JP2003098087A - Method and device for detecting fluorescent bead or fluorescent dot array, and dna testing method and device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve problems in a conventional method that the fluorescent detection is performed by scanning one-spot excitation light over the whole surface of a sample face when a bead array or a dot array with a fluorescent sign is detected; as a result, a ratio of the time for irradiating the excitation light to an internal part of the beads and the dots is increased, which causes the waste of time, whereby the high-speed detection becomes difficult; and the detection of a high sensitivity and wide dynamic range, which can be realized only by elongating the detection time of one bead and dot, becomes impossible. SOLUTION: The multi-spot excitation light of approximately equal to a diameter of the bead or dot array is applied, the bead or dot array and the spot excitation light are relatively scanned, and the obtained fluorescence is separated from the excitation light to be detected. Here, the spot excitation light is trailed and driven so as to generally constantly apply the spot excitation light to the bead or dot array within the time necessary for the relative scanning by one pitch of the bead arrangement. Or the fluorescent bead or dot array sample is moved by one pitch step to generally apply the spot excitation light to the bead or dot array within an average time necessary for the relative scanning by one pitch of the bead or dot arrangement.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for detecting or inspecting a fluorescent substance or a fluorescently labeled substance, especially a fluorescently labeled DNA.
In particular, the present invention relates to a method and apparatus for detecting and inspecting fluorescence arranged in the form of beads or dot arrays at high sensitivity, wide dynamic range and high speed.

[0002]

2. Description of the Related Art As a method for detecting a sample in which a fluorescent substance or fluorescently labeled DNA is arranged in the form of beads or a dot array, a conventional excitation laser beam is formed into one spot, and the sample and the excitation laser spot are relatively scanned. ,
The resulting fluorescence was detected. Further, a wide area of the sample was surface-illuminated with excitation light, and the obtained fluorescence was detected by a two-dimensional CCD or the like.

[0003]

When the above-mentioned one spot is irradiated and the relative scanning is performed for detection, the beads or dot array sample is scanned over the entire surface, so that it can be effectively used for fluorescence detection as shown below. The ratio of time spent is very small. That is, if the diameter of the beads or dots is D and the pitch of the beads or dots is P, and the array is in a square grid pattern, the ratio effectively used for fluorescence detection during the scanning time is πD ² / 4P ^2. . For example, if the ratio of D and P is 1: 2, this value will be 19.6%, and even if it is 1: 1.5, it will be 34.9%. Thus, more than half of the time will not be effectively used for detection.

Further, since one spot light is irradiated for detection, when a sample is composed of a large number of beads or dots, it takes a lot of time for fluorescence detection, and high-speed detection is difficult. Further, when trying to detect with high sensitivity and wide dynamic range, it takes some time to excite one bead or one dot, and in order to realize high speed detection, sensitivity and dynamic range must be sacrificed. The above wasteful time is the same when the surface irradiation is performed and the two-dimensional detection is performed, and it is difficult to achieve high sensitivity and a wide dynamic range.

[0005]

The present invention uses the following means in order to solve the above problems.

Spot-like excitation light having a beam diameter approximately equal to the diameter of the beads or dot array to which the fluorescent substance is attached is irradiated. Fluorescence obtained by relatively scanning this bead or dot array and this spot-shaped excitation light is separated and detected from the excitation light. At this time, the spot excitation light is driven to be tracked so that the spot excitation light irradiates the beads or the dot array almost constantly within the time required for relative scanning of one pitch of the bead array. By doing so, it becomes possible to utilize most of the scanning time, which has been a problem in the past, for fluorescence detection.

As means for obtaining the same effect, when fluorescence is detected by relative scanning of a bead or dot array and spot-shaped excitation light, the spot excitation light is generally bead or dot within an average time required for one pitch of relative scanning. The fluorescent beads or dot array sample is step-moved by one pitch step so as to irradiate the array.

Further, the spot excitation light is multi-spot. This allows a large number of beads or dots to be detected simultaneously. As a result, the time taken to detect one bead or dot becomes longer, and high sensitivity, wide dynamic range, and high speed detection can be realized.

