JP3675273B2 - Multi-spot light forming method, confocal detection method, fluorescence detection method, and DNA inspection method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for efficiently forming a plurality of spots from laser light, and further to a method for detecting confocal light using this method, a method for detecting fluorescence, and a method for examining DNA.
[0002]
[Prior art]
A method of irradiating an object to be detected with multi-spot light and detecting a confocal point uses a Nippon disc having a large number of openings on a disk. In this method, the entire disk is irradiated with light, and light passing through the opening becomes illumination light. Therefore, most of the light emitted from the light source is shielded at portions other than the opening, and the light energy contributing to the illumination is only a few percent of the light emitted from the light source.
[0003]
[Problems to be solved by the invention]
When forming multi-spot light from a beam emitted from a laser light source and trying to make the emitted light into multi-spot light without waste, for example, 50 rows of 0.04 mm diameter arranged in a row with a pitch of 0.4 mm are used. In order to form multi-spot light, the emitted laser beam is shaped into an oval shape with an aspect ratio of 1:50. When this oval beam is irradiated onto a microlens array arranged at a pitch of 0.4 mm, if the focal length of this lens is, for example, 60 mm, multi-spot light having a diameter of approximately 0.04 mm is 50 at a pitch of 0.4 mm. Can be obtained. Since the intensity distribution of the laser beam is a Gaussian distribution, the multi-spot light formed by such a method is bright in the vicinity of the center of the array of 50 multi-spot lights, and the periphery is dark. In order to make the intensities of the central and peripheral spots as equal as possible, the spread of the incident oval 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.
[0004]
An object of the present invention is to solve the problems that the light use efficiency in such a conventional multi-spot light forming method is poor and the intensity variation between the multi-spot lights is large, and to obtain multi-spot light with almost uniform intensity. It is to provide a method and apparatus thereof.
[0005]
It is a further object of the present invention to provide a method and apparatus for performing confocal detection, fluorescence detection, and DNA inspection using multi-spot light having substantially uniform intensity.
[0006]
[Means for Solving the Problems]
In order to solve the above-described problems and obtain close multi-spot light, the present invention has the following configuration.
[0007]
That is, a plurality of N laser beams which are directed in substantially the same direction and are adjacent to each other are made incident on a polarizing element, thereby separating the beams into two orthogonally polarized light beams. A plurality of 2N laser beams are used. Thereafter, a plurality of 2N laser beams that are directed in substantially the same direction and are adjacent to each other are formed.
[0008]
As a specific method of obtaining such a double adjacent beam, a plurality of N adjacent laser beams are divided into a polarization beam splitting plane, parallel to the polarization beam splitting plane, and L1 and L2 from the polarization beam splitting plane. The light is incident on the polarizing element including a multi-spot light doubling prism having a total reflection surface at a distance. In this way, the number of the adjacent laser beams can be doubled to 2N.
[0009]
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 separated into two mutually orthogonal polarization component beams. A 2N laser beam is used. In this way, it is possible to obtain a plurality of 2N laser beams that are directed substantially in the same direction and are adjacent to each other.
[0010]
Further, by using a plurality of M polarizing elements for doubling the beam as described above, 2 ^M N laser beams can be obtained.
[0011]
When a plurality of N adjacent laser beams incident on at least one of the one or more polarizing elements are converged laser beams, a minute multi-spot light can be obtained at the converged position of the converged beam. At this time, this convergent beam is formed by a plurality of lenses arranged to form multi-spot light.
[0012]
In addition, the convergence position of the convergent laser beam is 2 for an integer M ′ that satisfies 1 ≦ M ′ ≦ M. ^{M '} It is possible to obtain pure micro multi-spot light free from noise stray light by arranging a mask in which openings of about N or more multi-spot light convergence diameters are arranged and concentrating the multi-spot light on this opening. Become.
[0013]
The above-mentioned multiple N laser beams can be doubled even when they have an adjacent interval pitch of 4d or less with respect to the beam diameter d of the laser light source emitted beam or the parallel laser beam. To reduce the adjacent beam spacing by half ^{M '} It is possible to That is, multi-spot light having a pitch smaller than the diameter d of the laser beam of the parallel light beam can be formed without using a lens system.
[0014]
As a method for forming the plurality of N laser beams, the triangle has a cross section whose hypotenuse is approximately 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 slope. One or more second beam doubling prisms are provided. In this way, for example, N / 2 laser beams can be formed into a plurality of N laser beams by the second beam doubling prism.
