JP2005030919A - Light detector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a light detector capable of emitting sufficient light efficiently and capable of performing the detection of light with high reliability. <P>SOLUTION: The light detector is equipped with a laser beam source 14 for irradiating the carrier contained in a sample with a laser beam and a light detecting means having an object lens 5 for condensing the light emitted from the carrier by the laser beam and a photodetector 9 for detecting the light passed through the object lens 5. A laser beam passing light path and the light path of the light detecting means are separated. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a photodetection device applied to an inspection apparatus for a biological substance used for analysis of DNA, nucleic acid such as DNA, antigen-antibody reaction, protein binding reaction and the like.
[0002]
[Prior art]
Conventionally, the main genetic testing methods that have been put into practical use are the extraction of nucleic acids from biological samples and the basis of PCR (Polymerase Chain Reaction) or NASBA (Nucleic Acid Sequence-Based Amplification: nucleic acid sequences). A method of amplifying a target gene by amplifying a radioisotope or a nucleic acid labeled with a photopigment using a method such as a method for measuring the base sequence of the target gene or its concentration. .
[0003]
Electrophoresis is used for examination of gene expression levels and analysis of mutations. However, this electrophoresis method has problems such as time-consuming and time-consuming measurement, and limitations in measurement that can be performed at one time.
[0004]
Therefore, recently, capillary electrophoresis has been widely used in which photolabeled nucleic acids are reacted in a plurality of capillaries and many samples can be rapidly processed at once. According to this capillary electrophoresis method, it is possible to perform measurement easily and in a short time with a small amount of sample, compared with a method using a conventional electrophoresis method.
[0005]
Furthermore, recently, a test method using a DNA microarray capable of performing a plurality of gene tests simultaneously has been newly developed. Here, a DNA microarray is one in which a number of DNA probes are immobilized on the surface of a glass substrate, or a number of oligo probes synthesized on a small area on a silicon wafer by applying a semiconductor manufacturing process. is there.
[0006]
In such an inspection method using a DNA microarray, a plurality of DNA base sequences and expression levels in a sample can be determined simultaneously. In addition, the application of DNA microarrays enables various tests such as analysis of many gene expression levels and multiple mutations. Furthermore, from data obtained using a DNA microarray, many genes can be classified into a plurality of groups, and information on gene fluctuations associated with development and differentiation can be obtained.
[0007]
However, although the gene analysis method using a DNA microarray has the advantage of being able to perform a large number of tests at once, it requires a long test time, has many test steps throughout, and requires complicated operations. There was a problem that it was difficult to obtain a good detection result.
[0008]
Therefore, in order to overcome such problems, a porous filter is used as a substrate on which the probe of the DNA microarray is immobilized, and the same test as the DNA microarray can be performed in a short time with good reproducibility. Methods and methods for conducting hybridization reactions by electric force have been developed (for example, Patent Document 1, Patent Document 2, and Patent Document 3).
[0009]
Furthermore, a genetic testing device that performs genetic analysis using a DNA microarray has been considered. This device is based on a microscope, and causes a hybridization reaction between a target nucleic acid pre-labeled with a nucleic acid and a number of nucleic acid probes, and an optical signal from a light substance generated by this reaction is generated. An optical image is obtained based on the acquired image. Here, the DNA microarray has, for example, a structure in which a large number of minute reaction regions for spotting a probe for capturing a sample to be immobilized are arranged.
[0010]
In such a genetic testing method using a DNA microarray, it is possible to excite a light substance efficiently and generate a stable light from a reaction product generated in a reaction vessel of the DNA microarray. It is extremely important to do.
[0011]
2. Description of the Related Art Conventionally, an LED (Light Emitting Diode) excitation light source as disclosed in Patent Document 4, for example, is disclosed in which a fluorescent material is irradiated with excitation light from a light source to generate fluorescence. The light material is excited by the excitation light emitted from the light source, and the light signal is received by the solid-state imaging device through filter means that selectively transmits the wavelength of the incident light with respect to the light emitted from the excited light material. There is one in which an optical image is obtained based on a signal.
