JPH0572179A - Two-dimensional fluorescence image detector for nucleic acids and proteins - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To provide a two-dimensional fluorescence image detector for nucleic acid or protein, which detects a signal with high sensitivity from a gel for electrophoresis whose molecular weight has been separated by electrophoresis or its entire blotting membrane. [Structure] The electrophoresis gel 2 in which DNA fragments are distributed is irradiated with a laser beam 6, a two-dimensional fluorescence emission image is condensed by an imaging lens 7, and the two-dimensional fluorescence emission image is divided substantially evenly by a beam splitter 8. , Through the filters 9 and 10, two-dimensional fluorescence emission images of different wavelengths are detected by the two-dimensional photodetectors 11a and 11b.
Detect with. [Effect] It becomes possible to detect a two-dimensional image with high sensitivity by using any one of a cube prism, a semi-transmissive mirror and a dichroic mirror that can be divided after the two-dimensional luminescent image is condensed by a lens. In addition, it is possible to realize a highly sensitive, compact and inexpensive two-dimensional fluorescence image detecting device by using a commercially available imaging lens and a beam splitter without using a huge prism.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a two-dimensional fluorescent image detecting device for detecting nucleic acids and proteins labeled with a fluorescent substance, such as a DNA sequencer or gene diagnostic device.

[0002]

2. Description of the Related Art For the detection of DNA, methods such as base sequencing or fragmentation of several kinds of DNA to separate them by one-dimensional or two-dimensional electrophoresis and measure the pattern thereof are used. In addition, recently, attempts have been made to fragment DNA to obtain a three-dimensional electrophoresis pattern. In this case, it is necessary to measure the time change of the two-dimensional pattern for each of several fluorescences having different wavelengths. For DNA sequencing, DNA fluorescence labeling and optical real-time D
DNA sequencers that measure NA fragments are commercially available. There, there is proposed a device capable of detecting one color or multiple colors by performing line sensing on a part of the migration path.
For example, a method using a line-splitting prism has been proposed as a method for labeling different DNA samples with two fluorophores and performing DNA sequencing while wavelength separation. Regarding such a conventional technique, Japanese Patent Application Laid-Open No. 2-269936
No. 4, there is a description of a fluorescence detection type electrophoresis apparatus. This method is suitable when the measurement target is linear, but cannot be applied to a two-dimensional image. However, many of the blotting samples could not be used because it was necessary to measure their patterns after two-dimensional electrophoretic separation for analysis. That is, it could not be used for the purpose of measuring the difference between patterns by labeling two kinds of DNA with different fluorophores, fragmenting them with an enzyme, and then separating each fragment into a two-dimensional pattern. Therefore, there has been a demand for a method of equally dividing a two-dimensional image as an image and measuring a fluorescence emission image through an optimum filter for each phosphor.

[0003]

In the above-mentioned prior art, it was effective to use a prism to divide a linear luminescent image. However, the divided luminescent image is 1
When a plurality of lines or two-dimensional images are used instead of lines, the conventional method using the image division prism has a problem that the light receiving solid angle becomes small when the area of the subject is widened. On the other hand, in order to increase the light receiving solid angle, it is expensive to use a prism or an imaging lens of the same size as an electrophoretic gel (about 20 cm × 30 cm), and it is not practical because the device becomes huge. was there.

Further, since the light from the end of the electrophoretic gel has a large incident angle on the prism surface, there is a problem that the light is reflected or dispersed and the image is blurred and the sensitivity is lowered.

An object of the present invention is to solve the above problems by using a beam splitter such as a cube prism or a semi-transmissive mirror, and to realize a highly sensitive, compact and inexpensive protein and nucleic acid detection apparatus.

[0006]

To achieve the above object, first, the luminescent image of the gel electrophoresis plate is condensed to an appropriate size by using an imaging lens so that the solid angle of light reception is increased. Next, the collected luminescent image is split substantially evenly by using a beam splitter such as a cube prism or a dichroic mirror in which metal or the like is vapor-deposited on the diagonal surface of a semitransparent mirror or prism of a normal size. Furthermore, after passing through the bandpass filter specific to each phosphor, DN on the photodetector
This is achieved by identifying the position of fluorescence generation by the A fragment and measuring the fluorescence intensity.

