JPH04223261A - Reading device of fluorescent pattern - Google Patents

Abstract

PURPOSE:To make it possible to read a very weak fluorescent pattern with high sensitivity by a construction wherein a light source emitting an exciting light is provided at a position whereat the exciting light refracted through glass plates passes through a gel and turns to be an optical path wherein a fluorescent substance is excited. CONSTITUTION:An exciting light for a fluorescent substance emitted from a light source enters the end parts of glass plates 5b and 5c. The exciting light having entered is refracted through the glass plates and the refracted exciting light passes through a gel/5a and turns to be an optical path 61 wherein a fluorescent substance is excited. The exciting light entering the gel irradiates the gel 5a of a sample tagged by the fluorescent substance. Since the refractive index of the glass plates 5a and 5b is larger than the refractive index of the gel 5a, the optical path of the incident light from the end parts of the glass plates 5b and 5c is set so that the light from the light source gets out of the glass plates 5b and 5c at an angle being near a critical angle whereat the light advances from the glass side to the gel 5a. According to this constitution, the exciting light advances along the gel 5a in parallel with the glass plates 5b and 5c substantially and a very weak fluorescent pattern can be read with high sensitivity.

Description

[Detailed description of the invention]

0001

[Industrial Field of Application] The present invention relates to a fluorescence pattern reading device, and in particular, the present invention relates to a fluorescence pattern reading device, in particular, electrophoresis is performed on a sample labeled with a fluorescent substance to develop a migration pattern, and then excitation light is irradiated to form a migration pattern. The present invention relates to a fluorescence pattern reading device that excites a fluorescent substance to emit fluorescence and reads the emitted fluorescence pattern.

0002

[Prior Art] In general, electrophoretic analysis using radioisotope labels is used to perform various gene structural analyzes including DNA sequencing (gene base sequencing), mass spectrometry of proteins such as amino acids, and structural analysis of macromolecules. law is used. In this electrophoretic analysis method, fragments of a sample labeled with a radioactive isotope are subjected to electrophoresis using a gel, and the distribution pattern of the sample fragments developed by electrophoresis is transferred onto X-ray film and then analyzed. This is a method of analyzing a sample by performing the following steps.

On the other hand, with recent advances in light source technology such as lasers, an electrophoretic analysis method using a fluorescence method has been developed in which fluorescent substances are used instead of radioactive isotopes for labeling. Fluorescence-based electrophoretic pattern reading devices have the advantage of not requiring dangerous and expensive radioisotopes. However, in order to obtain a signal-to-noise ratio (SN ratio) equivalent to electrophoretic analysis using radioisotopes, advanced optical system technology and signal processing technology are required.

An example of a reading device that reads a light pattern with a very weak amount of light is a fluorescent electrophoresis pattern reading device that reads a distribution pattern of a fluorescent dye. Here, high-sensitivity optical sensors such as optical filters, two-dimensional image intensifiers, and photomultiplier tubes are used to distinguish between background noise and the weak light pattern to be detected, and to read the light pattern. .

[0005] As a typical example of an electrophoretic pattern reading device using a fluorescent label, it is possible to read the type of DNA that requires the most sensitivity.
A sequencing device will be explained as an example. When performing DNA sequencing using a DNA sequencing device, the DNA sample whose structure is to be determined is first processed by controlling the reaction rate of a chemical reaction specific to each base using restriction enzymes. is fragmented and labeled with a fluorescent substance to create fragments. This fragment has various lengths and contains adenine, cytosine, guanine, and thymine at the cut end.
e, Guanine, Thimine; hereinafter, A, C,
It is a fragment having one of four specific bases (abbreviated as G, T). Fragmented A, C, G, T
Each DNA sample can be separated by electrophoresis according to the length of its fragments, so after electrophoresis is performed and each fragment is separated, a laser beam is irradiated to detect the fluorescence labeled on each fragment. By exciting a substance and measuring the intensity distribution of fluorescence emitted from the fluorescent substance, the sequence of each base is read and the structure of DNA is determined.

FIG. 15 is a diagram showing an example of the distribution of DNA fragments subjected to electrophoresis. Difference in length of DNA fragments (
Because the distance of electrophoresis differs due to the difference in molecular weight, each DNA fragment becomes D of the same molecular weight over time.
Each NA fragment is assembled, and as shown in FIG. 15, each band 66 is formed corresponding to each molecular weight. The overall result is an electrophoretic pattern 70 having bands 66 developed in lanes 71, 72, 73, and 74 of each base. The amount of sample in each band is 1/1016 mo
1, which is a very small amount. This electrophoretic pattern 70 is
Each band 66 is developed and formed corresponding to each molecular weight. D of each base of A, C, G, T
Because NA fragments always have a molecular weight difference of one base or more,
The electrophoresed distance is in lanes 71 and 72 for each base.
, 73, and 74 are all different for each band. therefore,
In principle, each band 66 in lanes 71 to 74 of each base A, C, G, and T is not lined up horizontally with the band in the lane. In DNA sequencing, the DNA sequence is analyzed by reading a pattern that follows the order of band 66 from the bottom of lanes 71 to 74 of each base of A, C, G, and T.

