JP7085231B2 - Microstructure detection method and its equipment - Google Patents

Description

The present invention relates to a detection device for optically detecting microstructures, a detection method, and a disc for detecting microstructures.

Conventionally, the culture method is most often used as a method for detecting microstructures such as microorganisms existing in water or the like. However, in general, it takes about one day for culturing the microorganism and several more days for the test for identifying the type of the microorganism, so that the test result cannot be obtained at an early stage.

As an example of efforts for early detection, in a particle counter or flow cytometer, the water to be inspected is passed through a flow path and then irradiated with laser light to emit fluorescence or scattered light from microorganisms that pass through the condensing part. By measuring, the type and size of microorganisms and the like are specified (see Patent Document 1). One of the problems with the method using the flow path is that the inspection device is generally large and expensive.

An optical disc-based method has been proposed with the aim of realizing a compact and inexpensive inspection device (see Patent Document 2). Optical discs have been developed for recording information such as video and music, and are a technology that can read out recording pits with a length of several hundred nm to several μm at high speed. By measuring the recording pits as if they were microorganisms, it is possible to detect microorganisms using an optical disk.

The present inventors have conducted research and development on a method for detecting microstructures and the like (see Patent Document 3). Patent Document 3 proposes a method of forming a marker on a track structure for tracking to enable mapping of attached microstructures.

Japanese Unexamined Patent Publication No. 62-293143 Japanese Patent No. 4670015 Japanese Unexamined Patent Publication No. 2014-153165

Conventionally, a technique for early detection of microorganisms and the like has been desired, but there is a problem that it is large and expensive.

Further, in the conventional technique for detecting microstructures using an optical disc, a method for determining the presence or absence of microorganisms and the like in an environment with impurities and a method for specifying the type of microorganisms have not been established.

For example, Patent Document 2 describes a method in which a mechanism for specifically capturing a microorganism to be detected is provided on a substrate in advance, but in general, microorganisms unrelated to the reaction are also physically present on the substrate to a certain extent. It will be adsorbed. That is, there is a problem that the presence or absence of the target microorganism can be significantly determined only when the number thereof sufficiently exceeds the number physically adsorbed.

In Patent Document 3 of the present inventors, mapping of microstructures is possible, but the detection accuracy is insufficient in an environment with impurities.

The present invention is intended to solve these problems, and the present invention is to use an optical means in a liquid in a state where microstructures such as microorganisms existing in water or the like are mounted on a disk. It is an object of the present invention to provide a detection method, a detection device, and a detection disk for clarifying the presence or absence of microorganisms and identifying the type of microorganisms regardless of the number of microorganisms.

The present invention has the following features in order to achieve the above object.

In the microstructure detection method of the present invention, a laser beam is applied to one surface of a rotating disk on which a microstructure sample is mounted, and the light on the other surface side of the disk is detected from the sample. It is characterized by measuring the transmitted light intensity or the fluorescence intensity of. For example, in the measurement of the transmitted light intensity, it is preferable to measure the transmitted light in the state before and after dyeing the microstructure or in the state after dyeing. Alternatively, for example, in the measurement of the fluorescence intensity, it is preferable to measure the fluorescence from the fluorescently labeled microstructure or the autofluorescence of the microstructure. In the microstructure detection method of the present invention, it is preferable to adjust the focal position on the disk based on the intensity of the reflected light of the laser beam from the disk. In the measurement of the transmitted light intensity or the fluorescence intensity, the fluctuation of the measurement result can be removed by cutting the frequency portion of 20 times or less the frequency corresponding to one rotation of the disk from the measurement result.

The microstructure detection device of the present invention is based on a rotating means for rotating a disk, an irradiation means for irradiating one surface of the disk with a laser beam, and the intensity of the reflected light of the laser beam from the disk. A focal position adjusting means for adjusting a focal position on a disk and a measuring means for measuring transmitted light intensity or fluorescence intensity from a sample mounted on the disk using the laser beam as a light source on the other surface side of the disk. It is characterized by being prepared. For example, there is a signal processing means for specifying any one or more of the presence / absence, number, and type of microstructures by inputting and comparing the measurement results before and after staining the microstructures. You may.