[0015]
Further, two or more types of the second beam doubling prisms having different dimensions are used, and these are arranged in an n-stage cascade from the largest to the smallest in the beam traveling direction, and the number of beams is set to 2. ⁿ Double. That is, when the dimension of the beam doubling prism at the last stage is D, this number is N / 2, the dimension of the beam doubling prism at the preceding stage is 2D, the number is N / 4, and the dimension at the first stage is 2 ^n-1 D and the number is N / 2 ⁿ And In this way, N / 2 ^{n + 1} N laser beams can be obtained from the small number of beams.
[0016]
By the method described above, multi-spot light can be obtained from a laser beam emitted from one or a plurality of laser light sources. Moreover, since all the beam doubling methods use one beam by a polarizing element or a beam splitting prism, the total energy of the beam after bisection can be 92% or more of the original beam energy. Even when taking into account the lens for forming the convergent beam and the reflection loss on the surfaces of these polarizing elements, 70% or more of the total energy of the emitted laser energy can be made the total energy of the multi-spot light.
[0017]
Furthermore, by performing reflection loss on the surface of the optical component with an anti-reflection coating or the like, 90% or more of the total energy of the laser beams emitted from one or more laser light sources is added to the multi-spot light. Can be total energy.
[0018]
Compared with the conventional method of forming a mast spot from various parts of the Gaussian distribution, the beam can be divided easily and the variation of each spot energy of the multi-spot light can be within ± 20%. .
[0019]
Moreover, the intensity of the doubled beam can be made substantially equal by optimizing the polarization state of the light incident on the polarizing element or by optimizing the film thickness of the multilayer type polarizing beam splitter. The energy variation of each spot of the multi-spot light can be made within ± 10%.
[0020]
2 of the laser beam explained above ^{M '} By doubling, it becomes possible to form multi-spots that are close to each other, and thus the multi-spot light thus obtained is used for confocal detection, fluorescence detection, DNA detection, or the like. This makes it possible to perform detection with high light utilization efficiency and small variation between multi-spot lights, which has been impossible in the past. As a result, high-speed, high-precision confocal detection, fluorescence detection, DNA detection, etc. Is possible.
[0021]
A multi-aperture which projects the multi-spot light produced using the multi-spot light forming means of the present invention described above onto the object to be observed, and arranges the light reflected or transmitted by the object to be inspected at the position where the projection spot forms an image. Make it transparent. The transmitted light of each opening is individually detected. By performing confocal detection in this way, it is possible to perform confocal detection with high light utilization efficiency and little variation between multi-spot lights, thereby realizing high-speed and high-precision confocal detection.
[0022]
The multi-spot light produced by using the above-described multi-spot light forming means of the present invention is used as excitation light and projected onto an observation object including a phosphor. The fluorescence generated in the object to be inspected by the excitation light is separated from the excitation light, and the fluorescence at each multi-spot light position is detected by arranging a detector at the position where the fluorescence generated in the excitation light projection spot forms an image. . By doing so, it is possible to irradiate a plurality of positions with high-intensity excitation light at the same time, and since the variation of each spot is small, high-speed and high-precision fluorescence detection becomes possible.
[0023]
In the fluorescence detection described above, a detector that transmits the fluorescence generated at the excitation light projection spot through a multi-aperture disposed at a position where an image is formed and individually detects the fluorescence transmitted light of each aperture is disposed. In this way, fluorescence other than the observed object generated by the excitation light is eliminated, and fluorescence detection with a high signal-to-noise ratio becomes possible.
[0024]
The multi-spot light produced by 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 DNA to which a phosphor is added. The object to be observed has a known probe DNA attached to a predetermined position on a substrate such as glass, and the target DNA obtained by adding a fluorescent substance to one end of DNA purified and amplified from a biological sample to be inspected is added to the glass. A target DNA corresponding to the base sequence of the probe DNA is hybridized on a substrate. The object to be measured is irradiated with multi-spot light excitation light to excite the phosphor added to the DNA to generate fluorescence. This fluorescence is separated from the excitation light, and the fluorescence at each multi-spot light position is detected by placing a detector at the position where the fluorescence generated at the projection spot forms an image, and DNA is detected from the detected position and the detected signal intensity. inspect.
[0025]
In the above DNA inspection, the fluorescence generated at the excitation light projection spot is transmitted through a multi-aperture disposed at a position where an image is formed, and the fluorescence intensity at each multi-spot light position is detected by individually detecting the fluorescence transmitted light at each aperture. By testing the DNA by detecting the DNA, a DNA test with a larger signal-to-noise ratio can be performed.
