JP2001208688A - Method of forming multi-spot light, method of detecting common focus, method of detecting fluorescence, and method of examining dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To increase the speed and accuracy of detecting a common focus using multi-spot light beams, detecting fluorescence, and performing detection for DNA examination by obtaining from a laser beam proximate multi-spot light beams having little difference in intensity and allowing a high efficiency for utilizing light. SOLUTION: A plurality (N) of proximate beams are incident on polarization elements for doubling each beam, and the beams are each divided into two according to polarization to provide 2N beams. The polarization elements are used by M to provide 2MN beams. The incident beams are formed into converging beams and an array of pinhole openings is provided in a converging position. 70 or 90% of the energy of a source of laser beams can then be utilized for the multi-spot light beams. Variation in intensity between the spots of the multi- spot light beams thus formed can be held within ±20% or ±10%. The multi- spot light beams thus formed are used in the detection of the common focus and fluorescence and in DNA examination to permit detection and examination at high speed and accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently forming a plurality of spots from a laser beam, and more particularly to a method for detecting confocal light, a method for detecting fluorescence, and a method for inspecting DNA using this method.

[0002]

2. Description of the Related Art A method for irradiating an object to be detected with multi-spot light and detecting confocal points uses a Nippow disk having a large number of openings formed on a disk. In this method, the entire disc is irradiated with light, and the light passing through the opening becomes illumination light. Therefore, most of the light emitted from the light source is shielded except at the opening, and the light energy contributing to the illumination is only a few% of the light emitted from the light source.

[0003]

In forming a multi-spot light from a beam emitted from a laser light source, if the emitted light is to be converted into a multi-spot light without waste, for example, it is arranged in a line at a pitch of 0.4 mm. 0.04mm
When trying to form 50 multi-spot lights of diameter,
The emitted laser beam is shaped so that the aspect ratio becomes an oval shape of 1:50. 0.4m of this oval beam
By irradiating the microlens array arranged at m pitches, if the focal length of the lens ゛ is, for example, 60 mm, 50 multi-spot lights having a diameter of about 0.04 mm can be obtained at a pitch of 0.4 mm. Since the intensity distribution of the laser beam is a Gaussian distribution, the multi-spot light formed by such a method is bright near the center of the array of 50 multi-spot lights and dark at the periphery. In order to make the intensities of the center and peripheral spots as equal as possible, the spread of the incident elliptical beam must be sufficiently larger than the width of 50 microlenses. As a result, more than half of the energy of the laser beam emitted from the laser light source is wasted.

SUMMARY OF THE INVENTION An object of the present invention is to solve the problem that the light use efficiency in such a conventional multi-spot light forming method is low and the intensity of the multi-spot light varies widely, and a multi-spot light having a substantially uniform intensity is obtained. It is an object of the present invention to provide a method and an apparatus for obtaining light.

It is a further object of the present invention to provide a confocal detection, a fluorescence detection and a D
An object of the present invention is to provide a method and an apparatus for performing an NA test.

[0006]

SUMMARY OF THE INVENTION In order to solve the above-mentioned problems and obtain a multi-spot light in close proximity, the present invention has the following configuration.

That is, a plurality of N laser beams, which are directed substantially in the same direction and are relatively close to each other, are made incident on a polarizing element to separate them into two mutually orthogonal polarized component beams. By doing multiple 2N
Laser beam. After that, a plurality of 2N laser beams are directed to almost the same direction and are adjacent to each other.

As a specific method for obtaining such a twice as close beam, a plurality of N adjacent laser beams are divided into a polarized beam split surface and a parallel beam L1 from the polarized beam split surface. And L2
And incident on the above-mentioned polarizing element constituted by a multi-spot light doubling prism having a total reflection surface at a distance of. With this configuration, the number of the plurality of adjacent laser beams is reduced to two.
2N can be doubled.

Further, a plurality of N adjacent laser beams are incident on the above-mentioned polarizing element made of an anisotropic optical medium, and the inside of the anisotropic optical medium is separated into two mutually orthogonal polarized component beams of each beam. In this way, a plurality of 2N laser beams are obtained. In this way, it is possible to generate a plurality of 2N laser beams that are directed substantially in the same direction and are adjacent to each other.

Furthermore it is possible to obtain a laser beam of 2 ^{M N,} by using a plurality M of polarizing element for doubling the beam as described above.

If each of the plurality of N adjacent laser beams incident on at least one of the one or more polarizing elements is a convergent laser beam, a minute multi-spot light is obtained at the converging position of the convergent beam. At this time, a multi-spot light is formed by forming the convergent beam by a plurality of arranged lenses.

In addition, the converging position of the converging laser beam is 1
≦ M a mask arranged an opening 2 ^{M 'N} or more multi-spot light converging diameter of about arranged against' integer M satisfying ≦ M ', if such multi-spot light is condensed into the opening It becomes possible to obtain a pure minute multi-spot light without noise stray light.

The plurality of N laser beams can be doubled with respect to the beam diameter d of the laser beam emitted from the laser light source or the beam diameter d of the semiconductor laser converted into a parallel beam, even if the laser beam has an adjacent pitch of 4d or less. Further, it is possible to further double the distance between adjacent beams to 1/2 ^{M '} . That is, a multi-spot light having a pitch smaller than the diameter d of the parallel beam laser beam can be formed without using a lens system.

