JP2002005834A - Distribution measuring apparatus for fluorescence labeled substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a distribution measuring apparatus which corresponds to a high-density DNA probe array. SOLUTION: A plurality of irradiation spots 101a, 101b, and so on are formed on the DNA probe array 2, which is placed on a stage 20. The stage is moved in the X-direction and the Y-direction so as to be scanned, and nearly the whole probe array is irradiated. A plurality of fluorescence emissions, generated by the plurality of irradiation spot parts on the probe array, are condensed so as to be detected simultaneously by a multidetector 17, and detected signals are processed by a data processing means 19 so as to reconstruct an image.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for reading and analyzing a luminescence (fluorescence) pattern emitted from a sample on a flat surface, and more particularly, to a method for applying a biological sample such as DNA or RNA or oligonucleotide labeled with a fluorescent substance on a substrate. The present invention relates to a device that captures at a plurality of positions and reads the emission pattern of the fluorescent label.

[0002]

2. Description of the Related Art DNA and protein analysis techniques are important in the fields of medicine and biology, including gene analysis and genetic diagnosis. In particular, recently, a DNA probe array (or various names such as an oligo chip, a DNA chip, a biochip, etc., but hereinafter, collectively referred to as a DNA probe array) is used to collect various kinds of DNA sequence information and genes from one sample. Attention has been focused on methods and apparatus for simultaneously analyzing and analyzing information. The DNA probe array uses a substrate such as glass, divides the substrate into a plurality (hundreds to tens of millions) of regions, immobilizes target (usually different types) DNA probes on each, and This is a minute reaction area. By reacting this with the sample, the objective D in the sample
NA is hybridized and captured with the immobilized DNA probe, and further bound by a fluorescent probe or the like, so that the binding state (position, that is, the hybridized sequence) and the amount thereof can be measured by fluorescence intensity or the like. become,
It can be used for genetic diagnosis, sequencing and the like.

[0003] A microscope (a confocal fluorescence microscope) -like device usually called a scanner is used to read the fluorescence intensity emitted from the fluorescent label of the target DNA captured in each reaction region of the DNA probe array (for example, ,
See JP-A-11-315095). This device is
The array is irradiated with excitation light, such as laser light, in one minute spot, and the resulting fluorescence is separated from the excitation light using a spectral element such as an interference filter, and the fluorescence intensity is detected by a photodetector such as a photomultiplier tube. Detect with. At that time, the excitation light is shaken using a galvanometer mirror or the like to scan the minute spots formed on the array two-dimensionally, or the position of the minute spots is fixed, and the array is scanned two-dimensionally. By doing so, the fluorescence intensity distribution of the entire array, that is, the degree of binding to each DNA probe can be known.

[0004]

In the DNA probe array, the types of probes to be immobilized are increasing, and the density is increasing. Therefore, a high-speed and high-sensitivity in addition to a high resolution is desired for the fluorescent label distribution measuring device of the DNA probe array. However, in the above-mentioned apparatus, this point is not sufficiently considered. In other words, in order to increase the resolution, it is necessary to make the spot diameter of the excitation light smaller, in which case (when the exposure time per spot is the same)
The scanning time increases. Furthermore, since the spot diameter is reduced, the number of fluorescent molecules existing inside the spot is reduced, and the detection sensitivity is also reduced. In addition, to increase the scanning speed, it is necessary to increase the scanning speed or increase the spot diameter of the excitation light. However, when the scanning speed is increased, the excitation time for the phosphor is shortened, and the fluorescence intensity is reduced. Further, circuit noise increases and detection sensitivity decreases. In order to increase the sensitivity, it is necessary to increase the spot diameter and reduce the scanning speed. That is, high resolution, high speed, and high sensitivity are mutually contradictory, and it has been difficult to realize them.

Further, an apparatus has been proposed in which a plurality of minute spots are formed on a sample surface and light from the plurality of spots is simultaneously detected (for example, Japanese Patent Application Laid-Open No. H11-11844).
No. 6). In this configuration, the luminous flux of the excitation light source is expanded, and a two-dimensional microlens array is arranged there to form a plurality of irradiation spots, which are projected on the sample surface. However, in this case, especially when laser light is used as the excitation light, the light intensity of the spot at the center becomes strong due to the Gaussian distribution characteristic of the laser light, and the light intensity of the spot becomes smaller toward the periphery, so that a plurality of irradiation spots are formed. It is difficult to make all light intensities uniform. If the excitation light is spread over a light beam having a diameter sufficiently larger than the width of the two-dimensional microlens array, the problem is reduced, but in this case, the light use efficiency is low, the light intensity is low, and the detection sensitivity is low. There are issues such as letting them do it. An object of the present invention is to provide an apparatus for measuring the distribution of a fluorescently labeled substance in a DNA probe array or the like that can solve the above-mentioned problems and simultaneously achieve high resolution and high speed.