[0026]
In fluorescence detection and DNA inspection, the wavelengths of excitation light and fluorescence are often relatively close. In such a case, if multi-spot light is used, the field of view of detection and inspection becomes large, and the characteristics of the optical system that separates fluorescence from excitation light inserted in the middle of guiding fluorescence generated by each multi-spot excitation light to the detection system are poor. It will be enough. That is, the incident angle of the fluorescence incident on the optical system that separates the fluorescence from the excitation light such as a wavelength separation beam splitter or an interference filter that is input on the way from the imaging optical system to the detector is different for each spot. As a result, the excitation light to be shielded leaks depending on the location of the multi-spot light, and accurate fluorescence detection cannot be performed.
In order to solve the above problems, the method of separating the fluorescence from the excitation light uses a wavelength separation beam splitter, and the fluorescence generated by the excitation of each multi-spot light causes the fluorescence from each spot from the imaging optical system to the imaging position. A telecentric imaging optical system in which chief rays are parallel to each other is inserted, and an interference filter or a wavelength separation beam splitter is inserted between the imaging optical system and the imaging position. In this way, the fluorescence from any spot excitation light enters the interference filter or the wavelength selective beam splitter at the same incident angle. As a result, the excitation light that becomes the fluorescence and noise components from any spot can be selected under the same conditions, and therefore under the optimum conditions, respectively, and the excitation light can be removed, and the leakage of the excitation light is extremely small. Very little fluorescence detection is possible. As a result of the above, further using this fluorescence detection method, it is possible to accurately and accurately perform a DNA test that needs to detect fluorescence with a very weak intensity with respect to the reflected light intensity of the excitation light.
[0027]
DETAILED DESCRIPTION OF THE INVENTION
Examples of the present invention will be described below.
[0028]
FIG. 1 is a diagram showing an embodiment of a method and means for obtaining multi-spot light according to the present invention.
[0029]
The laser beam which is substantially parallel light emitted from one or a plurality of laser light sources becomes a plurality of (even number in the figure) N / 2 laser beams 81 directed in the same direction parallel to each other with a pitch of several mm by the method described later. N / 2 beams are doubled by N / 2 beam doubling prisms 411, 412 and the like. As shown in the enlarged perspective view of FIG. 1B, the beam doubling prism is arranged with the beam doubling prisms 411 and 412 in FIGS. 1A and 1B. Does not necessarily correspond one-to-one), and has a triangle having a cross section of approximately 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 slope. ing.
[0030]
About 50% of the light incident on one side of the parallelogram is reflected by the beam split surface of the triangular slope, and the remaining 50% is transmitted. The transmitted light passes through the triangular prism as it is, and the reflected light is totally reflected by the other surface of the parallelogram and passes through the parallelogram prism. In this way, the lights that pass through the triangular and parallelogram prisms are parallel to each other and have substantially the same intensity. The distance between the two lights is P. As a result, the beam interval P is half that of the laser beam 81 having a beam interval of 2P.
[0031]
With the beam doubling prism array 4 in which N / 2 beam doubling prisms are arranged at a pitch of 2P, the laser beam 81 has a pitch of P and faces substantially in the same direction, and the adjacent ones are close to each other. Multiple N laser beams 8 are obtained. By making the plurality of N laser beams incident on a polarizing element 11 described later, a plurality of 2N laser beams which are directed in substantially the same direction and are adjacent to each other are formed.
[0032]
As shown in FIG. 2, the polarizing element 11 is parallel to the polarizing beam splitting surface 110 and is formed from transparent prisms 111 and 112 having total reflection surfaces 1101 and 1102 at distances L1 and L2 from the polarizing beam splitting surface 110, respectively. And a first multi-spot light doubling prism. In the incident surface of the N beams of the polarizing element 11, strips such as 21 and 22 are obtained by cutting a spherical plano-convex lens into strips as shown in the enlarged view of FIG. 1 and the enlarged cross-sectional view of FIG. Convex lenses are arranged corresponding to the positions of N parallel beams. As shown in FIG. 2, the beams transmitted through the strip-shaped convex lens array 20 become convergent lights, and are incident on the polarization beam splitting plane in parallel with each other.
[0033]
Since each beam is circularly polarized light or linearly polarized light of 45 degrees on the incident plane of the beam split, as shown in FIG. 2, the S-polarized component of the incident light 82 is reflected by this surface and the P-polarized component is transmitted. As shown in FIG. 2, the reflected convergent light 822 passes through the prism 111 and enters the surface 1101 that is parallel to the beam split surface 110 at an incident angle of 45 degrees. Since the convergent beam 822 ′ totally reflected by the 1101 plane is S-polarized light, it is reflected again by the beam splitting plane 110. The reflected convergent beam 822 ″ passes through the prism 111 and exits.