As a method of forming a plurality of N laser beams, a triangle having a cross section in which the hypotenuse is substantially 45 degrees with respect to the emission surface and a parallelogram having a total reflection surface in contact with and parallel to the hypotenuse are provided. At least one second beam doubling prism having a cross section is provided. In this way, for example, N / 2 laser beams can be formed into a plurality N of laser beams by the second beam doubling prism.

Further, two or more types of second beam doubling prisms having different dimensions are used, and these are sequentially arranged in a cascade of n stages from the larger one to the smaller one in the traveling direction of the beam. Is multiplied by 2 ⁿ . That is, when the size of the beam doubling prism in the last stage is D, the number is N / 2, the size of the beam doubling prism in the preceding stage is 2D, the number is N / 4, and the size of the first stage is D / 4. 2 ^n-1 D
And the number is N / 2 ⁿ . In this way, N
N laser beams are obtained from / 2 ^{n + 1} few beams.

According to the method described above, multi-spot light can be obtained from a laser beam emitted from one or a plurality of laser light sources. In addition, in any method of doubling the beam, one beam is performed by a polarizing element or a beam splitting prism, so that the total energy of the beam after the division into two can be 92% or more of the original beam energy. Also, even if the reflection loss on the surface of the polarizing element and the lens that forms the convergent beam is considered, 70% or more of the total energy of the total of the emitted laser energies can be used as the total energy of the multi-spot light.

Further, by performing the reflection loss on the surface of the optical component with an anti-reflection coat or the like, 90% or more of the total energy obtained by summing the respective emitted laser energies of the laser beams emitted from one or more laser light sources is multiplied. It can be made to be the total energy of the spot light.

Compared with the conventional method of forming a mastic spot from various places of the Gaussian distribution, the beam can be divided easily and uniformly, and the variation of each spot energy of the multi-spot light is within ± 20%. be able to.

Furthermore, it is possible to make the intensity of the doubled beam almost equal by optimizing the polarization state of the light incident on the above-mentioned polarizing element or optimizing the film thickness of the multilayer type polarizing beam splitter. It becomes possible, and the energy variation of each spot of multi spot light is ± 10%
Within.

By multiplying the laser beam by ^{2M 'as} described above, it is possible to form a multi-small spot close to each other, so that the multi-spot light obtained in this way can be subjected to confocal detection, fluorescence detection, or Used for DNA detection and the like. This makes it possible to detect light with high light use efficiency and small variation between multi-spot lights, which was impossible in the past, and as a result, high-speed and high-precision confocal detection, fluorescence detection, DNA detection, etc. Becomes possible.

The multi-spot light generated by the multi-spot light forming means of the present invention is projected onto the object to be observed, and the light reflected or transmitted by the object to be inspected is arranged at a position where the projected spot forms an image. Through the selected multi-aperture. The transmitted light of each of the openings is individually detected. By performing confocal detection in this manner, confocal detection with high light use efficiency and little variation between multi-spot lights is possible, and high-speed and high-accuracy confocal detection is realized.

The multi-spot light generated using the above-described multi-spot light forming means of the present invention is used as excitation light and projected onto an object to be observed including a phosphor. The fluorescence generated at the object to be inspected is separated from the excitation light by the excitation light, and the fluorescent light at each multi-spot light position is detected by disposing a detector at a position where the fluorescence generated at the excitation light projection spot forms an image. . By doing so, it is possible to simultaneously irradiate a plurality of positions with high-intensity excitation light, and since the variation in each spot is small, high-speed and high-accuracy fluorescence detection becomes possible.

In the above-mentioned fluorescence detection, a detector which transmits light through the multi-aperture arranged at a position where the fluorescence generated at the excitation light projection spot forms an image and individually detects the fluorescence transmitted light of each opening is arranged. By doing so, fluorescence other than the object to be observed, which is generated by the excitation light, is excluded, and fluorescence with a high signal-to-noise ratio can be detected.

The multi-spot light generated using the multi-spot light forming means of the present invention is used as excitation light and projected onto an object to be observed including DNA to which a fluorescent substance is added. The object to be observed has a known probe DNA adhered to a predetermined position on a substrate such as glass, and a target DNA obtained by adding a fluorescent substance to one end of DNA purified and amplified from a biological sample to be inspected is applied to the glass. The target DNA corresponding to the base sequence of the probe DNA is hybridized by flowing onto a substrate. The object to be measured is irradiated with multi-spot light excitation light to excite the fluorescent substance added to the DNA, thereby generating fluorescence. This fluorescence is separated from the excitation light, and a detector is arranged at a position where the fluorescence generated at the projection spot forms an image. The fluorescence at each multi-spot light position is detected, and D is obtained from the detected position and the detected signal intensity.
Check NA.

In the above DNA inspection, the fluorescence generated at the excitation light projection spot is transmitted through a multi-aperture arranged at a position where the image is formed, and the fluorescence transmitted light of each aperture is individually detected to thereby determine the position of each multi-spot light. By detecting the DNA by detecting the fluorescence intensity of the above, a DNA test with a higher signal-to-noise ratio can be performed.

