JP6729894B2 - Fertilized egg test method and fertilized egg test device for fish - Google Patents

Description

TECHNICAL FIELD The present invention relates to an inspection method capable of highly accurately and quickly determining whether a fertilized egg of a fish such as a zebrafish is good or bad.

Zebrafish have the advantage that they lay eggs almost every day, and that the number of days until they reach adulthood is very short. Further, since the zebrafish is small in size, it is easy to handle, and a large amount of data can be easily obtained with a relatively small device. Therefore, zebrafish are attracting attention as an experimental material in fields such as gene research, pathology research, and drug research. For example, a zebrafish utilization method has been proposed in which a target substance derived from this genetic material is recovered from an adult fish by injecting the genetic material into a fertilized egg of the zebrafish. In addition, a method for utilizing zebrafish has also been proposed in which a drug or the like is introduced into a fertilized egg of zebrafish to evaluate its biological effect.

Conventionally, an optical method using visible light, infrared light, ultraviolet light, or the like has been used as a non-invasive observation method for living tissues. For example, Patent Document 1 proposes to determine the quality of a fertilized egg by using a visible light spectrum or an infrared light spectrum. Patent Document 2 proposes to detect the characteristic based on the fluorescence spectrum from the living tissue. Patent Document 3 proposes to perform fluorescence measurement of a large number of cells dispersedly arranged in a multi-well plate manufactured using a cycloolefin polymer which is a low fluorescence material.

Special table 2004-516475 gazette Japanese Patent Publication No. 2004-518124 Japanese Patent Publication No. 2002-515125

The current zebrafish technology has a problem that the ratio of zebrafish that normally hatch from a fertilized egg is considerably low. This is because a large number of fertilized eggs of the zebrafish obtained become defective eggs such as dead eggs and weakened eggs.

These dead and debilitated eggs can contaminate the water system if not eliminated immediately. As a result, other normal eggs may be adversely affected via this contaminated water. Once the water system containing a large number of fertilized zebrafish eggs is contaminated with pathogenic bacteria, viruses, and other substances, they attach to the surface of normal eggs and the like, resulting in serious consequences.

Further, when injecting a drug or the like into a fertilized egg before hatching, it is not economical to consume expensive test material for a defective egg or an abnormal egg such as a dead or weak egg. Further, it was not possible to determine whether the abnormality of the zebrafish hatched from the defective egg or the abnormal egg was caused by the drug injection or the original one. Furthermore, injection into a bad egg infected with bacteria or virus may cause infection of other normal eggs through the needle.

After all, it was found that in the industrial use of zebrafish, it is necessary to select and use only normal fertilized eggs from a large number of fertilized egg groups of zebrafish in a short time. However, considering that the zebrafish fertilized egg has a high cell division rate and the number of fertilized eggs to be inspected is very large, the inspection time allowed per fertilized egg needs to be extremely short.

However, a method for selecting fertilized eggs of zebrafish having a very small diameter of, for example, about 1 mm with high accuracy in a short time has not been reported yet. From these viewpoints, the inventors have realized that in order to realize industrial use of zebrafish, it is essential to establish a highly accurate and high-speed fertilized egg test method in industrial use of zebrafish. ..

Fluorescence spectrum analysis technology is promising for zybrafish fertilized egg test because it is a non-invasive test method. However, it has been unclear whether a known fast and highly accurate fertilized egg test of zebrafish can be realized using a fluorescence spectrum analysis technique. Furthermore, even if possible, the specific method for realizing highly accurate determination was unclear. In particular, a specific method for completing a large number of zebrafish fertilized eggs in a short time was unknown. In other words, no fluorescence spectrum analysis technique has been known that can realize the required inspection accuracy and inspection speed.

The present invention has been made in view of the above problems required for a fertilized zebrafish egg, and an object thereof is to provide an optical inspection method capable of accurately and rapidly determining whether a fertilized zebrafish egg is good or defective.

According to the present invention, of the fluorescence spectra obtained from fertilized eggs of zebrafish, the spectrum of the excitation light band or a very narrow band adjacent thereto is adopted as the inspection spectrum. According to the observations of the present inventors, it was found that this inspection spectral spectrum of normal eggs has a relatively narrow band or low intensity as compared with that of dead or weak eggs (hereinafter referred to as bad eggs). It was

In one example, the narrow band test spectrum adjacent to the excitation light spectrum was significantly less intense in normal eggs as compared to defective eggs. In another example, the inspection spectrum, which substantially overlaps with the band of the excitation light spectrum, had a significantly lower intensity in the normal egg than in the defective egg. Further, depending on the wavelength of the excitation light, if the narrow-band inspection spectrum adjacent to the short wavelength side of the excitation light spectrum is optimal, the narrow-band inspection spectrum adjacent to the long wavelength side of the excitation light spectrum is It has been found that, in the optimum case, the narrow-band inspection spectrum that overlaps with the excitation light spectrum is optimum.

