JPH0682097B2 - Photo-detection electrophoresis device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] The present invention relates to a photodetection-type electrophoresis apparatus for detecting fluorescence or scattered light, and more particularly to a DNA base sequence determination apparatus.

[Conventional technology]

As an apparatus for electrophoretically separating and detecting fluorescently labeled DNA, there is known an apparatus based on spot irradiation point detection as described in JP-A-60-220860. For simultaneous measurement of a large number of samples, scanning of a point irradiation / point detection unit can be considered, but a line irradiation line detection method has been proposed as another method (Japanese Patent Laid-Open No. 61-62843). According to this method, as shown in a part of FIG. 1 showing a schematic view of one embodiment of the present invention, a laser beam is incident from a side on a gel electrophoresis plate having a plurality of migration paths,
Linearly irradiate a certain distance from the migration origin and excite the fluorescently labeled DNA passing therethrough. The emitted fluorescence is imaged on an image amplifier through a filter and a condenser lens, amplified, and then detected by a diode array or a TV camera. In this measuring apparatus, the direction in which the laser beam 6 is irradiated (hereinafter, abbreviated as an irradiation axis), the optical axis of the light receiving system that is the light detecting means including the filter, the lens 5, and the two-dimensional sensor 7 (hereinafter, the light receiving optical axis). The horizontal line that passes through the center of the monitor image (screen) of the center line of the diode array (hereinafter abbreviated as the center horizontal axis) or the read horizontal line of the two-dimensional sensor in the TV camera on the plane formed by Is geometrically configured so that the axis of the read horizontal line (hereinafter, abbreviated as the central horizontal axis) that gives

[Problems to be solved by the invention]

In the above prior art, the excitation light beam and the central horizontal axis of the measuring section diode array or the two-dimensional sensor are kept parallel by fixing the laser unit and the light receiving measuring unit (including the sensor section) on the same optical substrate. But there was no special adjustment function. Normally, the laser light is deflected 90 ° using a mirror and irradiates the gel panel. Laser light is incident on the gel plate with multiple migration paths from the side, and the distance from the migration origin of the gel plate to the position irradiated with the laser light, that is, the migration path, over the entire measurement area of the gel plate irradiated with laser light. It is necessary to set the laser light so that the length is constant. This can be automatically achieved by attaching the electrophoresis plate, but there is an individual difference when preparing the gel, so fine adjustment is necessary. For this reason, the mirror can be swung horizontally and vertically, and the laser optical path can be adjusted. In order to prevent deterioration of sensitivity and deterioration of resolution, it is desired that the irradiation light beam and the measurement system, that is, the center line of the line sensor or the read horizontal line of the two-dimensional sensor be parallel to each other. However, the central horizontal axis of the diode array or the two-dimensional sensor in the TV camera is not always parallel to the laser beam applied to the electrophoretic plate, which causes a decrease in sensitivity and deterioration of resolution.

The object of the present invention is to solve the above problems.

[Means for solving problems]

The above-described object is achieved by rotatably configuring a table holding a detection unit equipped with a diode array or a two-dimensional sensor.

[Action]

By making the detection unit rotatable on a rotatable platform, the laser beam is largely deflected by the mirror, and the excitation light applied to the electrophoretic plate without rotating the electrophoretic plate,
It can be parallel to the central horizontal axis of the array sensor or the two-dimensional sensor of the detection unit.