In order to reliably perform the above-described detection, in the present invention, the spot excitation light is reflected or diffracted by the beads or dots to detect the deviation of the light distribution in the scanning direction, and the spot excitation light is detected based on this detection signal. Correct the position of the sample.

Further, the deviation in the tracking direction of the light distribution of the spot excitation light reflected or diffracted by the beads or dots is detected. Based on this detection signal, the position of the spot excitation light or the fluorescent bead or dot array sample position is corrected in the direction orthogonal to the bead or dot array arrangement direction. By doing so, it is possible to ensure that the excitation light irradiates the beads or dots almost always.

A photon count detection method is used as a method for detecting the fluorescence. This makes it possible to detect even beads or dots with weak fluorescence, in combination with the effect of increasing the detection time described above.

Further, the intensity of the excitation light is changed stepwise within the time required for relative scanning of the bead array for one pitch, and the fluorescence at each step is separated and detected. By doing so, even if the sample contains a large amount of fluorescence and a weak amount of fluorescence, both can be detected with high accuracy, and wide dynamic range detection can be realized.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The embodiments of the present invention will be described in detail below.

FIG. 1 shows that beads labeled with fluorescently labeled DNA
DNA beads array sample 7 attached to the surface of No. 1 and having the beads in the shape of a honeycomb on a two-dimensional plane in the closest packing arrangement (however, P> D) is excited by light beams 301, 302, 303 ...
It is the figure which irradiates with ... and detects fluorescence. Beads 711
1, 7121, 7131 ... Are the excitation light beams 301,
.. are irradiated with 3001, 3002, 3003 .. The DNA bead array sample 7 is scanned at a continuous uniform velocity Vs in the x direction by an x stage (not shown) as indicated by a thick arrow.

Therefore, as shown in FIG. 2A, when the state of FIG. 1 is time (time) t ₁ , beads 7111
At time t ₂ after Δt (= t _{n + 1} −t _n ) time, the position of 7112 in FIG. Therefore, the excitation light beams 301, 302, 303 ... Are scanned in the x direction at a velocity Vb as indicated by a thick arrow. At this time, scanning is performed so that Vb = Vs. 7
When the beam is scanned to a position near 112, the original position, that is, 301, 30 in FIG. 1 is displayed as shown in FIG.
2, 303, ... And the beam is scanned again at the speed Vb at time t2.

This operation is repeated every time the bead array pitch P advances, and when the last bead arrayed in the x direction is reached, the DNA sample 7 is formed by using the y stage (not shown).
By moving √3P / 2 in the y direction, the lines 71A1, 71A2, 71A3 of FIG.
The lines 71B1, 71B2 ... Next to .. can be detected by the above method. When the line B is detected, if the number of multi-spots is N, then √3P (N-
1/2) move and repeat the above detection. In this way, the beads on the entire surface of the sample are detected.

As is clear from the above description, the excitation light irradiates the beads most of the time during which one pitch of the bead array is scanned. Therefore, it becomes possible to detect fluorescence within this time. That is, when scanning the entire surface of the sample as in the conventional case, if the scanning speed Vs is the same,
Although the detection time was only the time D / Vs when the beam passed through the beads, it was almost
It can be used for Vs time detection. Further, in the case of the conventional whole surface scanning, the number of step movements in the y direction is increased by √3P / 2D times as compared with the present method because the scanning between adjacent beads is also performed, and the time for detecting all the samples becomes long.

By adopting this method in this way,
Since it takes a long time to detect one bead, and therefore the fluorescence is weak and many photons enter the detector when the fluorescence intensity is detected by the photon counting method, the sensitivity is increased. In addition, the time for full surface detection is shortened and high speed detection is possible.