[0007] As mentioned above, the electrophoretic analysis method is used in DNA sequencing equipment to analyze the base sequence of each DNA. It can also be used in the same way when electrophoresis is performed. In this case, electrophoresis is performed on the sample to be analyzed. When a sample to be analyzed is subjected to electrophoresis, the sample is separated according to its molecular weight or the amount of charge of the sample in the solvent, and bands are formed for each, so the distribution of the formed bands is read. Determine the difference in molecular weight of the samples. Furthermore, by measuring the migration distance of sample fragments by electrophoresis and determining the presence or absence of a band at a predetermined position, it is possible to estimate the molecular weight and determine the presence or absence of a predetermined molecule.

When performing such an analysis by electrophoresis, a sample labeled with a fluorescent substance is injected into a base gel, and electrophoresis is performed on the gel. After electrophoresis, the gel contains the sample. Since a band distribution is formed due to the difference in molecular weight of each, this band distribution is measured. Measurement of band distribution involves emitting light from a laser or lamp, which becomes excitation light that excites fluorescent substances, and irradiating this light onto the gel that has undergone electrophoresis, thereby measuring the fluorescence emitted from the gel. The band distribution pattern is measured by detecting it with a photoelectric conversion element. As the gel, for example, polyacrylamide gel, agarose gel, etc. are used.

An example of an electrophoresis apparatus using this type of fluorescence detection method is an electrophoresis apparatus described in Japanese Patent Application Laid-open No. 62843/1983.

Next, an electrophoresis apparatus using such a fluorescence detection method will be specifically explained. FIG. 11 is a perspective view showing the appearance of a conventional fluorescence electrophoresis device. Referring to FIG. 11, the electrophoresis device performs electrophoresis of a sample;
It is composed of a migration measurement device 51 that measures the distribution of fluorescence, a data processing device 52 that performs data processing based on the measured data, and a cable 53 that connects them. The electrophoresis measurement device 51 has a door 51a, and the door 51a is opened to inject a gel that becomes a base for electrophoresis of DNA fragments, and further inject a predetermined amount of a sample to be electrophoresed. When the door 51a is closed and the electrophoresis start switch on the operation display panel 51b is pressed, electrophoresis is started. When electrophoresis is started, the operating state of the electrophoresis measuring device 51 is displayed on the monitor on the operation display panel 51b. The measured data 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 commands from the user, a display device 56 for displaying processing status and results, and a printer 57 for recording processing results. has been done.

FIG. 12 is a block diagram showing the internal configuration of the electrophoresis measuring device. As shown in FIG. 12, the structure of the electrophoresis measuring device (51; FIG. 11) is composed of an electrophoresis device section 63 and a signal processing device section 64. It constitutes the entire device. The electrophoresis device section 63 includes a migration section 5 that performs electrophoresis, a first electrode 2a and a second electrode 2b for applying voltage to the migration section 5, and a migration section 5 and each electrode 2a, 2b.
a support plate 3 for supporting the electrophoresis, an electrophoresis power supply device 4 for applying voltage to the migration section 5, a light source 11 for emitting light to excite the fluorescent substance, and a support plate 3 for guiding the light from the light source 11. optical fiber 12 and fluorescence 1 generated from a fluorescent substance.
3, an optical filter section 15 that selectively passes light of a specific wavelength, and an optical sensor section 16 that converts the received light into an electrical signal. has been done. The signal processing unit 64 includes an amplifier 17 that receives and amplifies the electrical signal from the optical sensor unit 16, an analog-to-digital conversion circuit 18 that converts the analog electrical signal into digital data, and a circuit that converts the analog electrical signal into digital data. Signal processing unit 1 that performs preprocessing such as averaging processing
9, an interface 20 that performs interface processing to send preprocessed data to an external data processing device, and a control circuit 10 that controls the entire electrophoresis device section and signal processing system. The digital signal OUT outputted from the signal processing device 64 is sent to a data processing device (52; FIG. 11), where data processing such as analysis processing is performed.

Next, the operation of the electrophoresis apparatus configured as described above will be explained. Please refer to FIGS. 11 and 12. The door 51a of the electrophoresis measurement device 51 is opened, and the gel is injected into the electrophoresis section 5 located inside, and a sample of DNA fragments labeled with a fluorescent substance is further injected. When a switch on the operation panel 51b is operated to instruct electrophoresis to start, voltage from the electrophoresis power supply device 4 is supplied to the electrophoresis section 5 through the electrodes 2a and 2b, and electrophoresis is started. Samples labeled with a fluorescent substance by electrophoresis are electrophoresed in lanes 71, 72, 73, and 74 for each sample, as shown in FIG. 15, and the molecules contained in the sample are collected by molecular weight, Create band 66 for each. The lighter the molecular weight, the faster the migration speed, so the distance traveled in the same amount of time is greater.

Detection of these bands 66 is shown in FIG.
), by guiding light from a light source through the optical fiber 12 and irradiating the gel from the lateral direction of the migration section 5 on the optical path 61, the labels gathered in the band 66 in the gel can be detected. This is done by causing a fluorescent substance to emit fluorescence 13. The fluorescence generated depends on the extinction coefficient of the fluorescent material, quantum efficiency, intensity of excitation light, etc., but it is 1/1016 mo per band.
Since it contains only a very small amount of fluorescent material, about 1 liter, the light is extremely weak. For example, as a fluorescent substance, fluorescein isothiocyanate (Fluores
In the case of using fluorescein isothiocyanate, the excitation wavelength of the excitation light of fluorescein isothiocyanate has a peak of 490 nm and the fluorescence wavelength has a peak of 520 nm. The molar extinction coefficient is 7
×104/mol·cm, and the quantum efficiency is about 0.65.