The microstructure detection disk of the present invention comprises a substrate having a groove structure for focusing the laser beam, and is characterized in that a part of the laser beam is reflected and a part thereof is transmitted. The microstructure detection disk of the present invention preferably has a thin film on the substrate. Further, it is preferable that the thin film has a refractive index of 2.0 or more at the wavelength of the laser beam and a film thickness of 5 nm or more and 200 nm or less.

In the present invention, as the material constituting the microstructure detection disk, a material having light transmission with respect to the laser light wavelength is used. Further, the microstructures such as microorganisms to be detected are mounted on the surface where the laser beam is focused on the disk.

In the microstructure detection device of the present invention, for example, by having a mapping function, the number of microstructures existing on the microstructure detection disk can be counted.

In the microstructure detection method of the present invention, for example, first, the transmitted light intensity before dyeing is measured while rotating the disc for detecting microorganisms with the microorganisms mounted on the disc, and then the microorganisms mounted on the disc are dyed. After dyeing with the agent, the transmitted light intensity after dyeing of the same part is measured, and in the comparison of the transmitted light intensity before and after dyeing, the peak of the transmitted light intensity or the part where there is a certain difference in the dip part is the transmission accompanying the dyeing. By expressing the change in light intensity, the presence or absence of microorganisms is clarified, and the type of microorganism is specified.

In the microstructure detection method of the present invention, in a state where microorganisms and the like are mounted on a detection disk, microorganisms and the like are mounted in an environment where the microorganisms and the like have high light transmittance with respect to the wavelength of the laser light used in the irradiation means. By measuring the transmitted light intensity after dyeing while rotating the disk after dyeing the microorganisms with a dyeing agent in this state, a dip occurs in the transmitted light intensity of the portion where the microorganisms are present due to the light absorption accompanying the dyeing. From, the presence or absence of microorganisms, etc. is clarified, and the types of microorganisms, etc. are specified.

As the dyeing agent used in the present invention, it is preferable to select one having high light absorption characteristics with respect to the wavelength of the laser light used in the irradiation means. When a dyeing agent capable of staining a whole microorganism is used, the presence or absence of the microorganism is clarified, and when a dyeing agent capable of dyeing a specific microorganism is used, the type of the microorganism can be specified.

In the microstructure detection method of the present invention, the microstructure is rotated while the microstructure is mounted on the detection disk, and the presence or absence of fluorescence emission from the microstructure is observed using a laser beam as an excitation light source. The presence or absence of an object can be clarified, and the type of microstructure can be specified.

In order to obtain fluorescent light emission, a microorganism or the like may be fluorescently labeled with a fluorescent agent, or autofluorescence from the microorganism or the like may be used. The fluorescent agent is selected so that it can be excited by the wavelength of the laser light source used in the detection device. When a fluorescent agent capable of fluorescing all microorganisms is used, the presence or absence of microorganisms or the like is clarified, and when a fluorescent agent capable of fluorescing only specific microorganisms or the like is used, the type of microorganisms or the like should be specified. Can be done. When autofluorescence is used, the wavelength of the laser beam used in the detection device of the present invention selects a wavelength that can excite the fluorescence source originally provided in the microorganism.

When detecting transmitted light using the light detecting means, due to the surface runout during the rotation of the detection disc and the adjustment operation of the focal position adjusting means, even when microorganisms and the like are not mounted, the disc is within the range of one rotation. Fluctuations may occur in the intensity of transmitted light. When the fluorescence is detected by the photodetecting means, it is expected to be almost nonexistent or small in principle, but the intensity of the fluorescence may fluctuate within the range of one rotation of the disk. The fluctuation can be removed by cutting the low frequency component, and it becomes easy to count the microorganisms by counting the number of peaks or dips based on a certain standard. Specifically, assuming that the rotation speed of the disk for detecting microorganisms is v and the radial position of the disk is r, the frequency f corresponding to one rotation of the disk is given by v / (2 × π × r). The frequency may be cut up to 10 times the frequency f, and more reliably the frequency may be cut up to 20 times the frequency f.