FIG. 3 is an embodiment of a fluorescent bead detecting device or a DNA testing device using fluorescent beads of the present invention. Reference numerals 11 and 12 are excitation laser light sources, 11 is a laser having a wavelength of 488 nm, and 12 is a laser having a wavelength of 532 nm. Each laser beam is made to have a desired beam diameter by a bead shaping optical system shown by 1201 and 1202 in this figure, if necessary. Each laser beam having a desired beam diameter is transmitted by the AO deflection modulators (acousti modulators) 111 and 121.
incident on the c-optic deflector & modulator). The AO deflection modulator changes the diffraction angle of the emitted laser beam by changing the frequency of the high-frequency signal input to the quartz crystal ultrasonic oscillator mounted therein, and also changes the amplitude of the input high-frequency signal. Change the intensity of the laser beam.

Since the optical axes of both laser beams are slightly shifted in the direction perpendicular to the paper surface, the laser beams of 488 nm and 532 nm are slightly displaced in the -z direction (downward) by the mirrors 112 and 121, and the angle Also moves slightly out of parallel. Both lights enter the convex lens 101, travel in parallel with each other, and enter the pinhole plate having two holes. Both lights that have passed through the respective pinholes pass through the polarization beam splitter 21, and each of the beams becomes two beams with the interval W. Although the two beams of each wavelength are linearly polarized light which are orthogonal to each other, they pass through the quarter-wave plate 22 to be circularly polarized light to the right and counterclockwise, and are incident on the prism 23 that is trapezoidally laminated. The bonded part of this trapezoidal bonded prism is a polarization beam splitter, and the height of the two trapezoids is slightly different, so it is 1/4.
When entering the wave plate 24, the beams of the respective wavelengths are four linearly polarized beams with an interval of 1/2 W and alternately orthogonal to each other.

When passing through the quarter-wave plate 24, 4 at each wavelength
The beams of the book are alternately turned into right-handed and left-handed circularly polarized lights, and enter the second trapezoidal bonded prism 25. This prism has the same structure as 23, but the difference in height of the two trapezoids is half that of 23. Therefore, the beam that has passed through this prism is made up of eight beams with an interval of ⅛ W for each wavelength, and by passing through the ¼ wavelength plate 26, the right and left circularly polarized beams are alternately arranged. .

Eight beams at each wavelength travel in the z direction parallel to the front and rear in the x direction, pass through the wavelength separation beam splitter 30, and are guided by the tube lens 31 and the objective lens 35.
Eight beads arrays of DNA bead sample 7 are simultaneously illuminated as shown in FIG. Eight beams of two wavelengths are aligned on the sample 7 in the y direction as shown in FIG.
A row of beads, which is not shown in FIG. 1 and is separated by mP (m is an integer) in the x direction, is irradiated with the multi-beam having the other wavelength.
The tube lens 31 and the objective lens 35 have a pinhole 10.
Since the image of No. 2 is reduced and formed on the sample 7 at a magnification of 1 / M, as shown in FIG. 1, the pinhole diameter and the magnification 1 / are set so that the spot diameter is almost the same as the diameter D of the beads 71 or slightly larger. M is set.

The control circuit 8 controls the xy stage 6 as shown in FIG. 2A, as already described with reference to FIGS.
Scanning in the direction, and when it reaches the end of the bead on the sample, it moves stepwise in the y-direction, drives the AO deflection modulator, and moves the multi-beams of two colors to move the bead as shown in FIG. 2B. Simultaneously scan at the same time. Since the beam irradiates the beads for most of the time of one pitch movement, the fluorescent label on each bead emits fluorescence during this time. The generated fluorescence is the objective lens 35 and the tube lens 3
1, is reflected by the wavelength selective beam splitter 30,
The fluorescence image of the irradiation beads is formed on the multi-channel photomuls 501 and 502 through the lens 52, the light shielding plate 53 with the aperture 53 and the lens 54 through the window 51 of the fluorescence detection optical system 5.
Reference numeral 55 denotes a wavelength selective beam splitter, which is excited at 488 nm, transmits fluorescence near 510 nm, and guides it to 501 multi-channel photomul, which is excited at 532 nm and reflects fluorescence near 570 nm and guides it to 502 multi-channel photomul. .