On the other hand, the convergent beam 821 having transmitted the P-polarized component through the beam splitting surface 110 is transmitted through the prism 112 and is incident on the 1102 surface parallel to the beam splitting surface 110 at an incident angle of 45 degrees. The reflected light becomes a total reflection convergent beam 821 ′ and enters the beam splitting surface 110 again. Since the incident light is P-polarized light, it is almost 100% transmitted, and this transmitted light 821 ″ passes through the prism 111 and exits.
[0034]
Total reflection surfaces 1101 and 1102 of prisms 111 and 112 are at distances L1 and L2 from the beam splitting surface, respectively. The values of L1 and L2 are P / 2 where √2 (L2−L1) is half the pitch P of the N parallel beams 8. Therefore, when the light separated at the beam splitting plane passes through the side response element, the two lights are shifted by P / 2. As a result, the N convergent beams having a pitch P incident on the polarizing element 11 become 2N convergent beams having a pitch of P / 2 and pass through the polarizing element 11.
[0035]
On the exit surface of the prism 111 of the polarizing element 11, there is a pinhole array mask 2 in which 2N pinhole openings 211 having a diameter d shown in FIG. 3 are arranged at a pitch P / 2, and convergent light passes through this pinhole. . Since the portions other than the 2N pinhole openings shield the light, stray light that becomes noise other than the convergent light is shielded by the pinhole array mask 2. As a result, the light transmitted through the pinhole array mask is emitted as spot array light only from the pinhole portion.
[0036]
The light transmitted through the pinhole array 2 is linearly polarized light of P and S for each adjacent spot. A quarter wave plate 3 is disposed immediately after the pinhole array 2. This quarter-wave plate is set so that its optical axis is 45 degrees with respect to the P and S polarization directions. Therefore, the P and S polarized lights that pass through the quarter wavelength plate 3 become right and left-handed circularly polarized lights, respectively.
[0037]
The 2N parallel circularly polarized beams that have passed through the quarter-wave plate 3 enter the polarizing element 12. The polarizing element 12 is a polarizing beam splitter having substantially the same structure as the polarizing element 11 described above. That is, the P-polarized light is transmitted through the beam splitting surface 120 and the S-polarized light is reflected. Further, the total reflection surface 1201 of the prism 121 that passes after being reflected by the beam splitting surface, and the distances L1 ′ and L2 ′ between the total reflection surface 1202 of the prism 122 that passes after the reflection and the beam splitting surface have values of √2 ( L2′−L1 ′) = ± P / 4 is satisfied. By doing so, 2N parallel beams with a pitch P / 2 incident on the prism 12 become 4N parallel divergent beams with a pitch P / 4 and are emitted from the prism 12.
[0038]
The light emitted from the 2N spots on the pinhole array mask appears as if 4N multi-spot lights are emitted from the mask surface by the polarizing element 12. In other words, 4N multi-spot lights can be formed on the surface of the mask 2 when viewed from the side that has passed through the polarizing element 12 due to the mirror image effect of the reflecting surfaces 120, 1201, and 1202, and are emitted as diverging light therefrom. .
[0039]
As an example, if the number of N is 16 and P is 1.6 mm, 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 incident on an objective lens of an infinite focus microscope through an imaging lens, a 2 μm spot is formed on the sample to be inspected by 20 μm if the magnification of the optical system is 20 times. It is possible to irradiate 64 spots simultaneously at a pitch.
[0040]
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 does not have a large output as in the case of a He-Ne laser and a large intensity is required as irradiation light, for example, eight He-Ne lasers are prepared and the second light is emitted at a pitch of 1.6 mm. It is sufficient that the light beam is incident on the beam doubling prism array 4. Further, eight semiconductor lasers may be used, each of which is made into a parallel beam to form eight beams in the same manner as the above He-Ne, and enter 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 1 mm of the He—Ne laser beam, and can be incident in parallel from the eight laser light sources. is there.
[0041]
In this way, even if the distance P is 4 times the diameter d of the beam emitted from the laser light source by the second beam doubling prism, that is, the interval P is 4d or less, the light is incident on the multi-spot light forming means using the polarizing element. The interval can be halved, and if a plurality of n polarizing elements are used, ½ ⁿ Can be. As described above, 2 N laser beams incident at a pitch of 4 times or less the beam diameter d, which has been difficult in the past, are obtained. ^{M '} N pitches P / 2 ^{M '} The multi-beam can be formed efficiently into multi-spot light.