In the microstructure detection method of the present invention, the thickness of the thin film formed on the detection disk is controlled so that the transmitted light intensity increases and a peak is formed when there is a microstructure. Above, if the transmitted light intensity after dyeing is reduced as compared with the case where there are no microstructures to form a dip, the peak of the optical characteristics associated with the dyeing changes to a dip, and the dyeing effect is achieved. Can be confirmed more clearly.

In the method of performing optical detection with a microorganism or the like mounted on a substrate or the like as in the present invention, by measuring the transmitted light intensity or the fluorescence intensity of the microorganism or the like, regardless of the number of the microorganism or the contaminants. Microorganisms and the like can be detected.

According to the detection device of the present invention, the transmitted light and fluorescence characteristics of individual microorganisms can be evaluated by the detection device which is an improvement of the technique of the recording / playback device of the conventional optical disc, and the size is smaller than that of the conventional detection device. , The accuracy has improved.

According to the detection method of the present invention, the microorganism can be identified by evaluating the action state of the dyeing agent having light absorption with respect to the laser light wavelength to be used on the microorganism. According to the detection method of the present invention, a microorganism can be identified by measuring the fluorescence emitted from the microorganism as a result of being excited by the laser light wavelength used.

According to the microstructure detection disc of the present invention, it is possible to measure the transmitted light intensity or the fluorescence intensity of microorganisms and the like, so that the disc does not have any number of microorganisms and impurities as compared with the conventional disc. Microorganisms and the like can be detected.

The figure which shows typically the arrangement of the optical system of the detection apparatus in this invention. The figure which shows the result of having calculated the change of the transmittance accompanying the dyeing of the disk for microorganism detection in the calculation example 1 of this invention. The figure which shows the result of having calculated the transmittance and the reflectance of the disk for detecting microorganisms in the calculation example 4 of this invention. The figure which shows the transmitted light intensity signal of unstained Escherichia coli at the time of measuring the disk for microorganism detection in the Example of this invention. The figure which shows the transmitted light intensity signal of Escherichia coli after staining at the time of measuring the disk for microorganism detection in the Example of this invention. The figure which shows the reflected light intensity signal of unstained Escherichia coli at the time of measuring the disk for microorganism detection in the comparative example. The figure which shows the reflected light intensity signal of Escherichia coli after staining at the time of measuring the disk for microorganism detection in the comparative example.

Embodiments of the present invention will be described below.

FIG. 1 schematically shows the arrangement of the optical system of the microstructure detection device for microorganisms and the like of the present invention. The detection device of the present invention basically measures the light intensity transmitted through a microorganism or the fluorescence from a microorganism instead of the structure and operating principle of the conventional optical recording / reproduction device and the function of recording / reproducing information. Common except for doing. The rotating means 1 for rotating the disk, the irradiating means 2 for irradiating the laser beam, and the intensity of the reflected light reflected from the disk of the laser beam are measured, and the lens position and the like are set so as to focus on a certain surface of the disk. As the focal position adjusting means 3 to be adjusted, the technique in the conventional optical recording / reproducing device can be applied. The focal position adjusting means 3 includes, for example, a condenser lens 32 and a photodetector 31.

The microstructure detection device of the present invention uses the microorganism detection disc 4 instead of the information recording / playback disc used in the conventional optical recording / playback device. The disc 4 may be produced by forming a thin film on a substrate. However, as the material constituting the disk 4, a material having light transmittance with respect to the laser light wavelength of the irradiation means 2 is used.

The smaller the light spot diameter, the better the resolution when a plurality of microorganisms are present. Therefore, the laser light wavelength of the irradiation means 2 is preferably a shorter wavelength in the range of 350 nm or more and 800 nm or less.

