, and there is a problem that complicated work is required before measurement. In particular, when the amount of microorganisms in the sample is a very small amount, there is a problem that the measurement becomes more complicated because the operation of reliably acting the fluorescent reagent is important. In addition, when the fluorescent reagent is expensive, there is a problem that the measurement is expensive.

In addition, since the technique of the said patent document 1 was assumed that the test substance is a liquid, when trying to employ | adopt the technique of the said patent document 1 when the test substance is air | atmosphere, such as air, air | atmosphere was collected. Thereafter, there is also a problem that a complicated operation before measurement, such as dispersion in a test solution, mixing with a fluorescent reagent, and coating on a measurement substrate, is required.

The present invention has been made in view of such a problem, and an object of the present invention is to provide a detection apparatus and a detection method capable of easily detecting biologically-derived particles in a specimen.

In order to achieve the above object, according to one aspect of the present invention, a detection device includes a fixing means for fixing a collection substrate capable of collecting particles in a specimen on a surface, and a trap fixed by the fixing means. A light source for irradiating excitation light onto the surface of the collecting substrate and a plurality of two-dimensionally arranged images for photographing at least a partial area of the surface of the collecting substrate fixed by the fixing means as an imaging range An imaging means including the sensor, a heating means for heating the collection substrate, and a living organism among the particles collected on the surface of the collection substrate using a photographed image obtained by the imaging means after being heated by the heating means. And a calculating means for calculating the amount of the derived particles.

Preferably, the calculation means calculates the amount of biologically derived particles among the particles collected on the surface of the collection substrate by analyzing a photographed image obtained by the photographing means after being heated by the heating means.

Preferably, the calculation unit analyzes the captured image captured by the imaging unit in a state where the collection substrate is irradiated with the excitation light from the light source to obtain the luminance value of each pixel, and the heating unit acquires the luminance value of the captured image before heating. Based on the change between the luminance value of each pixel and the luminance value of each pixel of the captured image after being heated by the heating means, the biological particles collected on the surface of the collection substrate are discriminated and the amount is calculated. To do.

More preferably, the detection device further includes a storage unit for storing a pixel that is determined by the calculation unit as having a biological particle in the photographed image, and the calculation unit includes the biological particle from the photographed image. Based on the difference between the pixel determined to be present and the pixel stored in the storage means, the amount of biologically-derived particles among the particles collected on the surface of the collection substrate is calculated.

Preferably, the light source emits light in a wavelength region that can excite biological particles.

More preferably, the light source is a semiconductor laser.
Preferably, the light source emits light having a wavelength in the range of 300 nm to 450 nm.

According to another aspect of the present invention, a detection method includes a step of heating a collection substrate capable of collecting particles in a specimen on a surface, and excitation light from a light source on the surface of the collection substrate after heating. A plurality of sensors arranged in a two-dimensional manner with at least a partial region of the surface of the collection substrate as an imaging range in a state where the irradiation substrate and the collection substrate after heating are irradiated with excitation light from a light source A step of photographing using the photographing means, and a step of calculating the amount of biologically-derived particles among the particles collected on the surface of the collection substrate using a photographed image by the photographing means.

According to the present invention, it is possible to easily detect organism-derived particles in a specimen.