[0006]

According to the present invention, there is provided an apparatus for measuring the distribution of a fluorescent label according to the present invention, which scans the emission pattern of a fluorescent label captured on a sample surface on a substrate and reads the fluorescent label. In the distribution measurement device, a stage on which a substrate to be read is placed, means for forming a plurality of irradiation spots on a sample surface on the substrate at specified intervals larger than the diameter of the irradiation spot, a moving mechanism for moving the stage, and a sample Condensation detection means for condensing and detecting light emitted from a plurality of irradiation spots on the surface, and control means for controlling a moving mechanism to irradiate substantially the entire surface of the sample according to the formation positions of the plurality of irradiation spots And a processing means for processing a signal from the condensing detection means to reconstruct an image of the sample surface. The intensity of the plurality of irradiation spots is substantially substantially the same.

The means for forming a plurality of irradiation spots includes at least one laser light source as an excitation light source, and divides the light from the excitation light source so that the distance between adjacent irradiation spots on the sample surface is 1 mm or less. Images can be formed side by side. The means for splitting the light from the excitation light source may include a splitting prism, an optically anisotropic medium, an optical fiber or a polarizing mirror, or a combination thereof.

The means for forming a plurality of irradiation spots further comprises N (N ≧ 1) excitation light sources and M-stage (M> 2) splitting means to form N × 2 ^M irradiation spots. You can do it. As the two-way splitting unit, a splitting prism, an optically anisotropic medium, an optical fiber, or a polarizing mirror can be used.

The means for forming a plurality of irradiation spots further comprises a plurality of laser light sources and a plurality of optical fibers, and forms a plurality of irradiation spots by arranging emission ends of the plurality of optical fibers in a straight line. Can be configured. The plurality of irradiation spots are preferably located on a straight line at substantially constant intervals. The light-gathering detection means collects and detects almost simultaneously a plurality of fluorescent luminescence generated from a plurality of irradiation spots, includes a wavelength selection filter, and includes an excitation component and a fluorescence component among the luminescence generated from the plurality of irradiation spots. And the fluorescent component is detected. The light-gathering detecting means can be provided with the same number of pinholes as the number of irradiation spots at positions where light emission generated from the plurality of irradiation spots forms an image, and can detect light passing through the pinholes.

The control means moves the stage in a specific one axis direction stepwise n times for each first minute width, followed by a step movement 1 having a second width larger than the first minute width. The entire surface of the sample surface can be measured by a method in which the sample surface is moved in units of times and the sample surface is repeatedly scanned. More specifically, the movement of the irradiation spots in the arrangement direction is performed stepwise movement n-1 times for each width of 1 / n (n: integer) of the irradiation spot interval, followed by (both ends of the excitation spot group). The stage movement can be controlled such that the step movement is repeated in units of one step movement of (width of width + first width) to scan the sample surface.

It is desirable that the signal from the light condensing detection means be taken in when the stage is moving in a direction perpendicular to the direction in which the plurality of irradiation spots are arranged. The means for forming a plurality of irradiation spots needs to simultaneously form a plurality of irradiation spots, and in order to image light on the sample surface, a numerical aperture of 0.7 or more, an effective field of view of 1.0 mm or more, and a sample A condensing lens having a distance of 0.7 mm or more between the surface and the tip of the lens is used.

The means for forming a plurality of irradiation spots includes a plurality of optically anisotropic media having different lengths in the optical axis direction or optically anisotropic media having different crystal axis directions, and a medium between the optically anisotropic media. And a wave plate arranged at the same position. The means for forming a plurality of irradiation spots further comprises an element in which a plurality of optically anisotropic media and wave plates are alternately arranged, and the length of each optically anisotropic medium in the traveling direction of light is immediately before. It can be configured to be approximately half the length of the optically anisotropic medium.

[0013] The means for forming a plurality of irradiation spots further comprises: scanning the light from the excitation light source so as to sequentially enter one end of a plurality of aligned optical fibers, and arranging the emission ends of the optical fibers in a straight line. May form a plurality of irradiation spots. Further, it is preferable to provide a function of performing intensity correction for each detection signal of each irradiation spot.

[0014]

Embodiments of the present invention will be described below with reference to the drawings. Here, taking a DNA probe array as an example, a fluorescence intensity distribution emitted from a fluorescent labeling substance which is used to label a target DNA captured by being hybridized with DNA probes immobilized in a large number of small reaction regions of the DNA probe array. An apparatus and a method for measuring the temperature will be described. However, it goes without saying that the present invention is not limited to DNA but can be similarly applied to the inspection and analysis of other biological samples such as RNA, oligonucleotides, and proteins labeled with a fluorescent substance.

The DNA probe array for measurement is
For example, it is created as shown below. First, a washed glass substrate (such as a slide glass) is treated with a silane coupling agent (aminopropyltriethoxysilane) to introduce amino groups on the surface. Next, the oligonucleotide having the aldehyde group introduced therein is dropped into the minute region and reacted to immobilize the oligonucleotide on the glass substrate. A fluorescently labeled DNA is separately prepared as a specimen to be tested, spotted on a region of the substrate to which the oligonucleotide is fixed, hybridized, captured on the substrate, and an array to be measured is prepared. The oligonucleotide may be immobilized on the glass substrate by subjecting the slide glass to a surface treatment with a polycation such as polylysine, and electrostatically binding the DNA with the charge of the DNA. Oligonucleotides may be synthesized directly on a glass substrate using synthesis techniques. The DNA probe array is not limited to these, and can be prepared by various well-known methods, and can be similarly measured.