[0042]
FIG. 4 is a diagram showing another embodiment for forming multi-spot beam light according to the present invention, in which N multi-beams incident on a polarizing element are formed using one laser light source. . For example, when the output of the light source is large, such as a YAGSHHG laser, even 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 cascade.
[0043]
That is, one beam 80 emitted from one light source is incident on a beam doubling prism 441 having a size of 8 times, and two beams 801 and 802 having an interval of 8P are obtained. These two beams are incident on the beam doubling prisms 431 and 432 having a quadruple size, and four beams 8011, 8012, 8021, and 8022 having a pitch 4P are obtained. The four beams are incident on the beam doubling prisms 421, 422, 423, and 424 having a double size to obtain eight beams having a pitch of 2P. This is incident on the eight beam doubling prisms 411, 412, 413, 414, 415, 416, 417 and 418 shown in FIG. 1B, and 16 beams with a pitch P are obtained. In this way, 16 beams with a pitch of 1.6 mm can be obtained from one beam emitted from one laser light source.
[0044]
The method described in the above embodiment makes it possible to form 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 has been impossible in the past.
[0045]
Furthermore, it is emitted from the laser light source by applying an anti-reflection coating for optical components, reducing loss during beam separation, applying a multilayer coating, or aligning incident light to the pinhole array, etc. It has become possible to form multi-spot light having a total energy of 90% or more of the energy of the laser beam.
[0046]
Further, the method described in the above embodiment makes it possible to make the variation of each spot energy of multi-spot light within ± 20%, which was impossible in the past.
Furthermore, the condition of the multi-layer coating for beam separation and the production control, or the polarizer 11 in FIG. 1 (a), the strip-shaped convex lens array 20 and the pinhole array shown in detail in FIG. 1 (c). 2 and the optical components such as the wave plate 3 and the polarizer 12 are bonded to each other with an optical adhesive. By doing so, the variation in beam intensity caused by reflection and interference on the surface of the optical component is greatly reduced, and the formation of multi-spot light can be achieved within ± 10% of the variation in spot energy of multi-spot light. It became possible.
[0047]
In the above embodiment, the case of forming 64 or 16 multi-spot lights has been described. However, the present invention is not limited to these, and when N = 2, that is, eight or more multi-spot lights. It is effective when obtaining.
[0048]
Next, an example in which the beam obtained by the multi-spot light forming method described above is applied to fluorescence detection, particularly to DNA inspection using fluorescence detection, will be described with reference to FIG.
[0049]
In FIG. 5, reference numerals 8001, 8002, 8003,... Are laser light sources, which are constituted by a total of eight lasers (not shown). Beams emitted from the laser light sources are directed in parallel to each other at a pitch of about 1.6 mm by mirrors 8011, 8012, 8013. The eight beams are obtained from the polarizing prism 12 so that 64 multi-spot lights are emitted from the mask surface 3 at a pitch of 0.4 mm by the method described above. The 64 multi-spot lights pass through the imaging lens 61, pass through the mirror 63 and the wavelength selection beam splitter 70, and irradiate the inspection surface 51 of the DNA chip 5 through the objective lens 60.
[0050]
On the surface of the DNA chip, DNA predetermined for each predetermined position is probed. On the other hand, when a target DNA having a fluorescent substance added to DNA purified and amplified from a living body is flowed on the above probe DNA, it hybridizes if the base sequences of the probe DNA and target DNA correspond to each other. Thus, the target DNA to which the phosphor is attached binds to the probe DNA.
There are various kinds of phosphors to be added to the target DNA. In this embodiment, the phosphor absorbs light of 633 nm from a He—Ne laser and emits fluorescence of 670 nm. The 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 selective beam splitter 70 is a multilayer coat beam splitter that transmits 633 nm and reflects 670 nm.
[0051]
The fluorescence reflected by 70 is reflected by the mirror 71 and passes through the imaging lens 62. The imaging lens 62 is a telecentric optical system so that the fluorescent principal rays emitted from each multi-spot light irradiated to the phosphor on the surface of the DNA chip travel parallel to each other after passing through the imaging lens 62. As a result, the fluorescence from any multi-spot light is incident at the same incident angle on the wavelength separation beam splitter that reflects the light having the fluorescence wavelength. 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, a slight amount of 633 nm excitation light is incident on the wavelength separation beam splitter 72, but the light is transmitted and only the fluorescence is reflected toward the detectors 91 and 92.