As the dyeing agent used in the microstructure detection method of the present invention, a known dyeing agent having light absorption with respect to the laser light wavelength of the irradiation means 2 can be used. Known dyes are specifically Bismarck Brown, Brilliant Green, Coomassie Brilliant Blue, Safranin, Fuchsin, Victoria Blue, Chrysoydin and the like.

As the fluorescent agent used in the microstructure detection method of the present invention, a known fluorescent agent that can be photoexcited at the laser light wavelength of the irradiation means 2 can be used. Specific known fluorescent agents include DAPI (4', 6-diamidino-2-phenylindole), HOECHST34580, DRAQ5 (BioStatus Limited, registered trademark) and the like.

The disc 4 may be fixed to the detection device of the present invention, or may be removable. When fixed, the background light or fluorescence intensity, which is the background, is measured in advance, and the background component is subtracted from the measurement result of the transmitted light or fluorescence intensity when the detection test for microorganisms is performed. Etc. can be detected. If it is removable, by using an unused disk 4 when performing a detection test for microorganisms, etc., it is possible to save the trouble of subtracting the background portion, and the disk 4 in a form according to the content of the detection test for microorganisms, etc. as appropriate. Can be used.

The substrate of the microstructure detection disk 4 may be tough enough to maintain its planar shape at least during rotation. Specifically, the substrate material is preferably glass such as synthetic quartz, molten quartz or borosilicate glass, or plastic such as polycarbonate, polyethylene terephthalate or acrylic resin. It is preferable that the substrate is similar to the substrate of the recording / playback disc so that the existing production equipment and the like can be made at low cost, so that the shape is a disk shape and the thickness is in the range of 0.1 to 1.2 mm.

It is desirable that one side of the substrate of the disc 4 has a groove-shaped spiral structure in which the laser beam emitted from the irradiation means 2 is focused and the entire surface of the disc 4 can be scanned.

The thin film of the disk 4 is formed on the side where the laser beam emitted from the irradiation means 2 of the substrate is focused for the purpose of adjusting the reflectance of the disk 4 and controlling the wettability to the water to be inspected containing microorganisms and the like. .. Specifically, the thin film material is Al, Si, Ti, V, Cr, Mn, Co, Ni, Cu, Zn, Ga, Ge, Y, Zr, Mo, Pd, In, Sn, Sb, Te, Ce, It is preferably a nitride, oxide, carbide or sulfide such as Nd, Hf, Ta, W, Pt and Bi. The compound is not limited to the binary, and may be appropriately used as a ternary or quaternary compound. Further, the thin film is not limited to a single layer, and may be composed of a plurality of layers according to functions.

The thin film of the disk 4 may also be formed by controlling the film thickness of a thin film material having a refractive index of 2.0 or more for the purpose of detecting a change in transmitted light intensity due to dyeing with high sensitivity. .. Specifically, the thin film material is preferably an oxide such as In, In—Sn, Nd, Sb, Zr, Ce, Ti, Bi, or a nitride of Si.

The lower limit of the film thickness of each film constituting the thin film of the disk 4 is the film thickness at which the thin film material to be used can not cover one surface and becomes particulate at last. The upper limit of the total thickness of the thin film is preferably within the range of the depth of focus of the optical system including the irradiation means 2 and the focal position adjusting means 3 in order to distinguish individual microorganisms and the like. Specifically, it is desirable that the total thickness of the thin film is 5 nm or more and 3.9 μm or less.

The thin film of the disc 4 is also intended to use the optical interference with the substrate to adjust the transmittance and the reflectance of the disc 4, or to detect the change in transmitted light intensity due to dyeing with high sensitivity. The film thickness of the thin film may be designed for the purpose. Conditions suitable for the above purpose appear periodically with respect to the film thickness, but in order to prevent the substrate from being deformed when the film thickness is thick, the upper limit of the film thickness is preferably 500 nm or less, which is more preferable. Should be 200 nm or less.

