JP2003232733A - Method and device for fluorescent image detection and method and device for dna inspection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the problem that it is difficult to widen the dynamic range for fluorescence detection without sacrificing detecting time by maintaining detection sensitivity in a sufficiently high state although it is needed to increase the dynamic range to about 10,000 or higher at the time of performing DNA expression, analysis, etc., because the ordinary dynamic range for fluorescence detection is <1,000. <P>SOLUTION: While weak exciting light and strong exciting light are alternately projected in one pixel, fluorescence of both intensities is detected and the intensities of the fluorescence are detected from these two data. In addition, at the time of detecting a fluorescent image obtained from the exciting light of one intensity by branching the image, the intensities of the fluorescence are detected by multiplying the intensity ratio between detected light rays by ten to several hundreds times and the intensities of the fluorescence are detected from the data of both intensities. Thus fluorescence detection is performed over a wide dynamic range. In addition, the fluorescence detection is made possible in a three-digit or >4-digit dynamic range by making the detecting time longer by performing photon counting and detection and multi-spot detection. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting a fluorescence image and an apparatus therefor, and a method for detecting a fluorescently labeled DNA to perform a DNA inspection and an apparatus therefor.

[0002]

2. Description of the Related Art A device for reading a sample in which spots are spotted on a glass substrate by changing the place depending on the type of probe DNA, or a so-called DNA microarray obtained by a photolithography technique is hybridized with fluorescently labeled probe DNA. Is used in the world. These devices irradiate a fluorescent-labeled probe DNA with a constant-intensity 1-excitation laser spot beam, detect the fluorescence generated from the probe DNA with a single photomultiplier, and read the fluorescence intensity from the detected signal. To go to.

[0003]

When an attempt is made to use a DNA test for expression analysis of DNA in a target living tissue, a target mRNA or a cDNA which is a copy thereof is used.
The concentration ratio between the types may exceed 1: 10000. Even in such a case, it is very important to detect with a wide dynamic range in order to reliably detect the target.

However, it is not easy to detect in a wide dynamic range by the above conventional detection method. Further, there is a problem that it takes much time even if the detection can be performed in a wide dynamic range.

An object of the present invention is to provide a fluorescence image detecting method and a fluorescence image detecting method capable of stably detecting a fluorescence image with high sensitivity and high speed in a wide dynamic range even for samples having concentrations different by 10,000 times or more. To provide a device. Another object of the present invention is to provide a DNA inspection method and an apparatus therefor capable of stably detecting fluorescently labeled probe DNA in a wide dynamic range at high speed with high sensitivity.

[0006]

In order to achieve the above object, in the present invention, an inspection object in which a sample containing a phosphor is placed in each of a plurality of divided regions is irradiated with excitation light, In the method of detecting fluorescence generated by irradiation of excitation light, excitation light having different intensities is sequentially irradiated to each region divided into a plurality of inspection objects, and the fluorescence generated from each region of the object by this irradiation. Is detected according to the intensity of the excitation light, and the fluorescence value generated by the object is obtained using the information on the fluorescence detected according to the intensity of the excitation light.

In order to achieve the above object, according to the present invention, an inspection object in which a sample containing a phosphor is placed in each of a plurality of divided regions is irradiated with excitation light, and the excitation light is irradiated. In the method of detecting the fluorescence generated by, by irradiating each region that is divided into a plurality of inspection object excitation light, the fluorescence generated from each region of the object by this irradiation is condensed and branched, Each of the branched fluorescence images is detected, and the fluorescence value generated by the object is obtained using the information of each detected fluorescence image.

Further, in order to achieve the above object, in the present invention, an irradiation step of irradiating an object to be inspected having a sample containing a phosphor with excitation light, and an irradiation step of irradiating excitation light from the sample An image acquisition step of acquiring a plurality of images with different fluorescence intensities to be generated, and a processing step of obtaining a fluorescence value generated by the sample of the sample using the plurality of images of different fluorescence intensities acquired in this image acquisition step The fluorescence image was detected. Further, the present invention is characterized in that the dynamic range of the obtained fluorescence value is 1000 or more.

Further, in order to achieve the above-mentioned object, in the present invention, an inspection object in which a sample containing a phosphor is placed in each of a plurality of divided regions is irradiated with excitation light, and the excitation light is irradiated. The device for detecting the fluorescence generated by the excitation light generation means for generating excitation light having different intensities, and the excitation light having different intensities generated by the excitation light generation means are sequentially irradiated to respective regions of the inspection object. Excitation light irradiation means, and fluorescence detection means for detecting fluorescence generated from each region of the object by sequentially irradiating excitation light having different intensities by the excitation light irradiation means, corresponding to the intensity of the excitation light, and The fluorescence detecting means processes the detection signal of the fluorescence detected corresponding to the intensity of the excitation light to obtain the information on the fluorescence generation of the object.

Further, in order to achieve the above object, in the present invention, an apparatus for irradiating an object including a phosphor with excitation light and detecting fluorescence generated by the irradiation of the excitation light is used. Irradiating means for irradiating the object, a condensing means for condensing the fluorescence generated from the object by the irradiation of the irradiating means, and a branching means for branching the fluorescence condensed by the converging means into two fluorescences having different intensities. A fluorescent image detecting means for detecting respective fluorescent images of different intensities branched by the branching means, and generation of fluorescence of an object using information of the fluorescent intensity of each fluorescent image detected by the fluorescent image detecting means And a processing means for obtaining information.