[0052]
The excitation light is reflected by the DNA chip and travels toward the detector side, but is several orders of magnitude stronger than the intensity of the generated fluorescence. Therefore, as described above, it is impossible to separate the light only by the wavelength selective beam splitter 70. In particular, the angle at which light is incident on 70 differs depending on the position of the spot of the excitation light, and it is impossible to remove the excitation light in any spot. In contrast, the wavelength selective beam splitters 72 and 73 placed between the telecentric imaging lens 62 and the detectors 91 and 92 can reflect light from any spot position under the same incident condition as described above. . By removing the excitation light and the interference filters 74 and 75, the excitation light can be almost completely blocked and only the fluorescence can be taken into the detector.
[0053]
The detectors 91 and 92 are multi-channel photomultipliers, each having 32 light receiving apertures. The 64 spot images of the fluorescence are separated into two by a right angle mirror in front of the photomultipliers 91 and 92, and are formed almost at the photomultiplier opening position. There is a pinhole array in front of the photomultiplier aperture. This pinhole array has openings and pitches substantially equal to the dimensions at which the excitation multi-spot light having a diameter of 2 μm and a diameter of 20 μm irradiating the DNA chip is imaged at a magnification M of the objective lens and the imaging lens. That is, it has an opening with a diameter of 2M μm and a 20M pitch. This pitch 20M is equal to the light receiving aperture pitch of the multichannel photomultiplier.
[0054]
By doing so, the multi-spot light array is simultaneously irradiated and the resulting fluorescence is confocally detected, so that fluorescence detection with a high signal-to-noise ratio can be performed. The obtained 64 signals are amplified by a circuit (not shown), converted into digital information by an AD (analog / digital) converter, sent to a CPU (processing circuit) (not shown), and fluorescence intensity data at the irradiation position is stored. The
[0055]
The stages 501 and 502 are driven, and the detection of the fluorescence intensity at different positions is sequentially obtained by the above method. In order to detect fluorescence with a large signal-to-noise ratio, the pitch of the irradiation spot is set to several to several tens of times the irradiation spot diameter. Accordingly, in order to detect the entire inspection object, the xy stage is driven and detected not only in the direction perpendicular to the arrangement of the multi-spot light but also in the arrangement direction.
[0056]
FIG. 6 is a diagram of an embodiment of the present invention. The parallel beam 8 coming from the laser light source is circularly polarized light or linearly polarized light of 45 degrees on the paper surface. One of the plurality of parallel beams 8 is incident on one lens 21 in an array 20 of strip-like convex lenses, becomes a convergent beam, and enters 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 transmission interval between the ordinary light and the extraordinary light is half the arrangement pitch P of the incident beam light 8 composed of a plurality N. As a result, after passing through the polarizer 11 ', 2N convergent beams travel in parallel at a pitch P / 2.
[0057]
Each beam is alternately 90 degrees different in the polarization direction of linearly polarized light. By passing a quarter-wave plate having an optical axis of 45 degrees in this polarization direction or a half-wave plate 3 having an optical axis of 22.5 degrees, it has ± 45 degrees of polarization direction with respect to circularly polarized light or paper. Obtain linearly polarized light. The 2N beams are incident on the polarizer 12 '. The polarizer 12 'is a calcite having a thickness half of 11'. When transmitted through the polarizer 12 ', 4N focused beams are obtained at a pitch P / 4.
[0058]
By passing this light through the same wave plate 3 'as the wave plate described above, it becomes circularly polarized light or linearly polarized light tilted 45 degrees to the paper surface. The light beam is incident on the plate 2 formed of a multi-aperture array having the same aperture diameter as the multi-beam pitch, which is the same as the multi-beam pitch and determined by the beam diameter incident on the lens. In this way, noise-free multi-spot light consisting only of light transmitted through the opening can be obtained.
[0059]
FIG. 7 is an embodiment diagram of a multi-spot light forming method used for confocal detection according to the present invention. 7 and FIG. 1 having the same part number are the same. The beams emitted from one or a plurality of laser light sources are converted into eight parallel beams, and the second beam doubling prism 4 converts the beams into 16 parallel beams. These 16 parallel beams are converted into convergent beams by the convex lens array 20.
[0060]
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 by the first multi-spot light doubling prisms 11 and 12. A quarter-wave plate 3 ′ is attached to the exit surface of the multi-spot light doubling prism 12, and 64 multi-spot light is incident on a polarizing element 11 ′ made of calcite with circular polarization. The polarizer 11 ', the quarter-wave plate 3 ", the polarizer 12", the quarter-wave plate 3'", the polarizer 13 ', and the quarter-wave plate 3""are split into two beams as described with reference to FIG. The method is the same, and the polarizers 11 ', 12 "and 13' have calcite thicknesses as thin as t, 1 / 2t and 1 / 4t. The direction in which the beam is split is the y direction. Accordingly, 64 spot lights are generated in the x direction by the first beam doubling prisms 11 and 12 and 8 spot lights in the y direction by the calcite beam splitting method, and a total of 64 × 8 spot lights are shown in FIG. To occur.