In fluorescence detection and DNA inspection, the wavelengths of excitation light and fluorescence are often relatively close. In such a case, the use of the multi-spot light increases the visual field of detection and inspection, and the characteristics of the optical system that separates the fluorescence generated from each multi-spot excitation light from the excitation light inserted on the way to the detection system are not suitable. Will be enough. That is, the incident angle of the fluorescent light incident on the optical system that separates the fluorescent light from the excitation light, such as a wavelength separation beam splitter or an interference filter, which is input on the way from the imaging optical system to the detector, differs for each spot.
As a result, depending on the location of the multi-spot light, the excitation light to be shielded leaks, and accurate fluorescence detection cannot be performed. In order to solve the above-mentioned problem, the method of separating the fluorescence from the excitation light uses a wavelength separation beam splitter, and the fluorescence generated by each multi-spot light excitation emits the fluorescence from each spot from the imaging optical system to the imaging position. A telecentric imaging optical system in which principal rays are parallel to each other, and an interference filter or a wavelength separation beam splitter is inserted between the imaging optical system and an imaging position. In this way, the fluorescence from any spot excitation light enters the interference filter or the wavelength selection beam splitter at the same incident angle. As a result, the excitation light as a fluorescence component and a noise component from any spot can be selected under the same conditions, and therefore under optimum conditions, to select the fluorescence and to remove the excitation light, and the leakage of the excitation light is extremely small. Very little fluorescence detection is possible. As a result of the above, using this fluorescence detection method, it becomes possible to accurately and precisely perform a DNA test which needs to detect fluorescence having an extremely weak intensity with respect to the intensity of reflected light of excitation light.

[0027]

Embodiments of the present invention will be described below.

FIG. 1 is a diagram showing an embodiment of a method for obtaining a multi-spot light according to the present invention and its means.

A laser beam, which is substantially parallel light emitted from one or a plurality of laser light sources, is several meters by a method described later.
A plurality of (even in the figure) N / 2 laser beams 81 directed in the same direction parallel to each other with m pitches are formed, and N laser beams are formed by N / 2 beam doubling prisms 411, 412 and the like. The beam doubling prism is shown in FIG.
As shown in an enlarged perspective view in FIG.
14A and 14B, the beam doubling prism 411 is used.
And 412 do not necessarily correspond one-to-one. ), A triangle having a cross section that is approximately 45 degrees with respect to the exit surface, and a parallelogram cross section having a total reflection surface in contact with the slope and parallel to the slope.

Light incident on one side of the parallelogram is reflected by the beam splitting surface of the triangular slope approximately 50%, and the remaining approximately 50% is transmitted. The transmitted light passes through the triangular prism as it is, and the reflected light is totally reflected on the other surface of the parallelogram and passes through the parallelogram prism. The light that has passed through the triangular and parallelogram prisms is parallel to each other and has almost the same intensity. The interval between the two lights is P. As a result, the beam interval P is a half of the laser beam 81 having a beam interval of 2P.

Such a beam doubling prism is N / 2
By the beam doubling prism array 4 arranged at the pitch of 2P, the laser beam 81 becomes a plurality of N laser beams 8 having a pitch of P, substantially in the same direction, and adjacent to each other. By causing the plurality of N laser beams to be incident on a polarizing element 11 described later, a plurality of 2N laser beams heading in substantially the same direction and adjacent to each other are obtained.

As shown in FIG. 2, the polarizing element 11 is a transparent prism 1 having total reflection surfaces 1101 and 1102 parallel to the polarizing beam splitting surface 110 and located at distances L1 and L2 from the polarizing beam splitting surface 110, respectively.
It comprises a first multi-spot light doubling prism composed of 11 and 112. 1 is cut into a rectangular plano-convex lens as shown in the enlarged view in FIG. 1 and the cross-sectional enlarged view in FIG.
N strip-shaped convex lenses such as 1 and 22 are arranged corresponding to the positions of N parallel beams. The beams transmitted through the array 20 of strip-shaped convex lenses become convergent lights as shown in FIG. 2, and are incident on the polarization beam split surface in parallel with each other.

Since each beam is circularly polarized light or linearly polarized light of 45 degrees on the beam split incidence surface, as shown in FIG. 2, the S-polarized light component of the incident light 82 is reflected on this surface and the P-polarized light component is transmitted. As shown in FIG. 2, the reflected convergent light 822 transmits through the prism 111 and is incident on the 1101 plane parallel to the beam splitting plane 110 at an incident angle of 45 degrees. The convergent beam 822 'totally reflected on the surface 1101 is S-polarized light and is reflected again on the beam splitting surface 110. The reflected convergent beam 822 ″ passes through and exits the prism 111. On the other hand, the P-polarized light component
The convergent beam 821 transmitted through 10 is transmitted through the prism 112 and is a surface 1 parallel to the beam splitting surface 110.
The light is incident on the surface 102 at an incident angle of 45 degrees. The reflected light becomes a totally reflected convergent beam 821 ′ and reenters the beam split surface 110. The incident light is approximately 10
The transmitted light 821 ″ passes through the prism 111 and exits by 0%.

Total reflection surface 110 of prisms 111 and 112
1 and 1102 are at distances L1 and L2, respectively, from the beam splitting plane. The value of L1 and L2 is √2 (L2-
L1) is P which is half the pitch P of the N parallel beams 8
/ 2. Therefore, when light separated on the beam splitting plane passes through the edge responsive element, the two lights are shifted by P / 2. As a result, the N convergent beams having the pitch P incident on the polarization element 11 are converted into 2N convergent beams at a pitch of P / 2, and pass through the polarization element 11.