It is a block diagram showing the schematic structure of the detection apparatus concerning embodiment. It is a figure showing arrangement | positioning of the optical system contained in a detection apparatus. It is a figure showing the other example of arrangement | positioning of an optical system. It is a figure showing the outline | summary of the detection of the particle | grains (microorganism) derived from the living body in a detection apparatus. It is a figure showing the measurement result of the fluorescence spectrum before and behind heat processing when the green mold | fungi microbe as biological origin particle | grains are heat-processed at 200 degreeC for 5 minute (s). It is the fluorescence image which detected the blue mold | fungi as biological-derived particle | grains before heat processing with the detection apparatus concerning embodiment. It is the fluorescence image which detected the blue mold | fungi microbe as a biological-derived particle | grains after heat processing with the detection apparatus concerning embodiment. It is a figure showing the measurement result of the fluorescence spectrum of the dust which emits the fluorescence before heat processing. It is a figure showing the measurement result of the fluorescence spectrum after heat-treating the dust which emits fluorescence for 5 minutes at 200 degreeC. It is the fluorescence image which detected the dust which emits the fluorescence before heat processing with the detection apparatus concerning embodiment. It is the fluorescence image which detected the dust which emits the fluorescence after heat processing with the detection apparatus concerning embodiment. It is a figure showing the flow of operation | movement for detecting the particle | grains derived from a biological body among the particles collected on the surface of the collection board | substrate using the detection apparatus. It is a figure for demonstrating the calculation process in step S33 of FIG. It is a figure for demonstrating the calculation process in step S33 of FIG. It is a figure for demonstrating the calculation process in step S33 of FIG. It is a figure for demonstrating the calculation process in step S33 of FIG. It is a block diagram which shows the specific example of a function structure of the control part of a detection apparatus. It is a flowchart showing the flow of the separation process in step S31 of FIG. It is a flowchart showing the flow of the calculation process in step S33 of FIG. It is a figure which shows the fluorescence image of the blue mold | fungi after heat processing. It is a figure which shows the line profile showing the luminance value of the horizontal direction of the luminescent point of the blue mold after heat processing. It is a figure showing the image (binarized image) which performed the binarization process with respect to the fluorescence image of FIG. 16A. It is the figure showing the area | region determined to originate in the particle | grains derived from a living organism | raw_food based on the area (the number of pixels) and the shape of the bright spot of the image which binarized the fluorescence image of FIG. 16A. It is a figure for demonstrating the state of the collected particle | grains at the time of using the same collection board | substrate 200 for n times of collection operation | movement. It is a flowchart showing the modification of the flow of the separation process in FIG.8 S31.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following description, the same parts and components are denoted by the same reference numerals. Their names and functions are also the same. Therefore, these descriptions will not be repeated.

[First Embodiment]
<Device configuration>
FIG. 1 is a block diagram showing a schematic configuration of a detection apparatus 100 according to the present embodiment. Referring to FIG. 1, a detection apparatus 100 detects a control unit 10 for controlling the entire apparatus, and a collection substrate 200 such as a culture medium or a plastic substrate that can collect particles in a specimen on the surface thereof. The fixing unit 20 is fixed to a predetermined position in the apparatus 100, the excitation light source 30 is used to irradiate the surface of the collection substrate 200 fixed by the fixing unit 20, and the fixing unit 20 is fixed. The imaging unit 40 for imaging at least a partial region of the surface of the collection substrate 200 as an imaging range, and the heating unit 50 including a heater (not shown) for heating the collection substrate 200 are included. The control unit 10 analyzes the captured image obtained by the imaging unit 40, thereby calculating a biological unit-derived particle amount among the particles collected in the imaging range on the surface of the collection substrate 200. including.

The imaging unit 40 includes an area sensor in which image sensors such as a CCD (Charge Coupled Device) image sensor and a CMOS (Complementary Metal Oxide Semiconductor) image sensor are arranged two-dimensionally, and acquires a two-dimensional captured image. The imaging unit 40 is electrically connected to the control unit 10 and performs an imaging operation according to the control signal. Then, the captured image obtained by the imaging operation is input from the imaging unit 40 to the control unit 10.

The excitation light source 30 is electrically connected to the control unit 10 and emits and extinguishes according to the control signal. The excitation light source 30 corresponds to a laser such as a semiconductor laser, an LED (Light Emitting Diode) element, or the like. The wavelength may be in the ultraviolet or visible region as long as it excites biological particles and emits fluorescence. Preferably, the wavelength is from 300 nm to 450 nm at which fluorescent tryptophan, NaDH, riboflavin and the like are efficiently excited.

The optical system that is the excitation light source 30 and the imaging unit 40 may be further provided with a lens and a filter (FIG. 2).

