JPH05180755A - Fluorescence pattern reader - Google Patents

Abstract

PURPOSE:To enhance the reading sensitivity of fluorescence by detecting the shift of the optical axis of an optical scanning system with respect to a reading sample of various thicknesses and a reading sample of an optical refractive index to automatically correct the same. CONSTITUTION:When an optical scanning mechanism scans the irradiation light 31 from a light source 21 along a predetermined optical axis to irradiate a gel in the thickness direction thereof, a light detecting part sets a light detecting surface in a direction different from the optical axis to read the fluorescence of a migration pattern on the basis of the spatial positional relation of a light detecting route and separates the same from the scattered light of the light detecting surface. A photoelectric convertion part 24 performs the photoelectric conversion of the light signal detected by the light detecting part to output an electric signal. An amplifier 25 performs integrating operation to the electric signal from the conversion part 24 corresponding to the scanning of the irradiation light 31 to amplify the electric signal and successively outputs an electric signal of fluorescence from a migration pattern. Herein, an optical axis correcting part 40 is further provided and receives the electric signal from the amplifier 25 as an optical axis detection signal to correct the optical axis of the irradiation light 31.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescent pattern reading apparatus, and more particularly, it automatically detects misalignment of an optical axis of an optical scanning system with respect to reading samples having various thicknesses and reading samples having various optical refractive indexes. The present invention relates to a fluorescence pattern reading device that improves the reading sensitivity of fluorescence by making a correction.

[0002]

2. Description of the Related Art Generally, an electrophoretic analysis method using a radioactive isotope is used for structural analysis of various genes including diagnosis of genetic diseases and structural analysis of proteins such as amino acids. In such an electrophoretic analysis method, a fragment of a sample labeled or substituted with a radioisotope is subjected to electrophoresis using a gel, and the distribution pattern of the fragment of the sample developed by electrophoresis is analyzed. Is a method of analyzing a sample.

Genetic disease diagnosis will be described as an example of reading and analyzing an electrophoretic pattern. Human genomic DNA is
It is composed of about 3 × 10 ⁹ base pairs. The base sequence is almost constant throughout the human population, but when viewed in detail, there are variations among individuals. Such variations in the DNA sequence are called DNA polymorphisms. DN
The A polymorphism is found in both the gene region and the non-gene region, but the polymorphism in the gene region often appears as the polymorphism of the protein that is its phenotype. Such as blood type, tissue-compatible antigens, and differences in skin color and hair color between races,
The various varieties found in the human population stem from this polymorphism. DNA polymorphism is a germ cell D of human population from the time when human was established as an evolutionary species to the present.
Mutations generated in NA are accumulated in the population. Such a mutation occurs at a site having an important function for the existence of human, and the resulting phenotype indicates some pathological condition is called "genetic disease". It is said that there are over 3,000 genetic diseases in the human population.

The etiology of a genetic disease is a mutation that occurs on DNA, but by the time it is recognized as a disease, DNA →
There are various stages of mRNA → protein → phenotype (disease). Diagnosis as a disease is usually performed at the last level, but if the above flow has a simple linear relationship, diagnosis can be performed at the protein or DNA level.

The technique underlying DNA diagnosis is Southern.
This is called blotting. This method is basically divided into the following steps. (Step 1) Extraction of sample DNA, (Step 2) Decomposition of DNA by restriction enzyme, (Step 3) Fractionation by molecular weight of DNA using gel electrophoresis, (Step 4) To thin film filter of fractionated DNA (Step 5) Formation of a hybrid (hybridization) with a previously prepared probe DNA (a DNA having a homologous sequence with the gene to be detected is labeled with an isotope, etc.), (Step 6) autoradiography Detection of hybrids by the above 6 steps. A nylon membrane, a nitrocellulose membrane, or the like is used for the thin film filter in the fourth step.

When a genetic disease is targeted, any organ may be used for extracting the sample DNA. In the usual case, leukocytes are separated from a few milliliters of peripheral blood and DNA is extracted therefrom. Steps 1 to 6 usually require about 5 days. In diagnosing a genetic disease, fraction patterns of normal bodies are obtained and compared with each other based on the above steps. If the patterns are the same, it is determined to be normal.

Recently, in view of safety and the like, a probe labeled with a fluorescent dye instead of a radioactive isotope is used,
Attempts have been made to excite the fluorescent dye and read the electrophoretic pattern. For genetic diagnosis and DNA sequencing, the amount of sample is on the order of 10 ^-15 mol. Therefore, in order to obtain a signal-to-noise ratio equivalent to that of radioactive isotopes, advanced optical technology and signal processing technology are required. Need.

An electrophoretic device described in Japanese Patent Application Laid-Open No. 61-62843 is a device for fluorescently detecting a minute sample. Next, an electrophoretic device using such a fluorescence detection method will be specifically described.

FIG. 15 is an external perspective view of a conventional fluorescence type electrophoretic device. Explaining with reference to FIG. 15, the electrophoretic device performs electrophoresis of a sample and measures the fluorescence distribution, a migration measurement device 51, a data processing device 52 that performs data processing based on the measured data, and those. And a cable 53 for interconnecting Electrophoresis measurement device 5
A door 51a is opened in 1 to inject a gel serving as a base for electrophoresing DNA fragments, and a predetermined amount of a sample to be electrophoresed is injected. When the door 51a is closed and the migration start switch of the operation display panel 51b is pressed, electrophoresis is started. When the electrophoresis is started, in the electrophoretic measurement device 51, the operating state is displayed on the monitor on the operation display panel 51b. The data measured by the electrophoretic measurement device 51 is transferred to the data processing device 52, and predetermined data processing programmed in advance is performed. The data processing device 52 includes a computer main body 54, a keyboard 55 for inputting a command from a user, a display device 56 for displaying a processing state and a result, and a printer 57 for recording a result of the data processing. It is configured.

FIG. 16 is a block diagram showing the internal structure of the electrophoretic measuring device. As shown in FIG. 16, the electrophoretic measurement device (51; FIG. 15) is composed of an electrophoretic device section 63 and a signal processing section 64.
Together, these two parts make up the electrophoretic measurement device. The electrophoretic device unit 63 includes an electrophoretic unit 5 that performs electrophoresis, a first electrode 2a and a second electrode 2b for applying a voltage to the electrophoretic unit 5, an electrophoretic unit 5, and electrodes 2a and 2b.
A support plate 3 for supporting the electrophoretic part, an electrophoretic power supply device 4 for applying a voltage to the electrophoretic part 5, a light source 11 for emitting light for exciting the fluorescent substance, and for guiding light from the light source 11. Optical fiber 12 and fluorescent light 1 generated from the fluorescent substance
It is composed of an optical condenser 14 for collecting and receiving 3 light, an optical filter 15 for selectively passing light of a specific wavelength, and an optical sensor 16 for converting the received light into an electric signal. .. Further, the signal processing unit 64 includes an amplifier 17 that receives and amplifies an electric signal from the optical sensor 16, and an analog / analog converter that converts an analog signal of the electric signal into digital data.
Digital conversion circuit 18 and signal processing section 19 for performing preprocessing such as averaging processing on the digitally converted data
And an interface 20 for performing interface processing for sending preprocessed data to an external data processing device, and a control circuit 10 for controlling the entire electrophoretic device part and the signal processing system. The digital signal OUT output from the signal processing device 64 is sent to the data processing device (52; FIG. 15) and subjected to data processing such as analysis processing.