The exit surface of the prism 111 of the polarizing element 11 has a pinhole array mask 2 in which 2N pinhole openings 211 having a diameter d shown in FIG. Pass through. Since portions other than the 2N pinhole openings block light, stray light which is noise other than convergent light is blocked by the pinhole array mask 2. As a result, light transmitted through the pinhole array mask emits spot array light only from the pinhole portion.

The light transmitted through the pinhole array 2 is P and S linearly polarized light for each adjacent spot. A 波長 wavelength plate 3 is disposed immediately after the pinhole array 2.
The quarter-wave plate is set so that its optical axis is at 45 degrees with respect to the polarization directions of P and S. Therefore 1/4
The P- and S-polarized lights transmitted through the wave plate 3 become right and left-handed circularly polarized lights, respectively.

The 2N parallel circularly polarized beams that have passed through the quarter-wave plate 3 enter the polarizing element 12. Polarizing element 1
Reference numeral 2 denotes a polarizing beam splitter having substantially the same structure as the above-described polarizing element 11. That is, P-polarized light is transmitted and S-polarized light is reflected on the beam splitting surface 120. Also, the total reflection surface 120 of the prism 121 which passes after being reflected by the beam split surface
1 and distances L1 'and L1 between the total reflection surface 1202 of the prism 122 and the beam splitting surface which pass after being reflected.
2 ′ satisfies √2 (L2′−L1 ′) = ± P / 4. By doing so, 2N parallel beams having a pitch of P / 2 incident on the prism 12 can have a pitch of P /
4 4N parallel divergent beams are emitted from the prism 12.

The light emitted from the 2N spots on the pinhole array mask looks as if 4N multi-spot light is emitted from the mask surface by the polarizing element 12. That is, the reflecting surfaces 120, 1201, and 1202
When viewed from the side that has passed through the polarizing element 12, 4N multi-spot lights are formed on the surface of the mask 2 and emitted as divergent light from the mirror image effect.

As an example, the number of N is 16, and P is 1.6 mm.
Then, the number of multi-spot lights in the above embodiment is 64
And the pitch is 0.4 mm. As will be described in detail later, if this light is made incident on the objective lens of the afocal microscope through the imaging lens, if the magnification of the optical system is set to 20 times, a 2 μm spot on the sample to be inspected becomes 20 μm.
It becomes possible to simultaneously irradiate 64 spots at a pitch.

In the embodiment shown in FIG. 1, N / 2 multi-beams are incident on the second beam doubling prism array 4. When N is 8, if the laser light source is He-N
When a large intensity is required as the irradiation light instead of a large output like the e-laser, for example, eight He-Ne lasers are prepared and incident on the second beam doubling prism array 4 at a pitch of 1.6 mm. What should I do? Further, using eight semiconductor lasers, each of which is made a parallel beam,
What is necessary is just to compose eight beams in the same way as e-Ne, and to make it incident on the second beam doubling prism. In this case, since P is set to 1.6 mm, the interval 2P between the eight laser beams is 3 mm, which is larger than the beam diameter of the He-Ne laser beam of 1 mm, so that the laser beams can be incident in parallel from eight laser light sources. is there.

As described above, the diameter d of the beam emitted from the laser light source by the second beam doubling prism is four times the diameter d,
That is, even if the distance P is 4d or less, if the light enters the multi-spot light forming means using the polarizing element, the distance becomes 1
/ 2, and 1/2 ^{n when a} plurality of n polarizing elements are used.
Can be. Thus, a plurality of N laser beams incident at a pitch of four times or less the beam diameter d, which has been difficult in
The multi-beam ^'pitch P / 2 M of ^{N' M,} be formed efficiently on a multi-spot light, it has enabled.

FIG. 4 is a diagram showing another embodiment for forming a multi-spot beam light according to the present invention.
This is to form N multi-beams to be incident on the polarizing element by using one laser light source. For example, YAGS
When the output of the light source is large as in an HG laser, one light source is sufficient. In such a case, as shown in FIG. 4, in addition to the second beam doubling prism shown at 4 in FIG. A total of n = 4 stages are arranged in a cascade.

That is, one beam 80 emitted from one light source is made incident on a beam doubling prism 441 having an eight-fold size to obtain two beams 801 and 802 with an interval of 8P. The two beams are made incident on the beam doubling prisms 431 and 432 having four times the size, and four beams 4111, 8012, 8021 and 8022 having a pitch of 4P are obtained. These four beams are made incident on beam doubling prisms 421, 422, 423 and 424 of twice the size to obtain eight beams with a pitch of 2P. This is shown in FIG.
8 beam doubling prisms 411 and 41 shown in FIG.
2,413,414,415,416,417 and 41
8 and 16 beams with a pitch P are obtained. In this way, 16 beams of 1.6 mm pitch can be obtained from one beam emitted from one laser light source.

According to the method described in the above embodiment, it is possible to form a multi-spot light having a total energy of 70% or more of the energy of the laser beam emitted from the laser light source, which was impossible in the related art. Was.

Further, a laser light source is provided by applying an anti-reflection coating to optical components, applying a multilayer coating to reduce loss at the time of beam separation, or performing positioning of incident light to a pinhole array. It has become possible to form a multi-spot light having a total energy of 90% or more of the energy of the emitted laser beam.