When 1/1016 mol of fluorescent material exists in one band, the excitation light has a wavelength of 488 nm and an output of 1 m.
The amount of fluorescent light generated when using a W argon ion laser varies depending on the thickness of the gel, etc., but it is 1010 pieces/
S-order fluorescence photons are generated. Furthermore, since the generated fluorescence spreads in the entire circumferential direction, light corresponding to a solid angle looking into the condenser lens is photoelectrically converted. Therefore, general CCD
It is difficult to directly receive light with a solid-state image sensor camera or the like.

The front view of the electrophoresis section 5 is shown in FIG.
As shown in FIG. 13(b), which is a vertical cross-sectional view, the gel 5a is composed of a gel 5a made of polyacrylamide, and support plates 5b and 5c made of glass for narrowly supporting the gel 5a from both sides. Then, a sample of, for example, a DNA fragment is injected into the gel 5a of the electrophoresis section 5 from above, and the first electrode 2a and the second
Electrophoresis is performed by applying a migration voltage to the electrode 2b (FIG. 12). Light irradiated from a light source, for example, a laser beam, passes from the optical fiber 12 through an optical path 61 in the gel 5a, and then passes through the optical path 61.
Irradiate the fluorescent material on top. As a result, the fluorescent substance present on the optical path 61 is excited and emits fluorescence 13. Fluorescence 1
3 reaches a condenser 14 of an optical system made up of a combination of lenses, and after being condensed, it is selected by an optical filter section 15 and converted into an electrical signal by a one-dimensional optical sensor section 16.

In order to efficiently convert weak light into an electrical signal, the optical sensor section 16 amplifies the light by 104 to 105 times using an image intensifier or the like, and transmits the image to a one-dimensional optical sensor such as a CCD. is converted into an electrical signal. The electrical signal obtained by the optical sensor section 16 is sent to an amplifier 17.
The signal is amplified to a desired level by the analog-to-digital conversion circuit 18, converted from an analog signal to a digital signal, and sent to the signal processing section 19. The signal processing unit 19 performs signal processing such as averaging processing to improve the signal-to-noise ratio (S/N ratio). The digital signal data processed in this way is sent to the data processing device 52 via the interface 20.

FIGS. 14(a) and 14(b) are diagrams illustrating examples of fluorescence intensity pattern signals of DNA fragments sent from the electrophoresis measuring device 51. For example, FIG.
), when the laser beam is irradiated on the optical path 61 to the electrophoresis section 5 where electrophoresis has been performed, the fluorescent substances in the gel present on the optical path 61 are excited and emit fluorescence. This fluorescence is detected over time in the electrophoresis direction 62 at a predetermined detection position for each lane. As a result, fluorescence is detected when the band 66 of each lane passes a position on the optical path 61, and the pattern signal of fluorescence intensity in one lane is detected as shown in FIG. 14(b). be done. Therefore, the band 66 is
When passing the position above 1, a peak of fluorescence intensity is obtained. Therefore, the fluorescence intensity pattern signal shown in FIG.
This is the fluorescence intensity pattern signal.

In the data processing device 52, the computer main body 54
receives the data of the fluorescence intensity pattern signal of the DNA fragment sent from the electrophoresis measurement device 51, and performs data processing to compare the molecular weight and determine the base sequence of the DNA from the data of the fluorescence intensity pattern. The sequence of bases etc. determined through data processing is output as a symbol and displayed on a screen by a display device 56 or printed out by a printer 57. In addition, the data resulting from data processing is
It is recorded on a magnetic storage medium as necessary.

[0020]

[Problems to be Solved by the Invention] However, in the electrophoretic pattern reading device using the fluorescence detection method as described above, since the gel used for electrophoresis is very thin, about 0.3 mm, it is difficult to enter the laser beam from the side. In this case, the optical path must be aligned with high precision, and it is difficult to align the incident position and angle of incidence. Furthermore, since the light is directly aimed at the end face of the gel, a member such as a glass piece is required to flatten the end face of the gel.

An object of the present invention is to provide an inexpensive fluorescence pattern reading device that can read extremely weak fluorescence patterns with high sensitivity.

[0022]

[Means for Solving the Problems] In order to achieve the above object, the fluorescence pattern reading device of the present invention includes a gel serving as a base for electrophoresis sandwiched between glass plates of a gel support, and the gel is labeled with a fluorescent substance. This is a fluorescence pattern reading device that performs electrophoresis by injecting a sample into a gel, excites the fluorescent substance in the gel to emit fluorescence, and reads the emitted fluorescence pattern. A light source that emits excitation light is placed at a position where the excitation light enters from the edge of the glass plate, is refracted through the glass plate, and the refracted excitation light passes through the gel to become an optical path for exciting the fluorescent substance. It is characterized by

[0023]

[Operation] According to this, for the glass plate of the gel support,
Excitation light for the fluorescent substance from the light source enters from the edge of the glass plate of the gel support. The incident excitation light is refracted through the glass plate, and the refracted excitation light passes through the gel and becomes an optical path for exciting the fluorescent substance. The excitation light that enters the gel through such a light path irradiates the gel of the sample labeled with the fluorescent substance. Since the refractive index of the glass plate of the gel support is larger than the refractive index of the gel, the incidence from the edge of the glass plate is such that the light from the light source exits the glass plate at an angle close to the critical angle that proceeds from the glass side to the gel. Determine the optical path of light. Thereby, the excitation light travels along the gel substantially parallel to the glass plate. The angle of incidence of the excitation light on the glass plate is determined by moving the light source directly or by deflecting it using a mirror or the like. The deflection angle is controlled by providing a beam position detection sensor on the exit side of the gel and feeding back its output.