Next, the operation of the electrophoretic device thus constructed will be described. Please refer to FIG. 15 and FIG. The door 51a in the electrophoretic measurement device 51 is opened, the gel is injected into the electrophoretic section 5 inside, and the sample of the DNA fragment labeled with the fluorescent substance is further injected. When a switch on the operation panel 51b is operated to instruct to start electrophoresis, the voltage from the electrophoresis power supply device 4 is supplied to the electrophoresis unit 5 by the electrodes 2a and 2b, and the electrophoresis is started. The samples labeled with a fluorescent substance by electrophoresis are electrophoresed in lanes 71, 72, 73, 74 of the respective samples, for example, as shown in FIG. 19, and are collected for each molecular weight of the molecules contained in the sample. , Make band 66 for each. Molecules with a lighter molecular weight have a faster migration speed, and therefore have a longer migration distance within the same time. The detection of these bands 66 is shown in FIG.
As shown in (A), the light from the light source is passed through the optical fiber 12 to irradiate the gel on the optical path 61, so that the fluorescent substances of the labels gathered in the band 66 in the gel are fluorescent 13
Is generated and fluorescence 13 is detected.

The fluorescence 13 emitted here depends on the extinction coefficient, the quantum efficiency, the intensity of the excitation light, etc. of the fluorescent substance, but the fluorescent substance is contained in a very small amount of about 10 ^-16 mol per band. Therefore, the light becomes extremely faint. For example,
Explaining the case where Fluorescein Isothiocyanate is used as the fluorescent substance, the peak value of the excitation wavelength of the excitation light by fluorescein isothiocyanate is 490 nm, and the peak value of the fluorescence wavelength is 520 nm. Further, the molar fluorescence coefficient is 7 × 10 ⁴ mol ⁻¹ · cm ⁻¹ , and the quantum efficiency is about 0.65.

When 10 ⁻¹⁶ mol of a fluorescent substance is present in one band, calculation is performed assuming that an argon ion laser with a wavelength of 488 nm and an output of 1 mW is used for excitation light, but the thickness varies depending on the gel. The amount of fluorescent light generated is 10
Only ¹⁰ photons / S of fluorescent photons are generated. Therefore, very weak fluorescence must be detected with high sensitivity.

The front view of the electrophoretic unit 5 is shown in FIG.
As shown in FIG. 17 (B), the longitudinal sectional view is composed of a gel 5a such as polyacrylamide and glass support plates 5b and 5c for narrowing and supporting the gel 5a from both sides. A sample of, for example, a DNA fragment is injected into the gel 5a of the electrophoretic unit 5 from above, and the first electrode 2a and the second electrode 2a
By applying a migration voltage to the electrode 2b of FIG. 16 (FIG. 16) and performing electrophoresis, light emitted from the light source, for example, laser light is passed from the optical fiber 12 through the optical path 61 in the gel 5a,
The fluorescent substance on the optical path 61 is irradiated. As a result, the optical path 6
The fluorescent substance existing on 1 is excited to emit fluorescence 13. The fluorescence 13 reaches a condenser 14 of an optical system composed of a combination of lenses, and after being condensed, a desired fluorescence wavelength is selected by an optical filter 15 and converted into an electric signal by a one-dimensional photosensor 16. To be done. In the optical sensor 16, in order to efficiently convert the weak light into an electric signal, an image intensifier or the like is used to optically amplify the light by 10 ⁴ to 10 ⁵ times, and the image is electrically converted by a CCD one-dimensional optical sensor or the like. Convert to signal. The electric signal converted by the optical sensor 16 is amplified to a signal of a desired level by the amplifier 17, converted from an analog signal to a digital signal by the analog / digital conversion circuit 18, and sent to the signal processing unit 19. In the signal processing unit 19, the signal-to-noise ratio (S / N ratio)
In order to improve the signal processing, signal processing such as arithmetic mean processing is performed. The data of the digital signal subjected to the signal processing in this way is transferred to the data processing device 5 by the interface 20.
2 is sent.

18 (A) and 18 (B) are diagrams for explaining an example of the fluorescence intensity pattern signal of the DNA fragment sent from the electrophoretic measuring device 51. For example, in FIG.
As shown in (A), the electrophoretic section 5 subjected to electrophoresis
When the laser light is irradiated onto the optical path 61, the optical path 6
Since the fluorescent substance of the gel present on 1 is excited and emits fluorescence, this fluorescence is detected in a direction of the electrophoresis direction 62 at a predetermined detection position for each lane over time. As a result, fluorescence is detected when the band 66 of each lane passes through the position on the optical path 61, and the pattern signal of the fluorescence intensity in one lane is shown in FIG.
As shown in FIG. Therefore, when the band 66 passes through the position on the optical path 61, the peak of the fluorescence intensity is obtained. Therefore, the fluorescence intensity pattern signal shown in FIG. 18B is a fluorescence intensity pattern signal of band 66 in the direction of electrophoresis 62.

In the data processor 52, the computer main body 54
By receiving the data of the fluorescence intensity pattern signal of the DNA fragment sent from the electrophoretic measurement device 51, the data processing for comparing the molecular weights and determining the base sequence of the DNA is performed from the data of the fluorescence intensity pattern. The sequence of bases and the like determined by data processing is symbolically output and displayed on the screen of the display device 56 or printed out by the printer 57.

In the above example, an example of an apparatus using a fluorescent dye as a method for labeling a sample has been shown, but as another example, after labeling with a fluorescent dye in the same manner and performing electrophoresis, a fluorescence pattern based on an electrophoretic pattern is obtained. A reading device is Japanese Patent Laid-Open No. 1-16
It is disclosed in Japanese Patent No. 7649. The device of this other example is a type that reads the fluorescence pattern of the entire migration unit after the electrophoresis is completed, unlike the type that reads the distribution of the fluorescence pattern that passes through the reading unit at the same time while performing electrophoresis as described above. Has become.

[0018]

By the way, various types of samples can be read by the type in which the fluorescence pattern of the entire electrophoretic portion is read after the electrophoresis as described above is completed. The samples to be read are roughly classified into, for example, DNA injected into gel and DNA transferred to a membrane filter.

The gel and the membrane filter have different optical refractive indexes. Also, the type and thickness of gel differ depending on the experimental method. Since the optical refractive index and the thickness differ depending on the sample in this way, the fluorescent light emitted after the excitation light is irradiated passes through the condensing lens, and the optical axis until it is focused at the entrance of the light receiving part is deviated. .. Further, the optical axis is displaced due to the variation in the thickness of the gel glass support and the difference in the optical refractive index. Due to these deviations of the optical axis, the fluorescence to be detected is focused outside the entrance of the light receiving portion, and the amount of light reaching the optical sensor is reduced. Therefore, there is a problem that the detection sensitivity of the fluorescence pattern is lowered.

An object of the present invention is to detect misalignment of the optical axis of an optical scanning system and automatically correct it for read samples of various thicknesses and read samples of various optical refractive indices.
An object of the present invention is to provide a fluorescence pattern reading device that improves the fluorescence reading sensitivity.