FIG. 1 is a schematic diagram showing the overall configuration of an example of a fluorescent label distribution measuring apparatus (DNA probe array measuring apparatus) according to the present invention. The apparatus comprises a DNA probe array measuring apparatus main body 1, a data processing / control unit 19, and a monitor 21. The substrate (DNA probe array) 2 is fixed on the XYZ drive unit 20. The light from the light source 10 such as a laser is
By passing through one, it is emitted as a plurality of lights (100a, 100b,...) Evenly arranged on a straight line. These lights are collimated by the lens 12, reflected downward by the dichroic mirror 13, condensed by the objective lens 14, and excited on the surface of the substrate 2 by the excitation spots (101a, 101b,
…) Is formed. Although only two light and two excitation spots are shown in the drawing due to space limitations, 64 excitation spots are actually formed. The number of excitation spots was 16, 32, 6
It is more effective to increase the sensitivity and speed by forming as many as 4,. That is, when the total measurement time is the same, the irradiation time per individual excitation spot can be increased as the number of spots increases, and the sensitivity can be improved.
If the irradiation time per individual excitation spot is the same, the total measurement time can be shortened as the number of spots increases, and in this case, the speed can be increased 64 times.

Light emission (fluorescence, scattered light, reflected light) generated from the excitation spot on the substrate 2 is collected again by the objective lens 14 and is reflected by the dichroic mirror 13 (reflecting the excitation light wavelength component and transmitting the longer fluorescence wavelength component). A fluorescent component is extracted through a dichroic mirror), passed through an interference filter 15 designed to pass a fluorescent wavelength component, and condensed by a lens 16 to form an excitation spot (101a, 1).
01b,...) Are converted into fluorescent images (102a, 102b,...).
As an image.

The multi-detection unit 17 detects the fluorescence intensity generated from a plurality of excitation spots substantially simultaneously.
The optical signal from the multi-detection unit 17 is subjected to data processing and
The data is digitized by the A / D conversion unit 18 in accordance with the timing from the control unit 19, and the data processing / control unit 19 performs various calculations and the like. Further, the data processing / control unit 19 controls the movement of the XYZ drive unit 20, constructs a two-dimensional distribution of the fluorescence intensity on the surface of the substrate 2 together with the XY drive information, and displays it on the monitor 21. This makes it possible to measure the fluorescence intensity distribution on a predetermined region surface of the substrate 2, the total fluorescence intensity for each of a plurality of minute regions provided on the substrate 2, and the like, and calculate the amount of the sample DNA bound to the immobilized oligonucleotide. .

Here, the size of the excitation spot on the surface of the substrate 2 is 2 μm in diameter, and the interval between the excitation spots is about 20 μm. It is desirable to adjust the size of the excitation spot so as to be an integral multiple of the interval between the excitation spots. When 64 excitation spots are formed, the distance between both ends of the spot group is 1.26 mm.
A 1.3 mm field of view is created, the numerical aperture is NA = 0.70 in order to improve the fluorescence collection efficiency, and the distance between the lens tip and the sample position is 1.3 mm in consideration of the case of irradiating from the back of the glass. Used.

The XYZ drive unit 20 is operated as follows to measure the fluorescence of the entire area of interest on the substrate 2. FIG. 15 is an explanatory diagram of an operation example of the XYZ drive unit 20. The arrangement direction of the excitation spots is defined as the x-axis. The excitation spots S0, S1, S2,..., S63 are arranged at the initial positions so that the position of the excitation spot S0 is (x, y) = (0, 0) as shown in the figure. A region 700 indicates a region of interest to be measured including the oligonucleotide-immobilized region of the DNA probe array. In order to scan the entire surface of the region 700, the stage is moved two-dimensionally. In the figure, the area 70
The moving trajectory of the excitation spot S0 relative to 0 is shown by an arrow.

First, the entire width of the area 700 is scanned in the y direction. Next, the y position is returned to 0, and shifted in the x direction by the excitation spot diameter (2 μm in this example). The entire width of the area 700 is scanned again in the y direction. This operation is repeated a plurality of times until the excitation spot S0 reaches the first S1. In the figure, for simplicity, the interval between the excitation spots is reduced so as to correspond to 8 μm, and the entire width is scanned in the y direction four times by changing the position in the x direction. If the diameter of the excitation spot is 2 μm and the interval between the excitation spots is 20 μm, the processing is repeated 10 times. Thereby, x = 0 to 1
The entire surface of the 280 μm wide area can be scanned. The next action is
The y position is returned to 0 and shifted by 1260 μm in the x direction to a region next to the region where scanning has been completed with 64 excitation spots, that is, the spot of S0 moves to a position corresponding to S64, and Repeat the operation. By repeating this, the entire area 700 is scanned.

