JP2001108684A - DNA testing method and DNA testing device - Google Patents

Abstract

(57) [Summary] A conventional method of detecting one point at a time cannot detect a DNA chip composed of many cells at a high speed. Also, it is difficult to remove the influence of foreign matter, and the size of the pixel
In the case of a chip, it becomes smaller and it is necessary to focus on the phosphor screen, but it has been difficult in the past. Kind Code: A1 A DNA chip is excited and irradiated with multi-spot array light at the same time, and fluorescence is simultaneously detected. Further, the array light is moved in the array direction and the detection system and the chip are relatively moved at least in a direction perpendicular to the array, thereby performing high-speed and accurate inspection. Excitation light scattering from foreign matter is detected, and the fluorescence detection result is corrected. In addition, the fluorescent screen is irradiated with the second light obliquely, and the position of the reflected light is detected to focus on the fluorescent screen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for inspecting DNA by fluorescence detection. In particular, the present invention relates to a DNA testing method and apparatus for testing DNA of many living bodies at high speed.

[0002]

2. Description of the Related Art In a conventional DNA chip inspection, DNA
Irradiating a spot of excitation light on the chip at one point, and detecting confocal fluorescence generated by the irradiated excitation light,
By changing the relative position between the DNA chip and the detection spot to sequentially change the irradiation position on the chip, a desired position on the chip is sequentially detected.

[0003]

When a DNA test is applied to a biological test such as a conventional blood test, it is essential to perform a high-speed test on a large number of biological samples. However, in the conventional technique, if one excitation light spot corresponding to the required resolution of the DNA chip is irradiated and the obtained fluorescence is sequentially detected, the inspection time is greatly increased. This is related to the fact that the time required for detecting one point cannot be shortened without limit. That is, the time Δt _L from the irradiation of the excitation light to the end of the generation of the fluorescence takes about 10 nsec.
If the process moves to the next detection point without waiting for the end of the fluorescence, detection becomes impossible.

In addition, it is necessary to detect with high sensitivity even a state where several fluorescent molecules are present in the spot light size corresponding to the required detection resolution. However, not all the generated fluorescence is detected. That is, the light utilization efficiency of the detection optical system and the photomultiplier tube quantum efficiency used for light detection are not 100%. Further, the efficiency with which the excitation light is absorbed by the fluorescent object and the probability that the absorbed excitation light is converted into fluorescence is small. For this reason, it is necessary to perform detection at least several tens to several hundreds of times of Δt _L , and the longer this time is, the higher the detection accuracy for weak light near the photon count becomes.

In order to achieve such a high speed at a practical level, the influence of foreign substances composed of various proteins contaminating the DNA chip is removed or reduced, or the test surface obtained by hybridization with a target to which a fluorescent substance is added is used. In addition, it is necessary to always focus the detection system. In addition, it may be necessary to inspect a plurality of fluorescent lights at high speed.

[0006]

Means for Solving the Problems In order to solve the above problems, the present invention provides the following means.

A plurality of M excitation light beams having a spot diameter d equal to or smaller than the dimension D of each cell are simultaneously irradiated to different positions using an objective lens for a time Δt equal to or longer than the fluorescence decay time,
The obtained fluorescence is guided to a fluorescence detection optical path, detected on an image forming surface having a conjugate relationship with the irradiation spot of the DNA chip, and the DNA chip to be inspected is inspected from the position and intensity of the fluorescence. At this time, the spot diameter is set to approximately 1 / N with respect to the integer N of the cell size. Further, the irradiation spot position and the relative position of the DNA chip are sequentially changed j times to inspect all the inspection target positions LN ² = Mj. Further, N is set to 2 or more, the cell is divided into a plurality of parts, and only the significant data of the N ² data in one cell is selected and processed to perform an accurate inspection. By doing so, the total inspection target sample number LN ² of DNA chips, ^LN 2 /
Fluorescence is detected within a time of (6 × 10 ⁵ ) seconds.

The image forming apparatus has M light receiving apertures having an effective diameter substantially equal to that of the irradiation spot image on the image forming surface in a conjugate relationship with the irradiation spots of the M chips on the DNA chip. The outside of the opening is shielded, and by detecting each light transmitted through the light receiving opening, noise light from an irradiation spot or a surface other than the irradiation spot surface is removed, and an inspection with a high signal-to-noise ratio is performed. The M light-receiving apertures having an effective diameter substantially the same as the irradiation spot image are optical fiber light-receiving ends. By detecting light emitted from the light-emitting end of the fiber, detection with a larger signal-to-noise ratio can be performed. Is going.

A plurality of M irradiation spots on the DNA chip are arranged in a straight line at intervals of about kd with respect to the spot diameter d and an integer k. And irradiate for Δt time. By repeating this operation sequentially k times, inspection is performed over KM spot positions in the array direction, and D
The desired two-dimensional area of the DNA chip is inspected by relatively moving the NA chip and the inspection device at least in a direction perpendicular to the array. The spot array is moved using an acoustic light deflector. Further, it is desirable that the integer k is 2 or more, and furthermore, if k is 5 or more, it is more advantageous in terms of signal-to-noise ratio.

[0010] The spot array is formed by a microlens array. Further, the spot array can be formed by a hologram. In synchronization with the movement of the spot array in the array direction, a fluorescence detection deflecting means is provided in the fluorescence detection optical path so that the fluorescence generated by the excitation light comes to substantially the same location on the light receiving aperture. At this time, a deflection unit using a piezoelectric element is used as the fluorescence detection deflection unit. Further, the fluorescence detection / deflection means can be efficiently detected by being constituted by a wavelength selection beam splitter that transmits excitation light and reflects fluorescence. Further, in order to improve the separation from the excitation light, a filter that transmits only fluorescence and blocks the excitation light is used in a fluorescence detection optical path separated from the excitation light path.

The M multi-excitation spot lights pass through substantially the same position A on the pupil of the objective lens,
The excitation light specularly reflected by the DNA chip is configured to reach a position B ′ on the pupil of the objective lens. In this way, the means for shielding the reflected excitation light at the position B 'on the pupil of the objective lens or at the image position B' on the plane conjugate with the pupil of the objective lens in the fluorescence detection optical path is provided. The excitation light which becomes a noise component by the application is removed from the fluorescence detection signal.

In addition, the M multi-excitation spot lights pass through substantially the same position A on the pupil of the objective lens, and DNA
The excitation light specularly reflected by the chip is configured to pass through a position B on the pupil of the objective lens different from the position A. In this way, a member that blocks reflected excitation light having a desired diameter around B is arranged at a position conjugate with the pupil of the objective lens or the pupil of the objective lens in the fluorescence detection optical path. In this way, the excitation light that becomes a noise component is removed from the fluorescence detection signal.

Further, the reflected excitation light to be shielded is converted into regular reflection excitation light, and the excitation light scattered from the foreign matter in the DNA chip is extracted so as to be transmitted from outside the light shielding means or the light shielding member. The light is branched from the fluorescence detection optical path, and an image is taken at a position conjugate with the irradiation spot of the DNA chip, and detected. The fluorescence information detected by the fluorescence detection means is corrected using the imaging information of the detected scattered light image.
By doing so, the effect of scattered light from a foreign substance present in the DNA chip can be eliminated, and accurate detection becomes possible.

The M multi-excitation spot lights are formed by a laser light source. By doing so, excitation light with high intensity is realized on a minute spot. Further, by forming the M multi-excitation spot lights using a plurality of semiconductor laser light sources, a larger excitation irradiation can be realized with a small mounting volume. At this time, the light emitted from the plurality of semiconductor laser light sources is introduced into the optical fiber, and emitted from the emission ends of the optical fibers arranged at M desired pitches. This makes it possible to obtain M multi-excitation spot lights having a desired pitch arrangement.

An ultra-sensitive N as an optical signal storage type imaging means
Using a two-dimensional imaging device consisting of _x × _Ny pixels, _nx ,
The n _y is an integer, n _x excitation light spot diameter d in the x-direction
N _x × N _y spots are simultaneously irradiated at a pitch of n _y d in the d and y directions. The fluorescent spot image composed of N _x × N _y thus obtained is detected by an ultra-high sensitivity two-dimensional imaging device,
Further, the relative position between the detection device and the DNA chip is moved by _nx × _ny steps in the xy directions at a pitch d. This makes it possible to detect a desired region of the DNA chip.

The excitation light has a plurality of different wavelengths, and detects different targets to which a plurality of phosphors are added separately. Further, the excitation light having the plurality of wavelengths is simultaneously irradiated, and different targets to which a plurality of fluorescent substances are added are separated and detected simultaneously. This makes it possible to perform various detection targets at high speed.

The second light is obliquely incident near the excitation spot light on the inspection surface of the DNA chip to be inspected on which the target to which the desired fluorescent substance is added is hybridized, and the light reflected by the inspection surface is reflected by the second light. Focus detection is performed by detecting the position. Focusing can be performed by controlling the relative distance between the inspection surface and the objective lens based on this detection information. In addition, by configuring the second obliquely incident light to pass through the objective lens, focus detection and control can be performed with a simple configuration.

The second obliquely incident light is converted into S-polarized light with respect to the detection surface. By doing so, the reflectance on the fluorescence detection surface is increased, and correct focus detection can be performed.
Further, the second obliquely incident light has a wavelength that does not excite the phosphor. This enables accurate detection without superimposing noise on the fluorescence detection signal.