The collection mechanism for collecting particles in the specimen on the surface of the collection substrate 200 may or may not be included in the detection device 100. Therefore, the collection method may be any method. For example, a sample such as outside air may be introduced into a collection device (not shown) on which the collection substrate 200 is set, and particles that fall naturally may be collected on the surface of the collection substrate 200. Alternatively, the surface of the collection substrate 200 is connected to the electrode, and the charged particles in the specimen are collected on the surface of the collection substrate 200 using the electrostatic force generated by the potential difference with the other electrode. It may be. Of course, it is not limited only to these methods. And the fixing | fixed part 20 is fixed so that the collection board | substrate 200 may face the defined direction. The “fixing” includes a case where the collection substrate 200 is loaded at a specific position. The configuration is not limited to a specific configuration.

The heating unit 50 is electrically connected to the control unit 10, and the heating amount (heating time, heating temperature, etc.) is controlled according to the control signal. The heating unit 50 includes a heater such as a ceramic heater, a far infrared heater, and a far infrared lamp.

2A and 2B are schematic diagrams for explaining the arrangement of the optical system included in the detection apparatus 100. FIG. 2A shows the arrangement of the optical system included in the detection apparatus 100, and FIG. 2B shows the comparison. An example of the arrangement is shown. Preferably, as shown in FIGS. 2A and 2B, a light source lens 31 for adjusting the irradiation direction is arranged in front of the irradiation direction of the excitation light source 30, and fluorescence is arranged in front of the imaging direction of the imaging unit 40. A detection lens 41 and a filter 42 are arranged.

Referring to FIG. 2A, the excitation light source 30 and the imaging unit 40 are arranged on the oblique axis with respect to the surface of the collection substrate 200. That is, the excitation light source 30 and the imaging unit 40 have an angle αA formed by the irradiation direction from the excitation light source 30 with respect to the normal direction of the surface of the collection substrate 200 fixed by the fixing unit 20 and the above method. The angle αB formed by the photographing direction in the photographing unit 40 is different from the line direction. Specifically, referring to FIG. 2A, when the imaging unit 40 is arranged (directly above) (αB = 0) so that the imaging direction coincides with the normal direction of the surface of the collection substrate 200 (αB = 0). The excitation light source 30 is arranged at a position where the irradiation direction does not coincide with the normal direction of the surface of the collection substrate 200 (αA ≠ 0).

On the other hand, FIG. 2B shows a specific example when the excitation light source 30 and the imaging unit 40 are arranged coaxially with respect to the surface of the collection substrate 200. That is, the angle αA formed by the irradiation direction from the excitation light source 30 with respect to the normal direction of the surface of the collection substrate 200 fixed by the fixing unit 20 between the excitation light source 30 and the imaging unit 40, and the above method An example is shown in which the angle αB formed by the photographing direction in the photographing unit 40 coincides with the line direction. In the case of FIG. 2B, all the angles are 0, that is, they are arranged (directly above) so as to coincide with the normal direction of the surface of the collection substrate 200. In order to arrange excitation light and fluorescence having different wavelengths in this way, it is preferable to arrange a wavelength selective mirror 43 such as a dichroic mirror at a position where the main line of the excitation light optical path and the fluorescent light path main line intersect.

When the excitation light source 30 and the imaging unit 40 are arranged coaxially with respect to the collection substrate 200 as shown in FIG. 2B, the excitation light from the excitation light source 30 is partially reflected on the surface of the collection substrate 200, and the light is reflected. If the filter 42 and the wavelength selective mirror 43 cannot cut the light, the light enters the imaging unit 40. When the imaging unit 40 receives the reflected light, the detection background (background value) increases (the S / N ratio decreases), leading to a decrease in detection accuracy. Therefore, as shown in FIG. 2A, preferably, the excitation light source 30 and the imaging unit 40 capture the excitation light from the excitation light source 30 reflected on the surface of the collection substrate 200 by the imaging unit 40. Arranged in a positional relationship that does not match the direction. Thereby, the influence on the detection of the reflected light of excitation light can be suppressed, and detection accuracy can be improved.