In another example, in the determination of the inspection spectrum adjacent to the peak wavelength spectrum of a normal egg, the quality of the fertilized egg is determined by the width of the total peak wavelength spectrum of the peak wavelength spectrum and the inspection spectrum. You can also do it. That is, if the bandwidth of this total peak wavelength spectrum exceeds a predetermined threshold value, it is determined as a defective egg.

In another example, in the fluorescence spectrum of the fertilized egg, it is determined whether or not the intensity of the spectral spectrum in a band less than 30 nm away from the band of the excitation light exceeds a predetermined threshold value, and if it exceeds, the fertilized egg is defective. Can be determined.

In another example, in the fluorescence spectrum of the fertilized egg, it is determined whether or not the intensity of the inspection spectral spectrum that substantially overlaps with the excitation light band exceeds a predetermined threshold value, and if it exceeds, it is determined that the fertilized egg is defective. can do.

Furthermore, the present inventors have found that the intensity difference of the above-mentioned spectroscopic spectrum for inspection between a normal egg and a defective egg is 5-8 hours, more preferably 5.5-7.5 hours after fertilization. It was discovered that the accuracy was sufficiently high at the time of observation. Therefore, by performing selection at this observation time point, it becomes possible to eliminate defective eggs before the eggshells of fertilized eggs are destroyed by hatching.

According to a preferred embodiment, in order to rapidly test a large number of fertilized eggs of a zebrafish, a multi-well plate formed on the bottom plate is horizontally driven with a large number of wells for receiving the fertilized eggs. The two-dimensional address of the well containing the fertilized egg determined to be defective in the inspection is stored. After the inspection is completed, the fertilized eggs with the defective addresses are sequentially eliminated. As a result, the inspection of a large number of fertilized eggs can be completed in a short time. The multi-well plate formed on the bottom plate of the well is preferably in a state where the fertilized egg is stably accommodated and does not move, and preferably has a cylindrical or polygonal cylindrical bottom portion as close to the diameter of the fertilized egg as possible.

It is a block diagram which shows the test|inspection apparatus which implements the fertilized egg test|inspection method of the zebrafish of an Example. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg.

FIG. 1 is a schematic block diagram showing an inspection apparatus for a zebrafish fertilized egg. A fish egg tray 2 is placed on an XY table 1 that can be moved two-dimensionally. However, in FIG. 1, the fish egg tray 2 is schematically illustrated.

An excitation light source 3 is provided diagonally above the fish egg tray 2. A fluorescence detector 4 is provided directly above the fish egg tray 2. The signal voltage from the fluorescence detector 4 is input to the determination unit 5 for determining a fertilized egg.

The XY table 1 is movable by two stepping motors (not shown) in the horizontal X direction and the Y direction which is perpendicular to the horizontal X direction. The fish egg tray 2 has a large number of wells 20 each of which is composed of a recess whose upper surface is opened. The wells 20 are arranged in a matrix. Each well 20 contains one fertilized egg 10 of zebrafish together with water. The fish egg tray 2 is formed by using a low fluorescent induction resin material, but the invention is not limited thereto.

The excitation light source 3 is composed of a laser having a predetermined wavelength. The excitation light source 3 emits the laser light 30 in a direction of about 45 degrees with respect to the horizontal X direction. The excitation light source 3 has a built-in lens system for emitting laser light in parallel to almost the entire inspection well 20A which is the well 20 at the inspection position. As the excitation light source 3, a spectrum of a predetermined wavelength obtained by a diffraction grating from a xenon lamp may be adopted.

The fluorescence detector 4 detects the predetermined direction component 40 of the fluorescence spectrum emitted from the inspection well 20A and converts it into a signal voltage. The optical axis of the fluorescence detector 4 coinciding with the predetermined direction component 40 extends in a direction of about 45 degrees with respect to the horizontal Y direction. Therefore, the angle between the optical axis of the excitation light source 3 and the optical axis of the fluorescence detector 4 is 90 degrees. Thereby, the amount of the excitation light emitted from the excitation light source 3 entering the fluorescence detector 4 is reduced. The fluorescence detector 4 includes a lens system having the inspection well 20A as a focal point, a prism or a diffraction grating that disperses a substantially parallel fluorescence beam emitted from the lens system, and a spectrum of a predetermined band emitted from the prism or the diffraction grating. At least. It is also possible to add an optical element such as a slit for narrowing the beam width.