[0019]

FIG. 1 is a diagram showing an embodiment of the present invention. Reference numeral 1 denotes a multi-spot excitation light irradiation system for forming multi-spot excitation light and irradiating the DNA chip 2 for fluorescence detection, and reference numeral 3 denotes a fluorescence detection system for detecting fluorescence generated by the multi-spot excitation light. An excitation light source 11 includes an excitation light source and an excitation light beam shaping optical system. H
The e-Ne laser beam is shaped into a desired vertical / horizontal beam diameter ratio by two cylindrical lenses having different focal lengths.
000 through the AO deflector. The AO deflector has an input terminal of the low amplitude signal omega _V from this frequency and high-frequency voltage terminal of the frequency omega to be applied to the crystal oscillator.

[0020] The signal of frequency ω sent from the control circuit 4 has a frequency band in the range of ω ± ω _0. When the frequency changes, the diffraction angle of the excitation light incident on the AO deflector changes. Since the diffraction efficiency from the control circuit 4 and inputs the amplitude signal omega _V is changed, it is possible to control the intensity of the diffracted light. A
Diffracted light passing through the O deflector 0 order light is separated (not shown) (0-order light is blocked) by the lens system 13 consisting of the focal length f ₁ and two lenses 131 and 132 of f ₂ The microlens array 14 is irradiated with a desired beam system.
When the frequency of the AO polarizer is changed, the position of the excitation light incident on the microlens array 14 does not change and the angle changes.

FIG. 4 is an enlarged detailed view of the microlens array 14. A large number of 32 to 256 microscopic microlenses made of glass are arranged one-dimensionally, and the light incident on the microlenses 1010 indicated by solid lines, for example, passes through the microlenses and passes through the microlenses 141, 142,. The small spots 111 and 1 which are transmitted and are on the straight line L on the focal plane Σ
12 ... 11M is connected. When the frequency of the AO deflector is changed, the angle of the excitation light incident on the microlens changes as shown by the dotted line in FIG.
j ₂ 1, 1j ₂ 2... 1j ₂ M.

The minute spot arrays 111, 112,..., 11M formed on the focal plane の of the micro lens array 14 are formed by a lens 15 and an objective lens 16 as shown in FIG.
The fluorescent substance added to the hybridized target of the NA chip 2 is irradiated and excited. Glass surface # 1 or # 2 with this target (see FIG. 33 for details)
So that the objective lens 16 has a minimum beam diameter.
Focus is made.

FIG. 2 shows the details of the surface structure of the DNA chip 2. Square minimum units 201, 211, 202, 212 and the like shown by thin vertical and horizontal lines represent detected picture elements. In the figure, 5 × 5 picture elements (indicated by thick lines) are cells 20
It is. The same DNA information fragment is planted in one cell. Therefore, targets having the same DNA fragment structure are hybridized to this cell.

The reason why one cell is divided into a plurality of picture elements as described above is that, when a protein or the like as a foreign substance is mixed in one cell, fluorescence of high intensity is generated by excitation light incident on the protein. What happens is what happens.
Usually, the size of such a foreign substance is several μm at most, so that the pixel size is several μm and the cell size is, for example, 10 μm.
In the case of μm, if the position of a foreign substance in a 10 μm cell can be detected and separated by a method described later, the magnitude of the fluorescence can be accurately obtained from information other than the foreign substance part.

FIG. 3 is a side view of the DNA chip. In this picture, the hybridized target has a structure in which it is naked on a glass substrate. In such a case, it may be sandwiched between glass substrates as described later. In any case, the minute excitation light spot is focused on a certain surface of the target. This focusing diameter is shown in FIG.
Is approximately equal to the size of the picture element to be detected, which is the minimum unit of square shown by.

At the initial frequency of the AO deflector, diffracted light indicated by a solid line in FIG. 1 is obtained. At this time, as shown in FIG. 3, an excitation light spot array such as 101, 102, 103. Is the picture element 20 on the DNA chip
1, 202, 203... 20M, and the fluorescence of each picture element is detected as described later. The irradiation time of the excitation light is a time Δt (several to several hundred μs) which is equal to or longer than the fluorescence decay time, and is 60 μs in this embodiment.

After the elapse of 60 μs, when the frequency of the AO deflector is changed, the diffraction angle of the excitation light changes. As shown in FIG. 1 and FIG.
j ₂₀ is incident on the DNA chip of FIG.
The excitation light spot array is irradiated like 112, 113... 11M, and the fluorescence of the picture elements of 211, 212, 213. In this way, the M multi-spots are sequentially irradiated with a shift of the picture element pitch, and when the picture elements are shifted by j picture elements, all of the jM picture elements are detected.

Next, an embodiment of fluorescence detection will be described with reference to FIG. The beam splitter 30 between the lens 15 and the objective lens 16 is a wavelength splitting beam splitter. The wavelength of the excitation light source HE-Ne laser used in this embodiment is 6
The fluorescent material having a thickness of 33 nm and being added to the target on the chip is Cy5. The wavelength of the fluorescence to be detected is 670
nm.

The wavelength separation beam splitter 30 has 633n.
In the case of m, 45% incident light is transmitted almost 100%, and 670 nm
Is almost 100% reflected at 45 degrees incidence. However, 633 nm is very slightly reflected. Even this slight reflection is a problem because the fluorescence is very weak. Therefore, in the embodiment of FIG. 1, an interference filter 34 having a center wavelength characteristic at 670 nm and a reversal width of about 15 nm is inserted into the fluorescence detection system 3, and leakage of excitation light is shielded by this interference filter. Note that the reference numeral 34 is not limited to the interference filter, and a so-called color filter that transmits light having a certain wavelength or more and blocks light below may be used. Further, a color filter and an interference filter may be used in combination. In the following description of the embodiment, only the interference filter will be described for the sake of simplicity.

Next, when the position of the multi-spot excitation light on the DNA chip is changed using the AO deflector, it is necessary to perform the change in the position of the detected fluorescence image and the detection by the fixed detector. The correction of the position change will be described. The wavelength separation beam splitter 30 in FIG. 1 also corrects this position change. FIG. 5 shows the main parts of FIG. 1, and the same numbers as those in FIG. 1 represent the same parts. The wavelength splitting beam splitter 30 is driven by a piezo element 301 having a high resonance frequency characteristic of 5 to 10 kHz, and has a structure in which the beam is minutely rotated around the y axis.

FIGS. 6 and 7 show the role of the minute rotation. The same reference numerals as those in FIG. 1 denote the same parts. The position of each of the multi-excitation micro-spots 1011, 1021,..., 10M1 on the DNA chip 2 in FIG. , 1022, ..., 1
It is sequentially changed to 0M2. At this time, assuming that the wavelength separation beam splitter 30 does not rotate as shown in FIG.
Multiple fluorescent points 1211, 1221,..., 1 on _PH
2M1 is also 1212, 1222, one pixel at a time.
..., it changes to 12M2.

A fluorescent spot image 1212 on the fluorescent detection surface
As shown in the figure, 1212 points are imaged on one fiber end of the optical fiber bundle 322 by the fluorescence detection microlens array 321 and are incident on the fiber. One optical fiber has a core 3221 through which light passes and a portion 32 for protecting the core.
22. Diameter of the core through which light is slightly greater than the fluorescence detection surface sigma _PH on the (multi) fluorescence point 1211. However, if the wavelength separation beam splitter 30 does not rotate, 1211 points are driven by the AO deflector to 1212 points.
, 1213, 1214,..., Which are shifted from the fiber incident end, and cannot be detected. Then, as shown in FIG. 7, the wavelength separation beam splitter 30 is minutely rotated by a piezo element 301 having a high resonance frequency characteristic.

That is, in FIG.
Locations 1212, 1 where fluorescent minute spot image 1211 is different
Whereas moved to 213,1214, by micro rotary come spot at substantially the same position on the sigma _PH as shown in the dotted box in FIG.

FIG. 8 shows the OO deflector 12 described above.
N-OFF or intensity modulated signal S _A12 , also AO
The deflection signal S _B12 of the deflector, the driving signal S ₃₀ of the piezo element 301 for deflecting the wavelength separation beam splitter 30, the fluorescence detection signal S _33k detected by the k-th single photomultiplier 33 via the fiber 32, and the fluorescence The image accumulation (integration) signal S ' _33k for each picture element of the detection signal, and the final result of each picture element of this image accumulation (the image accumulation is performed, and the excitation spot light is shifted to the excitation of the next picture element). the S _'33k for samples formate de value) S _"33k at time represents the relative time change of each signal. the multi-spot excitation light in the embodiment of this graph is shifted one by one picture element by AO deflector Then, one spot is sequentially shifted up to 10 picture elements in this way.

The number of shifts is required to be 2 or more in order to improve the SN for detecting each picture element. The larger the number, the better in order to improve the SN. However, there is a natural upper limit due to restrictions on the components of the apparatus. However, when the number is set to 5 or more, the influence of the scattered light or the fluorescence due to the irradiation of the foreign matter on the optical path by the adjacent spot excitation light is greatly reduced.

[0036] (ultrasonic frequency applied to the ultrasonic crystal oscillator, i.e. the deflection angle of the polarizers) frequency of the AO deflector 12, as shown in FIG. 8 sequentially stepwise changing the signal S _B12, depending on this change during this time The driving signal S ₃₀ for the piezo element 301 that drives the wavelength separation beam splitter 30 to deflect is linearly changed. While the AO deflector 12 diffracts light due to a very small change in the refractive index of the transparent medium due to the propagation of ultrasonic waves, the piezo element 301 drives the entire beam splitter, and therefore has a different frequency response. signal _{S B12} early AO deflector step, the drive signal _{S 30} of the slow piezoelectric elements are linearly.