In this way, by directly entering the excitation light from the light source through the glass plate that is the gel support,
Excitation light can be incident on the end of the gel support without providing a new glass piece for preventing diffused reflection at the end of the gel. In addition, since the difference in refractive index between the glass plate and the gel is used to obtain excitation light that is approximately parallel to the gel, it is only necessary to control the angle of incidence on the glass plate, making it easy to position the optical path of the incident light. I can do it.

[0025]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a perspective view showing the structure of a light receiving section of a fluorescent electrophoretic pattern reading device according to an embodiment of the present invention. In FIG. 1, 5 is a migration section that performs electrophoresis. The electrophoresis section 5 is composed of a gel 5a made of polyacrylamide or the like, and glass plates 5b and 5c that serve as gel supports for narrowly supporting the gel 5a from both sides. Electrophoresis section 5
For example, a sample of a DNA fragment is injected into the gel 5a from the top, and electrophoresis is performed by applying a migration voltage between a first electrode provided at the top of the electrophoresis section 5 and a second electrode provided at the bottom. In order to perform electrophoresis on the electrophoresis section 5 and at the same time perform a reading operation, the electrophoresis section 5 is irradiated with light (excitation light) from a light source.
For example, the laser beam 12 processed so as to obtain irradiation light with a required beam diameter is deflected by the vibrating mirror 21, guided to the optical path 61 in the gel 5a, and the fluorescent substance of the DNA fragment on the optical path 61 is excited. As a result, the fluorescent substance present on the optical path 61 is excited and the fluorescent substance 13 is emitted.

Fluorescence 13 emitted on the optical path 61 of the migration section 5
The pattern consists of light receiving parts (30, 31,
32, 33, 34, 35) are received and read. The light receiving section is connected to an optical path 61 through which a laser beam for exciting fluorescent substances distributed in the gel of the migration section 5 is irradiated via a light guide such as an optical fiber.
It is provided corresponding to the position of the fluorescent light 13 that emits light on the top. The end of the optical fiber is treated to collimate the laser beam. The light receiving section includes a rod lens array 30 for condensing the fluorescence 13 generated from the fluorescent substance, and an optical shutter 3 for selectively passing the fluorescence depending on the emission position.
1, a signal cable 32 that supplies a drive signal to drive the optical shutter 31, an optical fiber array 33 that collects the fluorescence that has passed through the optical shutter 31 and guides it to the optical sensor, and excitation light that is mixed in the received fluorescence. It is composed of an optical filter section 34 for separating the scattered light component of the light, and a photoelectric converter 35 for converting the received light component into an electrical signal.

To further explain the configuration for reading the fluorescence pattern from the electrophoresis section 5 on the light receiving section configured as described above, a light source emits light that is irradiated onto the optical path 61 of the electrophoresis section 5. For example, a secondary high-frequency generating solid-state laser excited by laser light from a semiconductor laser is used. The laser medium for this solid-state laser is
A YAG (yttrium, aluminum, garnet) crystal is used, and the solid-state laser oscillates at a wavelength of 1.064 μm. By using KTP (KTiOPO4) as a crystal for obtaining second harmonic laser light from the oscillated laser light, laser light with a wavelength of 532 nm can be obtained.

The laser beam 12 from the light source is incident on the vibrating mirror 21 with a desired beam diameter, and the laser beam 12 deflected by the vibrating mirror 21 is incident on the migration plate 5b. The deflection angle at this time is controlled by the vibrating mirror 21 so that the laser beam travels along the glass 5b and gel 5a. An optical sensor 22, which is a semiconductor position detection element, is provided to detect the optical path position of the excitation light that has passed through the gel, and the laser beam 12
The control for causing the beam to proceed along the optical path 61 is performed by feedback-controlling the amount of deviation measured by the optical sensor 22, which is a semiconductor position detection element, to the vibrating mirror 21. As the semiconductor position detection element of the optical sensor 22, for example, a one-dimensional one manufactured by Hamamatsu Photonics Co., Ltd. can be used. The irradiated laser light passes through an optical path 61. Therefore, the gel 5a of the migration section 5 existing in the optical path 61
The fluorescent substance inside is excited and emits fluorescence. Examples of fluorescent dyes used here to label DNA fragments include rhodamine X, Texas red, and tetramethylrhodamine isothiocyanate. The emitted fluorescence 13 is collected by the rod lens array 30 and formed into an image on the optical shutter 31 . The rod lens array 30 is a flat lens that can collect light in the same way as an optical lens by providing a difference in refractive index within a cylindrical rod. For example, a liquid crystal shutter is used as the optical shutter 31.