<Overview of operation>
(Detection principle)
FIG. 3 is a diagram showing an outline of detection of biological particles by the detection apparatus 100. The biologically-derived particles include molds such as mold fungi exemplified in the following description, microorganisms such as bacteria, and yeasts and pollen. Referring to FIG. 3, when excitation light source 30 emits light according to a control signal from control unit 10, excitation light is irradiated on the surface of collection substrate 200 from the arrangement. The imaging unit 40 performs an imaging operation in accordance with a control signal from the control unit 10, so that at least one region of the surface of the collection substrate 200 that is irradiated with excitation light from the excitation light source 30. The part is taken as the shooting range. When biologically derived particles are present in the imaging range, the particles are excited by excitation light to emit fluorescence, and the fluorescence is imaged by the imaging unit 40.

FIG. 4 shows measurement results of fluorescence spectra before and after the heat treatment (curve 76) when the green mold as biological particles was heat-treated at 200 ° C. for 5 minutes. 5A and 5B are fluorescence images of green mold before and after heating photographed using the detection apparatus 100. FIG. Specifically, FIG. 5A is a fluorescence image before the heat treatment, and FIG. 5B is a fluorescence image after the heat treatment. From the measurement results shown in FIG. 4, it can be seen that the fluorescence intensity from the biological particles is significantly increased by the heat treatment. In addition, comparing the fluorescence images before and after the heat treatment shown in FIGS. 5A and 5B also shows that the fluorescence intensity from the biological particles is significantly increased by the heat treatment.

In contrast, FIG. 6A and FIG. 6B show the measurement of fluorescence spectra before and after heat treatment (curve 78) when the fluorescent dust is heat treated at 200 ° C. for 5 minutes, respectively. It is a result. 7A and 7B are fluorescence images of dust that emits fluorescence before and after heating photographed using the detection apparatus 100. FIG. Specifically, FIG. 7A is a fluorescence image before the heat treatment, and FIG. 7B is a fluorescence image after the heat treatment. With reference to FIG. 6A and FIG. 6B, these measurement results are substantially the same, and it can be seen that the fluorescence intensity from the dust emitting fluorescence does not change before and after the heat treatment. Further, comparing the fluorescence images before and after the heat treatment shown in FIGS. 7A and 7B also shows that the fluorescence intensity from the dust that emits fluorescence does not change before and after the heat treatment.

Using this principle, the detection apparatus 100 detects biologically derived particles among the particles on the surface of the collection substrate 200 based on at least the fluorescence intensity after heating. As shown in FIG. 4 to FIG. 7B, there is a large difference in fluorescence intensity after heating between biologically derived particles and non-biologically derived particles. That is, as shown in FIGS. 5A and 7A, before the heating, both the fluorescence intensity from the biological particles and the fluorescence intensity from the non-biological particles are small, and the difference cannot be distinguished in the fluorescence image. Degree. On the other hand, after heating, as shown in FIG. 5B and FIG. 7B, the fluorescence intensity from the biologically-derived particles is greatly increased, and no change is observed in the fluorescence intensity from the non-biologically-derived particles. That is, there is a marked difference in the change in fluorescence intensity between before and after heating between biologically derived particles and non-biologically derived particles. Therefore, it is possible to identify and detect biologically derived particles from the absolute value of the fluorescence intensity after heating. Preferably, biological particles among the particles on the surface of the collection substrate 200 are detected based on the difference in fluorescence intensity before and after heating. By utilizing this principle, the detection apparatus 100 can detect the particles collected on the surface of the collection substrate 200 by separating biological particles more accurately from non-biological particles. It becomes. Further, at that time, the detection apparatus 100 can detect without performing processing with a fluorescent staining reagent.

(Detection operation)
FIG. 8 is a diagram illustrating a flow of operations for detecting biologically derived particles among particles collected on the surface of the collection substrate 200 using the detection apparatus 100. Referring to FIG. 8, first, an operation for collecting particles in the specimen is performed using collection substrate 200 (step S <b> 1), and particles in the specimen are collected on the surface of collection substrate 200. The However, the operation of step S1 may be performed within the detection device or may be performed outside the detection device. Next, the collection substrate 200 is fixed to the detection device 100 by the fixing unit 20 (step S3).