FIG. 9 shows the fluorescence detection surface に よ り based on these two signals.
It is a figure showing the position of the fluorescent spot image which can be _{carried out} on _PH .
The change from the top to the bottom of this figure represents the passage of time t. The two graphs on the left show changes in S _B12 and S _{30. In} FIG. 8, the number of steps is 10, but in this figure, the number of steps is 5. The solid circles 、, 3211 ', 3221', 3231 '... On the right side of the graph in FIG. 9 indicate the fluorescence detection multi-spot images when the position correction by the piezo element is not performed as described with reference to FIG. This shows a position shift. Incidentally, the right direction (lateral direction) in the figure by the solid line represents the arrangement direction x _A spot array circle ○, 321
.., 2 ′, 3222 ′, 3232 ′... Indicate positional deviations of adjacent fluorescent spot images. The dotted circles ○ indicate the results of position correction performed by the piezo element.

That is, the AO deflector step-moves,
Even when the excitation light is stopped, the piezo element drives the deflection linearly, so that the fluorescence detection surface can be stopped while the excitation light is stopped.
Position of the fluorescent spot image can on _PH is 3211 _-, 32
11, 3211 ₊ (although there is a slight movement at the position of the adjacent fluorescent spot image, 3212 ₋ , 3212, 3
212 ₊ and there is only a will to move it).

While the excitation light is stepped to the next irradiation position on the DNA chip and stopped, the position of the fluorescent spot image moves slightly but to 3221 ₋ , 3221 and 3221 ₊ (next side). 3 at the position of the fluorescent spot image
222 _-, will move although it is 3222,3222 ₊ slightly). Slight to be detected by covering moves long the array direction x _A fluorescent detecting light-receiving surface (or light-receiving surface and a plane conjugate) 320 as shown in FIG. 10 below in Figure 9 oblong in fluorescence spot image Arrangement of openings 3201, 3202 ...
... 320M are provided. In the case of detection with the above-mentioned fiber, the incident end face of the fiber may include this ellipse.

FIG. 11 is a diagram showing an embodiment of the present invention. The same numbers as those in FIG. 1 indicate the same things or those having the same functions. In this figure, only the part related to the generation of the multi-spot excitation light in the whole apparatus of FIG. 1 is shown. Other configurations are basically the same as those in FIG. 14 'is a multi-spot generating hologram.

FIG. 12 is a diagram showing a method for producing this multi-spot generating hologram. As shown in FIG.
Multi pinhole array _1j 1 1 having a hole diameter corresponding to the dimensions of the spot excitation light _', 1j 1 _2' ......... 1j
The laser element is irradiated to the mask 18 having a _{1 M} 'opening, it focused onto the holographic recording medium transmitted light in Furye transform lens 15. The reference beam 1000 is incident on this focusing position at an incident angle φ.
Irradiation is performed slightly obliquely at = ₀ to form a Fourier transform hologram at the above condensing position.

In order to improve the light use efficiency, a phase modulation type recording medium is used. Also, in order to avoid that the intensity of the zero-order light (the position where the spatial frequency is 0) at the center position on the Fourier transform plane is increased by orders of magnitude, the SN and diffraction efficiency of the formed hologram are not deteriorated. Add random phases unrelated to each other.

The hologram thus produced is shown in FIG.
This is used for the multi-spot excitation light generation system of the DNA inspection apparatus of No. 1.

The laser beam entering the AO deflector 12 of FIG. 11, after passing through the lens system 13, incident angle phi ₀ irradiating a hologram 14 'made in the manner described above. AO deflector center (deflection angle of the center) in the next ₀ phi incident angle phi of the laser beam incident on the hologram of the driving frequency, the incident angle to the hologram surface of the reference beam 1000 when creating a hologram by the method of FIG. 12 keep the equal to phi _0. When the hologram is irradiated with the laser beam in this manner, as shown in FIG. 11, 1j _{1 1} , 1j ₁ 2... 1j
To reproduce the same spot array and the pinhole array during hologram production of 12 to _{1 M.}

Next, when the frequency of the AO deflector is changed,
Since the angle of incidence on the hologram changes slightly, the angle of the multi-spot reproduction light also changes slightly. As a result, the position corresponding to the adjacent picture element is excited and illuminated. AO
If the deflectors are sequentially driven, multi-spot excitation illumination can be performed by sequentially changing the position of the DNA chip at one pixel pitch in a stepwise manner.

FIG. 13 is a diagram showing an embodiment of the DNA testing apparatus of the present invention. The same numbers as those in FIG. 1 represent the same items. In the present embodiment, unlike the embodiment of FIG. 1, two-dimensional detection is performed simultaneously. That is, the excitation illumination light passes through an illumination system lens 13 'capable of simultaneously illuminating a two-dimensionally wide range, is reflected by a wavelength selection beam splitter 30', and illuminates a two-dimensional multi-lens array 14 '. 1
The 4 'two-dimensional microlens array is already shown in FIGS.
And has the same function as that described for the one-dimensional microlens array, and only the two-dimensional array arrangement is different. Therefore, as shown in FIG.
A two-dimensional minute spot 1410 'is formed at the focal position of.

In the case of the present embodiment, a pinhole opening is formed around the position of 1410 '. Thus, the light passing only through the pinhole opening irradiates the DNA chip. Small multi-spot light spot 1410 '
Is an integer of 2 or more, preferably 5 or more. As shown in FIG. 13, the two-dimensional multi-spot light transmitted through the pinhole aperture simultaneously excites and irradiates the DNA chip with the high-resolution lens 16 '. Pixels 2x11y11, 2x12y11, 2x to be irradiated with excitation
11y21, 2x21y21,... Are equal in pitch with four picture elements being skipped.

At the same time, the fluorescent light generated from the picture element, which is excited and irradiated, passes through the high resolution lens 16 'and passes through each pinhole 1410' on the lower surface of the micro lens array. The light passing through the pin pole spreads over the size of the upper surface (convex surface) of each micro lens by passing through the micro lens. The fluorescence intensity on the upper surface of the microlens is imaged on the image storage type high-sensitivity two-dimensional sensor 32 'via the wavelength selection beam splitter 30' and the imaging lens 31 '. Although an interference filter is not shown in this drawing, an interference filter or a color filter transmitting a wavelength longer than fluorescence is provided between the wavelength separation beam splitter 30 'and the two-dimensional sensor 32'.

In the one-dimensional and two-dimensional microlens arrays shown in FIGS. 4 and 14, there is a danger that the light incident on the intermediate region between the adjacent microlenses of each microlens becomes scattered light and becomes noise light. is there. Therefore, such a noise can be removed by masking the intermediate region with a light-shielding portion made of a material such as chromium oxide (not shown).

The above embodiment, FIGS. 1, 5, 7, and 13
FIGS. 15 and 16 show the spectral reflection characteristics and spectral transmission characteristics of the wavelength separation beam splitters 30 and 30 'and the interference filter 34 used in FIG. By using both, the influence of the excitation light can be reduced, and accurate detection can be performed. In the figure, λ is the wavelength of the excitation light, and the excitation wavelength bandwidth is usually narrow because laser light is used to increase the intensity per unit area of the spot irradiation. λ _L is the center wavelength of the fluorescence to be detected.

The DN to be hybridized
Several types of fluorescent substances are used as the fluorescent substance added to the fragment A. For example, a commonly used Cy5 (Cyan)
In Ine 5), the peak wavelength of the absorption of the phosphor is 649 n.
m, the peak wavelength of the fluorescence is 670 nm. On the shorter wavelength side, in Cy3 (Cyanine3), the peak wavelength of absorption of the phosphor is 550 nm, and the peak wavelength of fluorescence is 570 nm. Since the spectral absorption characteristics of the absorber have a bandwidth, the peak wavelength of absorption and the wavelength of the excitation laser light do not always need to be matched, and laser light close to the absorption peak wavelength is used.

In Cy5, the red light 63 of the He-Ne laser
A semiconductor laser light having a wavelength of 3 nm or a wavelength of 635 nm is used, and a green light 544 nm of a He—Ne laser or the like is used for Cy3. The interference filter that extracts only the fluorescence and the wavelength separation beam splitter select the center wavelength that is close to the peak wavelength of the fluorescence and easily separates the excitation light.

In the above-described fluorescence detection, it is very important to completely block the excitation light, especially when the fluorescence is weak.

FIG. 17 is an embodiment of the present invention for more completely blocking such excitation light. That is, it is performed when the above-described interference filter or the wavelength selection beam splitter alone is not sufficient, or when the band half-width of the interference filter is to be increased in order to increase the intensity of the fluorescence detection light. 17, the same reference numerals as those in FIG. 1 denote the same components or components having the same functions.

Σ _f with a microlens array or hologram
Multi-spot arrays 111, ..., formed on the surface
11M is DNA by lens 15 and objective lens 16.
An image is formed on the chip as excitation light. In this case the multi-spot light passes in the spread of generally radially RNA _sigma 'in the center of the entrance pupil EP ₀ of the objective lens. When the numerical aperture of the objective lens is NA and the focal length is f, the spot diameter Ds of the excitation light on the DNA chip is given by the following equation.