FIG. 2 is a schematic cross-sectional view for explaining the optical path of excitation light. In FIG. 2, 5a is D
The gel on which the NA fragment material is being electrophoresed, 5b and 5c are glass plates of gel support for narrowly supporting the gel from both sides, and 61 is the optical path through which the irradiation light to the gel passes. gel 5
The refractive index of a is larger than that of water and has a value of around 1.4. On the other hand, the refractive index of glass is around 1.5, which is higher than that of gel. If lead glass or the like is used, it is possible to further increase the refractive index to 1.7 or more. Here, the edges of the glass plate are cut to facilitate the incidence of laser light, but this is not always necessary.

The laser beam 12 incident on the cut surface of the glass plate 5b travels straight through the glass plate 5b and reaches the boundary with the gel 5a. By setting the incident light to the gel 5a to be slightly below the critical angle, an optical path 61 can be formed that travels straight along the gel 5a. The light that has passed through the gel reaches the optical sensor 22, which is a semiconductor position detection element. The semiconductor position detection element of the optical sensor 22 is an element that can obtain an output corresponding to the incident position of the beam as a change in resistance value by utilizing the optical effect of the semiconductor. The angle of the vibrating mirror 21 is changed according to the amount of deviation from the position centered on the gel 5a by the output of the semiconductor position detecting element of the optical sensor 22, and is controlled so as to always be at the desired position.

FIG. 3 is a schematic cross-sectional view for explaining the optical path in the light receiving section. In FIG. 3, 5 is the electrophoresis section, 5a is the gel on which the DNA fragment sample is being electrophoresed, and 5 is the electrophoresis section.
b, 5c are glass plates of a gel support for narrowly supporting the gel from both sides; 61 is an optical path through which light irradiates the gel;
13 is a fluorescence, 30 is a rod lens array, 31 is an optical shutter, and 33 is an optical fiber array that condenses light from the rod lens array. These elements correspond to the reference numbered elements in FIG. As shown in FIG. 3, the fluorescent light 13 emitted from the gel 5a on the optical path 61 is collected by the rod lens array 30 and formed into an image on the optical shutter 31. The fluorescence imaged on the optical shutter 31 is further guided to an optical fiber array 33, condensed by the optical fiber array 33, and guided to an optical filter section 34 and a photoelectric converter 35.

FIG. 4 is an explanatory diagram showing an example of the pixel configuration of the optical shutter. In this embodiment, since the purpose of reading the fluorescent pattern is to read one-dimensional pixels of the fluorescent pattern that emit light along the optical path 61, the optical shutter 31 is, for example, horizontally arranged as shown in FIG. 6 in direction
A liquid crystal shutter having 40 pixels and functioning as a one-dimensional line pixel shutter having one pixel in the vertical direction is used.

[0033] In order to actually measure the fluorescence pattern,
A drive signal is supplied to the optical shutter 31 via a signal cable (32; FIG. 1) by a control signal generated by a control circuit (not shown) of the signal processing device. This light shutter 3
According to the drive signal No. 1, the light transmittance of each section of the optical shutter 31 is controlled. For example, when controlling with binary values,
In the on state, the light transmittance is high, and in the off state, the light transmittance is low. Further, in order to control the light transmittance to an arbitrary value, a corresponding driving voltage is applied. Here, in order to simplify control, the optical shutter is assumed to be controlled with two values, and has two values: on state and off state.
Control the light shutter by value. The liquid crystal shutter that serves as the light shutter has a light transmittance of approximately 20% to 30% in a high transmittance state, and a transmittance of approximately 1/20 to 1/30th of the high transmittance state in a low transmittance state. There is. If higher contrast is required, it is possible to obtain a transmittance ratio of 100:1 or more by using a ferroelectric liquid crystal or the like.

As another form of controlling the optical shutter 31,
Instead of performing binary control on each section, it is also possible to perform filtering processing by appropriately weighting the temporal ratio of moderate intermediate transmittance or changing the transmittance of the section itself.

Each section of the optical shutter 31 is set so that its length in the vertical direction is longer than the imaging width of the rod lens array 30. In addition, regarding the on/off (transmission/blocking) of each section (pixel) of the optical shutter 31, each
Including a reading method that reads each pixel as an on state,
As will be described later, the light intensity of the fluorescent pattern is read while the pixels of several sections are turned on at the same time, and the light intensity for each pixel is later determined by calculation. This allows weak fluorescence patterns to be read with high sensitivity. As the optical shutter 31,
In addition to such a liquid crystal shutter, a mechanical shutter, a shutter using various optical properties such as the Pockels effect, etc. can be used.

The light transmitted through the optical shutter 31 is incident on an optical fiber array 33 arranged one-dimensionally. At the other end of the optical fiber array 33, a plurality of optical fibers in a one-dimensional array are all bundled into a circular shape, and the light is condensed and output. The light collected by this optical fiber array 33 is sent to an optical filter section 34.

FIG. 5 is a sectional view showing the structure of the main parts of the optical filter section. As shown in FIG. 5, the optical filter section 34
has an optical lens 34a and a pinhole 34 on one optical path.
b, an optical lens 34c, an optical filter 34d, and an optical lens 34e are arranged in sequence at a predetermined distance. The function of this optical filter section 34 is to remove the scattered light component of the excitation light contained in the light guided from the optical fiber array 33.