[0012]
Further, as disclosed in Patent Document 5, light from an LED is irradiated to a well as excitation light via a dichroic mirror and objective lens, and light emitted from the sample is pinhole unit via the objective lens and dichroic mirror. In some cases, a photomultiplier tube is used for detection.
[0013]
Further, as disclosed in Patent Document 6, a semiconductor light source is two-dimensionally arranged to illuminate a specimen through a condenser lens as a surface light source, and light transmitted through the specimen is incident on an objective lens. In other words, illumination light from a two-dimensionally arranged semiconductor light source is sent along the optical axis and incident on the objective lens in a state where it matches the optical axis of the objective lens so that an enlarged image can be obtained. There is something.
[0014]
Furthermore, as disclosed in Patent Document 7, a semiconductor excitation light source is used to illuminate the sample through the objective lens to excite the photopigment inside the sample, and the light from the sample is the same optical as the incident light. Some are detected by a photodetector through the system.
[0015]
Non-Patent Document 1 discloses resonance light scattering by metal fine particles.
[0016]
[Patent Document 1]
JP-T 9-504864
[0017]
[Patent Document 2]
Japanese National Patent Publication No. 2000-515251
[0018]
[Patent Document 3]
JP-T-2001-501301
[0019]
[Patent Document 4]
JP-A-10-132744
[0020]
[Patent Document 5]
JP 2002-116148 A
[0021]
[Patent Document 6]
Japanese Patent Publication No. 7-122694
[0022]
[Patent Document 7]
US Pat. No. 6,154,282
[0023]
[Non-Patent Document 1] Yguerabid J and Yguerabe E, Analytical Biochemistry, 262, p. 137
[0024]
[Problems to be solved by the invention]
However, in these conventional methods, a fluorescent substance photolabeled on a sample is excited with excitation light, and light emitted by the fluorescent substance is detected, so that a dichroic mirror, a color filter, etc. are in the optical path of light detection. These optical elements are arranged. However, when these optical elements are arranged in the optical path, the light intensity is attenuated by passing signal light through each optical element, and it may be impossible to detect light with high reliability.
[0025]
In addition, the fluorescent substance used as a photolabel has a problem in that the wavelength of excitation light is limited, and any light source having a high light intensity can be used.
[0026]
In addition, if a light source with high light intensity is used as excitation light, the time required for the fluorescent substance to fade is accelerated, and the measurement time is greatly limited. Conversely, if the intensity of the excitation light is too weak, the fluorescent substance is Although it takes a long time to fade, there is a problem that the detection light becomes small and stable light detection cannot be performed.
[0027]
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a photodetector that can efficiently irradiate sufficient light and can perform highly reliable light detection.
[0028]
[Means for Solving the Problems]
According to the first aspect of the present invention, there is provided light source means for emitting light for illuminating a carrier labeled on a sample, and light collection for collecting reflected light or scattered light emitted from the carrier by illumination light from the light source means. A light detection means having a lens and a light detector for detecting light that has passed through the condenser lens, wherein the optical path of the illumination light emitted from the light source and the light path of the light detection means are separated It is characterized by.
[0029]
According to a second aspect of the present invention, in the first aspect of the present invention, a plurality of the light source means are arranged around the condenser lens.
[0030]
A third aspect of the present invention is characterized in that, in the second aspect of the present invention, the light source means has substantially the same spectral characteristics of emission wavelengths.
[0031]
According to a fourth aspect of the present invention, in the invention according to any one of the first to third aspects, the light source means is a white light source.
[0032]
The invention according to claim 5 is the invention according to any one of claims 1 to 3, wherein the light source means is a laser light source.
[0033]
The invention according to claim 6 is the invention according to claim 1, wherein the light source means is inclined around the light source means with respect to the surface on which the carrier labeled with the sample is placed. It is characterized by having a position adjusting means capable of adjusting at least one of the directional positions.