In front of each of the multi-channel photomultipliers 501 and 502, an interference filter that transmits only the above-mentioned trend wavelength adjustment is installed, and excess noise light is cut by this filter. In addition, by installing a pinhole array that allows only 8 fluorescent spots to pass between this filter and Photomal, it is possible to prevent the noise light of fluorescent lamps generated from places other than the figure from entering each channel of Photomar. By performing confocal detection, detection with a higher SN can be performed.

If the window 51 is a color filter that cuts light having a wavelength away from the fluorescence wavelength, it will block stray light that becomes external noise. Furthermore, since the lens system including the tube lens and the objective lens is both telecentric, the principal rays of fluorescence obtained from each bead are parallel. Therefore, the light blocking plate 53 is placed at the focal position of the convex lens 52.
By arranging the
Therefore, even if external bright light enters, it is almost shielded.

Also, although 55 in FIG. 3 is one uniform wavelength selective beam splitter in the above description, it is x
Two wavelength selection beam splitters having different characteristics may be used, with the boundary between the positions of the two fluorescence beams just before and after the direction. That is, in the part near the image forming position of this bead,
Since the images of both fluorescences are completely separated, it becomes possible to put two wavelength selective beam splitters optimal for each fluorescence color around.

Eight bead images of each fluorescence wavelength are detected in each channel of the multichannel photomultiplier. Since the detection time of one bead is approximately P / Vs (= Δt), the control circuit 8 counts the number of photons that come during this time. The counted number of photons is stored in the memory of the control circuit for each bead address and each fluorescent color.

By comparing the data stored in this memory with the data stored separately, fluorescence detected D
The state of NA can be inspected and evaluated. Further, the information on the number of photons thus obtained may be displayed on a screen (not shown) together with the address information of beads on the sample so that the operator can view the result. Further, the obtained information on the number of photons can be used together with the address information of the beads on the sample for transmission to an analysis device, an analysis device, or another inspection device via a communication means. Some beads on the sample have a large number of fluorescent molecules attached. In such a case, too many photons arrive at the time of Δt, and if this number is Np and the time width Δt _p of the photon pulse is Np> Δt / Δt _p , the pulses will overlap. , It becomes difficult to count. For the sample including weak fluorescence to very strong fluorescence, the signal sent from the control circuit to the AO deflection modulator is controlled as follows to solve the above problem.

That is, the time Δt for detecting one bead is divided into 2 minutes, for example, 100% of the excitation light is irradiated in the first half Δt / 2, and several percent of the excitation light is emitted in the second half Δt / 2. The high frequency amplitude of the AO deflection modulator is changed so as to irradiate light. In this way, the fluorescences of the two types of excitation light of strong and weak are separately detected. By doing this,
It becomes possible to perform accurate fluorescence detection with a wide dynamic range from beads with very few fluorescent molecules to beads with very many fluorescent molecules. Moreover, it becomes possible to detect two fluorescent labels in a wide dynamic range by one scanning.

Reference numeral 34 in FIG. 3 denotes a beam splitter which reflects the excitation light with a low reflectance. The excitation light reflected by this beam splitter enters the lens 41. This lens 4
1 forms an image of the pupil of the objective lens on the sensor surface of the 4-division position sensor 40. In front of the four-division position sensor 40, there is a mask 40 shown in detail in FIG.

As described above, the eight multi-spot beams are incident parallel to the optical axis of the tube lens, and the respective beams converge at the center of the pupil of the objective lens. 8 through the objective lens
The beam of books illuminates the beads vertically as shown at 300 in FIG. The light 502 reflected on the surface of the bead deviates from the center of the bead, and when the incident angle becomes θ, the reflected light is reflected at a large angle 2θ from the perpendicular and does not enter the objective lens.

On the other hand, the excitation light refracted on the surface of the bead and entered into the bead is reflected on the lower surface of the bead and refracted on the upper surface of the bead, and emerges upward at an angle α with respect to the optical axis. As shown in 3001A of FIG. 5, even when the light refracted greatly deviates from the center of the bead, the angle α from the optical axis is small when returning to the objective lens, and most of the light is incident on the objective lens.