Ds = 2k ₁ fλ / RNA _σ ′
(However, k ₁ = 0.6) Assuming that the pixel resolution (pixel pitch) of DNA chip detection is p, this value is almost equal to Ds. Assuming that p is 2 μm and the excitation light wavelength λ is 633 nm, RNA _σ ′ / f is 0.1
It becomes 9. RNA _σ ′ / f is the NA of illumination for irradiating a 2 μm spot (the minimum NA of the objective lens required to image a 2 μm spot), where NA ′ is: NA ′ = sin (tan -1 (RNA _σ '/ f)) ≒ R
NA _σ ′ / f = 0.19. The NA of the objective lens 16 is 0.7 or more and 0.9 or less in order to detect weak fluorescence. Each excitation spot light having passed through the entrance pupil of the objective lens with the above-mentioned spot diameter irradiates the DNA chip with an excitation spot light of about 2 μm by the objective lens. Since this objective lens is both telecentric, about 4 to 8% of the excitation light vertically incident on the DNA chip is specularly reflected on the chip surface and returns to the objective lens. This specularly reflected light is transmitted by the wavelength splitting beam splitter, but is slightly reflected and directed toward the fluorescence detection system 3 while the excitation light is transmitted.

The fluorescence detection system has an interference filter 34,
The excitation light is blocked, but it is difficult to completely block the excitation light. That is, when the bandwidth of the interference filter is widened to detect as much fluorescence as possible, the excitation light leaks slightly. Therefore, the fluorescent spot light is converted to a fluorescence detection surface (or a surface conjugate to it).
A lens system 31 'for detecting an image is connected to a lens system 3 as shown in FIG.
11 and 312, and the pupil of the objective lens is 311 and 31
An image is formed at a position between the two. The spatial filter 35 is arranged at a position conjugate with the pupil. The spatial filter 35 has a structure as shown in FIG.

The magnification of imaging the pupil plane of the objective lens on the spatial filter 35 by the lens diameter 311 is set to 1. The spatial filter has a circular light-shielding portion 351 having a radius of RNA _σ from the center of the optical axis of the light-shielding portion 353 to the center of an opening having a radius of RNA equal to the pupil radius of the objective lens. The multi-spot excitation light specularly reflected by the DNA chip has a beam diameter RNA _σ 'on this spatial filter. Therefore RNA _σ
If it is> RNA _σ ', the specularly reflected light is shielded by the light shielding portion 351.

On the other hand, the fluorescent light excited and generated by the multi-spot excitation light is incident on the objective lens almost omni-directionally, and is incident on this spatial filter. Although the fluorescent light is also shielded by the light shielding unit 351, the ratio of the shielded fluorescent light to the fluorescent light incident on the spatial filter is (NA ′ / NA) ² , and when the above value is included, 7 (when NA = 0.7) to 4 (When NA = 0.9), and this loss is negligible.

As described above, unnecessary excitation light can be significantly reduced without reducing the amount of fluorescence to be detected. The fluorescence detection light transmitted through the spatial filter is
Through the interference filter 34, a spot image of the DNA chip is formed on the incident end of the fiber which is the detection surface, and the fluorescence is detected.

FIG. 19 is a diagram showing an embodiment of the present invention. A method for preventing excitation light from being guided to the fluorescence detection optical path will be described. The same number as FIG. 1 represents the same thing. In this embodiment, a polarization beam splitter 30 ″ is used as a beam splitter for splitting the excitation light path and the fluorescence detection light path. That is, the excitation light is applied to the split surface of the deflection beam splitter by P.
Irradiate with polarized light. The excitation light reflected back on the DNA chip surface keeps the P-polarized light, so it passes through the polarization beam splitter and does not enter the fluorescence detection optical path. On the other hand, since the polarization of the generated fluorescence is deviated from the excitation light, the S-polarized light is reflected by the polarization beam splitter 30 ″ and guided to the fluorescence detection optical path. This prevents the excitation light from entering the fluorescence detection optical path. it can.

FIG. 20 is a view showing an embodiment of the present invention, in which fluorescence is detected using an optical fiber and a multi-channel photomultiplier. FIG. 20 shows specific details of detection of a fluorescent multi-spot image in the embodiments shown in FIGS. 1, 5, 7, 17, and 19. FIG. The incidence end of the fiber system 32 having the number of multi-spots of M or more is a multi-lens array 321 of M one-dimensionally arranged as shown in FIG.
It is.

As described with reference to FIG. 7, the fluorescent multi-spot image is incident on the core through which the light of the fiber propagates by each microlens. When the multi-channel photomultiplier tube 33 has a two-dimensional light receiving aperture arrangement as shown in FIG. 20, the emission ends of the fibers are arranged in a two-dimensional arrangement. In the case of the embodiment shown in FIG. 20, the image of the fluorescence emitted from each emission end is formed by the imaging lens 324 so that the fiber emission end 323 corresponds to each light receiving opening 331 of the photomultiplier 33.

In the case of the embodiment shown in FIG. 21, a two-dimensional lens array 3241 is provided corresponding to the emitting end of the fiber, and the fluorescent light emitted from each fiber passes through each lens and directly passes through the multi-channel photomultiplier tube. The light is focused on the two-dimensional light receiving aperture (photoelectric surface) 3311. FIG.
In the embodiments shown in FIGS. 0 and 21, the two-dimensional multi-channel photomultiplier is used. However, the same method can be used for a one-dimensional multi-channel photomultiplier by arranging the emission ends in a one-dimensional array.

FIG. 22 is a diagram showing an embodiment of the present invention. In the present embodiment, a plurality of wavelengths are used as the excitation light. The light source systems 11A, 11B, and 11C shape light from the excitation light sources of different wavelengths λ _A , λ _B , and λ _C ,
A multi-lens array is arranged at the exit end, and a pinhole array is provided at a position where light is condensed substantially after exit. The excitation light emitted from the pinhole array is
It is configured to pass through the wavelength separation beam splitters 30A, 30B, and 30C. As shown in FIG. 22, when 11A is selected as the excitation light for fluorescence detection,
The excitation light of λ _A passes through the lens 15 and
0, passes through the objective lens 16 and passes through the DNA chip 2
Is irradiated with multi-spot excitation. Fluorescence generated from each spot passes through the objective lens 16, the deflection mirror 300, and the lens 15 (31), and passes through the wavelength selection beam splitter 30A.
20 and 21 through the interference filter 34A.
The fluorescence of each spot is detected by a multi-channel photomultiplier by the method described in the above section.

When the types of fluorescence to be detected are different,
The entire light source system is moved from the control device 4 'in the direction of the solid line arrow by a drive mechanism (not shown) to select any one of the different wavelengths λ _B and λ _C and perform fluorescence detection at this wavelength.
When a plurality of fluorescent lights are used for one DNA chip, the light source system is sequentially moved, and detection is sequentially performed using different excitation lights.

FIG. 26 shows another embodiment in which a plurality of fluorescent lights are used. Unlike FIG. 22, a plurality of excitation lights are irradiated simultaneously to shorten the inspection time. In FIG. 26, a plurality of wavelengths λ _A ′, λ _B ′, and λ _C ′
Are, for example, light source systems 11A 'and 11 of three colors of red, green and blue.
Light emitted from B 'and 11C' is efficiently combined into one optical path by the wavelength selective combining mirrors 51 and 52. That is, the wavelength selective combining mirror 51 transmits blue and reflects green. The wavelength selective combining mirror 52 transmits red and reflects green and blue.

The three combined colors are the beam splitter 3
The light passes through 0 ', passes through the lens 15, the deflecting mirror 300, and the objective lens 16, and simultaneously irradiates three colors with spot excitation light on the DNA chip. Each phosphor is excited at each wavelength and emits light in each fluorescent color. However, since the fluorescent light has a wavelength slightly longer than the excitation light, the fluorescent light of the three wavelengths excited by the light of the three wavelengths is The wavelengths can be separated by the wavelength splitting beam splitters 53 and 54. That is, the wavelength separation beam splitter 53 reflects blue fluorescence, transmits green and red, the wavelength separation beam splitter 54 reflects green,
Transmits red.

The interference filter 3 that transmits only the fluorescent light of each color with high purity in the optical path separated into each of the three colors as described above.
4C, 34B, and 34A, and a micro spot image of each fluorescent light is multiplied by a multi-channel photomultiplier tube 33C ', 33B', and 33 through a fiber according to the method described above.
A 'is detected simultaneously.

In the embodiment shown in FIGS. 22 and 26, scanning of the excitation multi-spot light in the array direction is performed by the deflection mirror 300.
Do with. As the deflecting mirror, a piezo drive type or galvano mirror type is used. In this case, unlike the embodiment described with reference to FIG. 1, the multi-spot is not a step movement but a continuous (linear) scan. The multi-spot on the DNA chip is scanned, but since the same (identical) deflection mirror is used in the detection optical path, an immovable multi-spot is formed at the end of the fiber for fluorescence detection. Therefore, the pixel address is determined based on the deflection angle of the deflection mirror.

Based on such a relationship between the pixel address and the deflection angle, a plurality of fluorescence detection signals obtained in parallel from the multi-channel photomultiplier tube are supplied to the control circuit 4 '(FIG. 22) or 4 ".
(FIG. 26). Of course, in such data processing, it is necessary to input measurement and detection conditions in advance, and the input information is input from the terminal 41 ′ or the information is input from the host computer 40, In response, the measurement / inspection result data is sent to the computer.

In the embodiment shown in FIG. 26, an example in which a plurality of excitation lights having different wavelengths are simultaneously irradiated has been described. For example, it is assumed that the plurality of excitation lights overlap with the respective wavelength bands of the target fluorescence. In such a case, such a problem can be avoided by using a shutter (not shown) near the light source for the overlapped light, or detecting the fluorescence with a time lag by ON / OFF of the light source itself.