In a state where the collection substrate 200 is fixed by the fixing unit 20, the surface thereof is imaged by the imaging unit 40, and the fluorescence image F1 is acquired (step S5). Specifically, in this operation, first, excitation light is applied to the surface of the collection substrate 200 fixed from the excitation light source 30 (step S21). The particles collected on the surface of the collection substrate 200 are excited and irradiated with excitation light to emit fluorescence. The fluorescence is received by the imaging unit 40 (step S23), and the image data is acquired (step S25). The image data acquired here includes information on the luminance value of each pixel.

After capturing in step S5, the collection substrate 200 is heated by the heating unit 50, whereby the particles collected on the surface are heated (step S7). Thereafter, in the same manner as in step S5, a fluorescent image F2 of the surface of the collection substrate 200 after heating is acquired (step S9). Then, the amount of microorganisms is calculated using at least the fluorescence image F2 after heating, preferably the fluorescence images F1 and F2 before and after heating (step S11).

The calculation process in step S11 is largely separated from the separation process for separating whether or not biological-derived particles are included in the fluorescence image (step S31), and is separated if biological-derived particles are included. And a calculation process (step S33) for calculating the amount of microorganisms based on the fluorescence image.

Specifically, in step S31, the absolute value of the fluorescence intensity of the fluorescence image F2 is captured by determining whether or not it falls within the previously stored range of the brightness value after heating the biological particle. It is determined whether or not organism-derived particles are included in the imaging range on the surface of the current collecting substrate 200. Preferably, the fluorescent images F1 and F2 are compared for each pixel, the luminance value changes for the same pixel, and the change degree of the luminance value is stored in advance before and after the heating of the biological particles. By determining whether or not the degree of change in luminance value is satisfied, it is determined whether or not organism-derived particles are included in the imaging range on the surface of the collection substrate 200. The degree of change in luminance value before and after the heating of the biological particles is obtained by conducting an experiment or the like in advance and stored in a memory (not shown) of the control unit 10.

9 to 12 are diagrams for explaining the calculation process in step S33. FIG. 9 is a diagram schematically showing the fluorescence image F2 that is determined to contain biological particles, and FIG. 10 is image data that schematically shows luminance information for each pixel of the fluorescence image of FIG. This is a specific example. In step S33, image data is binarized by applying a prescribed threshold value to the luminance information for each pixel. FIG. 11 shows the result of binarizing the image data of FIG. Prior to the binarization processing, the image data may be reduced in bit.

A bright spot is specified from the binarized image data, and it is determined whether or not the bright spot is derived from a biological particle based on its shape. Here, the number and shape of pixels constituting one bright spot caused by biological particles are stored in advance, and compared with the bright spot specified from the binarized image data, It is discriminated whether or not the bright spot is caused by a biological particle.

FIG. 12 shows bright spots included in the binarized image data of FIG. 11, and regions R1 to R4 surrounded by a thick frame in FIG. 12 correspond to bright spots. When the number of pixels constituting one luminescent spot due to biological particles is stored as 6 and the shape is stored as a square, among the regions R1 to R4 in FIG. 12, the regions R1 and R2 are biological particles. It is determined that it is a bright spot caused by. The other regions R3 and R4 are excluded from the subsequent calculation processing as electrical noise and stray light components. Then, by counting the bright spots determined to be caused by the biological particles, the biological particle amount (number of particles) is calculated. In addition, the range of the number of pixels and the shape constituting one luminescent spot caused by biological particles is stored, and when the luminescent spot specified from the binarized image data satisfies the range, the biological origin It may be discriminated as a bright spot caused by the particles.

<Functional configuration>
FIG. 13 is a block diagram illustrating a specific example of a functional configuration of the control unit 10 for performing the above operation. Each function of FIG. 13 is mainly formed on the CPU by reading and executing a program (Central Processing Unit) (not shown) included in the control unit 10 that is stored in the memory. A part may be realized by a hardware configuration such as an electric circuit.