Further, in order to achieve the above-mentioned object, in the present invention, a sample formed by adding a fluorescent label to DNA is irradiated with excitation light on an inspection target object arranged in a plurality of regions on a substrate, and this irradiation is performed. For detecting fluorescence emitted from a sample by
In the inspection method A, each sample arranged in a plurality of regions is sequentially irradiated with excitation light of different intensities, and the fluorescence emitted from each sample by this irradiation is detected according to the intensity of the excitation light. The information on the fluorescence generation of the inspection object is obtained by using the information on the fluorescence detected corresponding to the light intensity.

Further, in order to achieve the above-mentioned object, in the present invention, a sample formed by adding a fluorescent label to DNA is irradiated with excitation light on an object to be inspected arranged in a plurality of regions on a substrate. To detect fluorescence emitted from the sample by D
In the NA inspection method, excitation light is irradiated to each sample arranged in a plurality of regions of the inspection object, fluorescence generated from each sample of the object by this irradiation is collected, and the collected fluorescence is collected. Was branched into two fluorescences having different intensities, the respective branched fluorescences were detected, and the information on the fluorescence generation of the target object was obtained using the information on the respective fluorescences obtained by the detection.

The fluorescence image of the obtained sample has a dynamic range of 1000 or more.

Further, in order to achieve the above object, in the present invention, a sample formed by adding a fluorescent label to DNA is irradiated with excitation light on an inspection object arranged in a plurality of regions on a substrate, and this irradiation is performed. For detecting fluorescence emitted from a sample by
A: Excitation device for generating excitation light having different intensities and excitation light having different intensities generated by the excitation light generating device for sequentially irradiating the samples arranged in respective regions of the inspection object A light irradiation means, and a fluorescence detection means for detecting fluorescence generated from a sample arranged in each region by sequentially irradiating excitation light having different intensities by the excitation light irradiation means in correspondence with the intensity of the excitation light, and And a processing means for processing the detection signal of the fluorescence detected by the fluorescence detection means in correspondence with the intensity of the excitation light to obtain information on the fluorescence generation of the inspection object.

Further, in order to achieve the above-mentioned object, in the present invention, a sample formed by adding a fluorescent label to DNA is irradiated with excitation light on an inspection object arranged in a plurality of regions on a substrate. To detect fluorescence emitted from the sample by D
The NA inspection apparatus detects excitation light irradiating means for irradiating an object with excitation light and fluorescence emitted from a sample by irradiation of the excitation light irradiating means, which is collected and branched to detect each branched fluorescence. Fluorescence image acquisition means for obtaining a fluorescence image by
And a processing means for obtaining information on the fluorescence generation of the target object using the fluorescence image acquired by the fluorescence image acquisition means.

In the method for detecting fluorescence described above, when detecting fluorescence, it is possible to detect with high sensitivity and in a wide dynamic range by performing photon count detection for detecting each photon. Become.

By applying the above-mentioned means, when the photon count detection time for one picture element is T with respect to the pulse width ΔT of the photon count pulse signal, 0.5 · T / ΔT
It becomes possible to detect in the above dynamic range.

As a result, the dynamic range is 1000
As described above, it has become possible to detect even more than 10,000.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below with reference to embodiments.

First, the first embodiment of the present invention will be described with reference to FIGS.
An embodiment will be described. In FIG. 1, 2 is an excitation light source of wavelength λ1. The light emitted from the excitation light source is shaped into a laser beam by the excitation light beam shaping optical system 21 through the objective lens 3 so as to have a desired beam shape on the sample 5.
The excitation light beam with which the sample is irradiated is preferably a multi-spot beam, but may be a one-spot beam. A wavelength selection beam splitter 31 is arranged in the middle of the excitation light path. The wavelength selection beam splitter 31 reflects the excitation light of the wavelength λ1 and transmits the fluorescence of the wavelength λ2 emitted from the fluorescent substance on the sample 5.

As a specific example, Cy3 (Amersham Pharmacia Biotech (Amersham) is used as a fluorescent substance.
In the case of using the product name of Pharmacia Biotech), λ1 is 532 nm and the peak value of the fluorescence wavelength λ2 is 570 nm when the YAG second harmonic laser is used as the excitation light. Therefore, the wavelength selection mirror 31 has 5
It reflects 90% or more at 32 nm, and nearly 90% transmits fluorescence at 560 to 590 nm. The transmitted fluorescence passes through the above-mentioned fluorescence wavelength band by the interference filter 32 and leaks 53.
The excitation light of 2 nm is shielded almost completely.

The fluorescence transmitted through the interference filter 32 is detected by the high sensitivity detector 11 such as Photomul. The photon count pulse signal detected by the high-sensitivity detector 11 is sent to the control circuit 1, converted into digital information, and then stored in a memory together with position information on the sample that generated photons. In the present embodiment, an example in which Photomul is used as the fluorescence detector has been described, but a semiconductor detector such as a cooled CCD may be used. Alternatively, the excitation spot may be widely irradiated and a two-dimensional image may be detected by the cooled CCD.

The control circuit 1 has a function of changing the intensity of the excitation light. That is, for example, the intensity of the pump laser light is changed with time using a known AO modulator, as shown in FIG. The excitation laser light in this embodiment irradiates the sample surface with a beam diameter corresponding to the size of one picture element to be detected on the sample. As shown in FIG. 1, the sample 5 is scanned in the in-plane X direction of the sample by a stage (not shown) based on a control signal from the control circuit 1.