Further, according to the method described in the above-described embodiment, it is possible to make the spot energy variation of the multi-spot light, which was impossible in the past, within ± 20%. Furthermore, by setting the conditions of the multilayer coating for beam separation and controlling the production, or by using FIG.
The polarizer 11 shown in FIG. 1A and the optical components such as the strip-shaped convex lens array 20, the pinhole array 2, the wave plate 3, and the polarizer 12 shown in FIG. And glue them with an optical adhesive. By doing so, the variation of the beam intensity generated by the reflection and interference at the surface of the optical component is greatly reduced, and the variation of each spot energy of the multi-spot light is reduced by ± 10.
% Can be formed.

In the above embodiment, the case where 64 or 16 multi-spot lights are formed has been described. However, the present invention is not limited to these.
= 2, that is, when eight or more multi-spot lights are obtained.

Next, an example in which the beam obtained by the above-described method for forming a multi-spot light is applied to fluorescence detection, particularly to DNA inspection using fluorescence detection, will be described with reference to FIG.

In FIG. 5, 8001, 8002, 80
03 denotes a laser light source, not shown, but a total of 8
It consists of a book laser. Beams emitted from each laser light source are mirrors 8011 and 801 having a size of several mm.
Are directed in one direction parallel to each other at a pitch of about 1.6 mm. Eight beams are obtained from the polarizing prism 12 by the method described above so that 64 multi-spot lights are emitted from the mask surface 3 at a pitch of 0.4 mm. These 64 multi-spot lights are focused on the imaging lens 6
1, the mirror 63, the wavelength selection beam splitter 70
Irradiates the inspection surface 51 of the DNA chip 5 through the objective lens 60.

On the surface of the DNA chip, predetermined DNA is probed for each predetermined position. On the other hand, when a target DNA obtained by adding a fluorescent substance to DNA produced by purification and amplification from a living body is allowed to flow on the probe DNA, hybridization occurs if the respective base sequences of the probe DNA and the target DNA correspond to each other. As a result, the target DNA to which the fluorescent substance is attached binds to the probe DNA. There are various types of phosphors to be added to the target DNA. In the present embodiment, the phosphor absorbs light of 633 nm from a He—Ne laser and emits fluorescence of 670 nm.
This fluorescence is received by the objective lens 60, and the transmitted light is reflected by the wavelength selection beam splitter 70 and guided to the detection optical system. The wavelength selection beam splitter 70 transmits 633 nm,
This is a multilayer coat beam splitter that reflects 0 nm.

The fluorescence reflected at 70 is reflected at the mirror 71 and passes through the imaging lens 62. The imaging lens 62 is a telecentric optical system such that the principal rays of fluorescence emitted from each multi-spot light applied to the phosphor on the surface of the DNA chip pass parallel to each other after passing through the imaging lens 62. As a result, the fluorescence coming from any of the multi-spot lights enters the wavelength separation beam splitter that reflects the light of the fluorescence wavelength at the same incident angle. 633nm of excitation light to fluorescence emitted from any excited spot for this is the noise component contained slightly also can be transmitted without being reflected most. That is, the wavelength separation beam splitter 72
, A small amount of excitation light of 633 nm is transmitted, but the light is transmitted and only the fluorescence is reflected, and travels to the detectors 91 and 92.

The above excitation light is reflected by the DNA chip,
The light is directed toward the detector, but is several orders of magnitude stronger than the intensity of the generated fluorescence. For this reason, as described above, the angle of incidence of the excitation light differs depending on the position of the spot of the excitation light, especially in the case of 70 which cannot be separated only by the wavelength selection beam splitter 70, and it is impossible to remove the excitation light in any spot similarly. In contrast, telecentric imaging lens 6
As described above, the wavelength selective beam splitters 72 and 73 placed between the detector 2 and the detectors 91 and 92 can reflect light from any spot position under the same incident condition.
The removal of the excitation light and the interference filters 74 and 75 almost completely shield the excitation light and allow only the fluorescence to be taken into the detector.

The detectors 91 and 92 are multi-channel photomultipliers, each having 32 light receiving apertures. 64 at the right angle mirror in front of Photomaru 91 and 92
The spot image of the fluorescence is separated into two, and is formed substantially at the light receiving aperture position of the photomultiplier. There is a pinhole array in front of the light receiving aperture of the photomultiplier. This pinhole array has an aperture and a pitch substantially equal to the size at which the multi-spot light of 2 μm diameter and 20 μm pitch irradiating the DNA chip forms an image at a magnification M of the objective lens and the imaging lens. That is, it has an opening having a diameter of 2 M μm and a pitch of 20 M. This pitch 20M is equal to the light receiving aperture pitch of the multi-channel photomultiplier.

In this manner, simultaneous irradiation with the multi-spot light array is performed, and the resulting fluorescence is confocal-detected, so that fluorescence with a high signal-to-noise ratio can be detected. The obtained 64 signals are amplified by a circuit (not shown),
The data is converted into digital information by an (analog-digital) converter, sent to a CPU (processing circuit) (not shown), and the data of the fluorescence intensity at the irradiation position is stored.