The light from the optical fiber array 33 is first
The light enters the optical lens 34a, is condensed to a focal point, passes through the pinhole 34b, and then passes through the optical lens 34c.
Only the parallel light component of the incident light is extracted. The obtained parallel light component is incident on the optical filter 34d. The optical filter 34d extracts only the fluorescence wavelength component. Next, the light is further condensed by the following optical lens 34e and is incident on the next stage photoelectric converter 35. The reason why the optical filter unit 34 extracts the parallel light component from the incident light and inputs it to the optical filter 34d is to fully bring out the characteristics of the interference filter used as the optical filter 34d.

The electric signal corresponding to the intensity of light converted into an electric signal by the point-type photomultiplier tube of the photoelectric converter 35 is converted from an analog signal to a digital signal by an analog-to-digital converter, and is converted into a desired signal. Data processing is performed.

Next, a description will be given of the pixel on/off control process in the optical shutter 31 for reading the fluorescent pattern and the corresponding signal processing method in the signal processing section.

FIG. 6 is a diagram showing a coefficient matrix for controlling the light transmittance of pixels in each section of the optical shutter. Here, since the control of the optical shutter is binary control, in this coefficient matrix, each element Sij of the matrix is in an on state (transmitting state; 1) or an off state (light blocking state; 0). Indicates the state of one of the control patterns.

To read the fluorescence pattern, on/off of pixels in each section of the optical shutter 31 is set for each row of this coefficient matrix, and the output electrical signal detected from the photoelectric converter 35 is measured. The relationship between the coefficient matrix S [Sij] for turning on/off the pixels in each section of the optical shutter, the vector [xi] of the amount of light X for each pixel which is an unknown quantity, and the vector [mi] of the measured value M is as follows. This becomes a simultaneous linear equation and is expressed by the following equation. [Sij]・[xi]=[mi] Therefore, the optical shutter is controlled by the rows of the coefficient matrix,
After performing measurements to find the measured value of the light amount corresponding to that row and completing the measurement for all rows, by solving the above simultaneous linear equations (inverse matrix (by performing calculations), it is possible to know the amount of light at each pixel in each section.

In this example, this coefficient matrix is
A size of about 56 or 512 is used. For the value of each element of the coefficient matrix, a Hadamard matrix is used in which the rows or columns are orthogonal to each other in order to simplify the calculation of the inverse matrix operation, increase the sensitivity of light detection, and improve the measurement accuracy. Also, as an example of other types of matrices,
A Walsh function sequence or the like, which is an orthogonal function, can be used.

The obtained pixel-by-pixel data of each section is sent to a film printer in time series, and output as a film image while being reconstructed two-dimensionally. In addition to being output to a film printer, the image data is also stored as image data on a recording medium such as a magneto-optical disk or digital audio tape.

Next, a modification of this embodiment will be explained. First, another example of the optical path of the excitation light that irradiates the gel will be explained. By changing the exit part of the excitation light from the gel in the optical path, the excitation light can be incident on the gel between the glass plates while maintaining the spacer of the glass plate of the gel support provided during electrophoresis. The fluorescent material can be irradiated with the fluorescent substance.

FIG. 7 is a schematic cross-sectional view for explaining another example of the optical path of excitation light. In Figure 7,
75a is a gel into which a sample of DNA fragments is injected and electrophoresed; 75b and 75c are glass plates of a gel support for narrowly supporting the gel from both sides; and 77 is an optical path through which light irradiates the gel. This example is an example in which spacers 76a and 76b remain on both sides when gel 75a is injected. As shown in FIG. 7, the laser beam traveling through the gel 75a passes through the glass plate 7 in front of the spacer 76a.
5c while being refracted. Further, the laser beam that has traveled through the gel 75a reaches the glass plate 7 in front of the spacer 76b.
It emits while being refracted to 5b. Therefore, in this case,
Measurement can be performed with the spacers 76a and 76b at both ends attached. In this embodiment, in order to facilitate the incidence of laser light, the edges of the glasses 75b and 75c are cut at an angle of about 30 degrees at the incident and exit areas.

Next, another modification of this embodiment will be explained. Here, an example of a mechanical optical shutter used as an optical shutter will be described.

FIGS. 8 and 9 are diagrams each showing an example of the configuration of a mechanical optical shutter. The example of the mechanical optical shutter shown in FIG. 8 is an optical shutter configured by providing a disc shutter 36 with a slit 37 on a part of a concentric circle. By rotationally driving this disk shutter 36, an optical shutter is constructed in which each section of the corresponding position of the slit 37 is turned on for a time determined by the angle of view from the center of the disk and the rotational angular velocity of the disk. The position where the slit 37 is provided is shown in Figure 6.
Set using the coefficient matrix shown in . Further, another example of the mechanical optical shutter shown in FIG. 9 is a rectangular plate shutter 38.
This is an optical shutter configured by providing a slit 39 in the. The slits 39 are holes made at the positions of the sections of the optical shutter where high transmittance is desired. The position of the hole of the slit 39 is based on the coefficient matrix shown in FIG. The mechanical optical shutter of the rectangular plate shutter 38 is driven to move up and down. The mechanical optical shutters (36, 38) configured in this way are installed at the imaging position of the rod lens array, and each lateral slit is rotated or moved vertically. Measure the amount of light for each combination of 37 and 39. For the obtained measurement results, the amount of light for each pixel is determined by solving simultaneous linear equations using inverse matrix calculations in the same manner as described above.