〔Example〕

An embodiment of the present invention will be described below with reference to FIG. The apparatus is a laser light source 3, an electrophoretic separation unit having a migration plate 1 and a buffer tank 2, a filter and a lens 5, a two-dimensional sensor 7, a detection unit having a frame memory 8, and a calculator.
9. A data processing unit having an output device 10. In this embodiment, the case where the two-dimensional sensor 7 is used for the detection unit will be described, but a TV camera may be used instead. The laser light 6 passes through the mirror 4 and the focusing lens (not shown) and enters the gel 1 from the side surface. The center of the area of the electrophoretic plate 1 irradiated with the laser light is selected as the focus point of the ordinary beam, and the beam 6 incident on the electrophoretic plate 1 is narrowed down over a wide area in the incident direction of the laser light. The irradiation angle is set so that the distance from the migration origin to the position where the laser light is irradiated on the gel plate, that is, the length of each migration path is constant. Various substances electrophoretically separated pass through the irradiation part of the gel plate 1 irradiated with the laser light. If the detector does not have the resolution to detect position in the migration direction, the separation detectability is determined by the laser beam width. The length of the gel irradiation area (that is, the above irradiation part) is 20 cm,
The laser beam 6 is narrowed down, and the beam diameter becomes the smallest at the center of the irradiation area of the electrophoretic plate 1 irradiated with the laser beam, and the beam diameter at the entrance is equal to or less than the gel thickness (0.5 mm to 0.3 mm). There is a need to. Due to the aberration of the lens and the interference of light, the actual laser beam 6 is limited to 0.5 mm at the entrance to the electrophoretic plate 1 and 0.3 m at the center. That is, the separation detectability in this case is 0.3 to 0.4 mm. However, if the light receiving optical system has a high resolution, it is possible to obtain high separation and detection ability as a detection system regardless of the width of the excitation light. In fact, the optical resolution of the embodiment is 0.1 mm, which is smaller than the width of the laser beam 6, and a resolution equal to or smaller than the width of the laser beam is obtained.

Assemble the device in the usual way and observe fluorescence image 13
When is measured, the monitor image looks like Fig. 2, but
Not coincident with the center horizontal line 12, the center horizontal axis of the TV camera in the measurement system, that is, the center horizontal line 12 at the image center portion of the monitor image 15 and the observed fluorescence line image 13 do not necessarily coincide with each other. If you try to adjust only with mirror 4, the second
As indicated by a broken line 14 in the figure, the image is largely deviated from the center of the screen, which causes a problem. Further, the migration distance may be slightly shifted in each migration path, and if the irradiation angle is adjusted for this correction, it may be further displaced. For low resolution measurement, a region is set centering on the central part, and signals coming into this range are vertically integrated and used, so the central horizontal axis of the light receiving system and the irradiation axis are not parallel and are not parallel. But no problem occurs. However, when attempting to measure with resolution close to the resolution limit of the optical system, it is necessary to extract a signal corresponding to a single search line on a TV monitor. If the irradiation axis and the central horizontal axis of the measuring system do not coincide and are not parallel, the signal can be obtained from only a part of the area irradiated with the laser beam 6. Therefore, in the embodiment, by using the light receiving unit rotating mechanism equipped on the gantry 11 to make both axes parallel (the irradiation axis and the central horizontal axis of the measurement system), the total irradiation length of the laser beam 6 irradiated on the gel is reduced. A signal with high position resolution in the migration direction was obtained over the region.

〔The invention's effect〕

As described above, in the present invention, first, the irradiation system for irradiating the laser light is set so that the migration path length becomes constant,
Next, since the irradiation axis and the central horizontal axis of the light receiving system can be aligned, high position resolution measurement can be performed.

[Brief description of drawings]

FIG. 1 is a schematic diagram of an embodiment of the present invention, and FIG. 2 is a diagram showing a measurement image on a two-dimensional sensor display device. 1 ... Migration plate, 2 ... Buffer tank, 3 ... Laser light source, 4 ... Mirror, 5 ... Filter and lens, 6
...... Laser beam, 7 …… two-dimensional sensor, 8 …… Frame memory, 9 …… Calculator, 10 …… Output device, 11 ……
Frame with rotation function, 12 …… Central horizontal line, 13 …… Observation fluorescence line image, 14 …… Fluorescence line image when mirror is adjusted, 15 …… Monitor image.

Claims (1)

[Claims] 1. A gel electrophoresis section in which a plurality of migration paths for electrophoresis of fluorescently labeled DNA fragments are arranged on a plane,
A light source means for emitting a laser beam, and the laser beam from the side of the gel electrophoresis section so as to intersect the migration direction of the DNA fragment, sequentially illuminating each of the plurality of migration paths, fluorescent labeling of the DNA fragment In a photo-detection type electrophoretic device comprising a photo-detection means for detecting light emitted from
The light detection means includes a line sensor or a two-dimensional sensor, the irradiation axis of the laser light incident from the side surface, and the read line of the line sensor or a predetermined read line of the two-dimensional sensor on the same plane. A photodetection-type electrophoretic device comprising means for rotating the light-receiving part in the photodetection means for setting.