The stages 501 and 502 are driven, and the detection of the fluorescence intensity at different positions is sequentially obtained by the above method. To perform fluorescence detection with a large signal-to-noise ratio, the pitch of the irradiation spots is set to several times to several tens times the irradiation spot diameter. Therefore, in order to detect the entire inspection target, the xy stage is driven to detect not only the direction perpendicular to the array of the multi-spot light but also the direction of the array.

FIG. 6 is a diagram showing an embodiment of the present invention. The parallel beam 8 coming from the laser light source is circularly polarized or linearly polarized at 45 degrees on the paper. One of the plurality of parallel beams 8 is incident on one lens 21 of the array 20 of strip-shaped convex lenses, becomes a convergent beam, and is incident on a birefringent material such as calcite. The polarization component of ordinary light goes straight as it is, and the polarization component of extraordinary light is refracted. The thickness of the polarizing element is set so that the interval between transmission of the ordinary light and the extraordinary light is half the arrangement pitch P of the plurality of N incident light beams 8. As a result, after passing through the polarizer 11 ', 2N convergent beams advance in parallel at a pitch P / 2.

Each beam alternately has a linear polarization direction of 9
0 degrees different. A quarter-wave plate having an optical axis of 45 degrees in this polarization direction or 1 having an optical axis of 22.5 degrees
By passing through the / 2 wavelength plate 3, circularly polarized light or linearly polarized light having a polarization direction of ± 45 degrees with respect to the paper surface is obtained. This 2N
The beams are incident on the polarizer 12 '. The polarizer 12 'is calcite, half the thickness of 11'. This polarizer 12 '
, 4N focused beams are obtained at a pitch of P / 4.

By passing this light through the same wavelength plate 3 'as the above-described wavelength plate, it becomes a circularly polarized light or a linearly polarized light inclined at 45 degrees to the paper surface. The light is incident on the plate 2 composed of a multi-aperture array having the same aperture pitch as the pitch of the multi-beams and having an aperture diameter substantially equal to the converging diameter determined by the beam diameter incident on the lenses. In this way, a noise-free multi-spot light consisting only of the light transmitted through the aperture can be obtained.

FIG. 7 is an embodiment of a multi-spot light forming method used for confocal detection according to the present invention. 7 and 1
Are the same. A beam emitted from one or a plurality of laser light sources is converted into eight parallel beams, and the second beam doubling prism 4 converts the beams into sixteen parallel beams. These 16 parallel beams are converted into convergent beams by the convex lens array 20.

First multi-spot light doubling prism 1
By means of 1 and 12, 64 beams are obtained from the multi-spot light doubling prism 12 so that 64 multi-spot lights are effectively emitted from the opening of the mask 3. A 波長 wavelength plate 3 ′ is attached to the exit surface of the multi-spot light doubling prism 12, and 64 multi-spot lights are incident on a polarizing element 11 ′ made of calcite as circularly polarized light. This polarizer 11 ′, quarter-wave plate 3 ″, polarizer 1
The 2 ″, 波長 wavelength plate 3 ′ ″, polarizer 13 ′ and １ ／ wavelength plate 3 ″ ″ are the same as the beam splitting method described with reference to FIG. 6, and the polarizers 11 ′, 12 ″ and 13 ′ are used. The thickness of the calcite is reduced to t, 1 / 2t, 1 / 4t, and the direction in which the beam is split is the y direction, so the first beam doubling prisms 11, 12 are in the x direction. To 64
Eight spot lights are generated in the y direction by the beam and calcite beam splitting method, and a total of 64 × 8 spot lights are generated as shown in FIG.

FIG. 9 shows a confocal detection method using the above-mentioned 64 × 8 multi-spot lights. Components having the same reference numerals in FIG. 9 and other drawings represent the same components. 8 emitted from a laser light source to form eight beams
Eight beams from the beam optics 800 form 64 shown in FIG.
In the x8 multispot light forming optical system 100, 64 × 8 multispot light beams shown in FIG.

The multi-spot light is transferred to the half mirror 7
0 ′, the image is reduced and projected on the surface of the measured object 5 ′ by the imaging lens 61 and the objective lens 60. The projected multi-spot light is reflected by the surface to be measured, passes through the objective lens 60, the imaging lens 61, and the half mirror 70 ', and
An image is formed on the 64 × 8 pinhole mask 901 ′ arranged immediately before the imaging plane of 0 ′, and the light transmitted through this pinhole reaches the imaging plane of the detector. If the surface of the object to be measured matches the image plane of the projected multi-spot light, the reflected light forms an image on the pinhole of the pinhole mask,
Strong detection values are obtained. If they do not coincide with each other, the light becomes out-of-focus and spread to the pinhole of the pinhole mask 901 ', so that the intensity of the light transmitted through the pinhole decreases. Therefore, if the object to be measured is moved up and down by the z stage 503 'shown in FIG. 9 and the above detection is performed, the height at each spot position can be obtained as the maximum detection intensity at each position. It can be determined from the z value.