Referring to FIG. 13, the control unit 10 includes a calculation unit 101 for calculating the amount of biological particles collected in the imaging range of the imaging unit 40 on the surface of the collection substrate 200, and the excitation light source 30. And an imaging operation unit 102 for controlling the operation of the imaging unit 40 to cause the detection apparatus 100 to perform an imaging operation, and a heating control unit 106 for controlling the heating operation in the heating unit 50. The photographing operation unit 102 includes a light emission control unit 103 for controlling emission / extinction of excitation light of the excitation light source 30, a photographing control unit 104 for controlling photographing in the photographing unit 40, and photographing from the photographing unit 40. An image input unit 105 for receiving image input. Further, the calculation unit 101 determines whether or not organism-derived particles exist in the imaging range by analyzing the fluorescence image F2 after heating, or by comparing the fluorescence images F1 and F2 before and after heating. A separating unit 107 for separating a fluorescent image in which biological particles are present, and an image analyzing unit 108 for analyzing an image of the fluorescent image in which biological particles are present. The image analysis unit 108 specifies a bright spot from the binarization unit 109 for binarizing image data representing luminance value information for each pixel which is a fluorescent image, and binarized image data, And a discriminator 110 for discriminating whether or not the particle is derived from a biological particle.

<Operation flow>
14 and 15 are flowcharts showing the flow of control in the control unit 10, and in particular, are flowcharts showing the separation process in step S31 and the calculation process in step S33.

Referring to FIG. 14, in step S101, the control unit 10 compares the luminance values of the fluorescent images F1 and F2 from the photographing unit 40 for each pixel. And the pixel with a change in a luminance value is contained (it is YES at step S103), and the change degree of the luminance value corresponds to the change degree of the luminance value before and behind the heating of the biological particle memorize | stored beforehand. If so (YES in step S105), the control unit 10 determines that biologically derived particles (microorganisms) are present in the fluorescent images F1 and F2 (step S107). Even if a pixel with a change in luminance value is not included (NO in step S103), or even if there is a change in luminance value, the luminance value before and after the heating of the particles derived from living organisms If the degree of change does not correspond (NO in step S105), the control unit 10 determines that the biological images (microorganisms) are not present in the fluorescent images F1 and F2 (step S109).

As described above, the detection apparatus 100 can also calculate the amount of biologically derived particles using only the fluorescent image F2 after heating. In general atmospheric environment, fluorescent dust (generated from fibers, etc.) exists in the atmosphere. Therefore, as described above, both fluorescent images F1 and F2 before and after heating are analyzed, and fluorescent dust and biological particles are analyzed. Can improve the accuracy. However, in an environment such as a high-grade clean room, there is a high possibility that fluorescent dust is not present or very little. In such a case, it is highly likely that the bright spot can be distinguished from those derived from living organisms. Therefore, in such a case, analysis of both fluorescence images before and after heating is not necessarily required, and only analysis of the fluorescence image F2 after heating may be performed.

FIG. 19 is a flowchart showing a modified example of the flow of the separation process in step S31. That is, with reference to FIG. 19, for each pixel of the fluorescent image F <b> 2 after heating, the control unit 10 has the luminance value of the pixel within the range of the luminance value of the biological particle stored in advance. Whether or not (step S301). The range of the luminance value after heating the biological particles may be acquired in advance through experiments or the like and stored in a memory (not shown) of the control unit 10. When the luminance value after heating is within the above-described range, that is, a biological luminance value (YES in Step S303), the control unit 10 displays biological particles (microorganisms) in the fluorescent image F2. Is present (step S305). When the luminance value after heating is not a biological-derived luminance value (NO in step S303), the control unit 10 determines that biological-derived particles (microorganisms) are not present in the fluorescent image F2 ( Step S307).

Next, referring to FIG. 15, in step S201, the control unit 10 determines the luminance value information for each pixel that is the fluorescent image F2 that has been determined as having biologically derived particles (microorganisms) by the above-described processing. Is binarized (step S201). The control unit 10 identifies a bright spot from the binarized image data, and sets the number of pixels (area) and shape constituting one bright spot for each of the identified bright spots in advance to a biological particle. The number of pixels and the characteristics of the shape stored as the resulting bright spots are respectively compared. In other words, the number of pixels (area) constituting one bright spot matches the number of pixels (area) stored in advance as the number of pixels constituting one bright spot caused by biological particles (step) If the shape of one bright spot matches the shape stored in advance as the shape of one bright spot derived from a biological particle (YES in step S205), the control unit 10 discriminate | determines that the one luminescent spot originates in the particle | grains derived from living organisms. Then, the control unit 10 counts the bright spot as a biological particle (step S207).