FIGS. 16A and 16B are xy-axis operation curves of the XYZ drive unit 20, wherein FIG. 16A shows the movement of the XYZ drive unit in the x direction, and FIG.
3 illustrates a moving state in a direction. As described above, in the x direction, that is, in the arrangement direction of the excitation spots, at least two types of movement patterns of n steps (10 times in this example) having a very small width of 2 μm and one movement of a large width are used. Repetitive scanning of the entire measurement area is characteristic of the operation of the present drive unit. This is because the size of the field of view of the objective lens is limited, and the interval between a plurality of excitation spots is limited. If the interval between the excitation spots is set to a width obtained by dividing the entire width of the measurement region substantially by 64, it is not necessary to adopt the above-described moving method, and the x direction is a step of a minute width corresponding to the excitation spot diameter. All you have to do is repeat the basic movement. However, in this case, the interval between the excitation spots on both sides is wide and almost equal to the entire width of the measurement area, which exceeds the field width of a normal objective lens (an objective lens capable of forming an excitation spot of about 2 μm), and the measurement is not performed. It becomes difficult. Even if there is an objective lens having a sufficient field width, the numerical aperture becomes small or the objective lens becomes very expensive, which is not practical. That is, the scanning method shown in FIGS. 15 and 16 is an appropriate method for scanning the entire measurement region using a plurality of excitation spots.

There are two timings of data collection during scanning: movement in the x direction and movement in the y direction.
In principle, it is possible to obtain information on the entire surface in either case. However, it is particularly convenient to collect data in accordance with the movement in the y direction. This is because the stroke of one movement is long in the y direction, so that the connection with the adjacent pixels is clearer, and it is easy and reliable to reconstruct an image from the measured data.

Next, the configuration of the multi light source distribution unit 11 will be described. The size of the excitation spot is 2 μm in diameter,
When the interval between the excitation spots is 20 μm, the coupling magnification including the objective lens is set to 20 at the output end of the multi-light source distribution unit 11, the size of the light spot at the output end is 40 μm in diameter, and the distance between the light spots is Is 400 μm, and light needs to be emitted from 64 locations. It is usually impossible to arrange light sources at 400 μm intervals, and in the present invention, the following configuration was constructed. FIGS. 2 to 7 and FIGS. 17 to 1
This will be described with reference to FIG.

FIG. 2 is a diagram showing an example of the configuration of a multi-light source distribution unit 11 for distributing light from n laser light sources (two of which are shown) into 4n light beams. The light from the laser light sources (10a, 10b) is
0a, 200b) and condensed by an optical fiber (201a, 2b).
01b). The optical fiber has two branching points in the middle, whereby the light is split into four and 100a
, 100b4 and the excitation light can be split. In addition, the branch is not limited to the two-branch type as shown in FIG.
Various types such as a branch type can be selected. In such a branch, a fiber that divides light uniformly is used.
For example, the output end of an optical fiber having a core diameter of about 40 μm is 4
By arranging them on one straight line at intervals of 00 μm and projecting them at a reduction of 1/20 times, the structure of this example can be realized.

FIG. 3 shows a configuration of another type of multi-light source distribution unit 11 for simply arranging light from m laser light sources (four of them are shown) on a straight line at intervals of 400 μm. It is a figure showing an example. The figure shows the case where m = 4. Laser light sources (10a, 10b, 10c,
10d) from each lens (200a, 200b,
200c, 200d) and condensed by an optical fiber (202
a, 202b, 202c, 202d). By arranging the output ends of the optical fibers on a straight line at intervals of 400 μm, the structure of the present invention can be realized regardless of the size of the laser light source at intervals of 400 μm for the light (100a, 100b, 100c, 100d).

FIG. 4 is a diagram showing a configuration example of the multi-light source distribution unit 11 using a birefringent material (for example, calcite, other materials can be used). The birefringent materials 300 and 302 such as calcite and the quarter-wave plates 301 and 303 are combined with the birefringent material 300, the quarter-wave plate 301, the birefringent material 302 from the laser light source side as shown in the figure. 1
The distribution element 310 is formed by combining in order of the ３０３ wavelength plate 303. Light from the laser light source 10a is incident on the element through the lens 200a. At this time, the polarization plane of the laser beam is inclined by 45 degrees, or circularly polarized through a を 通 し て wavelength plate on the way. The laser beam incident on the birefringent material 300 is birefringent and separates into an ordinary ray and an extraordinary ray. The length of the birefringent material 300 is adjusted so that they are separated by w1. This light is further converted into circularly polarized light through a quarter-wave plate 301 and is incident on a birefringent material 302. The split light is likewise split further into two. The distance is w2
The length of the birefringent material 302 is adjusted so that w
By adjusting 1 to be twice w2, the laser beam can be divided into equal intervals. Note that w2 = 40
To make 0 μm, the lengths (optical axis direction) of the birefringent materials 300 and 302 are about 8 mm and about 4 mm, respectively (when the birefringent material is calcite and the crystal axis is 45 degrees with respect to the optical axis). What should I do? The length of each birefringent material varies depending on the material type, wavelength, and inclination of the crystal axis. However, when the direction of the crystal axis is the same, the length of the birefringent material is as described above. What is necessary is just to make it 1/2 of the length of a material.