FIG. 23 shows an embodiment in which a plurality of semiconductor lasers 111A2, 111A2,... Since semiconductor lasers are small in volume, have relatively high output, and are inexpensive, by using a large number of semiconductor lasers as shown in the figure and making a multi-spot light source,
It becomes possible to irradiate the DNA chip with strong multi-spot excitation light, and high-speed detection is realized. The light emitted from each semiconductor laser is taken into the incident end of the fiber 120A1, 120A2,... By the lenses 112A1, 112A2,.

As shown in FIG. 24, the laser light emitted from the emission end is condensed on pinhole arrays 111, 112,... Via a microlens array, and the transmitted light is used as multi-spot excitation light.

When a semiconductor laser cannot be used as the excitation light, a high-output solid-state laser excited by a semiconductor laser or a high-output gas laser is used. In such a laser light source, when the output beam is divided and used by a method as shown in FIG. 25, it is possible to form multi-spot excitation light having substantially the same intensity and substantially the same beam shape.

That is, the beam emitted from the laser light source 111A 'is divided into multi-divided beam splitters 1110A', 1
120A '... If the number of divisions is k,
2-stage from the first stage of the division, three-stage ...... k _r 1 the reflectivity of the beam split up _stage, r 2, and ... _r j ... _{r k.} r
_{Since k} is 1 and each beam split light has the same intensity, r _j = 1 / (k−j + 1) may be satisfied for any j of 1 ≦ j ≦ k. That is, k in 1110A 'is 4, i.e. with four divisions, _{r 1} is 1/4, _{r 2} is 1 /
3, r ₃ is 、, and r ₄ is 1. Similarly 1120
Since A ′ is divided into three, r ₁ is ３, r ₂ is 、,
r ₃ becomes 1. As described above, laser beams having the same beam intensity and the same beam shape enter the lens array 112A1 'and enter the core of the fiber.

In the embodiment shown in FIG. 23, a plurality of semiconductor lasers are used as a light source for generating a multi-beam spot, but a multi-spot excitation light is formed by using a plurality of gas lasers other than the semiconductor lasers or lasers using the second harmonic. This is also necessary when the available laser light power is insufficient. In such a case, by using a plurality of systems shown in FIG. 25 and arranging the fiber emission ends extracted from the plurality of systems one-dimensionally or two-dimensionally,
Multi-spot light with high excitation intensity can be obtained.

In the case of using a plurality of excitation lights, it is necessary to use various types of lasers such as a semiconductor laser, a gas laser, and a solid-state laser using the second harmonic of semiconductor excitation depending on the excitation wavelength. In such a case, the multi-spot excitation light may be formed by the method shown in FIGS.

FIG. 27 is a view showing an embodiment of the present invention, in which excitation multi-spot light removes the influence of fluorescent light and scattered light from a foreign substance mixed into a DNA chip or scattered light from a foreign substance in a detection optical system. It realizes highly accurate fluorescence detection. Reference numeral 11 'denotes an excitation light source which transmits the pinhole aperture array 14' through a fiber to produce an excitation multi-spot light. The multi-spot light forms an image on the DNA chip by the lens 15 and the objective lens 16. There is a spatial filter 161 at the position of the entrance pupil of the objective lens.

The excitation multi-spot light is set to pass through a position 163 shifted from the center of the pupil.
That is, each multi-spot light passes through a portion deviated from the optical axis of the objective lens as shown in FIGS. 29 and 30, becomes a light flux of Ii indicated by oblique lines in FIG. 30, and is incident on the DNA chip 2 in the oblique direction θi. Irradiate at the corner. The convergence angle θI of the irradiation convergent light is sin (NA ′) = θI / 2, as described above for the relationship between the spot diameter and NA ′.

The excitation light specularly reflected by the DNA chip passes through the objective lens, and passes on the pupil 161 at a symmetrical position (corresponding to 162 in the figure) with the lens optical axis as the center of symmetry with 163. .

Therefore, as shown in FIG. 28, if a member 162 for blocking the regular reflection light is formed here, the excitation regular reflection light is blocked by the member 162. By setting the position deviated from the center of the pupil in the optical path of the principal ray of the excitation light and satisfying the condition of θi> θI / 2 with respect to the incident angle θi of the principal ray, the excitation light is focused on the pupil. The excitation specular reflection light can be blocked on the pupil without blocking. This embodiment is shown in FIG.
Needless to say, it has the effect of removing noise by the excitation light described in the embodiment.

In addition to this effect, in the embodiment of FIG.
The effect of foreign substances on or in the DNA chip
Take as follows. That is, if there is a foreign substance such as various proteins mixed in at the time of preparing the DNA sample, the excitation light is scattered when irradiated with the excitation light because the size of such foreign substance is as small as several μm. In addition, in the case of a foreign substance such as the above-mentioned organic substance, strong fluorescence is emitted from the foreign substance, and the DN to be detected is
The fluorescence becomes stronger than that of the phosphor added to A.

As shown in FIG. 30, the scattered excitation light passes through the area L + S determined by the NA of the objective lens painted with dots, passes through the objective lens, passes through a portion other than the light shielding portion 163 of the spatial filter, and detects the fluorescence. Leaks into the system. Only the excitation light is extracted by the wavelength separation beam splitter 61 and the image is detected. The wavelength splitting beam splitter transmits the fluorescence and reflects the excitation light. The insertion position of the wavelength separation beam splitter is located in the optical path behind the wavelength separation beam splitter 30 ″ in FIG. 29, but may be placed in front of the wavelength splitter beam splitter. The excitation light scattering image S100 is detected as an image by the detector 62 in the same manner as the image is captured by the detector 33 'such as a multi-channel photomultiplier tube.

FIG. 31 shows the fluorescence image signal D _L and the excitation light image signal D _S detected in this way at the signal level of each picture element. That is, 1, 2,..., 25 indicate the addresses of the detected picture elements, and (1), (2), (3), (4), (5) are the addresses of the cells having the same DNA sequence. That is, for example, picture elements at addresses 1, 2, 3, 4, and 5 belong to the cell (1). Actually, the picture elements in the cell are two-dimensional, for example, 5 ×
Although there are five picture elements, they are made one-dimensional for easy understanding using figures. Scattered excitation light of the pixel if it takes a picture element is foreign matter exceeds the threshold D _ST.

[0086] In FIG. 31, the excitation light image signal _{D S} is beyond the threshold value _{D ST} picture elements addresses 9, 10, 11 and 16,17,18,19,20. Therefore, information of the corresponding address in the fluorescent image detection signal D _L since strongly influenced by foreign matter, to remove the information in the address obtaining an average value of fluorescence of each cell. That is, for cell number (2), picture element 9,
The average value is calculated using only the information at the remaining addresses 6, 7, and 8 without using the information at address 10. Similarly, for cell (3), an average value is obtained using information of only 13, 14, and 15. Cell (4) is invalid because all picture elements are scattered by foreign matter. By determining the average intensity in the cell as shown in the lower graph of FIG. 31 in this way, the influence of foreign matter is greatly reduced, and accurate fluorescence detection can be performed.

In the optical system of the embodiment for removing the influence of foreign matter described with reference to FIGS. 27 to 30, the excitation light passes through the objective lens to illuminate the DNA chip. A configuration may be adopted in which light is emitted obliquely from between the objective lens and the DNA chip without passing through, and scattered light is detected. In this case, since the specularly reflected light does not enter the objective lens, a spatial filter (corresponding to 161) is not required.

FIG. 32 is a diagram showing an embodiment of the present invention. Reference numeral 1 denotes a multi-spot excitation light irradiation system which has already described the detailed embodiment, and reference numeral 3 denotes a fluorescence detection system which detects fluorescence generated by the multi-spot excitation light which has also been described in detail. In order to irradiate the fluorescence attached to the DNA hybridized on the DNA chip 2 with a small spot excitation light of several μm by the objective lens 16, the objective lens and the fluorescence are adjusted so that the spot size is kept constant. The plane must always be maintained at regular intervals within the depth of focus. For this reason, the focus detection system 7 which is structurally integrated with the objective lens 16 in terms of rigidity is used. The focus detection system 7 includes an oblique beam spot irradiation system 71 and a spot position detection system 72. The oblique beam spot irradiation system 71 irradiates the fluorescent screen on the DNA chip with a small spot obliquely.

FIG. 33 shows an example of a sectional structure of a DNA chip. In this embodiment, the fluorescent screen is # 2.
That is, there is a fluorescent screen # 2 on a flat substrate 23 such as glass,
Between this surface and the surface # 1 on the glass substrate 21, there is a gap 22 for flowing a liquid containing the DNA to be inspected to which fluorescence has been added and for hybridization. In the stage of DNA inspection, the gap is filled with liquid. However, in some cases, this gap may be left empty. The fluorescent screen may be set to # 1, and the substrate 23 may be made of an opaque material or a material that absorbs light.

The light beam f ₀ radiated obliquely from the oblique spot irradiation system 71 so as to converge on the fluorescent screen is applied to the upper surface {3 and the lower surface # ₁ of the upper substrate 21 and the upper surface} of the lower substrate 23.
The light is specularly reflected on four surfaces 2 and the lower surface # 4. Oblique irradiation beam f ₀
Rays _l 3 principal ray _{f 0M} of regularly _{reflected, l 1,} l _2, l
₄ is a position sensor 72 by an imaging lens 721.
P ₃ , P ₁ , P ₂ , and P4 on a surface equal to the light receiving surface of _{No. 2}
The image is tied to each position. Since the positions of the phosphor screens are predetermined, if the planes # 3, # 1, # 2, and # 4 are separated to some extent, the plane to be detected (# 2 in this embodiment).
Light receiving position sensor 722 so that only the spot image position is received and the other spot images are outside the light receiving aperture.
By determining the dimensions of and the imaging magnification of the lens 721,
The height of only the desired detection surface can be detected.