The thin film of the disk 4 may basically have a mechanism that can capture an unspecified number of microorganisms, such as having adhesiveness on the surface of the thin film.

The mounting of microorganisms or the like on the disk 4 is performed on the side where the laser beam emitted from the irradiation means 2 on the substrate of the disk 4 is focused, and if there is a thin film on the focused side, it is mounted on the thin film. The mounting is performed, for example, by dropping the water to be inspected onto the disc 4. If the water to be inspected is dried before the microorganism detection test, the microorganisms and the like can be placed directly on the thin film, and the microorganisms and the like can be detected. Even when the water to be inspected is not dried, the microorganisms can still be detected as long as the microorganisms are within the depth of focus of the optical system including the irradiation means 2 and the focal position adjusting means 3.

In order to reduce impurities in the water to be inspected, the water to be inspected may be passed through a filter or the like in advance to remove structures larger than microorganisms. When the water to be inspected is dried at the time of mounting, the surface of the disc 4 may be washed with pure water, ethanol, or the like to reduce the amount of precipitated contaminants.

After mounting the microorganisms on the disc 4, in order to suppress the movement and scattering of the microorganisms and the like accompanying the rotation of the disc 4, a separate cover layer may be provided on the surface of the disc 4 on the side on which the microorganisms and the like are mounted. The surface of the cover layer may be covered with a resin such as an ultraviolet curable resin, or a flat structure such as a cover glass may be fixed with an adhesive or the surface tension of the water to be inspected.

The microstructure detection device of the present invention includes a light detecting means 5 for measuring light transmitted through a microstructure such as a microorganism mounted on a detection disk or fluorescence from a microstructure such as a microorganism. The photodetector 5 preferably has a photodetector 53 and a condenser lens 51 or the like in front of the photodetector 53 in order to efficiently acquire transmitted light and fluorescence.

As the photodetector 53 of the photodetection means 5, a photodiode, a photomultiplier tube, or the like can be used depending on the intensity of transmitted light or fluorescence.

When measuring the fluorescence, a filter 52 that blocks the laser light wavelength of the irradiation means 2 and allows a part of the light having a wavelength longer than the laser light wavelength to pass is arranged in front of the light detector 53. Is desirable.

Examples will be described later, but first, it will be described by a calculation example that the case of detecting the light transmitted through the disk is superior to the case of detecting the reflected light in the detection of microstructures. ..

(Calculation example 1)
A laminated structure having a microorganism-equivalent layer (layer thickness: 100 nm) on the thin film of a microorganism detection disk provided with a thin film (thickness: 0 to 200 nm) on a polycarbonate substrate was assumed. Light having a wavelength of 405 nm was irradiated from the side where the thin film of the laminated structure was not present, and the transmittance of the laminated structure was calculated. The refractive index at a wavelength of 405 nm was 1.63 (polycarbonate) and 1.45 (microorganism), respectively, and the refractive index of the thin film was varied in the range of 1.0 to 3.0.

FIG. 2 shows the results of calculating the change in transmittance when the extinction coefficient of the microorganism-equivalent layer at a wavelength of 405 nm was changed from 0 at the time of non-staining to 0.01 at the time of staining. Even when the film thickness of the thin film was changed in the range of 0 to 200 nm and the refractive index was changed in the range of 1.0 to 3.0, the transmittance was reduced by about 2 to 3% in all cases.

The largest change in transmittance in FIG. 2 was in the range indicated by "-3" in the figure. When the refractive index is 3.0, the film thickness is 4 to 12 nm, 72 to 80 nm and 140 to 148 nm, and when the refractive index is 2.5, the film thickness is 6 to 22 nm, 88 to 102 nm and 168 to 184 nm, and the refractive index. When was 2.0, it was in the range of 16 to 36 nm and 118 to 136 nm.

(Calculation example 2)
Although the laminated structure, film thickness condition, and refractive index condition are the same as those in Calculation Example 1, the change in transmittance when the extinction coefficient was changed from 0 at the time of non-staining to 0.1 at the time of dyeing was calculated. Even if the film thickness and the refractive index of the thin film were changed, the transmittance was reduced by about 18 to 26% in all cases.