[0034]
According to a seventh aspect of the present invention, in the first aspect of the present invention, the first polarizing element that converts the polarized light of the light emitted from the light source means into a linearly polarized light and the front of the photodetector are arranged, and is fixed. A second polarizing element that transmits only polarized light in the direction is further provided.
[0035]
The invention described in claim 8 is characterized in that, in the invention described in claim 1, the carrier is a metal particle or a dielectric particle.
[0036]
The invention according to claim 9 is the light intensity detection means according to the invention according to claim 1, wherein the light intensity detection means detects the intensity of light emitted from the light source means in the vicinity of the surface on which the carrier labeled on the sample is placed, Based on the intensity of light from the light source means detected by the light intensity detecting means, the driving means supplying driving current for driving the light source means and controlling the magnitude of the driving current. The drive means can control the drive current to the light source means.
[0037]
The invention according to claim 10 is the invention according to claim 1, further comprising: a light intensity detecting means for detecting the intensity of light emitted from the light source means in the vicinity of a surface on which a carrier labeled on the sample is placed; A moving unit that enables movement of the direction and position of the light source unit, and the position and direction of the light source unit are controlled by the moving unit based on the intensity of light from the light source unit detected by the light intensity detecting unit; It is characterized by having made it possible.
[0038]
As a result, according to the present invention, sample DNA is hybridized using a carrier, and scattered light or reflected light from the carrier is detected. Therefore, measurement can be performed easily and in a short time.
[0039]
Also, according to the present invention, a plurality of light source means are arranged along the periphery of the condenser lens, and the sample is irradiated with light simultaneously from these, so that scattered light or reflected light from the carrier is efficiently emitted. Can be caught.
[0040]
Furthermore, according to the present invention, the light detector is configured to irradiate the carrier with light emitted from the light source means as linearly polarized light, and to detect only a specific polarization component of the scattered light or reflected light from the carrier. Since the polarizing element is arranged in front of the light source, the background noise can be reduced and signal light detection with a high S / N ratio can be performed.
[0041]
Furthermore, according to the present invention, the direction of the light emitted from the light source means and the tilt direction with respect to the optical axis of the condensing lens can be adjusted, so that the light is condensed at a desired specific place of the carrier labeled on the sample. be able to.
[0042]
Furthermore, according to the present invention, the drive current to the light source means or the position and direction of the light source means can be individually controlled based on the light intensity detected in the vicinity of the surface on which the carrier labeled on the sample is placed. The light from the light source means can be aligned with the same brightness, and the sample can be illuminated with uniform brightness.
[0043]
DETAILED DESCRIPTION OF THE INVENTION
By the way, the “specific binding substance” used in the present invention is a hormone, tumor marker, enzyme, antibody, antigen, abzyme, other protein, nucleic acid, cDNA, DNA, RNA, etc. It is a substance that can specifically bind to and is called a probe.
[0044]
Further, the “biological substance” used in the present invention is a substance that specifically binds to a known specific binding substance arranged at a predetermined position on an antibody, and is extracted, isolated, etc. from a living body. In addition to those extracted directly from the living body, those obtained by chemical treatment, chemical modification, etc. are also included. For example, substances such as hormones, tumor markers, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, cDNA, DNA, and RNA.
[0045]
“Biological substance” and “specific binding substance” specifically bind to each other when, for example, a stable duplex is formed between complementary nucleotide sequences found in DNA, RNA, etc. ( Hybridization), or a highly specific bond that selectively reacts only with a specific substance, such as an antigen and an antibody, or piotin and avidin.
[0046]
Furthermore, the “carrier” used in the present invention refers to various particles labeled with a biological substance to be examined.
[0047]
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0048]
(First embodiment)
FIG. 1 shows a schematic configuration of an optical inspection apparatus based on a microscope structure to which the present invention is applied.
[0049]
In FIG. 1, reference numeral 1 denotes a sample stage. On the sample stage 1, a reaction region 4 installed in a DNA reaction container (not shown) is placed as an object.