FIG. 2B shows the relationship between the position of the sample and the time change associated with the scanning of this stage. The horizontal axes of (a) and (b) of FIG. 2 are the same, and one pixel passes through between time t ₀ and t ₁ , and the fluorescence intensity of one pixel is detected during this period. There are two time zones between this time t ₀ and t ₁ , namely
It is divided into time t ₀ to time t ₀₁ and time t ₀₁ to t ₁ . As shown in FIG. 2A, the first half irradiates the excitation light with a weak excitation light intensity αI, and the second half irradiates the excitation light with the intensity I. Next, the adjacent picture element is detected by scanning the stage. Similarly, two time zones, that is, time t ₁ to time t ₁₁ between the time t ₁ and t ₂ at which the adjacent picture element is detected are detected. Divide from t ₁₁ to t ₂ . As shown in FIG. 2A, the first half irradiates the excitation light with a weak excitation light intensity αI, and the second half irradiates the excitation light with the intensity I.

As described above, the intensity of the excitation light is changed while the excitation light beam passes through one picture element, so that
It becomes possible to detect in a wide dynamic range as shown in. FIG. 3 shows an example in which fluorescence detection when passing through three picture elements is performed by the photon counting method. The concentration of the fluorescent label in the three consecutive picture elements is normal between time t ₀ and t ₁ , very low between time t ₁ and t ₂ , and very high between time t ₂ and t _3. The case of high concentration is shown.
Since the concentration is normal between the times t ₀ and t _1, the photon pulse signal is sparse because the excitation light intensity is as weak as αI.
The photon pulse count number C ₀₁ has a small value. However, the excitation light intensity is high between times t ₀₁ and t ₁ , and the saturation is not reached but is close to saturation, and the photon pulse count number C ₀₂ has a large value.

FIG. 4 shows the fluorescence detection intensity on the horizontal axis and the photon pulse count number on the vertical axis. When the fluorescence detection intensity increases, adjacent photon pulses overlap each other, and the number of photon pulse counts apparently decreases. Therefore, as shown in the example of FIG. 4, the number of photon pulse counts reaches a maximum when the number of photons coming to the detector is about 2300, and the number of photon pulse counts decreases conversely when more photons come. Therefore, at time t ₀₁
The photon count number during t ₁ reaches the level indicated by the arrow C ₀₂ in FIG. 4, but there are two candidate values for the true photon pulse number corresponding to this count number, and it is not clear which is correct. Absent.

However, if the value of the photon count number C ₀₁ at the time of detection with a weak excitation light intensity αI between the times t ₀ and t ₀₁ before this detection is known, either of the above two candidate values will be obtained. I know if it's correct. If the value of C ₀₂ is smaller than a certain value, it is possible to obtain a more accurate result by using the detected value with the strong excitation light I at the found candidate value.
The detection value with the strong excitation light I is adopted. Conversely, if the value of C ₀₂ is larger than a certain value, it is better to use the detection value with the weak excitation light αI. Of course, since the graph is not linear as shown in FIG. 4, the true value is calculated by correction using the method described later.

Since the fluorescent label density of the detected picture element obtained between the times t ₁ and t ₂ in FIG. 3 is very small, the photon count number becomes 0 when the excitation light intensity is αI. However, when the excitation light intensity is I, although it is a small count number, C
A value of ₁₂ can be detected. Also at this time, the true number of photon pulses is calculated by correction using the value of C ₁₂ detected with strong excitation light.

The fluorescent label densities of the detection pixels obtained over the next time points t ₂ and t ₃ are very high. Therefore, a sufficient count number C ₂₁ can be obtained even with a weak excitation light intensity αI. On the contrary, when the excitation light intensity is I, photon pulses are overlapped, and the count number C _{22 at} this time is rather C
_It becomes smaller than ₁₂ . If the count number C _{22 for} such strong excitation light is small, the count number C ₂₁ for weak excitation light is adopted.

As described above, both the value when the excitation light is weak and the value when the excitation light is strong are recorded in the memory of the control circuit 1, and one of these two values or both values are used to obtain the true value. Ask for. Further, although the photon counting method is used in the above-described embodiment, when analog detection is performed as a detection method, more accurate detection with a wide dynamic range can be performed by using excitation lights having different intensities. . However, it is possible to detect even weak fluorescence by using the photon counting method, and it is advantageous to adopt the photon counting method when high-sensitivity detection is required.

In the case where the excitation light irradiating the sample is narrowed down to a spot having a size corresponding to the pixel size, the spot and the sample are relatively scanned, and the entire sample is scanned and detected, the multi-spot light is used. Is advantageous in realizing high speed detection. If the number of spots of the multi-spot light is M, for example, even if the time required to detect one picture element is multiplied by √M, the time required to detect the entire sample can be reduced to 1 / √M. When the photon count detection is performed, there is an effect of expanding the dynamic range in proportion to the time taken to detect one picture element, so that the longer the detection time is, the wider the dynamic range can be detected.

A second embodiment of the present invention will be described with reference to FIG. Since the basic configuration of FIG. 5 is the same as that shown in FIG. 1, the elements having the same numbers in FIG. 1 represent the same elements.
Unlike the embodiment of FIG. 1, the sample 5 is irradiated with a constant strong excitation light. The generated fluorescence passes through the objective lens 3, the wavelength selection beam splitter 31, and the interference filter 32,
It enters the beam splitter 111. The beam splitter 111 transmits, for example, 95% and reflects 5%. The fluorescence after transmission and reflection is detected by the photomuls 11 and 11 '. In this way, the photomultiplier which detects 5% is detected by the weak excitation light in the embodiment of FIG.
Photomal detecting 5% gives almost the same detection value as that in strong excitation light. As a result, similarly to the embodiment of FIG. 1, the detection results of both the photomultipliers 11 'and 11 are sent to and stored in the control circuit 1', and using these two detection results, in the same manner as described above, with high accuracy, A wide dynamic range can be detected.