That is, in this embodiment, only the specularly reflected light from the # 2 surface is captured by the position sensor, and the spot position on the position sensor, that is, the height position of the # 2 surface can be detected. The control circuit 4 allows the objective lens and the focus detection system 7 integrated with the objective lens to be finely moved up and down by the driving device 73 based on the detection information, thereby enabling the fluorescence to be always detected in a focused state. .

If the angle of incidence of the obliquely incident focus detection light in FIG. 33 is close to the Brewster's angle, if it is incident with P deflection, the reflection on the surface will be very small, making detection difficult. Therefore, S deflection is used. S-deflection is advantageous because the reflectance at the surface is higher than P-deflection at any angle of incidence.

FIG. 34 is an embodiment of the present invention, in which focus is detected through the objective lens 16. Laser light guided from a near-infrared semiconductor laser light source (not shown) by a fiber 74 passes through a beam splitter 76 after exiting the fiber, a lens 77, a wavelength separation beam splitter 300 ″,
The light passes through the objective lens 16 and is obliquely focused and irradiated on the fluorescent screen of the DNA chip 2. The incident angle θ of the irradiation light is a size corresponding to the aperture NA of the objective lens (sin θ = incidence angle slightly smaller than NA ≒ 0.8). The specularly reflected light passes through the objective lens again and forms an image on the position sensor 73 'at a position corresponding to the condensing point on the chip. In this way, the focus position of the phosphor screen of the chip can be detected as in the embodiment of FIG. If light having a longer wavelength than the detected fluorescence is used as the light used for focus detection as in the present embodiment, accurate focus detection can be performed without exciting the phosphor, that is, without generating detection noise.

The wavelength splitting beam splitter 3 shown in FIG.
Reference numeral 00 "transmits near-infrared light and reflects excitation light and fluorescence used for fluorescence detection. The one-dimensional excitation light spot array formed by the one-dimensional excitation light irradiation optical system 1 includes a lens 15 and a wavelength selection beam splitter 300. And a fluorescent screen of the DNA chip 2 through the objective lens 16. The generated fluorescence is detected by the fluorescence detection optical system 3. Movement of the irradiation position of the excitation light spot array in the array direction is performed by finely rotating the wavelength selection beam splitter.

The D described with reference to each example of the embodiment is described above.
A method for performing the fluorescence detection of the NA chip over all the cells of the DNA chip will be described below with reference to FIGS.

FIG. 35 shows the entire structure of the DNA chip. Reference numeral 204 denotes an entire case where a DNA chip is mounted. The DNA chip is a glass substrate inside the window 200 in this case, and is fixed to the case 204. A DNA fragment with a fluorescent object attached thereto is hybridized in the region 202 inside the window. An alignment mark 201 for positioning is drawn outside the region 202 and inside the window 200.

N × N described in FIG. 2 (5 × 5 in FIG. 5)
Cell 20 in which a fragment of DNA information having the same picture element (shown by a thick line) is planted (probed).
And the relative position of the alignment mark 201 is 1
It is designed and manufactured with an accuracy of a few μm.

A DNA chip is mounted on an inspection apparatus, and the position of at least two alignment marks 201 is detected by an alignment detection optical system (not shown) already mounted in the DNA inspection apparatus described in the embodiment. 2
It is detected as a position on the CCD by a dimensional CCD or the like. In addition, as shown in FIG. 32, for example, as shown in FIG. 32, the position of the DNA chip at the time of performing the mark detection is the length measuring devices 81 and 82 for position detection in the x and y directions provided on the chuck on which the DNA chip is mounted. To detect.

Since the distance between the optical axis of the above-mentioned CCD detection optical system and the detection optical axis of the above-mentioned various fluorescence detection optical systems is constant, this distance, the above-mentioned CCD alignment mark detection position, and the length measuring device It is possible to detect a picture element obtained by dividing each cell of the DNA chip more finely from the detection position at the correct position. At this time, if it is found that the DNA chip is rotating by detecting the positions of two or more alignment marks, the rotation is corrected by a rotating mechanism (not shown). The correct position detection after the rotation correction is performed as needed. Further, if the rotation amount is small without performing the rotation correction, the rotation amount may be detected by the above-described method, and the xy coordinates may be corrected based on the rotation detection amount. Further, it is also possible to detect the above-mentioned multi-spot light after correcting it by minute rotation of the optical system.

As described above, it is possible to detect a picture element in a cell on a DNA chip with accurate positioning accuracy by detecting an alignment mark and measuring the length of a DNA chuck. Can be sequentially detected at the speed of light for all the picture elements. 2A of FIG.
Irradiates M multi-spot array excitation light, sequentially scans N picture elements in the spot array direction, returns to the original operation position, and scans the chuck in the y direction, thereby causing one of the chucks to scan. Represents the area detected by scanning. That is, as shown in FIG. 36, the picture elements 2A101, 2A102,..., 2A10M on the DNA chip are first excited and illuminated simultaneously, and then one picture element pitch Δ is obtained by the above-described method.
The multi-spot moves by P and continues this one by one.

By moving by N (k) picture elements in this way, over the total MN (k) picture elements, that is, over the width of NMΔP,
They are detected in a one-dimensional array. When scanning of N (k) pixels is completed, the multi-spot array is returned to the initial position.
During this time, the DNA chip is _switched from time t ₀ to t
Since moving one pixel worth in the y direction such as between _1, it is repeated between the above operation from the pixel number of the time t ₀ in the y direction t _1, fluorescence detection over the entire pixel region 2A Ends.

When the above operation is performed by the DNA testing apparatus of the embodiment shown in FIG. 1, the driving signal of the multi-spot excitation light by the AO polarizer or the moving position of the spot is determined by S in FIG.
_px . More specifically, S in FIG.
It is stepping like _B12 . Also, the deflection signal of the wavelength selection beam splitter is changed similarly to _{Spx in} FIG. By repeating such a variation from the time t ₀ to t _1, can detect the entire region 2A.

[0103] Once detected until the bottom edge of the region 2A (time _{t 1)} only NMΔP the chuck of the DNA chip in the x direction as shown in _{x st} in FIG. 40 to step movement. After the step movement, scanning the chuck in the direction opposite to the y as shown in the y _st after time t ₁ in FIG. 39. The same time at regions 2A detects the deflection signal AO polarizer 12 and the wavelength-selective polarization beam splitter 30 into _{S px} so the time _t after ₁ of FIG. 38 (time _t 0 ~t ₁₎ is scanned in the opposite direction. In this way, as shown in FIG. 37, the area 2B can be detected continuously following the detection of the area 2A. By repeating the above operation alternately in the negative and positive directions in the y direction of the DNA chip, all the chips from 2A to 2J of the DNA chip can be detected at high speed.

The time required to detect all the DNA chips in this embodiment will be described. By dividing the number of cells in the chip by L and each cell by N × N, the influence of foreign matter or the like is avoided. In this way all the detecting number of picture elements in the chip becomes LN ^2. Assuming that the number of simultaneously irradiated spots of the multi-spot excitation light is M and that the time for simultaneous multi-spot detection is Δt, the time taken by the movement of the spot and the movement of the chuck in the x direction is ignored as being shorter than the time required for fluorescence detection. Then, the total fluorescence detection time T is given by the following equation.

[0105] For ^{T = LN 2 Δt / M =} LN 2 / (M / Δt) scan number k of multi-spot, the response time _{t p} of the wavelength separation beam splitter 30 is required to be more k.DELTA.t. Further, the power of the light source needs to be βM / Δt or more. Further, it is desirable that the interval between the excitation multi-spot lights is 5 or more in view of SN.

The quantum efficiency of the photomultiplier used for fluorescence detection is as low as 5 to 10% at 670 nm where the fluorescence wavelength is long. There is also a limit on the output of laser light used for excitation.
Considering such conditions, in order to detect as quickly as possible, for example, k (= N) = 10, M = 50, Δt = 50 μ
Assuming that the number of cells L = 1000 × 1000 and the number of divided picture elements in the cell N = 5, T = 25s. That is, LN ² /
The condition of (6 × 10 ⁵ ) seconds or less is satisfied. By the way, if the above L and N values are included in this condition, it will be 42 seconds. I do.

If the detection can be performed in one minute, it is effective when testing many specimens. For example, a test that took 5 minutes in the past can be done in 1 minute, so even if the pre-processing takes a little time, if the number of samples approaches 100, pre-processing can be performed on many samples in parallel. , 5 or 6 hours.

The present invention enables such high-speed and high-precision detection because multiple spots are simultaneously used as excitation light. In addition, when fluorescence is detected by excitation light of each spot, another excitation spot light is used. This is because the detection is performed with an interval between the spots so as not to be affected by as much as possible.

[0109]

According to the present invention described above, it has become possible to rapidly and accurately detect the fluorescence of the DNA to be inspected, which has been hybridized to various and many DNA probes on the DNA chip. As a result, it has become possible to perform infectious disease diagnosis, genetic testing, and the like on a large number of samples at high speed and with high accuracy.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating an embodiment of the present invention.