[0050]
Above the sample stage 1, an objective lens 5 is disposed as a condenser lens. The objective lens 5 has an optical axis that coincides with the center of the reaction region 4 and is positioned on a perpendicular line from the surface of the sample stage 1.
[0051]
The objective lens 5 is held by the objective lens holding mechanism 6. The objective lens holding mechanism 6 has a cylindrical shape, and the base end portion of the objective lens 5 is fitted in the hollow portion. In this case, the objective lens 5 is movable in the direction of the optical axis of the objective lens 5 in the objective lens holding mechanism 6 so that focus adjustment can be performed.
[0052]
On the optical axis a of the objective lens 5, a lens 8 and a photodetector 9 that constitute a light detection means together with the objective lens 5 are arranged. The lens 8 forms an image of the light transmitted through the objective lens 5 on the detection surface of the photodetector 9. The photodetector 9 detects the intensity of the light collected on the detection surface, converts it into an electrical signal, and outputs it to a computer 10 described later. Here, the lens 8 may be a commonly used single lens, and the material may be BK7, a diffractive optical element, a liquid crystal lens, or the like, an element or a material capable of condensing normal visible light. Can be used.
[0053]
Around the objective lens 5, a plurality (two in the illustrated example) of laser light source units 11 are arranged as light source means. In the laser light source unit 11, a laser light source 14 composed of a gas laser, a semiconductor laser, or the like is disposed, and a lens 12 and a shutter 13 are disposed on an optical path of light from the laser light source 14.
[0054]
The lens 12 expands the beam diameter of the light from the laser light source 14 and irradiates the reaction region 4 on the sample stage 1 evenly as parallel light. The shutter 13 is connected to a computer 10 to be described later, and an opening / closing operation is performed according to a command from the computer 10.
[0055]
Each of the laser light sources 14 used in the plurality of laser light source units 11 has the same oscillation wavelength.
[0056]
The laser light source unit 11 configured as described above is incorporated in a laser light source unit position adjusting mechanism 15 as position adjusting means. The laser light source unit position adjusting mechanism 15 includes a plurality of stepping motors having different movable axes (not shown), an inclination angle with respect to a surface of the reaction region 4 (a surface on which a carrier labeled on a sample to be described later is placed), a height position, Each of the horizontal positions can be adjusted, and each position can be controlled by a computer 10 described later. In this case, the range of the inclination angle possible by the laser light source unit position adjusting mechanism 15 is about 45 ° to 60 ° with respect to the optical axis of the objective lens 5.
[0057]
FIG. 2 shows a specific example of a DNA reaction vessel in which the reaction region 4 is installed. In this case, FIG. 2 shows a DNA slide glass reaction vessel 3 as the DNA reaction vessel. In this DNA slide glass reaction vessel 3, a large number of minute reaction regions 4 are arranged in a slide glass-like reaction vessel main body 3a, and a DNA microarray (not shown) is laid in these reaction regions 4.
[0058]
Then, when a sample solution (test sample) containing DNA labeled with a carrier is injected into these reaction regions 4, a nucleic acid probe immobilized on a DNA microarray, a target nucleic acid in the test solution, and A hybridization reaction occurs between the two, and this reaction generates scattered light from the carrier labeled with the target nucleic acid bound to the probe. In this case, the sample solution that has not contributed to the reaction is washed with the buffer.
[0059]
FIG. 3 shows a state during a hybridization reaction in a DNA microarray. A nucleic acid probe 7c previously immobilized on a substrate 7a is compared with a target nucleic acid 7b labeled with a metal particle 7d as a carrier. Hybridization reactions occur between them. When incident light 14a is irradiated here, scattered light 14b is generated. Here, the case where the hybridization reaction is performed after labeling the carrier with the target nucleic acid has been described. For example, the target nucleic acid is labeled with biotin, the hybridization reaction is performed, and then the carrier to which avidin is bound is added. In addition, the scattered light 14b can be generated with respect to the incident light 14a by setting the state shown in FIG. 3 by the reaction of biotin and avidin.