In the case of this system, it is necessary to use two photomulsers, but it is not necessary to control the intensity of the excitation light, and since the excitation light is not detected by changing the intensity with time division, it is almost 2
Since it is possible to detect the photon count by doubling the detection time, the dynamic range can be widened accordingly.

[0034] For pulse width ΔT of the photon pulse signal, when the one pixel worth of photon count time t _{n + 1} -t _{n (n} an integer) = T shown in FIGS. 2 and 3, in the conventional method As shown in Figure 4, the dynamic range is 0.5T
/ ΔT or less. By adopting the method according to the present invention shown in FIG. 1 or 5, it is possible to detect for the first time in a dynamic range of 0.5 · T / ΔT or more.
It was difficult to obtain 1000 or more as a specific value by the conventional method, but this method has made it possible.

Furthermore, by adopting a plurality of stages of excitation light intensity or branching fluorescence detection light into a plurality of intensity ratios of 1: several tens or 1: several hundreds, and detecting in parallel, the above dynamic range can be obtained. For the first time, it was possible to increase the value to 10,000 or more.

By using the photon counting method described above, even very weak fluorescence can be detected, the above dynamic range is satisfied, and the fluorescence detection sensitivity is 50 fluorescent molecules or less / pixel. It has become possible to detect. Also satisfy the above dynamic range,
In addition, it became possible to detect with a fluorescence detection sensitivity of 10 fluorescent molecules or less / mm ² .

FIG. 6 is a diagram for explaining the third embodiment of the present invention. 2A and 2B are excitation laser light sources having different wavelengths for exciting the DNA inspection target of the sample 5 '. 2A is a laser having a wavelength of 488 nm, and 2B is a laser having a wavelength of 532 nm. The output of each laser is 100-200m
W. Sample 5'is a so-called DNA microarray. Substrate 5'with target DNA to which a fluorescent label is added
It is a sample hybridized to the probe DNA above. Laser beams emitted from the two types of excitation laser light sources 2A and 2B form a multi-beam as shown in FIG. 10 below.

FIG. 10 shows the system of the excitation light 488 nm, but the excitation light 532 nm has the same structure. In FIG. 10, the laser light emitted from the light source 2A is the well-known AO modulator 211.
With A, it is possible to change the intensity of the laser light at a switching speed of microseconds based on a signal from the control circuit 1. The excitation light obtained as the first-order diffracted light of the AO modulator is mirror 212A, collimator lenses 213A and 2132A.
Then, the light passes through the pinhole mask 2131A, has a desired beam diameter and divergence angle, and enters the multi-beam generator described below.

2141A and 2142A are multi-beam generators made of calcite, and one beam is 2141A.
When it is incident on the beam, the 2142A splits into four beams and emits them. In addition, a quarter-wave plate (not shown) is inserted between the two rocks and on the exit surface of 2142A. The four beams emitted from the 2142A are alternately circularly polarized to the right and left. Two parallel prisms and a right-angled bisector triangle are adhered to these four beams, and the two adhering surfaces are two pairs of prisms 2151A and 2151A which are polarization beam splitters.
It is incident on 153A. The two sets of prisms have a size of 1: 2, and quarter wave plates 2152A and 5154A are provided between them and on the exit surface, respectively. The four incident beams become 16 beams by these two prisms.

The 16 beams are the microlens array 2
The 16 A narrows the spot down to a 40 μm spot, and two trapezoidal prisms having slightly different thicknesses (heights) are incident on the trapezoidal prism 2171 A bonded at the bottom polarization beam splitting surface. This trapezoidal prism 2171A
Due to this, each of the 16 beams is branched into two beams to become 32 beams with a half pitch. There is a quarter wave plate on the exit surface of the trapezoidal prism 2171A.
The two beams are alternately left and right circularly polarized.

The 32 beams emitted from the trapezoidal prism 2171A have the same structure as that of the trapezoidal prism 2171A described above, and the second trapezoidal prism 2 having a thickness difference of half.
The light beams are incident on the 173A, and each of the beams is further branched into two beams, and the beams are emitted as 64 beams with a half pitch. There is a quarter-wave plate 2174A on this exit surface, and by passing this, a total of 64 circularly polarized beams 2110A of clockwise rotation and counterclockwise rotation are obtained.

In this way, 6 emitted from the 488 nm light source 2A and obtained through the multi-spot optical system 21A.
Four multi-beams 2110A and 64 multi-beams 2110 emitted from the 532 nm light source 2B with the same optical system and obtained through the multi-spot optical system 21B.
B is incident on each of beam interval adjusters 22A and 22B including two prisms as shown in FIG. The two-color multi-beams that have passed through these are reflected by the wavelength selection beam splitter 31 ′, pass through the objective lens 3 having a large NA, and excite and illuminate the fluorescent label of the sample 5 ′ in a multi-spot state.

FIG. 7 shows a state in which the multi-spots of these two colors illuminate the sample. Reference numeral 51 is a field of view of the objective lens. 2110A is a multi-spot excitation light having 64 spots of 488 nm, and 2110B is a multi-spot excitation light having 64 spots of 532 nm. The sample 5'is scanned by a stage and a stage drive circuit (not shown) in the direction of the arrow in FIG. 5, that is, in the direction perpendicular to the array direction of the 64 multi-spot array in FIG. The spot diameter of one of the multi-spots is determined by the required resolution of the sample, but in this embodiment it is 2 μm and the spot pitch is 20 μm.