In the confocal detection method of FIG. 9 described above, if the object to be measured is fixed in the xy directions, for example, if the spot diameter on the surface to be measured is 1 μm and the spot interval is 8 μm,
Only 64 × 8 information at 8 μm intervals can be obtained. Therefore, it is necessary to move the xy stages 501 'and 502 "to sequentially detect confocal images at other positions. FIG. 10 is a diagram showing an embodiment of the present invention, and this two-dimensional confocal image is shown. The driving directions x ′ and y ′ of the xy stage in FIG. 9 and the arrangement direction of the multi-spot light projected onto the object to be measured are slightly shifted as shown in FIG. In the case of the multi-spot light, the angle θ between these two directions is set so that tan θ becomes 1/8, so that when the stage is scanned in the y ′ direction, the gap between spots 011 and 021 is lost. Spot 0
Seventeen spots of 12,013... 018 are illuminated, and a 1 μm spot is scanned every 1 μm over the entire surface.

When the above method is used, the object to be measured is irradiated with multi-spot light having a high light intensity, so that a 64 × 8 spot position can be detected in a short time. Therefore, the sample is rapidly moved in the y 'direction by the y stage.
The confocal signal of each height is sequentially detected at high speed by repeating scanning, scanning, slightly moving in the z direction, and scanning again. In this way, by obtaining the highest intensity at each point from the two-dimensional intensity data at each point obtained at each height z, it is possible to perform height measurement by a high-speed confocal detection method. If the scanning width is k times the pitch p of the multi-spot light, the number of pixels in the x direction is 6
4 × 8 = 512, two-dimensional height information of 8 k pixels in the y direction is obtained.

[0065]

According to the present invention, the laser light emitted from the laser light source can be converted into multi-spot light very efficiently, without waste, and the variation of each multi-spot light can be extremely reduced. As a result, high-speed, high-precision confocal detection can be performed using the multi-spot light. In addition, high-speed, high-precision fluorescence detection and DNA inspection can be performed using the multi-spot light.

[Brief description of the drawings]

FIG. 1 is a front view showing a configuration of an embodiment of a multi-spot light forming means according to the present invention.

FIG. 2 is a front view showing a configuration of one embodiment of a multi-spot light forming means according to the present invention.

FIG. 3 is a front view of the mask showing one embodiment of the mask according to the present invention.

FIG. 4 is a front view showing the configuration of an embodiment of a multi-spot light forming means according to the present invention.

FIG. 5 is a perspective view showing a configuration of an embodiment of a fluorescence detection and DNA inspection apparatus according to the present invention.

FIG. 6 is a front view showing one embodiment of a multi-spot forming means according to the present invention.

FIG. 7 shows a two-dimensional multi-spot forming means according to the present invention.
FIG. 1 is a front view showing an embodiment.

FIG. 8 is a front view showing an example of an arrangement of a two-dimensional multi-spot light according to the present invention.

FIG. 9 is a front view showing an embodiment of a confocal detection device according to the present invention.

FIG. 10 is a diagram showing the relationship between multi-spot light and stage scanning for confocal detection according to the present invention.

[Explanation of symbols]

11, 12, 11 ', 12', 13 '... polarizing element
2 Pinhole array mask 20 Lens array 3 3 ′ Wave plate 4 Beam doubling prism 5 Object under measurement 60 Objective lens 61
62 ... imaging lens 70, 71, 72, 73 ... wavelength selection beam splitter 90, 91, 92 ... detector 9 ... control circuit

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Kenji Yasuda 882 Ma, Hitachinaka-shi, Ibaraki F-term in the Hitachi Measuring Instruments Group (reference) 2G043 AA03 BA16 CA03 EA01 FA01 FA02 GA02 GA07 GB01 GB03 GB05 GB19 HA02 HA07 HA09 JA03 JA05 KA05 KA07 KA09 LA01 MA01 NA01

Claims (23)