[0021]

In order to achieve the above object, the fluorescence pattern reading apparatus of the present invention emits fluorescence by exciting a fluorescent substance labeling a sample after the sample is electrophoresed and developed. In a migration pattern reading device that reads a fluorescent pattern that emits light, a light source that emits irradiation light that excites the fluorescent substance of the migration pattern, and irradiation light from the light source is scanned along a predetermined optical axis in the thickness direction of the gel. An optical scanning mechanism for irradiating, and a light receiving section for setting the light receiving surface in a direction different from the optical axis of the irradiating light and separating the fluorescence of the migration pattern from the scattered light on the reading surface by the spatial positional relationship of the light receiving path, , A photoelectric conversion unit that performs photoelectric conversion of the optical signal received by the light receiving unit and outputs an electric signal, and an electric signal from the photoelectric conversion unit that performs an integration operation corresponding to scanning of irradiation light and amplifies and sequentially An amplifier for outputting an electric signal of the fluorescence from the dynamic pattern, characterized by comprising an optical axis correcting unit that performs electrical signal to correct the optical axis of the irradiation light by receiving an optical axis detection signal from the amplifier.

[0022]

In the fluorescent pattern reader of the present invention,
The light source emits irradiation light that excites the fluorescent substance in the migration pattern. When the light scanning mechanism scans the irradiation light from the light source along a predetermined optical axis and irradiates it in the thickness direction of the gel, the light receiving part sets the light receiving surface in a direction different from the optical axis of the irradiation light to set the space of the light receiving path. The fluorescence of the electrophoretic pattern is separated from the scattered light on the reading surface and received according to the physical positional relationship. The photoelectric conversion unit photoelectrically converts the optical signal received by the light receiving unit and outputs an electric signal. Then, the amplifier performs an integration operation on the electric signal from the photoelectric conversion unit in correspondence with the scanning of the irradiation light to amplify the electric signal, and sequentially outputs the electric signal of fluorescence from the migration pattern. Here, an optical axis correction unit is further provided.

The optical axis correction unit receives the electric signal from the amplifier as an optical axis detection signal and corrects the optical axis of the irradiation light. The correction of the optical axis of the irradiation light performed by the optical axis correction unit detects the deviation of the optical axis with respect to reading samples of various thicknesses and reading samples of various optical refractive indices, and detects the optical axis of the optical scanning system. Is an optical axis correction for correcting Therefore, for example, the optical axis correction unit includes a folding mirror that changes the optical axis of the light emitted from the light source, an optical axis correction actuator that changes the angle of the folding mirror, an actuator driver that controls the drive of the actuator, and an actuator driver. And a control circuit for giving a drive control signal to the chainer driver. The optical axis correcting actuator is, for example, a ceramic actuator, the control circuit is a feedback control circuit, receives an electric signal from the amplifier as an optical axis detection signal, and the direction in which the intensity of the optical axis detection signal becomes an extreme value. In addition, a drive control signal to the actuator driver is generated.

Accordingly, in the fluorescence detection of the electrophoretic pattern to be read, the optical axis of the folding mirror is set at an angular position where the amount of received fluorescence of the fluorescence emitted from the sample surface is maximized, and the fluorescence from the sample can be read with high sensitivity. You can Therefore, it is possible to correct the difference in the optical distance from the sample surface to the focusing lens due to the difference in the thickness and the optical refractive index of each sample, and it is possible to read the fluorescence pattern with high sensitivity.

[0025]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be specifically described below with reference to the drawings. FIG. 1 is a schematic diagram for explaining the overall configuration of a fluorescent pattern reading device according to an embodiment of the present invention, and FIG. 2 is a diagram for explaining the mounting position of an electrophoretic unit mounted on the measuring unit body of the fluorescent pattern reading device. is there. As shown in FIG. 1, in the fluorescence pattern reading apparatus, the electrophoretic unit 1 and the reading unit 6 are separated to constitute the entire apparatus. The electrophoretic unit 1 includes an electrophoretic unit 5 including a gel serving as a base for performing electrophoresis and a gel support that sandwiches and supports the gel with a glass plate or the like, and the electrophoretic unit 5 to which the electrophoretic unit 5 is attached. The first electrode 2a and the second electrode 2b that apply an electrophoretic voltage to the electrophoretic portion 5 and a support plate 3 that supports the electrophoretic portion 5 while supporting the first electrode 2a and the second electrode 2b; It is composed of an electrophoresis power supply device 4 for supplying a voltage. As described above, the electrophoretic section 5 is composed of a gel such as polyacrylamide serving as a base for developing a sample to be electrophoresed, and a glass plate gel support that sandwiches and supports the gel from both sides (described above). (See FIG. 17A and FIG. 17B)

In the electrophoretic unit 1, the electrophoretic unit 5 is attached, and DN is electrophoresed from above the gel of the electrophoretic unit 5.
A fragmented sample such as A fragment is supplied, and the first power supply 2 for electrophoresis and the second electrode 2b
An electrophoretic voltage is applied to the cells to cause electrophoresis. The electrophoretic unit 5 after the electrophoresis in the electrophoretic unit 1 is removed from the electrophoretic unit 1, and then the reading unit 6 is used.
Then, read the electrophoretic pattern.

In the reading unit 6, the electrophoretic unit 5 which has been electrophoresed in the electrophoretic unit 1 is left as it is (or in the gel state in which only the gel is taken out from the electrophoretic unit 5).
It is attached to the measuring unit main body 7, the electrophoresis pattern is read, and data processing is performed. As shown in FIG. 1, the reading unit 6 is configured with the measuring section main body 7 as a main part, and is further configured with a data processing device 8, an image printer 9, and the like. The data processing device 8 and the image printer 9 perform data processing, image processing, and image processing on the image data of the electrophoretic pattern read by the measuring unit body 7.
Judgment processing is performed, and the read electrophoretic pattern data is processed and output. As shown in FIG. 2, a reading stand 7c is attached to the measuring section main body 7 to read the electrophoretic section 1 (electrophoretic section unit including a gel and a gel support) which has already undergone electrophoresis in the electrophoretic unit 1. It is provided directly below the lid 7a on the upper part of the main body.

The electrophoretic unit 5 removed from the electrophoretic unit 1 has a measuring unit main body 7 with a lid 7a on the upper portion of the measuring unit main body.
Opened and attached to the reading stand 7c. Reading stand 7c
After mounting the gel to be read by the electrophoretic unit 5 on the
Is closed and the reading start switch of the operation display panel 7b of the measurement unit main body 7 is pressed, the measurement unit main body 7 moves to the migration unit 5
Begin reading the electrophoretic pattern of the gel. When the reading of the electrophoretic pattern is started, the measuring unit main body 7
Scanning of the irradiation light from the point light source built into the device is started, the excitation light is irradiated to the gel of the electrophoretic part 5 mounted, the fluorescent substance is excited, and the fluorescence emitted by this is received to detect the distribution of the fluorescent substance. Measure the pattern. The data processing device 8 performs data processing based on the read data measured by the measuring unit body 7, and also controls the measuring unit body 7. The data processed data is output and visualized by the image printer 9. In this image printer, the migration pattern is distinguished for each sample and the migration pattern is printed.