Fluorescence generated by being excited by such two excitation lights passes through the objective lens 3 and the wavelength selection beam splitter 31 ′ in FIG. 5, and is then detected by the fluorescence separation mirror 112 at each fluorescence wavelength. Therefore, the fluorescence is branched into two fluorescence wavelength bands. That is, the fluorescence in the vicinity of 500 nm generated by the excitation light of 488 nm is reflected by the fluorescence separation mirror 112, passes through the interference filter 321A having a center at 500 nm, and passes through the 64-channel photomultiplier 11A.
Incident on. Similarly, 57 generated by excitation light of 532 nm
The fluorescence in the vicinity of 0 nm passes through the fluorescence separation mirror 112 and becomes 5
The light passes through the interference filter 321B having a center at 70 nm and enters the 64-channel photomultiplier 11B.

As shown in FIG. 8, in the fluorescence separation mirror 112, the lower half 112 A portion reflects 500 nm and 570
nm is transmitted. The upper half 112B is 570n
m is transmitted and 500 nm is reflected. FIG. 9 is a detailed view of the photomultipliers 11A and 11B. In this figure, the Photomalu 11A and the Photomalu 11B are representatively described as the Photomul 11 for convenience. There is a mask 1102 in front of the photomultiplier 11 and an aperture array 110 of 64 channels.
1 is provided in front of the first slit, and a narrow slit 1103 is formed at the center. Fluorescence corresponding to each spot generated when the sample 5 ′ is excited by the multi-spot is imaged as a fluorescence spot image 1121 on each aperture of this multi-channel photomultiplier via the objective lens 3.

In this way, the fluorescence from each spot on the sample 5'is separated and detected, and the control circuit of FIG. 6 obtains 64 data of the photon count signal for each excitation light at the same time. As described above, the sample 5'is scanned in the direction orthogonal to the multi-spot array, and when reaching the scanning end, the multi-spot array is moved by the spot diameter in the direction in which the spots are arranged, and scanning is repeated in the opposite direction to repeat fluorescence. By performing the detection, it is possible to detect fluorescence in a desired region.

At this time, the time T for passing one picture element is divided into two, and as described above, the AO modulator 211A (21
It is possible to detect in a wide dynamic range by changing the excitation light intensity by using 1B).

A specific embodiment for detecting the excitation light intensity in two steps or three steps will be described below. The detection time T at each excitation light intensity stage is 40 μs, and the photon pulse width ΔT is 10 ns. This pulse width ΔT is equally divided into m (for example, 1000). Hereafter, ΔT / m is set as a time sampling point. During the detection time T, the average number of photon pulses is n.
Suppose that individual pieces are detected. Focusing on an arbitrary time point within the detection time T, it is assumed that the photon pulse signal has risen at this time point (time) t ₀ . When T / ΔT is N, the probability p that the photon pulse signal rises at each sample point is n / Nm, so the probability that the photon pulse does not rise at the m + 1 sample points before the point of interest is (1-p) becomes ^{m + 1} . That is, the probability p ₀ that the focused pulse does not overlap with the preceding pulse is given by the following equation.

[0049]

[Equation 1] p ₀ = (1-p) ^{m + 1} (Equation 1)

[Equation 2]     p = n / Nm (Equation 2) On the contrary, a pulse occurs in front of the pulse of interest and the probability of overlapping is
1-p₀Is. Therefore, two pulses are connected in succession.
The rate is 1-p within the time ΔT in front of the pulse of interest. ₀Probability of
There is a pulse rise at, and within the time ΔT in front of it.
Probability p₀Since there is no pulse rise at,
p₀(1-p₀). Similarly, the probability of overlapping m pulses is
p₀(1-p₀)^mBecomes

As a result, if there is no overlap, the average n pulses are counted during the detection time T, but in reality, overlap occurs and the pulse count number decreases. This reduction number d _n is given by the following equation.

[0051]

[Equation 3]

Therefore, the pulse count number n _e detected during the detection time T is

[0053]

[Equation 4]

It becomes This is almost as shown in FIG.

FIG. 4 shows the time T required to detect one picture element.
Is 40 μs, and the time width ΔT of the photon pulse signal is 10 n
s. Therefore, the above N value is N = T / ΔT =
It will be 4000. With m = 1000, the above equations (1) to
Using (4), the number of photon pulse counts n _e actually detected with respect to the average number of photon pulses n within the detection time T is shown in Table 1 (however, in Table 1, the number of photon pulse counts is abbreviated to It is described as a count number).

[0056]

[Table 1]

As can be seen from FIG. 4 and Table 1, the photons
The pulse n is around 2200 and the photon pulse count is
n_eIs maximum around 1515, and n becomes larger.
And n _eDecreases. Also, near the maximum value, the differential value of the graph
Becomes smaller and the accuracy decreases. Therefore, the photon pulse
In this example, up to about 1400 is used as the number of und
It

Since the photon pulse number when the photon pulse count number is 1400 or less has two candidate values, the smaller candidate value is used. Since the relationship between n and n _e is not linear, the relationship as schematically shown in Table 1 was prepared as a numeric table, the true from the photon pulse count number n _e detected Request photon pulses n. Without using the above numerical table, the n _e and n relations are approximated by mathematical expressions,
I.e. n is approximated as _{n e} function expression n _{(n e),} may be obtained through n _{n e} using this equation.

As the number of photon pulses increases in this way, the number of photon pulse counts decreases, so that the excitation light is detected in two or three stages, as will be concretely described below. Tables 2 and 3 show two stages and three, respectively.
An example of detection with the excitation light intensity in stages will be shown. The detection time T and the photon pulse time width ΔT use the values used in the description of Table 1 above.