In other words, this photoelectric sensor is arranged at a position where only the spectral spectrum of a specific wavelength of the fluorescent spectrum emitted from the prism or the diffraction grating is incident. Accordingly, the fluorescence detector 4 can detect only the fluorescence spectrum of a predetermined narrow band determined in advance.

The signal voltage output from the photoelectric sensor is converted into a digital signal by the A/D converter and input to the microcomputer 5. The microcomputer 5 determines the quality of the fertilized egg of the zebrafish in the inspection well 20A based on the input digital signal, and controls the intermittent light emission of the excitation light source 3 and the intermittent movement of the XY table 1. As a result, the quality of the fertilized zebrafish egg in each well 20 is sequentially determined. FIG. 1 shows the basic configuration of the inspection apparatus, and it is needless to say that various modifications can be made by using known techniques.

Next, examples of observation of fluorescence spectra obtained from normal and abnormal fertilized zebrafish eggs will be described. The dotted line shows the fluorescence spectrum of a normal egg, and the solid line shows the fluorescence spectrum of an abnormal egg which is a dead egg. In addition, it was found that the fluorescence spectrum from the debilitated egg that could not be hatched at the same time as the normal egg had an intermediate value between the dead egg and the normal egg in the band of the spectroscopic spectrum for inspection.

First Example FIG. 2 shows a fluorescence spectrum obtained by irradiating a fertilized egg 6 hours after fertilization with excitation light having a wavelength of 310 to 330 nm. It was found that the intensity difference of the fluorescence spectrum between the abnormal egg and the normal egg exists in the wavelength band of 280-300 nm. For example, at a wavelength of 280 nm, the relative fluorescence intensity value of dead eggs exceeded 10, but that of normal eggs was less than 2. Therefore, for example, if the fluorescence relative intensity value 6 is set as the classification threshold value, dead eggs including debilitated eggs can be discriminated with high accuracy.

Second Example FIG. 3 shows a fluorescence spectrum obtained by irradiating a fertilized egg 7 hours after fertilization with excitation light having a wavelength of 310 to 330 nm. The results were almost the same as in the first example.

Third Example FIG. 4 shows a fluorescence spectrum obtained by irradiating a fertilized egg 6 hours after fertilization with excitation light having a wavelength of 390-410 nm. It was found that the intensity difference of the fluorescence spectrum between the abnormal egg and the normal egg exists in the wavelength band of 360-380 nm. For example, at a wavelength of 370 nm, the relative fluorescence intensity value of dead eggs exceeded 10, but that of normal eggs was less than 2. Therefore, for example, if the fluorescence relative intensity value 6 is set as the classification threshold value, dead eggs including debilitated eggs can be discriminated with high accuracy.

Fourth Example FIG. 5 shows a fluorescence spectrum obtained by irradiating a fertilized egg 7 hours after fertilization with excitation light having a wavelength of 390-410 nm. The results were almost the same as in the third example.

Fifth Example FIG. 6 shows a fluorescence spectrum obtained by irradiating a fertilized egg 6 hours after fertilization with excitation light having a wavelength of 590-610 nm. It was found that the intensity difference of the fluorescence spectrum between the abnormal egg and the normal egg exists in the wavelength band of 600 to 620 nm. For example, at a wavelength of 610 nm, the relative fluorescence intensity value of dead eggs exceeded 10, but that of normal eggs was less than 2. Therefore, for example, if the fluorescence relative intensity value 6 is set as the classification threshold value, dead eggs including debilitated eggs can be discriminated with high accuracy.

Sixth Example FIG. 7 shows a fluorescence spectrum obtained by irradiating a fertilized egg 7 hours after fertilization with excitation light having a wavelength of 590-610 nm. The results were almost the same as in Example 5.

Seventh Example FIG. 8 shows a fluorescence spectrum obtained by irradiating a fertilized egg 6 hours after fertilization with excitation light having a wavelength of 790-810 nm. It was found that the intensity difference of the fluorescence spectrum between the abnormal egg and the normal egg exists in the wavelength band of 780-820 nm. For example, at a wavelength of 790 nm, the relative fluorescence intensity value of dead eggs exceeded 10, while that of normal eggs was approximately 2. Therefore, for example, if the fluorescence relative intensity value 6 is set as the classification threshold value, dead eggs including debilitated eggs can be discriminated with high accuracy.

Eighth Example FIG. 9 shows a fluorescence spectrum obtained by irradiating a fertilized egg 7 hours after fertilization with excitation light having a wavelength of 790-810 nm. In the wavelength band of 780 to 820 nm, which is the same as the excitation light, the relative fluorescence intensity value of dead eggs exceeded 10, but that of normal eggs was less than 4. Therefore, for example, if the fluorescence relative intensity value 7 is used as the classification threshold value, dead eggs including debilitated eggs can be discriminated with high accuracy.