When a sample is subjected to an electrophoretic analysis using a fluorescence pattern reader, as described above, first, the sample (DNA fragment) labeled with a fluorescent dye (fluorescent substance) is used by using the electrophoretic unit 1. Perform electrophoresis. After completion of electrophoresis for a predetermined time of about 5 hours, the electrophoretic unit 5 is removed from the electrophoretic unit 1. With the gel of the removed electrophoretic unit 5 in the state of the electrophoretic unit 5 as it is or in the state of removing the glass of the gel support, open the lid 7a on the upper part of the measuring unit main body 7 of the reading unit 6, and read the internal reading table. Place on top of 7c. Then, close the lid 7a,
The setting to the reading unit is completed. At this time, if the gel on which the electrophoresis has been performed is a sample that is not labeled with a fluorescent dye, the sample may be treated with a fluorescent dye at this stage. At this time, a treatment such as drying the gel is also performed.

Next, an operation for instructing to start reading the electrophoretic pattern is performed. The reading start operation is performed by a pressing start instruction of the reading start switch of the operation display panel 7b or by a reading start instruction from the data processing device 8. When the reading operation is started by the data processing device 8, the mounting state of the electrophoretic unit in the measuring unit main body 7 is sent to the data processing device 8 side through the control signal line, and the state is confirmed to confirm the data processing device 8
Controls the operation of the reading unit of the measuring unit main body 7. In this case, the parameter setting such as the reading speed during the operation is registered in advance on the data processing device 8 side, and the reading start operation is automatically performed. To be done.

The read fluorescent dye distribution data is sent to the data processor 8. The data processing device 8 performs desired processing that is programmed in advance, such as processing for detecting peaks of fluorescence intensity and processing for obtaining migration distance. As a result of the data processing, the image printer 9 prints out the intensity of the fluorescence as a grayscale image, or outputs the intensity of the fluorescence as a contour line format or an image in which the intensity is classified by color or density, as necessary. The image printed out as a grayscale image according to the fluorescence intensity is the same image as the radioactive X-ray film image electrophoresed by labeling with a conventionally used radioactive substance. If necessary, the result data obtained by the data processing is stored as digital data in the magnetic or optical recording device.

FIG. 3 is a block diagram showing the construction of the main part of the measuring section main body including the optical axis correcting mechanism section. FIG. 4 is a diagram illustrating a schematic configuration of a mechanical section of an optical axis correction mechanism that performs automatic optical axis correction, and FIG. 5 is a diagram illustrating a configuration of an actuator of the mechanical section of the optical axis correction mechanism. As shown on the upper side of the block diagram of FIG.
An optical axis correction mechanism 40 that corrects the optical axis of the irradiation light is provided. The schematic configuration diagram of the mechanical portion of the optical axis correction mechanism shown in FIG. 4 is a longitudinal sectional view of a part of the structure of the optical system of the mechanical portion of the optical axis correction mechanism 40 shown in FIG.

First, with reference to FIG. 3, the flow of the entire operation of the measuring unit main body 7 for reading the fluorescent pattern will be described. In the measurement unit main body, a laser beam 31 for exciting the fluorescent substance labeling the electrophoretic pattern after electrophoresis is emitted from the light source 21, and the mirror driver 30
The oscillating mirror 22 driven by illuminates the folding mirror 44 of the optical axis correction mechanism 40 while scanning in the right and left directions in the figure. In the folding mirror 44, the actuator 41 attached to the folding mirror 44 is driven during the operation of correcting the optical axis of the irradiation light (laser beam), and the folding mirror 44 is displaced to cause the laser beam 3 to move.
The optical axis of 1 is changed in the front-back direction of the drawing, and the displacement of the folding mirror 44 is positioned at a position where the sensitivity of receiving the fluorescence of the migration pattern from the gel 5 is optimum. As a result, the laser beam 31 scanned by the vibrating mirror 22
Is reflected by the folding mirror 44 displaced for optical axis correction, and then is irradiated in the gel thickness direction of the electrophoretic unit 5.

That is, the laser beam 31 from the light source 21
Is reflected by the vibrating mirror 22 so as to be scanned in the left-right direction in the drawing, further reflected by the folding mirror 44, and applied to the gel of the electrophoretic unit 5 to be read. Every time the laser beam 31 is scanned once, the electrophoretic unit 5 is moved as a whole in the direction perpendicular to the scanning direction of the laser beam 31, that is, in the front-back direction of the drawing. Can be read. This movement in the sub-scan direction is performed by the reading table 7
This is performed by moving the electrophoretic unit 5 placed on c by a reading drive mechanism (FIG. 2). An optical trap 32 is provided on the side of the migration unit 5 opposite to the irradiation surface of the migration unit 5 so that the laser beam 31 that has passed through the gel does not adversely affect as stray light.

By irradiating the spot light of the laser beam 31 scanned by the vibrating mirror 22, the fluorescence 13 emitted from the gel of the electrophoretic unit 5 is received through the condenser 23. Since the condenser 23 receives the fluorescent light 13, the optical axis of the light receiving path is different from the optical axis of the spot light that illuminates the electrophoretic unit 5, and the spatial position relationship of the optical path of the light receiving path is adjusted by the optical lens system. Is defined to form an optical path, the detection sensitivity from scattered light emitted from the irradiation surface of the migration unit 5 is increased, and the fluorescence 13 is received. The fluorescence 13 received and collected by the condenser 23 is further photoelectrically converted by a photoelectric conversion unit 24 including a condenser lens, an optical filter, and a photomultiplier tube to be converted into an electric signal. The converted electric signal is input to the amplifier 25. Then, the electric signal amplified by the amplifier 25 is input to an analog / digital converter (hereinafter abbreviated as A / D converter) 26 and converted into digital data. The detection signal converted into digital data is stored in the memory 28, and the data stored in the memory 28 is sent to the data processing device 8 through the interface 29. The overall control of such a series of signal processing is performed by the control circuit 27.

Next, the operation of the optical axis correction mechanism 40 will be further described with reference to FIG. FIG. 4 shows the schematic configuration of the optical system of the optical axis correction mechanism 40 as a center. In the fluorescence pattern reading device here, the gel 5 of the electrophoretic unit 5 is used.
Since a is read for each glass plate (such as the glass plate of the garum support plate 5b and the reading table 7c), the difference in glass thickness, the gel thickness not being uniform, and
The optical distance will vary from sample to sample due to differences in the thickness of gel used and the refractive index. For this reason, the focus position of the fluorescence 13 received by the condenser 23 is shifted, and the amount of received light differs for each sample. Therefore, by the operation of the optical axis correction mechanism 40 here, the folding mirror 44 is displaced by the actuator 41, and the gel 5 of the migration unit 5 is moved.
As shown in FIG. 4, by displacing the irradiation position of the laser beam applied to a to the left-right direction, the optical axis 45 from the folding mirror 44 to the condenser 23 is corrected to the optimum position, and the gel 5a The automatic optical axis correction is performed so as to stably receive the fluorescence 13 from.

For the optical axis correction, a usual optical technique in an optical system can be used. For example, a correction method using an acousto-optic element that uses the diffraction phenomenon of light or a method using another actuator is used. In this embodiment, a method is used in which the folding mirror 44 is controlled by the actuator 41 composed of a piezoelectric ceramic element.