[0060]

[Table 2]

Table 2 shows the two-step excitation light intensity I._sAnd I_wEncouraged by
When it happens, I_wStrength of I _s1/20 the strength of
Is. Excitation light intensity I_sThe count value by 1 is 12 or more
00 or less, excitation light intensity I_wCount value by 60 or more
At the bottom, in Table 2, the encircled box in the middle row
Luminous intensity I_sThe count value of is adopted. Excitation light intensity I _w
When the count value by is more than 60, in Table 2,
Excitation light intensity I surrounded by a thick frame in the right column_wThe count value of
To use. In this way, the photon pulse is 1 to 200.
It is possible to detect up to 00 in 80 μs. Conventional one
For detection with a constant level of excitation light intensity
Since the limit was around 3000, a large dynamic
It is being improved.

In the above description, 1 count is the lowest level, but since the detection of photons is random, the average count may be 0. Therefore, if the minimum count is set to 4, the dynamic range in this method is 5000, and the conventional method is 750.

[0063]

[Table 3]

Table 3 shows three stages of excitation light intensities I _s , I _m and I.
_w . The intensity ratio of each is 100: 10-1.

The count value by the excitation light intensity I _s is 1 or more and 1200 or less, and the count value by the excitation light intensity I _m is 1 or more.
When it is 20 or less, the count value of the excitation light intensity I _s surrounded by the thick frame in the second column from the left (column of I _s ) in Table 2 is adopted.

Excitation light intensity I_mCount value is 120
Excitation light intensity I above 1200 _sCount value by
When is less than 120, the thick frame in the middle row in Table 3
Excitation light intensity I surrounded by_mThe count value of is adopted. excitation
Light intensity I_mWhen the count value by is more than 120,
In Table 3, the excitation light intensity I surrounded by a thick frame in the right column_wMosquito
Und value is adopted. In this way 1-2000
Being able to detect up to 0 photon pulses
It

As in the case of Table 2 above, if 4 counts is the minimum detection value, the dynamic range becomes 50,000,
A wider dynamic range can be detected than the conventional one. Further, if the minimum count is 4 as described above, the dynamic range in this method is 12,500.

The improvement of the dynamic range described with reference to Tables 2 and 3 above is the detection by the change of the excitation light intensity in two and three steps. However, the same result can be obtained even if the detection excitation light described in FIG. 5 is branched at a ratio of 1:20 or 1: 10: 100 and detected by a plurality of photomuls.
At this time, since the detection is performed in parallel, the detection time is 1/2 in two stages and 1/3 in three stages.

In the above example, the value of only one of the excitation light intensities is adopted as the final value. However, a weighted average is performed from two pieces of information near the switching area of the adopted data,
It may be the final data. For example, 1000 to 20 in Table 2
00 boundary using the above-mentioned table the excitation light intensity with respect to the count value n _es of I _s is determined photons pulses n (n _es), it was determined using the above table for the count value n _ew of I _w Photon pulse number n
And (n _ew ) are used.

The photon pulse number n ₀ is obtained from both data by the following formula, and this value is adopted.

[0071]

N ₀ = (α × 20 × n ( _nes ) + β × n (n _ew )) / (α + β) ( _Equation 5) where 20 is the ratio of the excitation light intensity , Α and β are weighting coefficients. For example, the following formula is adopted as the weighting coefficient.

[0072]

[6] _{α = (1500-n es)} × n es / 100000 β = (n ew) 2/1000 ··· ( 6) excitation light intensity when Thus them if _{I s} is about 1200 or more Is suddenly weighted toward I _w , and conversely I _s is about 12
When it is 00 or less, the excitation light intensity is sharply weighted toward I _s .

As a result, the values in the thick frame portion of Table 2 are automatically mainly adopted. Although various weighting methods can be considered other than those described above, it may be determined by examining the relationship between the number of photon pulses and the number of pulse counts in Table 1 and FIG.

The photon pulse number n thus obtained
The information of ₀ is stored in the storage means together with the position information of the probe DNA on the sample. At this time, the test conditions, probe DNA information, sample information, etc. may be stored together with the above information.

By comparing the stored data with the separately stored data, it is possible to inspect / evaluate the state of the fluorescence-detected DNA. In addition, the information of the photon pulse number n ₀ obtained above is used as the probe DNA on the sample.
Displayed on the screen not shown together with the position information of
The operator may be allowed to browse the results.

Further, the obtained information on the number of photon pulses n ₀ may be used together with the positional information of the probe DNA on the sample to an analyzing device, an analyzing device, or another inspection device via a communication means for use. it can.

In the above embodiment, the excitation light is 2
Although the excitation light of one wavelength is used, three or more excitation lights may be used. Although 5'is described as a DNA microarray, any object containing a substance having a fluorescent property may be used, and the present invention can be applied to the inspection of proteins and the like. Further, the detection target does not have to be fixed on the substrate, and may be a sample of beads in a one-dimensional or two-dimensional array, or a liquid or solid containing a fluorescent substance in a capillary tube.

In the above-mentioned fluorescence detection, only the embodiment in which a two-dimensional image is detected by scanning has been described.
It is obvious that the present invention can also be realized by detecting with a high-sensitivity two-dimensional imaging device by using excitation light of steps or more.

[0079]

As described above, according to the present invention,
It becomes possible to detect quantitatively with a wide dynamic range even for samples in which the density of fluorescent molecules differs by 1,000 or more by 10,000 times depending on the location. It has become possible to detect with high sensitivity.

Further, by adopting a multi-spot beam for high-sensitivity detection with such a wide dynamic range, it becomes possible to shorten the time required to detect the fluorescence on the entire surface of one sample, and the throughput is improved. High speed detection can be performed.

As a result, the present invention is particularly effective in the fields that require wide dynamic range, high sensitivity, and high-speed fluorescence detection, such as the field of DNA expression analysis in the future.

[Brief description of drawings]

FIG. 1 is a front view showing a schematic configuration of a first embodiment of a DNA testing device according to the present invention.