With the above operation, the detection apparatus 100 can obtain the number of organism-derived particles (microorganisms) present in the imaging range by counting the bright spots determined to be caused by the organism-derived particles. The control unit 10 converts the obtained number of particles into the area of the entire surface of the collection substrate 200, and then the amount of the sample used when the collection operation is performed on the collection substrate 200 (for example, 1 m ^{3 in the} atmosphere). Etc.) can be used to calculate the concentration of biological particles.

<Effect of Embodiment>
By using the detection device 100 described above, it is possible to detect biologically-derived particles using fluorescence without using a fluorescent reagent. Therefore, the detection can be easily performed at a reduced cost because the fluorescent reagent is unnecessary.

In addition, the inventors verified that it is possible to count the number of organism-derived particles by the above calculation process using the fluorescence image F2 of the blue mold after the heat treatment shown in FIG. 5B. . FIG. 16B shows a line profile that represents the luminance value in the horizontal direction of the bright spot in the fluorescent image F2 of the blue mold after heat treatment, which is surrounded by white circles in FIG. 16A. FIG. 16B shows a large difference between the luminance value at the bright spot and the background value.

FIG. 17A shows an image (binarized image) obtained by binarizing the fluorescent image F2 of FIG. 16A using the luminance value 50 as a threshold value. In FIG. 17A, a black area represents a pixel having a luminance value larger than the threshold value, and a white area represents a pixel having a luminance value smaller than the threshold value. FIG. 17B is a diagram showing a region determined to be caused by biological particles based on the area (number of pixels) and shape of the bright spot of the binarized image of FIG. 17A. From FIG. 17B, the number of bright spots attributed to organism-derived particles was counted as 14 (chained fungi linked in a chain were counted as 1 group).

From the above, it was verified that bright spots caused by biological particles can be counted by image processing. Therefore, it was verified that the biological particles can be easily detected using the detection apparatus 100.

[Second Embodiment]
When the collection substrate 200 is fixed and detected by the fixing unit 20 of the detection device 100 after the collection operation, the biological particles collected by the collection operation are detected. At this time, if the surface of the collection substrate 200 is not refreshed before collection, the particles collected in the previous collection operation remain on the surface of the collection substrate 200 and are collected in the subsequent collection operation. Only the collected biological particles cannot be detected. However, refreshing every collection operation becomes complicated. Therefore, even when the same collection substrate 200 is used for the collection operation a plurality of times using the detection apparatus 100, a counting method for detecting the biological particles collected in each collection operation. explain.

FIG. 18 is a diagram for explaining a state of collected particles when the same collection substrate 200 is used for n collection operations. Referring to FIG. 18, when the same collection substrate 200 is used for the n-th collection operation, the particles on the surface after the n-th collection operation ((C) in FIG. 18) Particles collected in the collecting operation, particles collected in the second collecting operation,... (Omitted), (n-2) particles collected in the second collecting operation ((A) in FIG. 18) ), And (n-1) particles collected in the collection operation ((B) in FIG. 18) are added together. Therefore, the particles collected in the n-th collection operation are excluded from the particles collected in the n-th collection operation until the (n-1) -th collection operation. Become particles.

Therefore, the detection apparatus 100 stores the detection result of the previous (n-1) th collection operation, and calculates the difference from the detection result of the current (nth) collection operation, The amount of organism-derived particles collected by the collection operation is calculated. Thereby, in the detection apparatus 100, even if it is a case where the same collection board | substrate 200 is used for collection operation | movement several times, the particle | grains derived from the organism collected by each collection operation | movement can be detected. This eliminates the need for refreshing for each collection operation and facilitates detection.