In this configuration, one laser beam can be arranged equally on four lines with almost the same intensity, and the emission ends are arranged on one straight line. Therefore, the laser beam is set to 0.4 × 4 = 1.6 m
If 16 light spots are arranged at m intervals, 64 light spots will be 0.4
They can be arranged on one straight line at mm intervals. By setting the focal position of the lens 200a at the position of the pinhole 304, extra light such as scattered light can be removed.
The shape of the excitation spot on the substrate 2 can be made clear, and the resolution can be improved. Furthermore, it can be used as one focus position for confocal measurement.

Although the distribution element 310 in the drawing has a structure in which it is divided into two stages, it can be divided into three, four, and six stages, in which case one laser beam is 8, 16, 64, respectively.
Therefore, the number of laser beams required to obtain 64 light spots can be reduced to 8, 4, and 1. For example, in the case of a six-stage structure using 32,16,8,4,2,1 mm length of each birefringent material in order from the light incident side, 0.1 mm from one laser.
It can be divided into 64 lights at intervals.

FIG. 5 is another configuration diagram of the multi-light source distribution unit 11. The element consists of four large trapezoidal glass prisms 351, 352, 353, 354 and a quarter-wave plate 3
57. Glass prisms 351 and 35
2, 353 and 354 are joined as shown in FIG.
5,356 is a polarizing beam splitter. Each glass prism has a slightly different size, and the incident light in the circularly polarized state is split into two at the bonding surface 355 and comes close again after total reflection. At this time, by making a difference in the optical path length, two parallel laser beams can be obtained. 1
Through the ３５ wavelength plate 357, the glass prism 353 is returned.
And 354 into the element section, the same principle as above,
Further, the light is split into two, and a total of four light beams can be created. The distance between the light beams can be arbitrarily set by adjusting the height of the trapezoidal glass prism, and it is easy to obtain a light beam having a very close distance of 0.4 mm. The figure shows a case where the number of outgoing light beams is 32 for 8 incident light beams. Note that a pinhole plate may be arranged close to the quarter-wave plate 357.

FIG. 6 is a diagram showing another example of the configuration of the multi light source distribution unit 11. Prisms 500a, 50 as shown having polarizing beam splitter and total reflection mirror
By using 0b..., The beam can be split into two, and by using eight of them, the light from the eight laser light sources is reduced to sixteen. The polarizing beam splitter may be a half-mirror beam splitter having transmittance = reflectance (= about 50%).

FIG. 7 is a diagram showing another example of the configuration of the multi light source distribution unit 11. A prism 510 is used in which prisms like the prisms of FIG. 6 are arranged, and further, prisms of increasing size are sequentially stacked thereon. The laser light is split by a half-mirror beam splitter. This is 4
By repeating the steps, one laser beam can be divided into 16 laser beams.

In the multi-light source distribution unit having the structure shown in FIGS. 6 and 7, there is a limit in reducing the size of the prism. The interval between laser beams to be split can be up to about 1 mm, but it becomes increasingly difficult to further narrow it. Therefore, in such a case, by using in combination with the multi-light source distribution unit of FIG. 4 or FIG.

In general, as in the configuration example of the multi light source distribution unit shown in FIG. 17, another multi light source distribution unit can be constructed by combining a plurality of multi light source distribution units 550 and 551. That is, a multi-light source distribution unit can be constructed by combining FIGS. 2 to 7 and FIGS. 18 and 19 described later. The number of laser light sources is appropriately selected based on the number of divided light sources, the light output of the laser light source, and the light intensity of the excitation spot required on the surface of the substrate 2.

FIG. 18 is a diagram showing another example of the configuration of the multi light source distribution unit 11. Light from a laser light source 2000 passes through a lens 2001, and is scanned by a beam scanning unit 2002 such as a galvanometer mirror or a polygon mirror, and optical fibers 3001, 3002, whose end faces are arranged on a circular arc at the focal position of the lens 2001. ... is incident on the end face. By arranging the other end surfaces of the optical fibers 3001, 3002,... At regular intervals, the light can be split into a large number of light beams, and the structure of the present invention can be realized.

FIG. 19 is a diagram showing another example of the configuration of the multi light source distribution unit 11. The light from the laser light source 2000 is scanned by a beam scanning unit 2003 such as a galvanometer mirror or a polygon mirror, passes through a lens 2004, and is incident on end faces of optical fibers 3001, 3002,. The split laser light can be extracted from the other end face of the optical fiber, and the structure of the present invention can be realized.