FIG. 2A is a graph showing a time change of the excitation light intensity according to the present invention, and FIG. 2B is a graph showing a time change of the stage scanning amount.

FIG. 3 is a diagram illustrating a photon count signal.

FIG. 4 is a graph showing a relationship between a photon pulse signal and a photon count number.

FIG. 5 is a front view showing a schematic configuration of a second embodiment of the DNA testing device according to the present invention.

FIG. 6 is a front view showing a schematic configuration of a third embodiment of the DNA testing device according to the present invention.

FIG. 7 is a plan view of the field of view of an objective lens showing an irradiation spot array on a sample with two-color excitation light.

FIG. 8 is a front view of a fluorescence separation mirror that separates two fluorescences.

FIG. 9 is a perspective view of a multi-channel photomultiplier that detects fluorescence from a multi-spot.

FIG. 10 is a front view showing a basic configuration of an optical system that obtains a multi-spot from an excitation laser light source.

[Explanation of symbols]

1 ... Control circuit 2 ... Excitation light source 3 ... Objective lens 5 ... Sample 11 ... Fluorescence detector 21 ... Multi-spot optical system
31 ... Wavelength selection beam splitter 32 ... Interference filter

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/50 G01N 33/53 M 5B047 33/53 37/00 102 5B057 37/00 102 G06T 1/00 295 G06T 1 / 00 295 400B 400 C12N 15/00 AF (72) Inventor Kenji Yasuda 882 Ichige, Hitachinaka City, Hitachinaka City, Ibaraki Prefecture Incorporated, Hitachi High-Technologies Corporation, Design and Manufacturing Headquarters, Naka Works (72) Inventor Tasaku Seino, Hitachinaka, Ibaraki Prefecture 882 Ichige Ichima Stock Company Hitachi High-Technologies Corporation Design / Manufacturing Headquarters Naka Term F-Term (Reference) 2G043 AA04 BA16 CA03 DA02 EA01 FA01 FA06 GA02 GB01 GB21 HA02 JA03 KA02 KA05 KA09 LA03 2G045 AA40 DA13 FA11 FB12 4B024 CA01A11A HA12 HA13 4B029 AA07 AA23 BB20 CC03 CC08 FA15 4B063 QA01 QQ42 QR32 QR56 QR66 QR84 QS34 QS36 QX02 5B047 AA17 AB02 BB01 BC07 CA19 DA01 5B057 AA10 BA02 CA02 CA08 CA11 CA16 CB02 CB08 CB12 DB01 CB12 CB01 DB12 CB16 DB09 DC05 DC22

Claims (26)