Return from the beads at the pupil position of the objective lens
The position of the light is the angle formed by the optical axis of the light emitted from the beads.
Angle θ corresponding to the NA of the objective lens_NA(= S
in ^-1Angle smaller than (NA) passes through the objective lens,
Large light does not pass. Within this pupil C as shown in FIG.
White part, that is, from the center O of the pupil to radially A,
Of the light coming from A to B, excluding B to C
Only detect it. Light passing through this white area
What is reflected by the spherical surface of the beads is the arc portion 300 of FIG.
The light 3002 is incident on 2A and is refracted and returned by the beads.
What comes is the light 30 incident on the arc portion 3001A of FIG.
Only 01.

The light 501 'refracted and returned by the beads returns from the wide range indicated by the arrow W1 upon incidence and returns to the white part in the pupil of FIG. 6, whereas the light reflected on the surface is At the time of incidence, only light from a narrow range indicated by W2 returns to the white part in the pupil of FIG. Moreover, the light reflected on the surface refracts and returns to the opposite side of the pupil from the returning light.

By using the above-described nature of the return light on the pupil, the four-division position sensor 40 placed at the position where the pupil is imaged as shown in FIG. 7 is equivalent to the white portion in the pupil of FIG. A mask 44 having a transparent portion 4401 and another portion as a company height portion is set as shown in FIG. On the back surface of the mask 44, there is a 4-division position sensor 40 as shown in FIG. The four-divided position sensor 40 has four independent sensors 401, 402, 403, and 404 arranged with a gap 400 in between, and the upper and lower terminals A1,
A2 and left and right terminals B1 and B2 are projected diagonally.

The directions of A1 and A2 are the array directions of the excitation multi-spots, and B1 and B2 are the directions perpendicular to the array. As described with reference to FIGS. 4 to 6, if the excitation spot light deviates from the beads in the array direction, A1, A
Since a voltage change occurs between the two terminals, this difference (including the sign) signal is amplified as shown in FIG. The displacement signal of the excitation spot in the array direction is AD-converted and input to the control circuit 8. Similarly, the voltage difference between B1 and B2 is differentially amplified,
A shift signal in a direction orthogonal to the D-converted array is input to the control circuit 8.

If the deviations in the two directions are detected, FIG.
As shown in Fig. 3, the direction orthogonal to the array is shown in Fig. 3A.
The frequency of the high-frequency signals input to the O-deflection modulators 111 and 121 is changed by the control circuit so as to reduce the deviation, and the y stage of the xy stage 6 is controlled by the control circuit 8 in the direction of the array, and the deviation is to correct.

FIG. 9 is an embodiment of the fluorescent labeled dot array inspection apparatus of the present invention. Reference numeral 72 is a dot of DNA which is spotted by a spotter or an ink jet. The dot diameter is several tens of microns, which are arranged in a square shape with a pitch P which is several times the dot diameter. The excitation optical system for irradiating a plurality of dots at the same time is almost the same as that in FIG. 1, but the design is such that the irradiation beam diameter is several tens of microns because the dot system is large. FIG. 10 shows an optical system in which the above multi-excitation spot forming optical system is omitted. The multi-spot excitation light 3 is the same as in FIG.
The dot array on the sample 7 is simultaneously irradiated by the objective lens of. The fluorescence generated by the excitation light is detected by the fluorescence detection system 5 in the same manner as in FIG.

The positional relationship between the sample and the excitation multi-spot in this embodiment will be described below with reference to FIG. Note that the operation of the similar positional relationship is also performed in the detection of the bead array shown in FIG. 16 described later.

FIG. 17A shows the change over time in the position of the bead array or dot array sample, and FIG. 17B shows the position of the excitation light. The stage moves stepwise over a distance corresponding to the array pitch P of beads or dot arrays, and t
Detection is performed within a time slightly shorter than _{n + 1-} t _n = Δt. Therefore, the excitation light is basically stationary as shown in FIG.

In the case of the bead detection shown in FIG. 16, the center of the bead and the excitation light spot are made uniform by the four-division position sensor, which is installed in a position where the reflected light from the bead described in FIG. It is detected that it is doing, and if there is a deviation, it is controlled.