When the change in the extinction coefficient is large as described above, the large change in the transmittance is shown regardless of the refractive index and the film thickness of the thin film, so that the dyeing effect can be sufficiently confirmed in the transmitted light measurement.

(Calculation example 3)
The disk for detecting microorganisms and the film thickness and refractive index are the same as those in Calculation Example 1, but when the layer thickness of the microorganism-equivalent layer is 1 μm and the extinction coefficient is changed from 0 at the time of non-staining to 0.01 at the time of staining. The change in transmittance was calculated. Even when the film thickness of the thin film was changed in the range of 0 to 200 nm and the refractive index was changed in the range of 1.0 to 3.0, the transmittance was reduced by about 13 to 29% in all cases.

When the microorganism is thick as described above, a large change in transmittance is exhibited regardless of the refractive index and the film thickness of the thin film, so that the dyeing effect can be sufficiently confirmed in the transmitted light measurement.

(Calculation example 4)
A laminated structure having a microorganism-equivalent layer (layer thickness: 100 nm) on the thin film of a microorganism detection disk having a TiO ₂ thin film (thickness: 40 nm or 80 nm) on a polycarbonate substrate was assumed. It was decided to scan the groove of the substrate, and an air layer (layer thickness: 34 nm) was inserted between the thin film and the microorganism-equivalent layer. Light having a wavelength of 405 nm was irradiated from the side where the thin film of the laminated structure was not present, and the reflectance and transmittance of the laminated structure were calculated. The refractive index at a wavelength of 405 nm is 1.63 (polycarbonate), 2.7 (TiO ₂ ), 1.0 (air), and 1.45 (microorganisms), respectively.

FIG. 3 shows the calculation results when the extinction coefficient of the microorganism-equivalent layer at a wavelength of 405 nm was changed from 0 at the time of non-staining to a value larger than 0 at the time of staining. It is clear that the reflectance decreases with increasing extinction coefficient, while the reflectance remains almost unchanged.

In the microstructure detection method of the present invention, it is necessary to control the film thickness of the thin film formed on the detection disk so that the transmitted light intensity increases and a peak is formed when there is a microstructure. good. If the transmitted light intensity after dyeing is reduced as compared with the case without microstructures to form a dip, the peak of the optical characteristics associated with the dyeing changes to a dip, and the dyeing effect becomes clearer. Can be confirmed in.

For example, in the method of measuring reflected light as described in Patent Document 2, the optical change associated with microbial staining cannot be obtained with high sensitivity, whereas the transmitted light is obtained by the optical arrangement in the microstructure detection device of the present invention. Only by measuring, highly sensitive detection becomes possible.

(Example)
A TiO ₂ thin film (film thickness: 40 nm) was formed on a polycarbonate substrate having a groove periodic structure to prepare a disk 4 for detecting microorganisms. Escherichia coli was used as a microorganism, and water to be inspected containing Escherichia coli was dropped onto the disc 4, and then a detection test of Escherichia coli was carried out in a dried state.

It consists of an irradiation means 2 and a focal position adjusting means 3 consisting of an optical system having a wavelength of 405 nm, a laser light power of 1.0 mW, and a numerical aperture of 0.65, an objective lens having a numerical aperture of 50 times and a numerical aperture of 0.50, and a photoelectron multiplying tube. An experiment was carried out using the light detection means 5, and the microscopic detection disk was rotated at a linear speed of 5.0 m / s. Further, the signal obtained by the photodetector means 5 was amplified five times by a signal amplifier. The photomultiplier tube has a mechanism in which a negative voltage is output when light is detected.

As a dyeing method, Bismarck brown powder adjusted to a concentration of about 10% with pure water was used as a dyeing agent, and an amount sufficient to cover the part of E. coli on the disk 4 was dropped and left for 1 minute. After that, the disc 4 was washed with water to remove excess dyeing agent. Using an optical microscope, it was confirmed that Escherichia coli was stained by the above staining method.