Next, the configuration of the multi-detection unit 17 will be described with reference to FIGS. As in this example, the size of the excitation spot is 2 μm in diameter, and the interval between the excitation spots is 20.
An image of μm is formed at an appropriate magnification. Magnification is determined by the pitch of the detector. Detectors 600, 601,... Such as photomultiplier tubes and photodiodes are arranged in accordance with the image forming position (FIGS. 8 and 9), and sensors such as a photodiode line sensor or a multi-anode type photomultiplier tube, a CCD line sensor, etc. 602 (see FIGS. 10 and 1).
1). In the sensor 602, the interval between the individual detectors can be narrowed as compared with the method shown in FIG. For example, in a multi-anode type photomultiplier, the distance between individual anodes is about 1 mm, and the optical system is adjusted so that the imaging magnification is 50 times. It should be noted that the pinholes 610 in FIGS.
Is used, it becomes a confocal detection system, the position resolution and the depth resolution are improved, and the accuracy of the fluorescence measurement is improved. If a photodiode or a CCD line sensor is used, an image may be formed at an arbitrary position and the signal intensity of the pixel at the image forming position may be detected.
The same effect as a pinhole can be obtained by excluding a signal of a pixel in a portion other than the image of the excitation spot.
Further, it can also be detected via the optical fibers 620 and 621 (FIGS. 12 and 13).

FIG. 14 is a diagram showing an example of an image measured using the present apparatus. The figure shows a fluorescent spot image captured on a slide glass. A spot was formed on the slide glass to which the fluorescence-labeled DNA was bound by the method described above. Each spot had a size of about 200 μm and was formed on a grid with an interval of about 0.4 mm. The entire area in which the spot is created is approximately 12 mm in length and width, and FIG. 14 shows a part thereof. In the figure, each spot is obtained by increasing the sample concentration in order from the top, and as a result, the fluorescence intensity is detected from the top to the bottom in order.

Next, data detection and image reconstruction (operation of the data processing unit) will be described. In order to detect signals in a 12 mm square area in units of 2 μm, the 12 mm square area is divided into 6000 × 6000 areas. Let them be area (ij). i (= 0-5999) is the X direction, j (= 0-5999)
Is a position in the Y direction. The measurement of the fluorescence intensity distribution by scanning the irradiation spot is performed as described with reference to FIG.
Scanning by each irradiation spot (S0, S1,..., S63) will be described.
(0,0), then scan according to the Y direction area
Scan the area from (0,1) to area (0,5999). The irradiation spot S1 also irradiates area (10,0) first, and then scans area (10,1) to area (10,599) in accordance with scanning in the Y direction.
Scan the area up to 9). The same applies to the other irradiation spots S3 to S63.
irradiate ea (630,0), then scan in Y direction
Scan the area from area (630,1) to area (630,5999).

At this time, the signal of the photodetector corresponding to each spot is sampled and AD-converted every time the light passes through the area. The signal is represented as sig (k, j), where k corresponds to the spot position number (0-63) and j is the position in the Y direction, 0-599.
Take the value of 9. That is, the signal of the spot S1 is sig (1,
j), j = 0-5999. These signals are stored in an image image storage memory corresponding to the area of area (ij). Image image storage memory is image (i, j), i (= 0-6399) is X direction,
Assuming that j (= 0-5999) is a position in the Y direction, first, a signal obtained by the above one scan is converted into an image (k × 10 + X, j) = sig (k, j) (X
= 0, k = 0-63, j = 0-5999). Note that X represents the X position of the spot S0 (in this case, X = 0).

Next, as shown in FIG.
And the X position is moved by +2 μm (X = 1), and measurement is performed in the same manner. The obtained signal is expressed as image (k × 10 + 1, j) = sig (k, j)
(k = 0-63, j = 0-5999). When this operation is repeated a total of ten times, the area of i = 0-639 and j = 0-5999 in image (i, j) is replaced with the measured signal.

Next, the X position is moved by +1260 μm (X = 640), and the above operation is performed.
e (k × 10 + X, j) = sig (k, j) (X = 640-649, k = 0-63, j = 0-5999)
To be stored. With this operation, i = 640-12 of image (i, j)
The area of 79, j = 0-5999 is replaced with the measured signal. Further, the X position is moved by +260 μm (X = 1280 position), and the above measurement is repeated. By repeating these, X
The position is moved to the position of X = 5760, and scanning is further performed 10 times to measure the entire area of i = 0-6399 and j = 0-5999 of image (i, j) (rather than area (ij)). Although the area is large, it is only necessary to take out as much as necessary). The image (i, j) is converted into an image and displayed on a monitor.

In the present embodiment, there are excitation spots and photodetectors corresponding to each spot. It is sufficient if all the excitation spot intensities and the sensitivity characteristics of the photodetector are uniform, but otherwise, a stripe pattern occurs in the obtained image. In such a case, a mechanism for correcting the sensitivity characteristics of each spot is required. In FIG. 1, an intensity correction value storage unit 22 is provided. To perform the correction processing, first, correction data is created. A sample having a uniform fluorescence intensity is prepared (or a light source that emits light uniformly at each spot position), and the intensity at that time is measured for each spot. Further, the intensity when the light was shielded was also measured for each spot, and the background value back (k) for each spot measurement and the signal intensity value when the reference light intensity was detected (the background value was subtracted) value)
The inverse value of corr (k) (k is the spot position number (0-6)
3)) is stored in the intensity correction value storage unit 22.
For the measured signal sig (k, j), sig (k, j) = (sig (k, j)
By outputting back (k)) corr (k) and the corrected signal value sig (k, j), it is possible to correct the sensitivity variation between spots.