[0060]
In this case, metal particles or dielectric particles are used as the carrier. For example, in addition to the gold fine particles 7d, fine particles such as silver, platinum, silicon, and latex particles can be used. In particular, fine particles of metal such as gold, silver, platinum, etc., having a particle size of 10 to 100 nm are particularly preferable because the speed of particles in a moving state is optimized. Further, latex particles having a particle size of 0.1 to 1 μm are particularly preferable because the speed of particles in a moving state is also optimal. The appropriate particle size is determined by the specific gravity of the particles and the speed of the Brownian motion. Here, examples of the motion state of the particles include Brownian motion and vibration.
[0061]
The main part of the optical inspection apparatus configured as described above is installed in the light shielding box 16 and is shielded from the outside.
[0062]
FIG. 4 shows a block diagram of the entire optical inspection apparatus.
[0063]
In the figure, reference numeral 10 denotes a computer, and a monitor 17 is connected to the computer 10 as display means. Connected to the computer 10 are a photodetector 9 constituted by the main part of the apparatus described in FIG. 1 and a laser light source unit driving device 18 as a driving means for driving each laser light source unit 11. Upon receiving a control command from the computer 10, the laser light source unit driving device 18 determines which one of the laser light source units 11 is to be driven, and simultaneously sets the magnitude of the drive current to supply a necessary current to the laser light source. Like to do.
[0064]
Next, the operation of the embodiment configured as described above will be described.
[0065]
Now, when a control command is sent from the computer 10 to the laser light source unit driving device 18, the laser light source unit driving device 18 determines what is to be driven from the laser light source unit 11 according to the command content at this time, At the same time, the magnitude of the drive current is determined and supplied.
[0066]
Here, it is assumed that all (two) laser light source units 11 are driven at the same time. When light is emitted from the laser light source 14, light is irradiated toward the reaction region 4 on the sample stage 1. In this case, the light from each laser light source 14 does not cause uneven irradiation on the surface of the reaction region 4 (the surface on which the gold fine particles 7d labeled on the sample 7 are placed), that is, in FIG. As shown, the center axis of the light from each laser light source 14 is made to coincide with the approximate center of the reaction region 4.
[0067]
As described with reference to FIG. 3, scattered light 14b is emitted from the gold fine particles 7d labeled on the sample 7 as a carrier by light irradiation by the laser light source 14. This light is collected by the objective lens 5, passes through the lens 8, and forms an image on the detection surface of the photodetector 9. The light detector 9 detects the light intensity, converts it into an electrical signal, and then outputs it to the computer 10.
[0068]
The computer 10 performs image processing such as contour enhancement, contrast correction, and color correction, signal analysis, and the like, and displays the image on the monitor 17 as an optical image. Alternatively, the signal light intensity value is displayed numerically or graphically.
[0069]
Therefore, with such a configuration, since the lens 12 is disposed on the optical path of the light emitted from the laser light source 14 as the laser light source unit 11, the collimated light without surface unevenness in the reaction region 4 is irradiated. Can do. Thereby, it becomes easy to specify the irradiation position in the surface where the gold fine particle 7d labeled on the sample 7 is placed, and it is possible to easily compile a program for automatically adjusting the irradiation position by computer control. Prediction of the irradiation surface position and its illuminance is easy to make and useful. Further, even when the position to irradiate the laser beam is manually set, the operation of the laser beam is accurately performed at a desired irradiation position on the surface on which the gold fine particle 7d labeled on the sample 7 is placed. Can be done. Further, since the light emitted from the light source is not diffused, light is erroneously irradiated including a portion in the reaction region 4 other than the surface on which the gold fine particle 7d labeled on the sample 7 is placed, for example, a side wall. Etc. can be prevented. In this case, the mounting position of each laser light source unit 11 needs to irradiate the surface on which the gold fine particle 7d labeled on the sample 7 is placed with uniform brightness. Irradiation unevenness is prevented from occurring due to the incident angle, and shadows and irradiation unevenness are also prevented from occurring on the sample 7 having a three-dimensional part in part or on the entire surface.