[0061]
FIG. 9 shows a confocal detection method using the above 64 × 8 multi-spot lights. Components having the same component numbers in FIG. 9 and other drawings represent the same thing. Eight beams are emitted from a laser light source and form eight beams from an eight-beam optical system 800 that forms eight beams. The 64 × 8 multi-spot light forming optical system 100 shown in FIG. Multi-spot light shown in FIG. 8 is effectively emitted from the mask surface 3.
[0062]
The multi-spot light is reduced and projected onto the surface of the object to be measured 5 ′ by the half mirror 70 ′, 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 is provided with 64.times.8 pins arranged immediately before the imaging surface of the detector 90'. The light that forms an image on the surface of the hole mask 901 'and passes through the pinhole reaches the imaging surface of the detector. If the surface of the object to be measured coincides with the projected surface of the projected multi-spot light, the reflected light forms an image on the pinhole of the pinhole mask, and a strong detection value is obtained. If they do not coincide with each other, the pinhole of the pinhole mask 901 ′ becomes spread light that is out of focus, and the intensity of the transmitted light through the pinhole is reduced. 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 carried out, the maximum detected intensity at each position can be obtained at the height of each spot. It can be determined from the z value.
[0063]
In the confocal detection method of FIG. 9 described above, if the object to be measured is fixed in the xy direction, for example, if the spot diameter on the surface to be measured is 1 μm and the spot interval is 8 μm, 64 × 8 with 8 μm intervals. Only point information can be obtained. Accordingly, it is necessary to move the xy stages 501 ′ and 502 ″ to sequentially detect confocal images at other positions. FIG. 10 shows an embodiment of the present invention, and this two-dimensional confocal image. 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 multi-spot light, the angle θ formed by these two directions is set so that tan θ is 1/8, so that when the stage is scanned in the y ′ direction, the gap between the spots 011 and 021 is detected. Seven spots of spots 012, 013... 018 irradiate the position, and a 1 μm spot is scanned over every 1 μm.
[0064]
When the above method is used, since the multi-spot light having a high light intensity is irradiated onto the object to be measured, it is possible to detect a 64 × 8 spot position in a short time. Therefore, the sample is scanned at a high speed in the y ′ direction by the y stage, moved slightly in the z direction after scanning, and the scanning operation is repeated again, so that confocal signals of each height are sequentially detected at a high speed. Thus, by obtaining the highest intensity at each point from the two-dimensional intensity data of each point obtained at each height z, the height can be measured by the high-speed confocal detection method. If the scanning width is k times the pitch p of the multi-spot light, two-dimensional height information with the number of pixels in the x direction of 64 × 8 = 512 and the number of pixels in the y direction of 8k is obtained.
[0065]
【The invention's effect】
According to the present invention, the laser light emitted from the laser light source can be converted into multi-spot light very efficiently and without waste, and variation in each multi-spot light can be extremely reduced. As a result, high-speed and high-precision confocal detection can be performed using the multi-spot light. Further, this multi-spot light can be used for high-speed and high-accuracy fluorescence detection and DNA inspection.
[Brief description of the drawings]
FIG. 1 is a front view showing a configuration of an embodiment of a multi-spot light forming unit according to the present invention.
FIG. 2 is a front view showing a configuration of an embodiment of a multi-spot light forming unit according to the present invention.
FIG. 3 is a front view of a mask showing one embodiment of a mask according to the present invention.
FIG. 4 is a front view showing a configuration of an embodiment of a multi-spot light forming unit according to the present invention.
FIG. 5 is a perspective view showing a configuration of one embodiment of a fluorescence detection and DNA testing apparatus according to the present invention.
FIG. 6 is a front view showing an embodiment of multi-spot forming means according to the present invention.
FIG. 7 is a front view showing an embodiment of a two-dimensional multi-spot forming unit according to the present invention.