[0049] Such a mechanical optical shutter may also be configured to open and close each section (pixel) using electromagnetic force.

Further, as another modification, an example will be described below in which a mirror whose reflectance can be controlled for each pixel section is used instead of the optical shutter 31.

FIG. 10 is a diagram illustrating a fluorescence pattern reading method using a mirror that controls reflectance for each pixel section. Referring to FIG. 10, fluorescence 13 emitted from a sample in which DNA fragments and the like have been electrophoresed reaches a rod lens array 30. The rod lens array 30 collects the received fluorescence, and the collected light passes through the deflection plate 40,
It reaches the liquid crystal cell 41. Here, in the section that receives fluorescent light, the mirror is made to have a high reflectance so that light is sufficiently reflected from the mirror 42. Therefore, the light is allowed to pass through this high reflectance section without changing the deflection angle of the light, is reflected by the mirror 42, and is made to reach the optical fiber array 33. In addition, for sections that do not receive fluorescence,
By rotating the deflection angle by 45 degrees in one direction within the liquid crystal cell 41, the reflected wave from the mirror 42 is reflected by the liquid crystal cell 4.
1 twice, and a 90 degree deflection is performed in both directions. Therefore, it cannot pass through the deflection plate 40 and does not reach the optical fiber array 33. Thereby, similar to the case where an optical shutter is used, it is possible to control the amount of received light and detect a similar optical signal.

[0052]Also, even in the method using a mechanical optical shutter as shown in Fig. 8 or 9, the slit part is made into a high reflection state such as a mirror, and conversely, the low reflection part is made into a slit shape. Then, it can be used in a similar manner.

In the embodiments and modifications described above, the explanation has been limited to the method of entering the laser beam from the side of the migration section 5 and reading the one-dimensional fluorescence pattern generated from the fluorescent substance. However, instead of this, the present invention can be similarly applied to a method of reading a two-dimensional light distribution pattern other than a fluorescent light emission pattern. Further, in this case, for example, an optical shutter having two-dimensionally controllable sections is used and applied in the same manner. It goes without saying that two-dimensional reading can be performed by moving a one-dimensional optical shutter relative to the sample to be read. Furthermore, as an optical coupling method between the optical shutter and the sample to be read, it is possible to use a so-called fiber optic plate that bundles fibers in a two-dimensional manner and guides the incident image to the opposite side. Furthermore, it is clear that instead of using a fiber array, a mirror can also be used to focus the light.

Although the present invention has been specifically explained above based on examples, it goes without saying that the present invention is not limited to the above-mentioned examples, and can be modified in various ways without departing from the gist thereof. .

[0057]

As described above, according to the present invention, in a fluorescence pattern reading device for reading fluorescence patterns of weak light, as a method of injecting excitation light into a fluorescent substance,
The light enters through a glass plate that is used as a gel support and has a higher refractive index than the gel, and excitation light along the optical axis along the gel can be easily obtained by refraction when it enters the gel from the glass plate. Excitation light can be incident on the gel. Thereby, the gel can be reliably irradiated with excitation light, and therefore the distribution pattern of the fluorescent substance can be read stably and reliably.

[Brief explanation of the drawing]

FIG. 1 is a perspective view showing the configuration of a light receiving section of a fluorescent electrophoretic pattern reading device according to an embodiment of the present invention.

FIG. 2 is a schematic cross-sectional view for explaining the optical path of excitation light.

FIG. 3 is a schematic cross-sectional view for explaining an optical path in a light receiving section.

FIG. 4 is an explanatory diagram showing an example of a pixel configuration of an optical shutter.

FIG. 5 is a sectional view showing the main part configuration of an optical filter section.

FIG. 6 is a diagram showing a coefficient matrix that controls the light transmittance of pixels in each section of the optical shutter.

FIG. 7 is a schematic cross-sectional view for explaining another example of the optical path of excitation light.

FIG. 8 is a diagram illustrating an example of the configuration of a mechanical optical shutter.

FIG. 9 is a diagram showing another example of the configuration of a mechanical optical shutter.

FIG. 10 is a diagram illustrating a fluorescence pattern reading method using a mirror that controls reflectance for each pixel section.

FIG. 11 is a perspective view showing the appearance of a conventional fluorescence electrophoresis device.

FIG. 12 is a block diagram showing the internal configuration of the electrophoresis measuring device.

FIGS. 13A and 13B are a front view and a longitudinal cross-sectional view of the electrophoresis unit, respectively, showing the operating principle of electrophoresis pattern detection using a fluorescence method.

FIGS. 14A and 14B are diagrams each illustrating an example of a fluorescence intensity pattern signal of a DNA fragment sent out from an electrophoresis measurement device.

FIG. 15 is a diagram showing an example of the distribution of DNA fragments subjected to electrophoresis.