Referring again to FIG. 3, the actuator 4
The control system for driving 1 will be described. The fluorescent light 13 generated from the gel is received by the condenser 23 and collected, converted into an electric signal by the photoelectric converter 24, amplified by the amplifier 25, and further converted into digital data by the A / D converter 26. Therefore, this digital data is used as a detection signal for generating a control signal for optical axis correction. That is, digital data proportional to the detected fluorescence intensity is used as an optical axis detection signal, and a feedback loop control system is configured so that the detected signal intensity is maximized. In the processing of this control system, the macro computer constituting the control circuit 27 processes a series of control signals and sends the control signals to the actuator control circuit 43, whereby the control operation is performed. The actuator control circuit 43 sends a control signal to the actuator driver 42, and the actuator driver 42 drives the actuator 41. A folding mirror 44 is attached to the actuator 41, and the actuator 4
When 1 is driven by the actuator driver 42, the reflection angle of the folding mirror 44 is displaced. The small displacement of the folding mirror 44 changes the optical axis of the laser beam with which the electrophoretic unit 5 is irradiated. The change of the optical axis is controlled so that the magnitude of the optical axis detection signal becomes maximum.

In the control operation of the actuator for displacing the folding mirror 44, first, in reading the gel of the electrophoretic section 5, the laser beam is scanned, and the voltage applied to the actuator 41 is changed each time the gel is read. The mirror 44 is displaced. Data of the magnitude of the electric signal detected from the photoelectric conversion unit 24 each time the scanning is performed is received via the amplifier 25, the A / D converter 26, and the memory 28, and sequentially compared, and the magnitude of the electric signal is maximum. The voltage applied to the actuator 40 at that time is stored. A control signal for generating a voltage applied to the actuator when the magnitude of the electric signal is maximum is used as a reference control signal for setting the optimum position of the folding mirror 44. When changing the voltage applied to the actuator, first, the variable width and the step width are set. First, the variable width and the step width of the applied voltage are set to be large, and the value of the applied voltage having a high electric signal is roughly grasped. Then, the value of the applied voltage obtained next is set as a reference, the variable width and the step width are set small, and the applied voltage is determined. By supplying the applied voltage determined in this way to the actuator 41, control of the optical axis correction of the laser beam 31 as the irradiation light is performed.

Next, the actuator 41 of the optical axis correction mechanism 40 will be described. Referring to FIG. 5, there is shown the structure of the actuator 41 to which the folding mirror 44 of the mechanism portion of the optical axis correction mechanism is attached. As shown in FIG. 5, a piezoelectric actuator having a structure in which two piezoelectric ceramic plates 41a and 41b made of a piezoelectric material are bonded to each other is used as the actuator 41 that constitutes the main part. The piezoelectric type actuator 41 here is called a bimorph type actuator, and the bimorph type actuator is characterized by a large displacement expansion rate in addition to a simple preparation method in which two ceramic plates are bonded with an adhesive. is there. Basically, it is a structure in which two piezoelectric ceramic plates that expand and contract in the length direction are bonded together, and when one expands, the other contracts, causing bending displacement as a whole. To increase the response frequency and increase the driving force generation force,
An elastic plate 41c called a shim is sandwiched between the ceramic plates 41a and 41b. As the elastic plate 41c, a phosphor bronze plate or the like is used. In the bending operation, an electric field is applied using the shim (elastic plate) 41c as a negative pole and the two ceramic plates 41a and 41b as a positive pole. Due to the applied bending displacement of the actuator 40, the folding mirror 4 to which the actuator 41 is attached
4 is displaced.

As described above, in the mechanical portion of the optical axis correcting mechanism 40, the ends of the shims 41c sandwiched between the two ceramic plates 41a and 41b of the actuator 41 are attached to the back side of the mirror surface of the folding mirror 44, and the folding is performed. The end surface of the actuator 41 on the side where the mirror 44 is not attached is fixed. Further, the shim 41c here is bent in a U shape. This enables the folding mirror 4 without bending the shim.
It is possible to obtain a larger displacement amount than when mounting No. 4. Regarding the drive control of the actuator 41,
The variable speed of the applied voltage and the scanning speed of the laser beam 31 are synchronized.

Next, an optical scanning mechanism for irradiating the irradiation surface of the gel of the migration unit 5 with the laser beam will be described. FIG. 6 is a diagram illustrating an optical scanning mechanism that scans a gel surface with a laser beam using a vibrating mirror, and FIG. 7 is a diagram illustrating a relationship between a rotation angle of the vibrating mirror and a moving distance of spot light of the laser beam. Is. In the optical scanning mechanism here, the migration unit 5
And the arrangement positions of the light source 21 and the vibrating mirror 22 have a positional relationship as shown in FIG.
When 2 is driven by the mirror driver 30 so as to oscillate at a constant angular velocity, in the migration unit 5, the moving speed of the light spots at both ends becomes faster than in the vicinity of the central part (X = 0). Therefore, the detection sensitivity of the fluorescence detected from the sample of the electrophoretic unit 5 differs between the central portion and the end portion. On the other hand, in this embodiment, the moving speed of the laser spot light on the gel of the electrophoretic unit 5 is constant,
The speed of driving the vibrating mirror is corrected and controlled. That is,
The relationship between the angle X of the mirror and the position X of the spot light is
The relationship is as shown in FIG. 7, and using the distance Z between the rotation center of the vibrating mirror and the center of the electrophoretic unit 5, the mirror angle θ is expressed by the following equation. θ = arctan (X / Z) where Z is the distance from the center of rotation of the vibrating mirror 22 to the gel of the electrophoretic unit 5, and X is a perpendicular line from the center of rotation of the vibrating mirror 22 to the gel surface of the electrophoretic unit 5. It is the distance in the plane direction of the gel with the lowered point as the origin.

As a method of correcting the relationship between the rotation angle and the moving distance in this type of optical scanning mechanism, fθ
Although there is a method of using a lens, since the fθ lens is expensive and the device becomes heavy because the fθ lens is mounted, here, the correction between the rotation angle of the vibrating mirror of the optical scanning mechanism and the moving distance is performed by the mirror driver. 30 includes a control circuit for variably controlling the rotational angular velocity of the vibrating mirror 22.
This is performed by correcting and controlling the rotational drive speed of No. 2.

FIG. 8 is a block diagram showing a main configuration of a control circuit of a mirror driver for rotationally controlling the oscillating mirror. A galvanometer scanner is used as the actuator of the vibration mirror, and the rotation angle control of the vibration mirror is
Control is performed by applying a voltage proportional to the rotation angle. In order for the spot light of the laser beam to move at a constant velocity on the irradiation surface of the gel, the distance X of the irradiation surface and the time t may be controlled to have a proportional relationship. Since the relationship between the rotation angle θ of the vibrating mirror and the movement distance X of the spot light is as shown in FIG. 7, the horizontal axis of the graph in FIG. 7 corresponds to the time axis and the vertical axis corresponds to the voltage axis. A signal having a different voltage waveform is generated and used as a drive control signal for driving the vibrating mirror. The generation of such a drive control signal is performed by the control circuit in the mirror driver 30, and the generated drive control signal is supplied to the actuator of the vibrating mirror 22 to control the drive of the vibrating mirror 22.