In the case of the dot array shown in FIG. 9, the optical system shown in FIG. 10 is used to detect the positional deviation between the excitation light and the dot and correct the deviation. This will be described in detail below with reference to FIG. The half mirror 34 in FIG. 10 reflects a part of the excitation light reflected from the sample dot and guides it to the lens 411. Reference numeral 440 is a 0th-order light cut filter whose detailed view is shown in FIG. 11. The 0th-order light of the light reflected from the sample is shielded by 4402 in FIG. That is, the 0th-order optical filter is installed at the image forming position of the pupil of the objective lens 35 by the lens 411, and the specular reflection light of the excitation light reflected by the sample is shielded by the filter 4402 and only the diffracted light is emitted. It passes through 4401 of this filter.

Each dot of the dot array is a probe DNA
A liquid containing a solvent and a solvent is spotted by a spotter or an inkjet mechanism, and then fluorescently labeled target DN
It is a hybridized form of A. Therefore, the dot portion is higher than the surrounding glass substrate. Therefore, when each laser beam is irradiated, the light is refracted at this raised portion, and the reflected light is diffracted in a direction different from the 0th order light. As a result, only this diffracted light passes through the 0401th light cut filter 440 4401.

FIG. 12 shows that the multi-spot excitation light 3 irradiates the sample dot array 72 in a shifted state. When the light is emitted in such a shifted state, it is reflected, and the light passing through the 0th-order cut filter is displayed on the surface of the image sensor 430 by the lens 412 in FIG. 10 as shown in FIG. Forms a dark image. Therefore, as an image sensor, for example, four-division sensors are arranged at a pitch equal to the array pitch of the 0th-order cut filter transmission image 721 of dots as shown in FIG. Further, the center of the four-divided sensor is arranged so as to coincide with the center of the image if the excitation light and the dot coincide with each other.

Therefore, if there is a deviation as shown in FIG.
As shown in FIG. 4, the 0th-order cut filter transmission image 721 is formed with a deviation from the center of the 4-division sensor. As a result, the detection intensity is strong in the order of 433 and 431 of the four-division sensor,
Almost no light will come to 4. As a result, by using the differential amplifier shown in FIG. 15 described above, the two-dimensional direction of the shift can be known, and by finely controlling the stage 6 via the control circuit 8, the shift is corrected and the correct position is excited. It can be illuminated by light.

[0046]

As described in detail above, according to the present invention, it becomes possible to irradiate excitation light only to a portion where a fluorescent substance consisting of beads or dot arrays or a DNA sample attached with a fluorescent label is to be detected, and fluorescence can be detected. High sensitivity, wide dynamic range and high speed detection are now possible. As a result, it is very effective for high-accuracy and high-speed inspection in a region handling a large amount of samples in future medical inspections and the like.

[Brief description of drawings]

FIG. 1 is a perspective view of a bead array sample for explaining a state of fluorescent bead array detection according to the present invention.

FIG. 2 (A) is a graph showing a change in position of a bead array,
(B) is a graph showing a change in position of multi-spot excitation light.

FIG. 3 is a front view showing a schematic configuration of a fluorescent bead array detection device according to the present invention.

FIG. 4 is a cross-sectional view of beads for explaining the relationship between excitation light and reflected light with which the beads are irradiated.

FIG. 5 is a cross-sectional view of beads for explaining a relationship between excitation light and reflected light with which the beads are irradiated.

FIG. 6 is a plan view showing a state of bead reflected light on a pupil of an objective lens.

FIG. 7 is a plan view of a four-division sensor that detects bead reflected light.

FIG. 8 is a plan view of a four-division sensor that detects bead reflected light.

FIG. 9 is a perspective view of a bead array sample for explaining a state of fluorescent dot array detection according to the present invention.

FIG. 10 is a front view showing a schematic configuration of a fluorescent dot array detection device according to the present invention.

FIG. 11 is a plan view of a 0th-order light cut filter.

FIG. 12 is a front sectional view of a bead array sample irradiated with multi-spot excitation light displaced.