[Explanation of symbols]

2a First electrode 2b Second electrode 3 Support plate 4 Electrophoresis power supply 5 Migration section 5a Gels 5b, 5c Glass plate 11 Light source 12 Optical fiber 13 Fluorescence 14 Concentrator 15 Optical filter section 16 Optical sensor section 17 Amplifier 18 Analog・Digital conversion circuit 19 Signal processing section 20 Interface 21 Oscillating mirror 22 Semiconductor position detection element (optical sensor) 30 Rod lens array 31 Optical shutter 32 Signal cable 33 Optical fiber array 34 Optical filter section 35 Photoelectric converter 36 Disc shutter 37 Slit 38 Rectangle Plate shutter 39 Slit 40 Deflection plate 41 Liquid crystal cell 42 Mirror 51 Migration measurement device 51a Door 51b Operation panel 52 Data processing device 53 Cable 54 Computer body 55 Keyboard 56 Display 57 Printer 63 Electrophoresis device 64 Signal processing device 61 Optical path 62 Scanning line 66 Bands 71, 72, 73, 74 Lane 75a Gel 75b, 75c Glass plate 76a, 76b Spacer 77 Optical path

Claims (12)

[Claims] Claim 1: A gel serving as a base for electrophoresis is sandwiched between glass plates as a gel support, a sample labeled with a fluorescent substance is injected into the gel, electrophoresis is performed, and the fluorescent substance in the gel is excited. This is a fluorescence pattern reading device for emitting fluorescence and reading the emitted fluorescence pattern, in which excitation light of a fluorescent substance is incident from an end of a glass plate of a gel support, the incident excitation light is refracted through the glass plate, A fluorescence pattern reading device characterized in that a light source that emits excitation light is disposed at a position where the refracted excitation light passes through a gel and becomes an optical path for exciting a fluorescent substance. 2. Further, an optical sensor for detecting the optical path position of the excitation light passing through the gel is provided on the opposite side of the incident position of the excitation light entering the glass plate of the gel support, and the output of the optical sensor is 2. The fluorescence pattern reading device according to claim 1, wherein the signal controls the position at which the excitation light from a light source that emits excitation light enters the end of the glass plate of the gel support. 3. The glass plate of the gel support is a glass plate with a cut end, and the excitation light of the fluorescent substance is incident on the cut surface of the glass plate of the gel support. Fluorescence pattern reading device as described. 4. Further, an optical shutter capable of controlling the light transmittance of a plurality of sections arranged in the reading direction of the fluorescent pattern, and controlling the reading of the fluorescent pattern by changing the light transmittance of each section of the optical shutter. The fluorescence pattern reading device according to claim 1, comprising a reading control device and a photoelectric converter that collects and receives light from each section of the optical shutter. 5. The fluorescence pattern reading device according to claim 4, wherein the optical shutter is a liquid crystal shutter whose light transmittance can be controlled, and the photoelectric converter is a point-type photomultiplier tube. 6. The fluorescent pattern reading device according to claim 4, wherein the optical shutter is a mechanical shutter having a slit corresponding to each section. 7. The fluorescence pattern reading device according to claim 4, wherein a combination of a liquid crystal cell and a mirror whose light reflectance can be controlled is used in place of the optical shutter. 8. The reading control device changes the light transmittance of each section of the optical shutter in accordance with the reading position, collects the light from the optical shutter, receives the light with a photoelectric sensor, and collects the light of each section. 5. The fluorescence pattern reading device according to claim 4, wherein the amount of light received in each section is determined by solving simultaneous linear equations from the transmittance and the amount of light received by condensing the light. 9. The reading control device controls the light transmittance of each section of the optical shutter according to a set coefficient matrix, collects the transmitted light from the optical shutter, receives the light by the optical sensor, and collects the transmitted light from the optical shutter according to the coefficients of the coefficient matrix. , wherein the amount of light received in each section is determined by performing an inverse matrix calculation from the amount of light received focused on each row of the matrix.
The fluorescence pattern reading device described in . 10. The coefficient matrix for setting the light transmittance of each section of the optical shutter by the reading control device is arranged in an orthogonal relationship, and a plurality of sections are set to have high transmittance at the same time.
The fluorescent pattern reading device according to claim 9, wherein the fluorescent pattern reading device collects and receives transmitted light from a plurality of sections from an optical shutter. 11. A gel serving as a base for electrophoresis is sandwiched between glass plates as a gel support, a sample labeled with a fluorescent substance is injected into the gel, electrophoresis is performed, and the fluorescent substance in the gel is excited. This is a fluorescence pattern reading device that emits fluorescence and reads the emitted fluorescence pattern.The glass plate of the gel support is a glass plate with both ends cut, and the excitation light of the fluorescent substance is emitted from the glass plate of the gel support. A light source that emits excitation light is arranged at a position where the excitation light that enters one cut surface is refracted through the glass plate, and the refracted excitation light passes through the gel and becomes an optical path for exciting the fluorescent substance. A fluorescence pattern reading device characterized by: 12. Further, an optical sensor for detecting the optical path position of the excitation light that has passed through the gel and is located opposite to the cut surface opposite to the incident position where the excitation light enters the glass plate of the gel support. 12. The fluorescent light source according to claim 11, wherein the fluorescent light source according to claim 11 is provided with an output signal of the optical sensor to control the position at which excitation light from a light source that emits excitation light enters one cut surface of the glass plate of the gel support. pattern reader.