[0007]

The cube prism, the semi-transmissive mirror or the dichroic mirror in which metal is vapor-deposited on the diagonal surface of the prism used in this method can be used after condensing the two-dimensional luminescent image. Therefore, a large light receiving solid angle can be obtained, and detection can be performed with high sensitivity. Further, the above-mentioned semi-transmissive mirror, cube prism, and dichroic mirror can split the incident light substantially evenly, regardless of the incident angle of the incident light. Therefore, there is no defocusing due to the spectral distribution of light that occurs when a prism is used, and no reduction in sensitivity. Further, it can be used in a state of being condensed by a lens, and a small beam splitter can be used. Further, since the beam splitter splits the light into angles separated by 90 degrees, it is easy to attach a filter to each emission position.

[0008]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A first embodiment according to the present invention will be described below with reference to FIGS. FIG. 1 is a perspective view showing the overall configuration and optical path of a two-dimensional fluorescence image detecting device according to a first embodiment of the present invention. FIG. 2 is a cross-sectional view showing the structure and optical path of the fluorescent light emission image detecting portion of the first embodiment according to the present invention.
This apparatus comprises an electrophoretic unit composed of a migration plate 1, a migration gel plate 2, etc., and a fluorescence excitation optical unit composed of a laser light source 4, a mirror 5, a laser beam 6 for irradiating the gel 2, etc. A beam splitter 8 which is an element part of the present invention,
It is composed of a photo-detecting section including a photo-detector for detecting a fluorescence emission image. Description will be made regarding the case where the DNA is two-dimensionally developed and detected in this device. First, a DNA fragment is labeled with a fluorophore and cut to an appropriate length with a restriction enzyme. Next, when these DNA fragments are mounted on a fine agarose gel and a voltage is applied in the longitudinal direction of the agarose gel by a DC high-voltage power source, the DNA fragments move toward the positive electrode and are separated according to the fragment length. Further, in order to further cleave the DNA fragment in the separated molecular weight in the agarose gel, the agarose gel is reacted with a restriction enzyme again. In order to obtain information on the fragments generated by this, an agarose gel is placed on top of the acrylamide gel 2 for electrophoresis sandwiched between the electrophoresis plates 1, and a voltage is applied again to separate the two-dimensional molecular weight in the acrylamide gel 2. do. The fluorescence-labeled DNA fragment 3 thus electrophoretically dispersed on the entire electrophoretic plate is excited by the laser light 6 scanning and entering from the side surface of the electrophoretic plate 1 to emit fluorescence. It is also possible to use a plurality of these laser light sources 4 and coaxially collect laser light composed of several laser beams having wavelengths that excite the fluorescence of each of a plurality of types of fluorescent substances that label DNA fragments, and irradiate the gel. The two-dimensional fluorescence emission image on the electrophoresis gel plate 2 is first focused by the imaging lens 7. As a result, in a state where the fluorescence emission image is sufficiently small, the beam splitter 8 such as a cube prism in which a metal is vapor-deposited on the diagonal surface is substantially evenly divided into two images having different 90 ° directions. A semitransparent mirror may be used in the beam splitter 8 instead of the cube prism. The divided fluorescence emission images are further transmitted through the bandpass filters 9 and 10 specific to the fluorescence wavelength bands to be identified, and then transmitted to the two-dimensional photodetectors 11a and 11b such as a video camera with an image amplifier or a cooled CCD camera. It arrives and is detected. The signal of the transmitted fluorescence emission image is output as data by the output device 13 via the signal processing device 12, and the monitor 1
4 is displayed. As the phosphor used here, Texa
s Red (emission wavelength 610 nm) and TRITC (tetramethy)
l rhodamine isothiocyanate; emission wavelength 573 nm) and the like. Examples of the excitation light source include a helium neon laser (594 nm) and an argon laser (488 nm).

Next, a second embodiment according to the present invention will be described with reference to FIG. By using the total reflection mirrors 15a and 15b, the two images divided by the beam splitter 8 as shown in FIG.
This is a method in which 0 is transmitted and made incident on one of the two-dimensional photodetectors 11a so as not to overlap with each other, thereby performing detection.
In this embodiment, one two-dimensional photodetector can detect fluorescence emission images of different wavelengths.

FIG. 4 shows a third embodiment according to the present invention for detecting the fluorescence emission image from the blotting film. UV rays
(UV) light irradiation system 18, imaging lens 7, beam splitter 8, filters 9, 10, 20, two-dimensional detector 11
It is possible to detect the luminescent image by irradiating the blotting film 16 on the brotting film holding table 17 with UV light 19 by combining the optical systems consisting of a and 11b. In this example, a UV lamp was used, but it is also possible to excite fluorescence using a laser. The signal processing device 12, the output device 13, and the monitor 14 are omitted in FIG. Needless to say, even if two total reflection mirrors are used, the light emission image from the blotting film can be divided and detected by the same method as in FIG.