FIG. 13 is a plan view of a bead array sample irradiated with the multi-spot excitation light displaced.

FIG. 14 is a plan view of a 4-division sensor array showing a state of a 0th-order cut filter transmission image of excitation light projected on the 4-division sensor.

FIG. 15 is a block diagram of a circuit that processes a signal of a 4-division sensor.

FIG. 16 is a perspective view of a bead array sample illustrating a state of fluorescent bead array detection according to the present invention.

FIG. 17A is a graph showing a change in the position of a bead array, and FIG. 17B is a graph showing a change in the position of multi-spot excitation light.

[Explanation of symbols]

5 ... Fluorescence detection optical system 6 ... XY stage 7 ...
DNA bead array sample 8 ... Control circuit 1
1, 12 ... Excitation laser light source 30 ... Wavelength separation beam splitter 31 ... Tube lens 35 ... Objective lens 40 ... 4-division position sensor 55 ...
Wavelength selection beam splitter 71 ... Beads
301, 302, 303 ... Excitation light beam 11
1,121 ... AO deflection modulator 501,502 ... Multi-channel photomultiplier

Continued front page    (72) Inventor Satoshi Takahashi             882 Ichimo, Hitachinaka City, Ibaraki Stock Association             Company Hitachi Ltd. measuring instrument group (72) Inventor Taisaku Seino             882 Ichimo, Hitachinaka City, Ibaraki Stock Association             Company Hitachi Ltd. measuring instrument group F-term (reference) 2G043 AA03 BA16 CA09 EA01 FA01                       GA06 GA07 GB01 HA01 HA07                       HA09 JA02 JA03 KA02 KA05                       KA07 KA09 LA02 MA01

Claims (17)