[Claims] 1. A method in which a plurality of N laser beams, which are substantially in the same direction and are adjacent to each other, are incident on a polarizing element to be separated into two mutually orthogonal polarized component beams of each beam. A plurality of 2N laser beams, and then a plurality of 2N laser beams heading in substantially the same direction and adjacent to each other. 2. A method according to claim 1, wherein the plurality of N adjacent laser beams have a polarizing beam splitting surface and a total reflection surface parallel to the polarizing beam splitting surface and at a distance of L1 and L2 from the polarizing beam splitting surface. 2. The multi-spot light forming device according to claim 1, wherein the light is incident on the polarizing element constituted by the multi-spot light doubling prism, and the number of the plurality of adjacent laser beams is doubled to 2N. Method. 3. A plurality of N adjacent laser beams are incident on the polarizing element made of an anisotropic optical medium, and the inside of the anisotropic optical medium is converted into two mutually orthogonal polarized component beams of each beam. 2. The multi-spot light forming method according to claim 1, wherein, after separation, the plurality of 2N laser beams are directed to substantially the same direction, and the plurality of 2N laser beams are adjacent to each other. 4. The use of a plurality of polarizers, ^2M,
4. The multi-spot light forming method according to claim 1, wherein N laser spots are obtained. 5. The method according to claim 1, wherein the plurality of N adjacent laser beams incident on at least one of the one or more polarizing elements are convergent laser beams.
The method for forming a multi-spot light according to the above. 6. A multi-spot light forming method according to claim 5, wherein said plurality of convergent laser beams are formed by a plurality of arranged lenses. 7. The method according to claim 1, wherein the converging position of said converging laser beam is 1 ≦
Characterized in that a mask arranged an opening 2 ^{M 'N} or more multi-spot light converging diameter of about relative' integer M satisfying ≦ M 'M arranged to obtain a noise stray-free pure fine multi-spot light The method for forming a multi-spot light according to claim 1, wherein: 8. The multi-beam laser according to claim 1, wherein said plurality of N laser beams have an adjacent pitch of 4d or less with respect to a beam diameter d of a laser beam emitted from a laser light source or a parallelized semiconductor laser. Spot light forming method. 9. The plurality of N laser beams have a triangular section having a cross section where the hypotenuse is substantially 45 degrees with respect to the emission surface, and a parallelogram cross section having a total reflection surface in contact with and parallel to the hypotenuse. 9. The multi-spot light forming method according to claim 1, wherein the multi-spot light forming method is formed using one or more second beam doubling prisms. 10. The second beam doubling prism has different dimensions, and the prisms are arranged in a cascade of n stages in order from a larger beam to a smaller beam in the traveling direction of the beam to double the number of beams by 2 ^n. 10. The multi-spot light forming method according to claim 9, wherein the plurality of N laser beams are obtained. 11. A multi-spot light forming method, wherein 70% or more of the total energy of laser beams emitted from one or more laser light sources is the total energy of the multi-spot light. 12. The multi-spot light forming method according to claim 11, wherein 90% or more of the total energy obtained by summing the respective emitted laser energies of the laser beams emitted from the one or more laser light sources is the total energy of the multi-spot light. 13. A multi-spot light is projected onto an object to be observed, and light reflected or transmitted by the object to be inspected is transmitted through a multi-aperture arranged at a position where the projected spot forms an image. 13. A confocal detection method for performing confocal detection by detecting at a time, wherein:
A confocal detection method characterized by using the multi-spot light forming method according to any one of the above. 14. A multi-spot light formed by using the multi-spot light forming method according to any one of claims 1 to 13 as excitation light, and projecting the excitation light to an object to be observed including a phosphor, It is characterized in that the fluorescence generated at the object to be inspected is separated from the excitation light, and the fluorescence at the position of each multi-spot light is detected by disposing a detector at a position where the fluorescence generated at the projection spot forms an image. Fluorescence detection method. 15. A multi-spot light formed by using the multi-spot light forming method according to claim 1 as excitation light, and projecting the excitation light onto an object to be observed including a phosphor, The fluorescence generated in the object to be inspected is separated from the excitation light, and transmitted through a multi-aperture disposed at a position where the fluorescence generated at the projected location forms an image. A fluorescence detection method comprising detecting fluorescence at a multi-spot light position. 16. The method for separating fluorescence from excitation light as described above,
In addition to using a wavelength separation beam splitter, a telecentric imaging optical system in which the fluorescence generated by each multi-spot excitation light from each spot from the imaging optical system to the imaging position is parallel to each other, 2. A method of inserting an interference filter or a wavelength separation beam splitter between an image optical system and an image forming position.
16. The method for detecting fluorescence according to 4 or 15. 17. A multi-spot light formed by using the multi-spot light forming method according to any one of claims 1 to 13, which is projected as excitation light onto an object to be observed including a DNA to which a fluorescent substance is added, and Separating the fluorescence generated in the object to be inspected by the projection from the excitation light reflected by the object to be inspected,
By locating a detector at the position where the fluorescence generated at the projection spot forms an image, the fluorescence generated at the position where each multi-spot light is projected is detected, and the DNA is detected from the information of the detected position and the detected signal intensity. A DNA test method, comprising: testing DNA. 18. A multi-spot light formed by using the multi-spot light forming method according to any one of claims 1 to 13, which is projected as excitation light onto a test object containing DNA to which a fluorescent substance is added, and Separating the fluorescence generated by the object to be inspected by the projection from the excitation light reflected by the object to be inspected, passing the fluorescence through a plurality of openings arranged at positions where the fluorescence forms an image, A DNA inspection method, wherein the fluorescence intensity at the position where the multi-spot light is projected is detected by individually detecting the transmitted fluorescence. 19. The method for separating fluorescence from excitation light as described above,
While using a wavelength separation beam splitter, the telecentric imaging optical system in which the principal rays of the fluorescence from each spot from the imaging optical system to the imaging position are generated by excitation by each multi-spot light and parallel to each other, 19. The DNA inspection method according to claim 17, wherein an interference filter or a wavelength separation beam splitter is inserted between the imaging optical system and the imaging position. 20. A laser beam emitted from a laser light source is branched to form eight or more beams, and the branched and formed eight or more beams are irradiated on an inspection surface of a DNA chip.
Fluorescence generated from the DNA chip by the irradiation is divided into eight or more beams corresponding to each of the irradiated beams, separated from the reflected light of the beams and detected, and the fluorescence detected corresponding to the respective beams is detected. DNA based on information
A DNA inspection method, comprising inspecting a chip. 21. A laser beam emitted from a laser light source is branched into a plurality of beams having substantially equal intensities, and images of the plurality of branched beams are projected onto an inspection surface of a DNA chip. A DNA inspection method comprising: capturing an image of fluorescence generated from the DNA chip using the image of the DNA chip; and inspecting the DNA chip based on information of the captured fluorescence image. 22. The DNA chip and the beam are
The beam is moved to the DN while being relatively moved in dimension.
22. The DNA inspection method according to claim 20, wherein the DNA chip is inspected by irradiating an A chip. 23. The DNA inspection method according to claim 20, wherein the branched beams are two-dimensionally arranged and irradiated on the DNA chip.