As shown in FIG. 7, the mirror driver 30 includes a read-only memory 30a storing a function waveform,
Digital that converts the read function data into a voltage signal
An analog conversion circuit 30b, a driver 30c that amplifies the converted voltage signal and outputs it as a drive control signal, a counter 30d that gives a read address to the memory in time series, and an oscillation circuit 30e that gives a clock signal to the counter. It consists of

The oscillator circuit 30e operates according to an instruction from the control circuit 27 of the measuring section main body, the clock signal from the oscillator circuit 30e is input to the counter 30d, and the counter 30d.
d counts the clock signal, and the read-only memory 3
The read address to be supplied to 0a is generated in time series. When the read addresses sequentially generated from the counter 30d are time-sequentially supplied to the read-only memory 30a, the prestored function data are sequentially read from the read-only memory 30a. Function data (FIG. 7) regarding the rotation angle of the vibrating mirror is written in the read-only memory 30a in advance, and such function data is read in time series. In this example, the number of bits of function data is 12 bits. The read function data is converted into a voltage signal of an analog signal for controlling the rotation angle of the vibrating mirror in the digital / analog conversion circuit 30b. This voltage signal is filtered by the driver 30c to remove step noise, and further power-amplified, and the vibration mirror 22 is used as a drive control signal.
Is supplied to. Thus, the vibrating mirror can be vibrated at a desired rotation angular velocity such that the moving speed (scan speed) of the spot light of the laser beam in the electrophoretic unit becomes constant.

Further, the scanning speed here is 0.5 Hz, 1 Hz, 2 Hz, 5 Hz, 10 Hz, 20 so that they are logarithmically divided into equal parts.
It is configured to be variable at each speed of Hz, 50Hz, 100Hz, and 200Hz. This is because the reading speed can be changed according to the amount of the fluorescent substance labeled on the sample to be electrophoresed and the difference in the quantum efficiency of the fluorescent substance, so that the reading can be performed efficiently. In this case, the scan speed is designated from the operation display panel 7b or the data processing device 8. From the control circuit 27 to the mirror driver 30,
When the scan speed instruction data is sent, the counter 30
d and the oscillation circuit 30e are controlled to drive the vibrating mirror 22 at a desired scan speed.

In this way, the laser beam from the light source 21 is scanned by the drive control of the vibrating mirror 22 and is emitted as spot light that moves at a constant speed in the migration unit 5. As a result, the fluorescent substance of the gel of the electrophoretic unit 5 in the portion irradiated by the irradiation light of the laser light is excited, and fluorescence 13 is emitted (FIG. 3).

FIG. 9 is a diagram showing the configuration of the main part of the condenser and the photoelectric conversion part for receiving the fluorescence generated from the gel, centering on the optical path. As described above, the gel 5a of the migration section
Are sandwiched and supported by glass gel supports 5b and 5c. As the gel supports 5b and 5c here, in the case of the electrophoretic section 5 of this example, the gel supports 5b and 5c are made of boron silicate glass with relatively little fluorescence. Besides this,
As the gel supports 5b and 5c, quartz glass or various optical glasses can be used.

In the electrophoretic unit 5, when the irradiation light of the laser beam 31 which is scanned and moved is applied, the irradiation light of the laser beam 31 passes through the gel support 5c, the gel 5a and the gel support 5b in the thickness direction. , Reach the gel 5a.
In the gel 5a, the light of the laser beam 31 advances in the thickness direction. The thicknesses of the gel supporting plates 5b, 5c and the gel 5a are about 5 mm and about 0.35 mm, respectively, and the light of the laser beam 31 irradiated in the thickness direction of the gel supporting plates 5b, 5c and the gel 5a migrates. The light intensity reaching the gel is almost equal at any position of the portion 5. Further, the spread and intensity decrease of the laser beam 31 due to the light scattering generated on the light incident surfaces of the gel 5a and the gel supports 5b and 5c are also largely because the excitation light is incident perpendicularly to the surface in the thickness direction. Less. The laser beam 31 transmitted through the gel 5a enters the optical trap 32 and is attenuated so as not to adversely affect as stray light.

As described above, by scanning the laser beam 31, the irradiation light is irradiated on the gel 5a, and the fluorescence generated from the inside of the gel 5a by the irradiation of the irradiation light (excitation light of fluorescence) is the excitation light. The light is condensed by the light collector 23 together with scattered light by itself. The scattered light generated in the gel supports 5b and 5c is geometrically and optically separated by the spatial positional relationship of the light receiving path by forming an optical path as shown in FIG. 7, and only the fluorescence from the gel is extracted. And is sent to the photoelectric conversion unit 24. In the photoelectric conversion unit 24,
The scattered light and fluorescence generated in the gel are separated by using an optical filter, and the weak fluorescence is converted into an electric signal by the photomultiplier tube. The configuration of the optical path of the condenser 23 is
As shown in FIG. 9, the light is introduced into the photoelectric converter 24 by an optical fiber array 23b having a single light entrance.

The configuration of the optical system in the condenser 23 and the photoelectric conversion section 24 will be described with reference to FIG.
As shown in FIG. 9, the optical path is configured such that the fluorescent light from the gel 5a of the electrophoretic unit 5 and the scattered light of the excitation light generated from the gel supports 5b and 5c are received and condensed by the cylindrical lens 23a. There is.

Referring to FIG. 9, fluorescence 1 from the electrophoretic unit 5
3 and the scattered light of the excitation light generated from the gel supports 5b and 5c reach the cylindrical lens 23a, and the scattered light and fluorescence are imaged on the opposite side by the cylindrical lens 23a as illustrated. A in the figure
The points are the focal points for the fluorescence from the gel 5a and the scattered light of the excitation light generated from the gel 5a. Further, in the case of scattered light of the excitation light generated on the surface of the light incident surface of the gel support 5b, 5c, for example, in the case of scattered light from the surface of the gel support 5c, an image is formed at the point A'in the figure. .. Here, the optical fiber array 23b is arranged at the position of the image forming point A so that the light entrance port receives only the fluorescence from the gel 5a. To
The fluorescence is separated from the scattered light from the gel support.

As described above, in the method of irradiating the excitation light in the thickness direction, the refractive index of the gel and the glass as the gel support is relatively close to about 1.4 to 1.5, and the gel and the gel support are supported. Due to the close contact of the body glass interface, very little scattered light is generated at this interface. Therefore, the light received at the point A has less scattered light of the excitation light generated on the surface of the gel 5a,
Only the fluorescence from the inside of the gel 5a is largely received.

By the way, in the case where either one of the gel supports 5b and 5c or both of the gel supports is removed and the gel 5a is directly scanned with the irradiation light as the excitation light, the gel as described above is used. Although almost the same amount of scattered light as that generated from the glass surface that is the support may be generated from the gel surface, in this case, the reading stand (7c; FIG. 2) of the measuring unit main body is placed. Since the thickness of the glass has the same effect as the thickness of the glass of the gel support as described above, the fluorescence from the gel surface can be reliably detected without lowering the detection sensitivity. The gel supports 5b, 5
When either or both of c are removed, the gel 5a is treated with a dye at this point. In particular, when it is not necessary to remove the gel supports 5b and 5c, the signal-to-noise ratio can be improved by reading the gel 5a with the gel supports 5b and 5c attached.

In the configuration of the condenser 23 here, only one cylindrical lens 23a is used, but a position symmetrical with respect to the scanning plane of the laser beam, the side opposite to the sample, and the like. You may make it mount a cylindrical lens on it. In particular, when the amount of fluorescence to be detected is insufficient, for example, a cylindrical lens and an optical fiber array are placed in four directions surrounding the scanning line of the gel that generates fluorescence, and fluorescence that emits weak light is placed. The amount of detected light is increased by condensing. In that case, it is effective to shift the optical axes so that the surface reflections of the opposing cylindrical lenses are not affected by each other.