From these observations, the following facts were found. First, it was found that the peak wavelength of the fluorescence spectrum emitted from the abnormal egg is almost constant regardless of the individual difference of the fertilized egg or the elapsed time from fertilization. Therefore, it was found that the quality of the fertilized eggs of the zebrafish can be determined with the highest accuracy by selectively detecting only the fluorescence spectrum in a very narrow band depending on the wavelength of the excitation light.

Next, when the excitation light wavelength is short, the intensity of the fluorescence spectrum of the abnormal egg is significantly increased in comparison with that of the normal egg in a narrow band adjacent to the short wavelength side of the excitation light wavelength. When the excitation light wavelength becomes longer, the intensity of the fluorescence spectrum of the abnormal egg increases significantly in a narrow band adjacent to the long wavelength side of the excitation light wavelength as compared with that of the normal egg. When the wavelength of the excitation light is further increased, the intensity of the fluorescence spectroscopic spectrum of the abnormal egg increases significantly in the same band as the excitation light as compared with that of the normal egg. Furthermore, the intensity difference between the fluorescence spectra of normal eggs and abnormal eggs increases with the elapsed time from fertilization, but when the elapsed time exceeds 8 hours, the intensity difference is almost saturated or rather decreases. I found out that Furthermore, it was found that sufficient discrimination accuracy was obtained when the elapsed time from the time of fertilization was 5 to 7 hours, more preferably 5.5 to 6.5 hours.
In the above description, the description has been centered on the zebrafish, but the present invention is not limited to this and can be applied to fishes although the position of the spectrum is slightly different. Further, the reflected light may be used instead of the fluorescent light, or the ratio of the reflected light contained in the fluorescent light may be increased.

Claims (8)

A fertilized egg inspection method for optically inspecting fertilized eggs of fish,
Of the fluorescence spectrum obtained from the fertilized egg, a fish fertilized egg test characterized in that the quality of the fertilized egg is determined based on an inspection spectrum composed of a spectrum of an excitation light band or a narrow band adjacent thereto. Method. Of the fluorescence spectra obtained from the fertilized eggs, it is determined whether or not the intensity of the inspection spectrum including the spectrum of a band less than 30 nm away from the band of the excitation light exceeds a predetermined threshold value. The method for inspecting a fertilized fish egg according to claim 1, wherein is determined to be defective. Of the fluorescence spectrum obtained from the fertilized egg, it is determined whether or not the intensity of the inspection spectrum including the spectrum of the excitation light band exceeds a predetermined threshold value, and if it exceeds, it is determined that the fertilized egg is defective. The method for inspecting a fertilized fish egg according to claim 1. The method for inspecting a fertilized fish egg according to claim 1, wherein the inspection spectrum is detected at an observation time point 5 to 8 hours after the fertilization time point. The method for inspecting a fertilized fish egg according to claim 4, wherein the inspection spectrum is detected at an observation time point after 5.5-7.5 hours from the time of fertilization. Fertilized fish eggs are stored with water in each well formed on the upper surface of the bottom plate of the multi-well plate,
After matching the center of the well with the optical axis of the excitation light, by irradiating the fertilized egg in the well with the excitation light, a predetermined spectral spectrum of the fluorescence spectrum obtained from the fertilized egg is detected,
After determining the quality of the fertilized egg based on the spectral spectrum, by horizontally moving the multi-well plate, the same detection operation is sequentially repeated for the fertilized eggs of the adjacent wells,
The obtained result of judgment and fish fertilized egg inspection method according to any one of claims 1 or et 4, wherein sequentially stores pairs of two-dimensional addresses of each well of each well. Each well formed on the upper surface of the bottom plate of the multi-well plate that contains fertilized eggs of fish together with water,
A light source that is incident with the center of the well aligned with the optical axis of excitation light, and by irradiating the fertilized egg in the well with excitation light, a light receiver that receives the reflected light obtained from the fertilized egg,
A measuring device for measuring the wavelength and intensity of the fluorescence spectrum of the light receiver which is incident while changing the wavelength of the light source, and a memory for recording the measured spectrum,
A fish fertilized egg inspection device having a discriminator for discriminating the quality of the fertilized egg based on a predetermined spectral spectrum recorded in the memory Said multiwell plate and a light source and means for relatively horizontally moving the position of the light receiver, for each of the wells obtained by measuring the same detection operation is sequentially repeated with respect to embryo of the well adjacent the determination result the fish embryo inspection apparatus according to claim 7, further comprising a second memory for the sequentially stores pairs of two-dimensional addresses of each well.

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