If image data is saved as a detection result for each detection operation, the amount of data becomes enormous, and a storage device such as a hard disk or RAM (Random Access Memory) included in the control unit 10 (or included in a connected PC or the like). It will squeeze the memory. Therefore, preferably, the detection apparatus 100 may store a detection result for each pixel instead of the image data. For example, it is possible to reduce the amount of data stored in the memory by binarizing the image data by setting the value of the pixel region where the biological particles are present to 1 and the value of the non-existing pixel region to 0.

For example, when the imaging unit 40 includes an area sensor of 10 million pixels and acquires 16-bit luminance information per pixel, the data amount of the entire captured image is 20 MB. On the other hand, if the value for each pixel is binarized with 0 or 1 according to the discrimination result as described above, the information amount per pixel becomes 1 bit (0 or 1), and the data amount is reduced to 1/16. Reduced.

Furthermore, in order to calculate the difference as described above, it is only necessary to store at least the previous (n−1) th collection operation detection result, and the previous collection operation detection result is not necessarily the same. It does not have to be stored. Therefore, preferably, the detection apparatus 100 stores the detection results as few times as possible including at least the detection result of the previous (n−1) collection operation by overwriting the detection results, for example. Also good. In this way, the amount of data stored in the memory can be reduced. Furthermore, at this time, as described above, the amount of data can be further reduced by storing the information obtained by binarizing the image data with the value for each pixel according to the determination result as described above.

The embodiment disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

10 control unit, 20 fixing unit, 30 excitation light source, 31 lens for light source, 40 imaging unit, 41 lens, 42 filter, 43 wavelength selective mirror, 50 heating unit, 75-78 curve, 100 detection device, 101 calculation unit, 102 imaging operation unit, 103 light emission control unit, 104 imaging control unit, 105 image input unit, 106 heating control unit, 107 separation unit, 108 image analysis unit, 109 binarization unit, 110 discrimination unit, 200 collection substrate.

Claims (8)

Fixing means for fixing a collection substrate capable of collecting particles in the specimen on the surface;
A light source for irradiating excitation light onto the surface of the collection substrate fixed by the fixing means;
An imaging means including a plurality of two-dimensionally arranged sensors for imaging at least a partial area of the surface of the collection substrate fixed by the fixing means as an imaging range;
Heating means for heating the collection substrate;
A detection means comprising: a calculation means for calculating an amount of biologically-derived particles out of particles collected on the surface of the collection substrate using a photographed image obtained by the photographing means after being heated by the heating means. apparatus. The calculation means calculates the amount of biologically-derived particles among the particles collected on the surface of the collection substrate by analyzing a photographed image by the photographing means after being heated by the heating means. The detection device according to claim 1. The calculation unit analyzes the captured image captured by the imaging unit in a state where the collection substrate is irradiated with the excitation light from the light source, obtains a luminance value of each pixel, and before heating by the heating unit Derived from the organism collected on the surface of the collection substrate based on a change between the luminance value of each pixel of the captured image and the luminance value of each pixel of the captured image after being heated by the heating means. The detection device according to claim 2, wherein particles are discriminated and the amount thereof is calculated. A storage means for storing the pixels determined in the calculation means that the organism-derived particles in the photographed image are present;
The calculation means is collected on the surface of the collection substrate based on a difference between a pixel determined from the captured image that the biological particle is present and a pixel stored in the storage means. The detection device according to claim 3, wherein the amount of biological particles among the particles is calculated. The detection device according to any one of claims 1 to 4, wherein the light source emits light in a wavelength region that can excite biological particles. The detection device according to claim 5, wherein the light source is a semiconductor laser. The detection device according to claim 5 or 6, wherein the light source irradiates light having a wavelength in a range of 300 nm to 450 nm. Heating a collection substrate capable of collecting particles in the specimen on the surface;
Irradiating excitation light from a light source to the surface of the collection substrate after the heating;
A plurality of sensors arranged in a two-dimensional manner with at least a partial region of the surface of the collection substrate as an imaging range in a state in which the collection substrate after the heating is irradiated with the excitation light from the light source; Photographing using a photographing means;
And a step of calculating an amount of biologically-derived particles among particles collected on the surface of the collection substrate using a photographed image obtained by the photographing means.

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