[Claims] 1. An excitation object is irradiated with an excitation light in which a sample containing a phosphor is arranged in each of a plurality of divided regions,
A method of detecting fluorescence generated by irradiation of the excitation light, wherein excitation light having different intensities is sequentially irradiated to each of the regions divided into a plurality of inspection objects, and the irradiation is performed to each region of the object. Fluorescence generated from the fluorescence is detected in association with the intensity of the excitation light, and the fluorescence value generated by the object is obtained using information about the fluorescence detected in association with the intensity of the excitation light. Image detection method. 2. The fluorescence detection method according to claim 2, wherein the excitation light is composed of a plurality of beams, and the plurality of beams are simultaneously irradiated to different regions of the object. 3. A plurality of fluorescence detection signals are obtained from the object by detecting the fluorescence corresponding to the intensity of excitation light,
The fluorescence image detection method according to claim 1, wherein the fluorescence image of the object is obtained by using information of a fluorescence detection signal corresponding to any excitation light intensity from the obtained plurality of fluorescence detection signals. . 4. An inspection object in which a sample containing a phosphor is placed in each of a plurality of divided regions is irradiated with excitation light,
A method of detecting fluorescence generated by irradiation of the excitation light, wherein excitation light is irradiated to each of the divided regions of the inspection target, and the irradiation causes generation of light from each region of the target. A fluorescence image characterized by collecting and branching fluorescence, detecting each of the branched fluorescence images, and obtaining the fluorescence value generated by the object using the information of each detected fluorescence image. Detection method. 5. The fluorescence image detection method according to claim 4, wherein the excitation light is composed of a plurality of beams, and the plurality of beams are simultaneously irradiated to different regions of the object. 6. An irradiation step of irradiating an inspection object having a sample containing a phosphor with excitation light, and acquiring a plurality of images of different fluorescence intensities generated from the sample by irradiating excitation light in the irradiation step. And a processing step of obtaining a fluorescence value generated by the sample of the sample using a plurality of images having different fluorescence intensities acquired in the image acquisition step. 7. The inspection target is arranged in a region where a sample containing a phosphor is divided into a plurality of regions, and in the irradiation step, the samples in the plurality of regions of the inspection target are simultaneously irradiated with excitation light, 7. The fluorescence image detection method according to claim 6, wherein images of a plurality of regions irradiated with the excitation light at the same time are simultaneously acquired in the image acquisition step of the previous term. 8. The fluorescence image according to claim 6, wherein the plurality of images having different fluorescence intensities are obtained by sequentially irradiating the inspection object with a plurality of excitation lights having different intensities in the irradiation step. Detection method. 9. The fluorescence image detecting method according to claim 1, wherein a dynamic range of the obtained fluorescence value is 1000 or more. 10. A device for irradiating an inspection object, in which a sample containing a phosphor is placed in each of a plurality of divided regions, with excitation light, and detecting fluorescence generated by the irradiation of the excitation light, Excitation light generation means for generating excitation light having different intensities, excitation light irradiation means for sequentially irradiating the respective regions of the inspection object with the excitation light having different intensities generated by the excitation light generation means, and the excitation light Fluorescence detection means for detecting fluorescence generated from each region of the object by sequentially irradiating excitation light having different intensities by the irradiation means, and the excitation light by the fluorescence detection means. And a processing unit for processing the detection signal of the fluorescence detected corresponding to the intensity of the object to obtain information on the fluorescence generation of the target object. 11. The fluorescence image detection according to claim 10, wherein said excitation light generating means emits a plurality of beams as excitation light and irradiates the plurality of beams to different regions of said object simultaneously. apparatus. 12. A plurality of fluorescence detection signals are obtained from the object by detecting the fluorescence corresponding to the intensity of the excitation light, and one of the plurality of fluorescence detection signals obtained has a desired excitation light intensity. 11. The fluorescence image detection device according to claim 10, wherein a fluorescence image of the object is obtained using information of the corresponding fluorescence detection signal. 13. An object containing a phosphor is irradiated with excitation light,
A device for detecting fluorescence generated by the irradiation of the excitation light, the irradiation means irradiating the object with the excitation light, the condensing means for condensing the fluorescence generated from the object by the irradiation, and the collecting means. A branching unit for branching the fluorescence collected by the light unit into two fluorescences having different intensities, a fluorescence image detecting unit for detecting the respective fluorescence images having different intensities branched by the branching unit, and detection by the fluorescence image detecting unit And a processing unit that obtains information on the fluorescence generation of the object using the fluorescence intensity information of each fluorescence image. 14. The object is divided into a plurality of regions, each of the divided regions contains a phosphor, and the excitation light is sequentially irradiated to each of the plurality of divided regions. The fluorescence image detection device according to claim 13, which is characterized in that. 15. The fluorescence image detection device according to claim 13, wherein the excitation light is composed of a plurality of beams, and the plurality of beams are simultaneously irradiated to different regions of the object. 16. A DNA test in which a sample formed by adding a fluorescent label to DNA is radiated with excitation light to a test object arranged in a plurality of regions on a substrate, and fluorescence emitted from the sample is detected by the irradiation. A method, which sequentially irradiates the respective samples arranged in the plurality of regions with excitation light of different intensities, and detects fluorescence generated from each of the samples by the irradiation in association with the intensity of the excitation light, A DNA inspection method, wherein information on fluorescence generation of the inspection object is obtained by using information on fluorescence detected corresponding to the intensity of the excitation light. 17. The DNA inspection method according to claim 16, wherein the excitation light is composed of a plurality of beams, and the plurality of beams are simultaneously irradiated to the samples arranged in the different regions, respectively. 18. A DNA test in which a sample formed by adding a fluorescent label to DNA is radiated with excitation light to a test object arranged in a plurality of regions on a substrate, and fluorescence emitted from the sample is detected by the irradiation. A method, irradiating excitation light to each sample arranged in a plurality of regions of the inspection object,
Fluorescence generated from each sample of the object by the irradiation is collected, the collected fluorescence is branched into two fluorescences having different intensities, each of the branched fluorescences is detected, and obtained by the detection. A method for inspecting DNA, characterized in that information on fluorescence generation of the object is obtained by using information on each fluorescence. 19. The DNA according to claim 18, wherein the excitation light is composed of a plurality of beams, and the plurality of beams simultaneously irradiate the samples arranged in different regions of the inspection object. Inspection method. 20. The dynamic range of the fluorescence image of the obtained sample is 1000 or more.
20. The DNA test method according to any one of 6 to 19. 21. A DNA test in which a sample formed by adding a fluorescent label to DNA is radiated with excitation light to a test object arranged in a plurality of regions on a substrate, and fluorescence emitted from the sample is detected by the irradiation. In the apparatus, excitation light generating means for generating excitation light having different intensities and excitation light having different intensities generated by the excitation light generating means are sequentially irradiated to the samples arranged in respective regions of the inspection object. Excitation light irradiation means,
Fluorescence detection means for detecting fluorescence generated from a sample arranged in each of the regions in correspondence with the intensity of the excitation light by sequentially irradiating the excitation light irradiation means with excitation light having different intensities, and the fluorescence detection means. 2. A DNA inspection device, comprising: processing means for processing a detection signal of fluorescence detected corresponding to the intensity of the excitation light to obtain information on fluorescence generation of the inspection object. 22. The excitation light generating means emits a plurality of beams as the excitation light, and simultaneously irradiates the plurality of beams to the samples arranged in different regions of the object, respectively. DNA tester. 23. A plurality of fluorescence detection signals are obtained from the object by detecting the fluorescence corresponding to the intensity of the excitation light, and one of the plurality of obtained fluorescence detection signals is set to an excitation light intensity. 22. The fluorescence image of the object is obtained by using the information of the corresponding fluorescence detection signal.
NA inspection device. 24. A DNA test in which a sample formed by adding a fluorescent label to DNA is placed on a plurality of regions on a substrate and the test object is irradiated with excitation light to detect fluorescence generated from the sample by the irradiation. An apparatus, which is an excitation light irradiating means for irradiating an object with excitation light, and collects and branches the fluorescence generated from the sample by the irradiation of the excitation light irradiating means and detects each of the branched fluorescences. And a processing means for obtaining information on the fluorescence generation of the object using the fluorescence image acquired by the fluorescence image acquisition means. . 25. The DNA inspection apparatus according to claim 24, wherein said excitation light irradiating means irradiates excitation light composed of a plurality of beams to different regions of said object at the same time. 26. The DNA inspection apparatus according to claim 24, wherein said fluorescence image acquisition means simultaneously acquires each fluorescence image of a plurality of regions of said sample.