[Claims] 1. A fluorescent substance is attached to a minute bead or dot array, and a fluorescent bead or dot array sample in which the bead or dot array is one-dimensionally or two-dimensionally arranged is irradiated with excitation light to obtain fluorescence. In the method of detecting, irradiating spot-shaped excitation light having a beam diameter approximately equal to the diameter of the beads or dot array, and relatively scanning the beads or dot array and the spot-shaped excitation light,
When separating and detecting the obtained fluorescence from the excitation light, the spot excitation light should be tracked and driven so that the spot excitation light irradiates the beads or dot array almost constantly within the time required for relative scanning for one pitch of the bead array. A method for detecting fluorescent beads or an array of fluorescent dots. 2. A fluorescent substance is adhered to minute beads or dot arrays, and fluorescent beads or dot array samples in which the beads or dot arrays are one-dimensionally or two-dimensionally arranged are irradiated with excitation light to obtain fluorescence. In the method of detecting, irradiating spot-shaped excitation light having a beam diameter approximately equal to the diameter of the beads or dot array, and relatively scanning the beads or dot array and the spot-shaped excitation light,
When separating and detecting the obtained fluorescence from the excitation light, fluorescent beads or dot array samples are so irradiated that the spot excitation light is generally applied to the beads or dot array within the average time required for relative scanning of one pitch of the beads or dot array. The method for detecting a fluorescent bead or a fluorescent dot array is characterized in that the step is moved by one pitch step. 3. The detection of fluorescent beads or fluorescent dot array according to claim 1, wherein the fluorescent beads or dot array are arranged two-dimensionally, and the spot excitation light is composed of multiple spots. Method. 4. The method for detecting fluorescent beads or fluorescent dot arrays according to claim 1, wherein the tracking drive is performed in synchronization with the relative scanning drive state by using an optical deflector. 5. The tracking drive is performed by detecting a deviation in the tracking direction of a light distribution in which spot excitation light is reflected or diffracted by beads or dots, and correcting the position of spot excitation light based on this detection signal. The method for detecting fluorescent beads or fluorescent dot arrays according to claim 1, wherein 6. The tracking drive detects a deviation between the arrangement direction of beads or dot arrays and the relative scanning direction, and a deviation in a tracking direction of a light distribution of spot excitation light reflected or diffracted by beads or dots. 3. The fluorescent beads or fluorescent dots according to claim 1, wherein the position of the spot excitation light or the fluorescent bead or dot array sample position is corrected in a direction orthogonal to the arrangement direction of the beads or dot array based on the detection signal. Array detection method. 7. The method for detecting fluorescent beads or fluorescent dot arrays according to claim 1, wherein the method for detecting fluorescence is a photon count detection method. 8. The intensity of the excitation light is changed stepwise within the time required for relative scanning of one pitch of the bead array,
3. The method for detecting fluorescent beads or fluorescent dot arrays according to claim 1, wherein the fluorescence at each stage is detected separately. 9. An excitation light source, an excitation light irradiation optical system for narrowing the excitation light emitted from the excitation light source to a fluorescent bead or dot array sample to a spot diameter approximately equal to the diameter of the bead or dot array, and fluorescent bead or dot array. A fluorescence detection optical system for detecting fluorescence obtained from
Light detection means for receiving the fluorescence and converting it into an electric signal, a drive stage for scanning the fluorescent bead or dot array sample in the direction of the array in general, and a stage for the beads or dots in the excitation light irradiation optical system. A fluorescent bead or fluorescent dot array detection device comprising a deflecting unit for tracking a change in position due to scanning and changing an excitation light spot, and a control circuit for controlling scanning of the stage and tracking of an irradiation spot. 10. An excitation light source, an excitation light irradiation optical system for narrowing the excitation light emitted from the excitation light source to a fluorescent bead or dot array sample to a spot diameter approximately equal to the diameter of the bead or dot array, and fluorescent bead or dot array. Fluorescence detection optical system for detecting the fluorescence obtained from, the light detection means for receiving the fluorescence and converting it into an electrical signal, a driving stage for scanning the fluorescent beads or dot array sample in the direction of the array in general, beads or From the control circuit that controls the fluorescent beads or dot array sample to move stepwise by one pitch step so that the spot excitation light irradiates the beads or dot array within the average time required for the relative scanning of one pitch of the dot array. Fluorescent beads or fluorescent dot array detector. 11. The excitation light irradiation optical system is provided with a multi-spot generation optical system which makes the excitation light spot a multi-spot and simultaneously irradiates a plurality of beads or dots of a bead or dot array sample. 9. The fluorescent bead or fluorescent dot array detection device according to 9 or 10. 12. A scanning direction deviation detecting means for detecting a deviation in a scanning direction of a light distribution in which spot excitation light is reflected or diffracted by beads or dots, and relative to the spot excitation light and beads or dots based on the detection signal. The fluorescent bead or fluorescent dot array detection device according to claim 9 or 10, further comprising scanning control means for correcting the position. 13. A tracking direction deviation detecting means for detecting a deviation of a light distribution of spot excitation light reflected or diffracted by beads or dots in a tracking direction orthogonal to the scanning direction, and spot excitation light based on the detection signal. The fluorescent bead or fluorescent dot array detection device according to claim 9 or 10, further comprising tracking control means for correcting the relative position of the bead or dot. 14. The fluorescent bead or fluorescent dot array detection device according to claim 9, wherein the light detection means is a photon count detector. 15. The excitation light irradiation optical system is provided with a modulation means for changing the excitation light intensity in a stepwise manner, and the photodetection means is provided within the time required for relative scanning of one pitch of the bead array. The fluorescent bead or fluorescent dot array detection device according to claim 9 or 10, further comprising means for separately detecting and detecting fluorescence at each step obtained by changing the intensity of the excitation light stepwise. 16. The fluorescence detection method according to claim 1, which is a method for inspecting DNA by fluorescence-detecting a DNA sample consisting of an array of fluorescent beads or fluorescent dots having fluorescent-labeled DNA attached to the surface thereof. DNA inspection method using any of the above. 17. The fluorescence detection device according to claim 9, which is a device for inspecting DNA by fluorescence-detecting a DNA sample consisting of an array of fluorescent beads or fluorescent dots having fluorescent-labeled DNA attached to the surface thereof. A DNA testing device using any of the above.