The fluorescence collected by the entrance (point A) of the optical fiber array is guided into the optical fibers of the fiber array 23 and supplied to the photoelectric conversion section 24 from the light exits in which the optical fibers are bundled. To be done. The fluorescence input to the photoelectric conversion unit 24 is converted into the first lens 24a, the diaphragm 24b, and the second lens 24a.
Only the parallel component is extracted using the lens 24c of No. 2 and is incident on the optical filter 24d. And the optical filter 2
4d removes the scattered light component, and the third lens 24
The light is collected by e, guided to the photomultiplier tube 24f, and the detected fluorescence is converted into an electric signal.

As described above, in the photoelectric conversion section 24, the incident light for improving the wavelength separation property of the optical filter 24d is made into the parallel light component only by the second lens and is incident on the optical filter 24d at a right angle. Then, the optical filter 24d separates the scattered light of the excitation light generated in the gel to improve the signal-to-noise ratio and the third lens 24e.
The light is condensed by and guided to the photomultiplier tube 24f.

In this way, the fluorescent light is received and collected, the scattered light is removed by the optical filter, and the fluorescent light guided to the photomultiplier tube 24f is converted into an electric signal by the photomultiplier tube 24f. The converted electric signal is input to the amplifier 25. Then, in the amplifier 25, the weak signal is sufficiently amplified in the amplification stage including the integrating circuit.

FIG. 10 is a circuit diagram showing the structure of an amplifier including an integrating circuit. As shown in FIG. 10, the amplifier 25 is provided with an integrating circuit composed of an operational amplifier 25a in the previous stage and an output amplifying circuit composed of the operational amplifier 25b in the next stage, and is provided with an integrating amplifier stage. I am configuring. The electric signal from the photomultiplier tube 24f is supplied to the operational amplifier 25a.
Entered in. The operational amplifier 25a constitutes an integration circuit together with the capacitor 25c and the switch 25d for controlling the integration operation, and the output of the integration circuit is input to the subsequent operational amplifier 25b to perform amplification with a gain determined by the external resistor, It is sent to the next analog-to-digital conversion circuit.

The operation of the amplifier 25 including the integrating circuit configured as described above will be described with reference to the timing chart of FIG. Since the output of the photomultiplier tube 24f has a very large output impedance, it can be regarded almost as a current source. The operational amplifier 25a includes an FET
(Field effect transistor) An input type one having a high input impedance is used, and when the switch 25d is in the off state, the output current ip of the photomultiplier tube 24f is
Becomes the current flowing through the capacitor 25c as it is. Due to this current, the output voltage of the operational amplifier 25a becomes a ramp function-like output, as shown in FIG. In the integration operation here, the integration is performed for a time corresponding to one pixel, and the sampling circuit in the analog-digital conversion circuit 26 samples at the timing of the S / H clock and holds it as it is. The conversion circuit 26 converts the signal into a digital signal.

After being held, the charge accumulated in the capacitor 25c is discharged by activating the C / D clock which is the C / D control signal applied to the switch 25d. Hereinafter, similarly, the integration operation is repeated for a time corresponding to one pixel. The amplification stage using the integration circuit of such an operational amplifier is different from a pseudo integration circuit composed of only a resistor and a capacitor, and the charge from the photomultiplier tube 24f can be almost completely integrated. Therefore, a high signal-to-noise ratio can be obtained. Also, the integration time can be arbitrarily changed by changing the C / D clock of the C / D control signal for the switch 25d. For this reason, it is possible to easily adjust the amplification degree for totally amplifying the weak signal. In the case of this example, by synchronizing with the scanning operation of the mirror driver 30 shown in FIG. 6, it is possible to control according to the size of the area of the sample to be read, and it is possible to eliminate the dead time of reading. it can. Further, since the scanning speed of the irradiation light (excitation light) and the integration time of the amplifier on the light receiving side can be freely set according to the intensity of the fluorescence from the sample, a very flexible device can be configured.

The electric signal thus amplified by the amplifier 25 (FIG. 3) is passed to the next analog / digital conversion circuit 26.
Is input to and converted into digital data. The fluorescence detection signal converted into digital data is stored in the memory 28, and the data stored in the memory 28 is sent to the data processing device 8 through the interface circuit 29. The control circuit 27 controls the entire series of such signal processing.

Next, a modification of the constituent elements of the fluorescent pattern reading apparatus of this embodiment will be described. Another example of the actuator 41 in the above-described optical axis correction mechanism 40 will be described. FIG. 12 shows another configuration example of the actuator in the optical axis correction mechanism. As shown in FIG. 12, the actuator 46 according to another configuration example has an elastic plate shim 4 that is sandwiched between two ceramic plates 46a and 46b.
The actuator 6c has a flat structure. The mechanism part of the optical axis correction mechanism 40 includes a folding mirror 44.
The structure is such that the actuator 46 is attached from the back side to the side direction, and the inverse piezoelectric effect due to the voltage applied to the actuator 46 causes a distortion proportional to the applied voltage, and the effect of causing this distortion is used as the driving force. Use
The folding mirror 44 is displaced. As with the actuator 41 (FIG. 5) described in the above embodiment, the shim 41c
A large amount of displacement can be obtained by bending the U-shaped type, but it may be difficult to accurately control the amount of displacement in proportion to the applied voltage. On the contrary,
In the piezoelectric actuator 46 according to another configuration example, the shim 4
The folding mirror 44 is attached without bending 6c. As a result, the optical axis correction mechanism using the piezoelectric actuator 46 can accurately control the displacement amount in proportion to the applied voltage. When a piezoelectric actuator is used as the actuator for obtaining the driving force for displacing the folding mirror 44, in order to improve the responsiveness,
When the driving force is insufficient, two piezoelectric actuators may be used to obtain the driving force for displacing the folding mirror 44.

FIG. 13 is a diagram showing another example of the structure of the mechanical portion of the optical axis correcting mechanism composed of two piezoelectric actuators. Like the actuator 46 shown in FIG. 12, the two actuators according to this configuration example are actuators having a structure in which the shim of the elastic plate sandwiched between the two ceramic plates is flat. The optical axis correction mechanism uses the folding mirror 44 and a plurality of actuators 4
In the mechanical part having the structure in which 6 is attached, the folding mirror 44
The respective actuators 46 must be driven at the same displacement and the same frequency so that the left and right of the displacement can be balanced, but a large driving force can be obtained with a relatively small actuator.

Further, in still another configuration example shown in FIG.
The mechanical portion of the optical axis correction mechanism is configured using four piezoelectric actuators. The four actuators according to this configuration example are configured by using an actuator 47 having a structure in which a shim of an elastic plate sandwiched between two ceramic plates is bent in a U shape. In the mechanical section having such a structure in which the four actuators 47 are attached to the folding mirror 44, it is necessary to drive the respective actuators 47 so that the displacement of the folding mirror 44 can be balanced in the front, rear, left, and right. A large driving force can be obtained with a relatively small actuator, and the displacement of the folding mirror 44 can be finely adjusted by individually adjusting the voltage applied to each actuator 47.