FIG. 2 is an embodiment of the present invention, showing multi-spot irradiation.

FIG. 3 is an embodiment of the present invention, showing the movement of multi-spot irradiation.

FIG. 4 is an embodiment of the present invention, showing generation of a multi-spot by a microlens array.

FIG. 5 is an embodiment diagram of the present invention, illustrating the embodiment of FIG. 1;

FIG. 6 is a view for explaining the movement of a spot fluorescent image when the wavelength selection beam splitter is fixed.

FIG. 7 is a view for explaining the movement of a spot fluorescent image when a wavelength selective beam splitter is deflected.

FIG. 8 is a diagram illustrating an operation of each component in FIG. 1, a detection signal, and the like according to the embodiment of the present invention.

FIG. 9 is a diagram illustrating a motion of an image on a fluorescence image detection surface in the embodiment of FIG. 1;

FIG. 10 is a diagram showing a light receiving aperture of a fluorescence detection surface in the embodiment of FIG.

FIG. 11 is a diagram showing an embodiment of the present invention, in which a multi-spot is formed by a hologram.

FIG. 12 is a diagram showing a method of creating the hologram.

FIG. 13 is a diagram showing an embodiment of the present invention.

FIG. 14 is a microlens array for generating a two-dimensional multi-spot used in the embodiment of FIG. 13;

FIG. 15 is a diagram illustrating characteristics of a wavelength separation beam splitter according to the embodiment of the present invention.

FIG. 16 is a diagram showing characteristics of an interference filter according to the embodiment of the present invention.

FIG. 17 is a diagram of an embodiment of the present invention, in which excitation light is shielded by a spatial filter.

FIG. 18 is a diagram of the spatial filter.

FIG. 19 is a diagram showing an excitation light shielding method using deflection in the embodiment of the present invention.

FIG. 20 is a diagram showing an embodiment of the present invention, in which a one-dimensional spot array is detected by a two-dimensional multi-channel photomultiplier.

FIG. 21 is a diagram showing the input / output end of the fiber and the capture of a spot image in the embodiment of FIG. 20;

FIG. 22 is a diagram of the embodiment of the present invention, in which excitation light is detected at a plurality of wavelengths.

FIG. 23 is a diagram of an embodiment of the present invention, in which a plurality of semiconductor lasers are used for excitation light.

FIG. 24 is a view of the embodiment of the present invention, in which multi-spot light is obtained from the emission end of the fiber.

FIG. 25 is a diagram showing an embodiment of the present invention, in which a multi-spot is obtained from one laser beam.

FIG. 26 is a diagram of an embodiment of the present invention, in which excitation light is set to a plurality of wavelengths and detected simultaneously.

FIG. 27 is a diagram of the embodiment of the present invention, in which multi-spot light is irradiated obliquely and specular reflection excitation light is blocked on a pupil.

FIG. 28 is a view showing a light-shielding portion on the pupil in FIG. 27;

FIG. 29 is a diagram illustrating a method of detecting excitation scattered light to obtain a foreign substance position and correcting a fluorescence detection result in the embodiment of the present invention.

FIG. 30 is a diagram showing excitation specular reflection light and scattering excitation light between the objective lens and the DNA chip of FIG. 29.

FIG. 31 is a diagram showing a method of correction based on a detection signal of foreign matter scattered light in FIG. 29;

FIG. 32 is an embodiment of the present invention, in which focus detection and
The figure which shows control.

FIG. 33 is a diagram showing the reflection response of the DNA chip in the focus detection of FIG. 32;

FIG. 34 is a diagram illustrating focus detection through an objective lens in the embodiment of the present invention.

FIG. 35 is a diagram showing a method for detecting the entire inspection target area of the DNA chip in the embodiment of the present invention.

FIG. 36 is an enlarged explanatory view of the embodiment of FIG. 35;

FIG. 37 is an enlarged explanatory view of the embodiment of FIG. 35;

FIG. 38 is a view showing a movement of a multi spot in the embodiment of FIG. 35;

39 is a view showing the movement of the DNA chip chuck in the x direction in the embodiment of FIG. 35.

40 is a view showing the movement of the DNA chip chuck in the y direction in the embodiment of FIG. 35.

[Explanation of symbols]

1 ... excitation light irradiation optical system, 10 ... multi-spot forming optical system, 11 ... light source system, 12 ... AO deflector, 14 ...
..Microlens array, 16... Objective lens, 14 '
···· Multi-spot generating hologram, 2 ··· DNA chip, 20 ··· cell, 201, 202, 212 ··· fluorescence detection picture element, 30 ···· wavelength selective beam splitter deflector, 3
00: mirror deflector, 32: optical fiber, 33:
Photomultiplier tube, 4, 4 '... control circuit, 41' ... input terminal, 51, 52, 53, 54 ... wavelength selection beam splitter, 7 ... focus detection system, 73 .. Indicate a fine movement mechanism of an objective lens and a focus system, 80... A DNA chip chuck, 81 and 82... .

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) G01N 33/50 G01N 33/53 M 33/53 33/566 33/566 C12N 15/00 A (72) Inventor Tomoaki Sakata 292 Yoshida-cho, Totsuka-ku, Yokohama-shi, Kanagawa Prefecture Inside Hitachi, Ltd.Production Technology Laboratory (72) Inventor Kenji Yasuda 882-Momo, Hitachinaka-shi, Ibaraki Prefecture Hitachi, Ltd.Measurement Equipment Group (72) Inventor Satoshi Takahashi 882 Ma, Hitachinaka-shi, Ibaraki F-term within the measuring instruments group of Hitachi, Ltd. CA25 DA13 FA12 FA15 FA29 FB02 FB07 FB12 GC15 JA07 JA08 4B024 AA11 AA19 CA04 HA14 4B029 AA07 AA23 FA10 4B063 QA01 QQ08 QQ42 QR32 QR66 QS34 QX02

Claims (46)