As shown in the above embodiments, according to the present invention, it is not necessary to handle a light emission image in the adjacent optical paths as in the case of using a polygonal prism, and a simple optical path system allows a compact and inexpensive two-dimensional fluorescence. An image detecting device can be obtained.

[0012]

According to the present invention, since the two-dimensional fluorescence emission image is substantially evenly divided after the light is condensed by the lens, the light receiving solid angle can be made large and the detection can be performed with high sensitivity.

Further, according to the present invention, when a two-dimensional fluorescence emission image is divided, a cube prism or a semi-transmissive mirror in which metal is vapor-deposited on a diagonal surface is used instead of an image division prism, so that wavelength dispersion is performed. Can be split without.

Further, according to the present invention, an inexpensive light splitting mechanism can be realized because not a huge prism but a cube prism or a semi-transmissive mirror in which a metal is vapor-deposited on a diagonal surface which is commercially available is used.

[Brief description of drawings]

FIG. 1 is a perspective view showing a first embodiment according to the present invention.

FIG. 2 is a sectional view best representing the characteristics of the first embodiment according to the present invention.

FIG. 3 is a sectional view showing a second embodiment according to the present invention.

FIG. 4 is a sectional view showing a third embodiment according to the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Electrophoresis plate, 2 ... Electrophoresis gel plate, 3 ... Fluorescence labeled DNA fragment, 4 ... Laser light source, 5 ... Mirror (for laser), 6 ... Laser light irradiated in gel, 7 ... Imaging lens, 8 ... Beam splitter for splitting the two-dimensional fluorescence emission image collected by the lens, 9 ... Filter 1, 10 ... Filter 2,
11a, 11b ... Two-dimensional photodetector, 12 ... Signal processing device, 13 ... Output device, 14 ... Monitor, 15a, 15b
... total reflection mirror, 16 ... blotting film, 17 ... blotting film holder, 18 ... UV lamp, 19 ... UV
Light, 20 ... Filter 3.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Satoshi Takahashi 1-280, Higashi Koikekubo, Kokubunji, Tokyo Metropolitan Research Center, Hitachi, Ltd.

Claims (4)

[Claims] 1. At least molecular weight separation by electrophoresis,
A gel for electrophoresis in which a fluorescently labeled sample fragment is distributed, or a blotting membrane in which the distribution information of the sample fragment is copied from the gel for electrophoresis, and the gel for electrophoresis or the blotting film is irradiated with light to excite fluorescence. In the two-dimensional fluorescence image detecting device for nucleic acid or protein, which comprises a light source for detecting light and a light detecting section for collecting and detecting a two-dimensional fluorescence image generated by irradiation of the light, the light detecting section includes an imaging lens, An optical image dividing means, an optical filter, and a two-dimensional photodetector. The two-dimensional fluorescence image is condensed by the imaging lens, divided into almost equal parts by the optical image dividing means, and passes through the optical filter. A two-dimensional fluorescence image detecting device for nucleic acids and proteins, which is imaged and detected on the light detection surface of the two-dimensional photodetector. 2. The optical image splitting means is any one of a semi-transmissive mirror, a cube prism and a dichroic mirror, and each of the two-dimensional fluorescent images split substantially evenly passes through an optical filter having different transmission characteristics. 2 for each
The two-dimensional fluorescence image detecting device for nucleic acid or protein according to claim 1, wherein the two-dimensional fluorescence image is detected by being incident on a three-dimensional photodetector and having different wavelengths. 3. The optical image dividing means is any one of a semi-transmissive mirror, a cube prism and a dichroic mirror,
Each two-dimensional fluorescence image, which is composed of a total reflection mirror and is divided into almost equal parts, passes through an optical filter having different transmission characteristics, enters the same two-dimensional photodetector, and has two-dimensional fluorescence with different wavelengths. The two-dimensional fluorescence image detecting device for nucleic acid or protein according to claim 1, which detects an image. 4. The method according to claim 1, wherein the fluorescence is excited by a laser beam or an ultraviolet lamp.
A two-dimensional fluorescence image detecting device for nucleic acid or protein according to any one of 1.