According to the present invention, while the ordinary apparatus uses only one irradiation spot for fluorescence excitation, a plurality of (for example, 64) irradiation spots for fluorescence excitation can be simultaneously applied to the substrate. Therefore, the following effects are obtained.

When the entire area is detected in a fixed time, the irradiation time per one irradiation spot is increased (6 in this example).
4 times), and the fluorescence detection sensitivity is improved. Also, 1
By increasing the irradiation time per irradiation spot, the moving speed of the stage can be reduced, and the mechanical stability and durability can be improved. Further, if the irradiation time per one point is the same, the time for measuring the entire predetermined region of the substrate can be shortened, and the speed can be increased. In particular, when the detection resolution is increased, that is, when the size of the irradiation spot on the substrate is further reduced, for example, when the measurement at a resolution of 10 μm is conventionally performed at a resolution of 1 μm to 2 μm, the magnification is 100 to 25 times. Although it is necessary to sequentially measure the number of irradiation spot areas, high resolution and high speed are achieved by simultaneously irradiating a plurality (64) of irradiation spots for fluorescence excitation and detecting fluorescence as in the present invention. be able to. When the laser beam is divided and radiated, the intensity of each of the divided laser beams is reduced correspondingly, but this problem can be easily solved by using a laser device having a larger output.

[0046]

According to the present invention, a DNA probe array or the like can be measured with high resolution, high speed, and high sensitivity.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the overall configuration of an example of a fluorescent label distribution measuring apparatus according to the present invention.

FIG. 2 is a diagram showing a configuration example of a multi light source distribution unit according to the present invention.

FIG. 3 is a diagram showing another configuration example of the multi light source distribution unit according to the present invention.

FIG. 4 is a diagram showing another configuration example of the multi light source distribution unit according to the present invention.

FIG. 5 is a diagram showing another configuration example of the multi light source distribution unit according to the present invention.

FIG. 6 is a diagram showing another configuration example of the multi light source distribution unit according to the present invention.

FIG. 7 is a diagram showing another configuration example of the multi light source distribution unit according to the present invention.

FIG. 8 is a diagram showing a configuration example of a multi-detection unit according to the present invention.

FIG. 9 is a diagram showing another configuration example of the multi-detection unit according to the present invention.

FIG. 10 is a diagram showing another configuration example of the multi-detection unit according to the present invention.

FIG. 11 is a diagram showing another configuration example of the multi-detection unit according to the present invention.

FIG. 12 is a diagram showing another configuration example of the multi-detection unit according to the present invention.

FIG. 13 is a diagram showing another configuration example of the multi-detection unit according to the present invention.

FIG. 14 is a diagram illustrating an example of an image to be measured.

FIG. 15 is an explanatory diagram of an operation example of the XYZ drive unit.

FIG. 16 is an xy axis operation curve diagram of the XYZ drive unit.

FIG. 17 is a diagram showing another configuration example of the multi light source distribution unit.

FIG. 18 is a diagram showing another configuration example of the multi light source distribution unit.

FIG. 19 is a diagram showing another configuration example of the multi light source distribution unit.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... The body of the distribution measuring apparatus of a fluorescent label, 2 ... Substrate, 10 ...
Light source, 10a, 10b, 10c, 10d ... laser light source,
11 ... Multi light source distribution unit, 12 ... Lens, 13 ...
Dichroic mirror, 14 objective lens, 15 interference filter, 16 lens, 17 multi-detection unit,
18 A / D conversion unit, 19 data processing / control unit, 20 XYZ drive unit, 21 monitor, 2
2. Intensity correction value storage unit, 100a, 100b, 1
00c, 100d: light, 100a1, 100a2, 10
0a3, 100a4, ..., 100b4 ... light, 101a,
101b: excitation spot, 102a, 102b: fluorescent image, 200a, 200b, 200c, 200d: lens, 201a, 201b, 202a, 202b, 202
c, 202d, 620, 621 ... optical fiber, 300,
302: birefringent material, 301, 303, 357 ... 1 /
4 wavelength plate, 304, 610: pinhole, 310: distribution element, 351, 352, 353, 354: glass prism, 355, 356: bonding surface, 400a1, 400a
2,400b1,400b2,400a, 400b ...
Light, 500a, 500b, 510 ... prism, 550,
551: Multi light source distribution unit, 600, 601: Photodetector, 602: Sensor, 700: Measurement target area, 10
00: light, 2000: laser light source, 2001, 2004
... Lens, 2002, 2003 ... Beam scanning unit, 300
1,3002 ... optical fiber.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C12Q 1/68 C12Q 1/68 A G01N 33/58 G01N 33/58 A (72) Inventor Kenji Yasuda 882, Ichimo, Hitachinaka-shi, Ibaraki F-term within the measuring instruments group of Hitachi, Ltd. DA12 DA13 DA14 FA11 FA12 FA29 FB07 FB12 GC15 JA07 JA08 4B063 QA01 QA13 QA17 QA18 QQ42 QQ52 QR55 QR82 QS34 QX02