Next, another modification of the optical axis correcting mechanism will be described. In the entire optical axis correction control system of the optical axis correction mechanism described in FIG. 5, the condenser 23 receives the fluorescent light,
Feedback control is performed to correct the deviation of the optical axis from the optimum detection position. In this case, for example, an optical axis correction mechanism that corrects the optical axis by moving the condenser 23 side is used. You can That is, the migration unit 5
If the correction of the optical axis of the laser beam applied to the laser beam cannot be corrected by the displacement of the folding mirror 44 by the actuator 41, the focus position of the fluorescence received by the condenser 23 deviates from the light receiving surface position B in the condenser 23. .. Therefore, here, the optical axis 45 is corrected by providing a drive mechanism for directly moving the condenser 23 side. For example, by attaching an actuator to the light collector 23, the light collector 23 is displaced in a direction in which more fluorescence to be received is detected. Further, the direction and arrangement position of the cylindrical lens 23a in the condenser 23 may be changed. Further, the light receiving surface position B in the condenser 23 may be moved. In any case, the feedback control is performed to displace the fluorescent substance in a direction and a position where more fluorescence to be received is detected. Also, such a light collector 2
Regarding the actuator control method for displacing 3, the control method similar to that for displacing the folding mirror as described above is used. Needless to say, when an acousto-optic device is used, the same effect can be obtained by inserting it between the galvanometer scanner and the light source and changing the incident angle of the laser beam with respect to the galvanometer scanner mirror.

[0070]

As described above, according to the fluorescence type electrophoretic pattern device of the present invention, the optical axis position that automatically optimizes the fluorescence detection sensitivity is set, and the fluorescence of the electrophoretic pattern is read. Therefore, various samples can be read with high sensitivity without worrying about the thickness and optical refractive index of the sample.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram illustrating an overall configuration of a fluorescence-type electrophoretic pattern reading device according to an embodiment of the present invention,

FIG. 2 is a diagram for explaining a mounting position of a migration unit mounted on the main body of the measuring unit;

FIG. 3 is a block diagram showing a configuration of a main part of a measuring unit main body including an optical axis correcting mechanism,

FIG. 4 is a diagram illustrating a schematic configuration of a mechanical portion of an optical axis correction mechanism that performs automatic optical axis correction;

FIG. 5 is a diagram illustrating a configuration of an actuator of a mechanism portion of an optical axis correction mechanism,

FIG. 6 is a diagram illustrating an optical scanning mechanism that scans a gel surface with a laser beam using an oscillating mirror.

FIG. 7 is a diagram for explaining the relationship between the rotation angle of the vibrating mirror and the moving distance of the spot light of the laser beam;

FIG. 8 is a block diagram showing a configuration of a main part of a control circuit of a mirror driver that controls rotation of a vibrating mirror.

FIG. 9 is a diagram showing a detailed configuration of an optical system of a condenser and a photoelectric conversion unit,

FIG. 10 is a circuit diagram showing a circuit configuration of an amplifier including an integrating circuit,

FIG. 11 is a time chart showing the timing of the reading operation of the amplifier,

FIG. 12 is a diagram showing another configuration example of the actuator in the optical axis correction mechanism,

FIG. 13 is a diagram showing another configuration example of the mechanical section of the optical axis correction mechanism configured using two piezoelectric actuators,

FIG. 14 is a diagram showing another example of the configuration of the mechanism section of the optical axis correction mechanism configured by using four piezoelectric actuators;

FIG. 15 is a perspective view showing an appearance of a conventional fluorescence type electrophoretic device,

FIG. 16 is a block diagram showing an internal configuration of the electrophoretic measurement device,

17 (A) and 17 (B) are a front view and a vertical cross-sectional view of a migration unit showing the operation principle of electrophoresis pattern detection by a fluorescence method, respectively.

18 (A) and 18 (B) are views for explaining an example of a fluorescence intensity pattern signal of a DNA fragment sent from an electrophoretic measurement device,

FIG. 19 is a diagram showing an example of distribution of DNA fragments subjected to electrophoresis.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Electrophoresis unit 2a ... 1st electrode 2b ... 2nd electrode 3 ... Support plate 4 ... Electrophoresis power supply device 5 ... Electrophoresis part unit (migration part) 6 ... Reading unit 7 ... Measuring part main body 8 ... Data processing device 9 Image printer 10 Control circuit 11 Light source 12 Optical fiber 13 Fluorescence 14 Concentrator 15 Optical filter 16 Optical sensor 17 Amplifier 18 Analog / digital conversion circuit 19 Signal processing unit 20 Interface 21 ... Light source 22 ... Vibration mirror 23 ... Concentrator 24 ... Photoelectric converter 25 ... Amplifier 26 ... Analog / digital conversion circuit 27 ... Control circuit 28 ... Memory circuit 29 ... Interface 30 ... Mirror driver 31 ... Laser beam 32 ... Optical trap 40 ... Optical axis correction mechanism unit 41 ... Actuator 42 ... Actuator driver 43 ... Actuator Control circuit 44 ... Folding mirror 46 ... Actuator 47 ... Actuator 51 ... Electrophoresis measuring device 51a ... Door 52 ... Data processing device 53 ... Cable 54 ... Computer main body 55 ... Keyboard 56 ... Display 57 ... Printer 63 ... Electrophoresis unit device 64 ... Signal processing device 61 ... Optical path 62 ... Scan line 66 ... Band 71, 72, 73, 74 ... Lane.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hitoshi Fujimiya 6-81 Onoue-cho, Naka-ku, Yokohama-shi, Kanagawa Hitachi Software Engineering Co., Ltd. In-house (72) Inori Nasu, Naganori 6-chome, Onoue-cho, Naka-ku, Yokohama-shi, Kanagawa Address 81 Hitachi Software Engineering Stock Association In-house

Claims (4)

[Claims] 1. An electrophoretic pattern reading device that excites a fluorescent substance labeling a sample after the sample has been electrophoresed and developed to emit fluorescence to read a fluorescent pattern that emits the fluorescent substance having the electrophoretic pattern. A light source that emits irradiation light to be excited, an optical scanning mechanism that scans irradiation light from the light source along a predetermined optical axis and irradiates in the thickness direction of the gel, and a light receiving surface in a direction different from the optical axis of the irradiation light. A light receiving part that sets and separates the fluorescence of the migration pattern from the scattered light on the reading surface according to the spatial positional relationship of the light receiving path, and a photoelectric conversion that photoelectrically converts the optical signal received by the light receiving part and outputs an electrical signal Section, an amplifier that performs an integration operation on the electrical signal from the photoelectric conversion section in response to scanning of irradiation light, amplifies, and sequentially outputs a fluorescent electrical signal from the electrophoretic pattern, and an optical signal from the amplifier is output. axis A fluorescent pattern reading device comprising: an optical axis correction unit that receives a detection signal and corrects the optical axis of irradiation light. 2. The optical axis correction unit includes a folding mirror that changes the optical axis of light emitted from a light source, an actuator for correcting the optical axis that changes the angle of the folding mirror, and an actuator driver that drives the actuator. 2. The fluorescence pattern reading device according to claim 1, further comprising an optical axis correction mechanism including a control circuit that gives a drive control signal to the actuator driver. 3. The optical axis correcting actuator is a ceramic actuator, and the control circuit receives an electric signal from an amplifier as an optical axis detection signal, and the magnitude of the optical axis detection signal is in an extreme value direction. The fluorescence pattern reading device according to claim 2, wherein the fluorescence pattern reading device is a feedback control circuit that generates a drive control signal to the actuator driver. 4. The optical axis correcting actuator is a plurality of ceramic actuators, and the plurality of ceramic actuators constitute a drive system for displacing the optical axis reflection angle of one folding mirror. The fluorescence pattern reading device according to claim 2.