4 and 5 show transmitted light intensity signals before and after staining E. coli, respectively. “E. coli spot 6” in both FIGS. 4 and 5 indicates a place where the water to be inspected containing E. coli is dropped, and the horizontal axis represents the time of laser light scanning. In FIG. 4, it is observed that the transmitted light intensity of the portion with E. coli increases or decreases with respect to the portion without E. coli. In FIG. 5, it is observed that the transmitted light intensity at the portion with E. coli is almost reduced with respect to the portion without E. coli. From the comparison between FIGS. 4 and 5, it can be seen that most of the transmitted light intensity of E. coli began to decrease with staining.

(Comparative example)
6 and 7, respectively, show the reflected light intensity signals before and after staining E. coli at the same sites as in FIGS. 4 and 5, respectively. The reflected light intensity was measured by the photodetector of the focal position adjusting means 3. In FIGS. 6 and 7, the reflected light intensity of the portion with E. coli is both decreased with respect to the portion without E. coli, and the effect of staining is not clear in the comparison between the two figures.

The results of FIGS. 4 to 7 are consistent with the calculation results when the film thickness of Calculation Example 4 is 40 nm. Therefore, it is clear that the presence or absence of a microorganism can be accurately determined by measuring and comparing the transmitted light intensity signal before and after staining a microorganism such as Escherichia coli.

It should be noted that the examples shown in the above embodiments and the like are described for the sake of easy understanding of the invention, and are not limited to this embodiment.

Using the detection disk or detection device of the present invention, the intensity of transmitted light from microorganisms or the like or fluorescence from microorganisms can be measured, and the presence or absence of microorganisms and their types can be specified by the detection method of the present invention. Since it can be used, it is suitable for early detection of microorganisms existing in water, and is therefore industrially useful.

1 Rotating means 2 Irradiation means 3 Focus position adjusting means 4 Microorganism detection disk 5 Light detection means 6 E. coli spot 31, 53 Photodetector 32, 51 Condensing lens 52 Filter

Claims (5)

While scanning one surface of the rotating disk on which the sample is mounted on the upper side of the spiral groove for focusing the laser beam with the laser light so as to be from the lower side of the spiral groove , the said A method of detecting the presence or absence of a specific microstructure in a sample by detecting the transmitted light from the other surface of the disk with a photodetector.
The sample is mounted directly on the spiral groove and
The microstructure to be detected is processed so as to have autofluorescence or emit fluorescence at the laser wavelength of the laser beam.
With respect to the transmitted light from the rotating disk, the laser light wavelength is blocked, a filter is provided to pass a part of the light on the wavelength side longer than the laser light wavelength, and the light detector is used. A method for detecting a microstructure, which comprises detecting a change in fluorescence intensity from a microstructure over time . The method for detecting a microstructure according to claim 1, wherein the spiral groove has a reflectance adjusting film on the surface and the sample is directly mounted on the film. The method for detecting a microstructure according to claim 2, wherein the reflectance adjusting film is made of any one of a nitride, an oxide, a carbide, and a sulfide. The method for detecting a microstructure according to one of claims 1 to 3, wherein the microstructure is a microorganism. While scanning one surface of the rotating disk on which the sample is mounted on the upper side of the spiral groove for focusing the laser beam with the laser light so as to be from the lower side of the spiral groove , the said A device that detects the presence or absence of a specific microstructure in the sample by detecting the transmitted light from the other surface of the disk with a photodetector.
The sample is mounted directly on the spiral groove and
The microstructure to be detected has autofluorescence or is processed so as to emit fluorescence at the laser wavelength of the laser beam.
With respect to the transmitted light from the rotating disk, the laser light wavelength is blocked, a filter is provided to allow a part of the light on the wavelength side longer than the laser light wavelength to pass through, and the microstructure is provided by the light detector. A microstructure detection device characterized by detecting a change in fluorescence intensity from an object over time .
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