Claims (14)

[Claims] An apparatus for measuring the distribution of a fluorescent label, which scans and reads a light emission pattern of a fluorescent label captured on a sample surface on a substrate, comprising: a stage on which a substrate to be read is placed; Means for forming a plurality of irradiation spots at specified intervals larger than the diameter of the irradiation spots, a moving mechanism for moving the stage, and a collection for condensing and detecting light emitted from the plurality of irradiation spots on the sample surface. Light detection means, control means for controlling the moving mechanism so as to irradiate substantially the entire surface of the sample in accordance with the formation positions of the plurality of irradiation spots, and And a processing means for reconstructing an image of the sample surface. 2. The fluorescence label distribution measuring apparatus according to claim 1, wherein the means for forming the plurality of irradiation spots includes at least one laser light source as an excitation light source, and divides light from the excitation light source. An apparatus for measuring the distribution of a fluorescent label, wherein an image is arranged side by side so that the distance between adjacent irradiation spots on the sample surface is 1 mm or less. 3. The distribution measuring apparatus according to claim 2, wherein the means for splitting the light from the excitation light source includes a splitting prism, an optically anisotropic medium, an optical fiber or a polarizing mirror, or a combination thereof. An apparatus for measuring the distribution of a fluorescent label, comprising: 4. The distribution measuring apparatus according to claim 1, wherein the means for forming the plurality of irradiation spots comprises N (N ≧ 1) excitation light sources and M stages (M> 2). A fluorescent marker-shaped distribution measuring apparatus, comprising: N × ^2M irradiation spots; 5. The apparatus for measuring distribution of a fluorescent label according to claim 1, wherein the means for forming the plurality of irradiation spots includes a plurality of laser light sources and a plurality of optical fibers.
A plurality of irradiation spots are formed by arranging emission ends of the plurality of optical fibers on a straight line, and a distribution measurement apparatus for a fluorescent label is provided. 6. The fluorescent labeled object distribution measuring apparatus according to claim 1, wherein the plurality of irradiation spots are located on a straight line at substantially constant intervals. Distribution measurement device. 7. The fluorescence label distribution measuring apparatus according to claim 1, wherein the light-concentration detecting means comprises:
A distribution measuring device comprising: a position where light emitted from a plurality of irradiation spots forms an image; and the same number of pinholes as the number of irradiation spots, and detects light passing through the pinholes. 8. The fluorescence label distribution measuring apparatus according to claim 1, wherein the control means moves the stage in a specific one axis direction in n steps in a stepwise manner every first minute width. An apparatus for measuring the distribution of a fluorescently labeled substance, wherein the sample is scanned by repeating a single step movement of a second width larger than the first minute width, followed by scanning the sample surface. 9. The apparatus for measuring the distribution of fluorescent labels according to any one of claims 1 to 8, wherein the light-condensing detection is performed when the stage moves in a direction perpendicular to a direction in which a plurality of irradiation spots are arranged. A signal from the means for taking in a signal; 10. The apparatus for measuring the distribution of a fluorescent label according to claim 1, wherein the means for forming the plurality of irradiation spots includes a numerical aperture for imaging light on the sample surface. An apparatus for measuring the distribution of a fluorescent label, comprising: a condensing lens having a diameter of 0.7 mm or more, a diameter of an effective field of 1.0 mm or more, and a distance between a sample surface and a lens tip of 0.7 mm or more. 11. The fluorescent label distribution measuring apparatus according to claim 1, wherein the means for forming the plurality of irradiation spots comprises a plurality of optically anisotropic media or crystal axes having different lengths in an optical axis direction. An apparatus for measuring the distribution of a fluorescent label, comprising: optically anisotropic media having different directions; and a wave plate disposed between the optically anisotropic media. 12. The distribution measuring apparatus for fluorescent labels according to claim 1, wherein the means for forming the plurality of irradiation spots comprises an element in which a plurality of optically anisotropic media and a wave plate are alternately arranged. A distribution measuring apparatus for a fluorescent label, wherein the length of each optically anisotropic medium in the light traveling direction is about half of the length of the optically anisotropic medium immediately before. 13. The apparatus for measuring distribution of a fluorescent label according to claim 1 or 2, wherein the means for forming the plurality of irradiation spots includes one end of a plurality of optical fibers arranged by scanning light from an excitation light source. Characterized in that a plurality of irradiation spots are formed by sequentially arranging the emission ends of optical fibers on a straight line. 14. The fluorescent labeled object according to claim 1, further comprising a function of performing intensity correction for each detection signal of each irradiation spot. Distribution measuring device.