FIG. 8 is a front view showing an example of an array of 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 for confocal detection and stage scanning 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 to be measured 60 ... Objective lens 61, 62 ... Imaging lens 70, 71, 72, 73 ... Wavelength selection beam splitter 90, 91, 92 ... Detector 9 ... Control circuit

Claims (15)

By using a polarizing beam splitter having at least one polarizing beam splitting surface and two total reflection surfaces arranged at different distances with respect to the polarizing beam splitting surface across the polarizing beam splitting surface, A method of forming a multi-spot light, comprising: dividing an incident laser beam and outputting twice as many laser beams as the incident laser beam . 2. The multi-spot light forming method according to claim 1 , wherein a laser beam incident on the polarization beam splitter is incident on the at least one polarization beam split plane from a direction of 45 degrees . 2. The multi-spot light forming method according to claim 1, wherein the laser beam incident on the polarizing beam splitter is circularly polarized light or linearly polarized light of 45 degrees with respect to the polarization beam split plane . 4. The multi-spot light forming method according to claim 3, wherein the laser beam emitted from the polarization beam splitter is transmitted through a quarter wavelength plate to be a circularly polarized laser beam . 2. The multi-spot light forming method according to claim 1, wherein the laser beam incident on the polarization beam splitter is a convergent laser beam formed by a plurality of lenses arranged . A laser beam incident on the polarization beam splitter by combining a plurality of polarization beam splitters having at least one split surface and two total reflection surfaces arranged at different distances with respect to the split surface across the split surface Are sequentially divided to output a plurality of laser beams more than twice the incident laser beam . 7. The multi-spot light forming method according to claim 6, wherein a quarter wave plate is inserted between the combined polarizing beam splitters . 7. The multi-spot light forming method according to claim 6, wherein the plurality of laser beams to be output are parallel to each other at equal intervals . By projecting multi-spot light onto the object to be observed, allowing the light reflected or transmitted by the object to be inspected to pass through the multi-aperture located at the position where the projection spot forms an image, and detecting the transmitted light at each aperture individually A confocal detection method for performing confocal detection, wherein the multi-spot light formed by the multi-spot light forming method according to claim 1 is used. The multi-spot light formed by using the multi-spot light forming method according to any one of claims 1 to 8 is used as excitation light, the excitation light is projected onto an observed object including a phosphor, and is generated at the inspection object. A fluorescence detection method characterized in that the fluorescence at the position of each multi-spot light is detected by separating the detected fluorescence from the excitation light and disposing a detector at a position where the fluorescence generated at the projection spot forms an image. The multi-spot light formed by using the multi-spot light forming method according to any one of claims 1 to 8 is used as excitation light, the excitation light is projected onto an observed object including a phosphor, and is generated at the inspection object. The fluorescent light separated from the excitation light is transmitted through a multi-aperture arranged at the position where the fluorescent light generated at the projected location forms an image, and the fluorescence transmitted light at each aperture is individually detected to detect the position of each multi-spot light. A fluorescence detection method characterized by detecting fluorescence. The method of separating the fluorescence from the excitation light uses a wavelength separation beam splitter, and the fluorescence chief rays from the spots where the fluorescence generated by each multi-spot excitation light reaches the imaging position from the imaging optical system are parallel to each other. The fluorescence detection according to claim 10 or 11 , wherein a telecentric imaging optical system is formed, and an interference filter or a wavelength separation beam splitter is inserted between the imaging optical system and the imaging position. Method. A multi-spot light formed by using the multi-spot light forming method according to any one of claims 1 to 8 is projected as an excitation light onto an observed object including DNA to which a phosphor is added, and the object to be inspected by the projection The fluorescent light generated in step 1 is separated from the excitation light reflected by the object to be inspected, and is generated at a position where each multi-spot light is projected by arranging a detector at a position where the fluorescent light generated in the projection spot forms an image. A DNA test method comprising: detecting fluorescence and examining DNA from information on a position where the fluorescence is detected and detection signal intensity. A multi-spot light formed by using the multi-spot light forming method according to any one of claims 1 to 8 is projected as an excitation light onto an object to be inspected including DNA to which a phosphor has been added, and the projection causes the inspection to be performed. Fluorescence generated in an object is separated from the excitation light reflected by the object to be inspected, and the fluorescence is allowed to pass through a plurality of openings arranged at positions where the fluorescence is imaged, and the fluorescence that has passed through each opening is individually And detecting the fluorescence intensity at the position where the multi-spot light is projected. The method of separating the fluorescence from the excitation light uses a wavelength separation beam splitter, and the fluorescence generated from the excitation by each multi-spot light causes the principal rays of the fluorescence from each spot from the imaging optical system to the imaging position to be mutually connected. The DNA according to claim 13 or 14 , wherein the method is a method of inserting a telecentric imaging optical system that is parallel and inserting an interference filter or a wavelength separation beam splitter between the imaging optical system and the imaging position. Inspection method.

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