[Claims] 1. A DNA chip comprising a plurality of L cells, each of which is a micro area, in which a plurality of types of desired DNA fragments are arranged according to a predetermined rule,
DNA prepared by pretreatment from the DNA to be tested
In a DNA testing method for irradiating excitation light having a desired wavelength to a DNA chip to be tested obtained by hybridizing a target obtained by adding a desired fluorescent substance to a fragment and analyzing the obtained fluorescence, Spot diameter d
And simultaneously irradiating the plurality of M multi-spot excitation lights to different positions using an objective lens for a time Δt longer than the fluorescence decay time, guiding the obtained fluorescence to a fluorescence detection optical path, and multi-spot excitation to the DNA chip. A DNA inspection method in which fluorescence from each multi-spot light generated by light is separated and detected, and a DNA chip to be inspected is inspected based on the position and intensity of the fluorescence. 2. The DNA inspection method according to claim 1, wherein a plurality of M excitation light spots are linearly arranged one-dimensionally or two-dimensionally at a constant pitch. 3. The DNA inspection method according to claim 1, wherein the spot diameter is approximately 1 / N with respect to an integer N of the cell size. 4. An accurate inspection by dividing the cell into a plurality of parts by setting N to 2 or more and selecting and processing only significant data among N ² data in one cell. The method for testing DNA according to claim 3, wherein: 5. The number L of all samples to be inspected on a DNA chip
The method according to any one of claims 1 to 4, wherein fluorescence is detected within a time period of LN ² / (6 × 10 ⁵ ) seconds with respect to N ² . 6. A plurality of M irradiation spots on the DNA chip are linearly arranged at intervals of about kd with respect to a spot diameter d and an integer k. By moving in the array direction and irradiating Δt time successively k times, inspection is performed over KM spot positions in the array direction, and the DNA chip and the objective lens are relatively moved at least in the direction perpendicular to the array. 2. The DNA according to claim 1, wherein a desired two-dimensional area of the DNA chip is inspected by moving the DNA chip.
Inspection methods. 7. The DNA testing method according to claim 6, wherein the integer k is 2 or more. 8. The DNA testing method according to claim 6, wherein the integer k is 5 or more. 9. A fluorescent light detecting and deflecting means in a fluorescent light detecting optical path so as to synchronize with the movement of the spot array in the array direction so that fluorescent light generated by the excitation light comes to substantially the same position on the light receiving aperture. The DNA testing method according to any one of 1, 6, 7, and 8. 10. The DNA inspection method according to claim 9, wherein said fluorescence detecting and deflecting means is constituted by a wavelength selective beam splitter which transmits excitation light and reflects fluorescence. 11. The DNA testing method according to claim 1, further comprising a filter that transmits only fluorescence and blocks excitation light in a fluorescence detection optical path separated from the excitation light path. 12. The M multi-excitation spot lights pass through substantially the same position A on the pupil of the objective lens, and the excitation light specularly reflected by the DNA chip reaches a position B 'on the pupil of the objective lens. Means for blocking the reflected excitation light at the position of B 'on the pupil of the objective lens or on the image position of B' on the plane conjugate with the pupil of the objective lens in the fluorescence detection optical path. The DNA testing method according to claim 1, further comprising: 13. The M multi-excitation spot lights pass through substantially the same position A on the pupil of the objective lens, and the excitation light specularly reflected by the DNA chip is different from the A on the pupil of the objective lens. It is configured to pass through the position B, and a pupil of the objective lens or a member that blocks the reflected excitation light of a desired diameter around B at a position conjugate with the pupil of the objective lens in the fluorescence detection optical path is disposed. D according to claim 1, characterized in that
NA inspection method. 14. The reflection excitation light is specular reflection excitation light,
The excitation light scattered from the foreign matter in the DNA chip is taken out of the light shielding means or the light shielding member, further branched from the fluorescence detection optical path, imaged at a position conjugate with the irradiation spot of the DNA chip, and using the imaging information, 14. The DNA inspection method according to claim 12, wherein information detected by separating fluorescence from each multi-spot light generated by the multi-spot excitation light is corrected. 15. The apparatus according to claim 1, wherein said M multi-excitation spot lights are formed by a plurality of laser light sources.
The DNA testing method according to the above. 16. Light emitted from the plurality of laser light sources is introduced into an optical fiber, and emitted from the emission ends of the optical fibers aligned at M desired pitches, thereby obtaining M multi-excitation spot lights. 16. The method according to claim 15, wherein
The DNA testing method according to the above. 17. A means for separating and detecting fluorescence from each multi-spot light generated by the multi-spot excitation light is a two-dimensional imaging apparatus having an ultra-sensitive N _x × N _y pixel number, and having a spot diameter of the excitation light d and _n x, a _{n y} an integer, _{n x} × x direction _n x d, y directions with a pitch of _{n y} d
The N _y spots are simultaneously illuminated, and the resulting fluorescent spot image composed of N _x × N _y pixels is detected by the ultra-high sensitivity two-dimensional imaging device, and the DNA detection device and the DNA chip are relatively moved at a pitch d in the xy directions. 2. The DNA detection method according to claim 1, wherein a desired area of the DNA chip is detected by moving by _nx * _ny steps. 18. The DNA testing method according to claim 1, wherein said excitation light has a plurality of different wavelengths, and separates and detects different targets to which a plurality of fluorescent substances are added. 19. The DNA testing method according to claim 18, wherein the excitation light having a plurality of wavelengths is simultaneously irradiated, and different targets to which a plurality of fluorescent substances are added are separated and detected at the same time. 20. A second light is obliquely incident on the inspection surface of the DNA chip to be inspected on which a target to which a desired fluorescent substance is added is hybridized, in the vicinity of the excitation spot light, and is reflected by the inspection surface. 2. The DNA inspection method according to claim 1, wherein a focus is detected by detecting a position of light, and focusing is performed by controlling a relative distance between the inspection surface and the objective lens based on the information. 21. A DNA inspection apparatus comprising an excitation light irradiation system for irradiating a DNA chip with excitation light from an excitation light source and a fluorescence detection system for detecting fluorescence generated from a fluorescent substance on the DNA chip. Dimension D of many cells L
Multiple M excitation lights having the following spot diameter d
An optical system for generating a multi-spot excitation light simultaneously generated on a chip, an objective lens for simultaneously irradiating the multi-spot light to a fluorescent object added to a DNA fragment on a DNA chip with the dimension d, and an objective lens for obtaining the obtained fluorescence. A beam splitter that guides the fluorescence from the multi-spot light generated by the multi-spot excitation light to a fluorescence detection optical path through the multi-spot excitation light;
The position of the multi-spot light and the position of D are set so that the multi-spot light is irradiated over a desired area of the NA chip to detect fluorescence.
Driving means for relatively changing the position of the NA chip, DNA detected by the driving means and the fluorescence detecting means
The DN to be inspected is determined based on the fluorescence intensity and the fluorescence position of the desired area of the chip.
A DNA testing apparatus comprising a control system for obtaining and testing DNA information of an A chip. 22. The multi-spot excitation light generating optical system according to claim 21, wherein a plurality of M excitation light spots arranged one-dimensionally or two-dimensionally on a straight line at a constant pitch are simultaneously generated. DNA testing equipment. 23. The method according to claim 21, wherein the spot diameter is substantially 1 / N of an integer N of the cell size D.
The DNA inspection apparatus according to claim 1. 24. The control system according to claim 24, wherein N is set to 2 or more, the cell is divided into a plurality of parts, and only significant data is selected and processed from N ² data in one cell. 24. The DNA testing device according to claim 22, wherein: 25. The DN according to claim 24, wherein fluorescence detection is performed within a time period of LN ² / (6 × 10 ⁵ ) seconds with respect to all the sample points LN ^{2 of the} DNA chip to be inspected.
A inspection device. 26. The fluorescence detecting means according to claim 1, wherein said fluorescence detecting means has an effective diameter substantially equal to the diameter of a spot image formed on a surface having a conjugate relationship with a plurality of M irradiation spots on said DNA chip.
22. The DNA testing apparatus according to claim 21, wherein the DNA testing apparatus has two light receiving openings. 27. M light receiving apertures having an effective diameter substantially equal to the irradiation spot image are optical fiber light receiving ends,
27. The DNA inspection apparatus according to claim 26, wherein the DNA inspection apparatus is a fluorescence detection unit that separates and detects light emitted from the emission end of the fiber. 28. A plurality of M irradiation spots on the DNA chip are linearly arranged at intervals of about kd with respect to the spot diameter d and an integer k, and after irradiating the spot array with the Δt time, almost d. By moving in the array direction and irradiating Δt time successively k times, inspection is performed over KM spot positions in the array direction, and DNA
22. The DNA inspection apparatus according to claim 21, comprising the control system for inspecting a desired two-dimensional area of the DNA chip by relatively moving the chip and the objective lens at least in a direction perpendicular to the array. 29. The DN according to claim 28, wherein the movement of the spot array is performed using an acoustic light deflector.
A inspection device. 30. The DNA testing apparatus according to claim 28, wherein said integer k is 2 or more. 31. The DNA testing apparatus according to claim 28, wherein said integer k is 5 or more. 32. The DNA testing apparatus according to claim 21, wherein the spot array is formed by a micro lens array. 33. The DNA inspection apparatus according to claim 21, wherein the spot array is formed by a hologram. 34. The DNA test according to claim 21, further comprising a deflecting means configured to synchronize the movement of the spot array in the array direction so that fluorescence generated by the excitation light comes to substantially the same position on the light receiving aperture. apparatus. 35. The DNA testing apparatus according to claim 34, wherein said deflecting means is constituted by a wavelength selection beam splitter that transmits excitation light and reflects fluorescence. 36. The DNA testing apparatus according to claim 21, further comprising a filter that transmits only fluorescence and blocks excitation light in a fluorescence detection optical path separated from the excitation light path. 37. The M multi-excitation spot lights pass through substantially the same position A on the pupil of the objective lens, and the excitation light specularly reflected by the DNA chip reaches a position B 'on the pupil of the objective lens. And the pupil of the objective lens or a plane conjugate with the pupil of the objective lens in the fluorescence detection optical path is configured between the fluorescence detection means and the objective lens, and the position of B ′ on the pupil or 22. The DNA inspection apparatus according to claim 21, further comprising means for shielding reflected excitation light at an image position of B 'on the pupil conjugate plane. 38. The M multi-excitation spot lights pass through substantially the same position A on the pupil of the objective lens, and the excitation light specularly reflected by the DNA chip is different from the A on the pupil of the objective lens. It is configured to pass through the position B, and a pupil of the objective lens or a member that blocks the reflected excitation light of a desired diameter around B at a position conjugate with the pupil of the objective lens in the fluorescence detection optical path is disposed. The DNA testing apparatus according to claim 21, wherein: 39. The reflection excitation light is specular reflection excitation light,
Excitation light scattered from foreign matter in the DNA chip is taken out of the light-shielding means or the light-shielding member, further branched from the fluorescence detection optical path, and provided with scattered image detection means for imaging at a position conjugate with the irradiation spot of the DNA chip. 39. The DNA testing apparatus according to claim 37, further comprising a control system that corrects information obtained by the fluorescence detecting means using imaging information obtained by the scattered image detecting means. . 40. The M number of multi-excitation spot lights are formed by a plurality of laser light sources.
2. The DNA testing device according to 1. 41. The DNA testing apparatus according to claim 33, wherein said plurality of laser light sources are formed by a plurality of semiconductor laser light sources. 42. Light emitted from the plurality of laser light sources is introduced into an optical fiber, and emitted from the emission ends of the optical fibers arranged at M desired pitches, thereby obtaining M multi-excitation spot lights. 42. The DNA testing device according to claim 40, further comprising: means. 43. The fluorescence detecting means according to claim 1, wherein said high sensitivity N _x × N
_{This is a} two-dimensional imaging device having _y pixels, and has a spot diameter d.
The excitation light _n x of the _{n y} an integer, _n x d in the x direction, y
N _x × N _y spots are simultaneously irradiated at a pitch of n _{yd in the} direction, and a fluorescent spot image composed of the obtained number of N _x × N _y pixels is detected by the ultra-sensitive two-dimensional imaging device. The chip is relatively n _{x in the} xy direction at the pitch d.
22. The DNA detection device according to claim 21, further comprising the control system for detecting a desired region of the DNA chip by moving by _ny steps. 44. The DN according to claim 21, wherein said excitation light source comprises a plurality of different wavelength light sources, and separates and detects different targets to which a plurality of phosphors are added.
A inspection device. 45. A second light is obliquely incident near the excitation spot light on the inspection surface of the DNA chip to be inspected on which the target to which the desired fluorescent substance is added is hybridized, and is reflected by the inspection surface. 22. A focus detection system for detecting a focus by detecting a position of light, and performing focusing by controlling a relative distance between an inspection surface and the objective lens based on the information.
The DNA inspection apparatus according to claim 1. 46. The method according to claim 4, wherein the second obliquely incident light has a wavelength that does not excite the phosphor.
6. The DNA testing device according to 5.