[0070]
Furthermore, according to such a configuration, a plurality (two in the illustrated example) of laser light source units 11 each having a laser light source 14 having a spectrum of the same emission wavelength are arranged around the objective lens 5 and light is simultaneously emitted from these. Therefore, it is convenient to obtain scattered light or reflected light from the carrier.
[0071]
Further, the central axis of each light is placed on the sample 7 so that the light from the laser light source 14 is uniform on the surface on which the gold fine particle 7d labeled on the sample 7 is placed. Since it is set to coincide with the approximate center of the placed surface, shadows and irradiation unevenness on these surfaces can be minimized, and scattered light generated in the reaction region 4 can be detected with good S / N. Can do.
[0072]
Furthermore, since the light from the laser light source 14 is irradiated obliquely from above toward the surface on which the gold fine particle 7d labeled on the sample 7 is placed, the specular reflection light from the surface of the sample 7 is directly used as the objective lens 5. It is possible to reduce the noise light by passing through the light receiving optical path on the side and entering the photodetector 9 as noise light as much as possible.
[0073]
If a semiconductor laser is used as the laser light source, the output light intensity can be easily adjusted electrically, and it is inexpensive, has a long service life, generates little heat, and further reduces power consumption. In addition, the device configuration can be made small and portable. For example, a semiconductor laser having an oscillation wavelength of 635 nm or 670 nm may be used. Of course, a gas laser such as a helium neon laser or an argon laser can also be used as the laser light source. Further, the number of laser light sources 14 is not limited to two, and may be three or more. When a plurality of light sources are arranged in this way, it is preferable to arrange them at a substantially rotationally symmetric position.
[0074]
Further, since the laser light source unit 11 has a configuration in which only the lens 12 is used as an optical element other than the laser light source 14, the configuration is simple, the assembly and the adjustment of the optical axis are easy, and the cost is reduced. It is also useful because it leads to a reduction in The lens 12 may be configured by combining a plurality of two or more lenses.
[0075]
In the first embodiment, the case where a plurality of laser light source units 11 are arranged around the objective lens 5 has been described. However, when the sample 7 is extremely small, the objective lens 5 is surrounded by the objective lens 5 in FIG. Only one laser light source unit 11 may be arranged. Even in this case, the same effect as described above can be expected.
[0076]
From these facts, in the first embodiment, as used in a method of fluorescently labeling and detecting a sample DNA that is usually used, an extra optical element such as a dichroic mirror or a colored glass filter is used. There is no need to install an element, and a simple device configuration can be achieved in this respect. In addition, since there is no optical element such as a dichroic mirror, light intensity is attenuated due to reflected light or scattered light generated on the surface of these optical elements, or absorption of light caused by light passing through these optical elements. Therefore, the loss of light intensity detected by the photodetector can be minimized. Further, since these optical elements are not necessary, the optical axis adjustment of the entire apparatus can be simplified. Therefore, from these, it is possible to always easily and reliably perform light detection.
[0077]
(Modification 1)
Next, the form of the modification 1 of this invention is demonstrated.
[0078]
FIG. 5 shows a schematic configuration of the first modification of the first embodiment, and the same parts as those in FIG.
[0079]
In the first modification, the light beam emitted from the laser light source unit 11 is focused light, and the laser light source unit position adjusting mechanism 15 further tilts the light beam with respect to the surface on which the gold fine particle 7d labeled on the sample 7 is placed. By changing the angle, the light irradiation angle can be adjusted.
[0080]
In this case, in FIG. 5, a condensing laser light source unit 19 is used in place of the laser light source unit 11 shown in the first embodiment.
[0081]
A laser light source 14 is disposed inside the condensing laser light source unit 19, and a lens 20 and a shutter 13 are disposed on the optical path of light from the laser light source 14.
[0082]
The lens 20 is a commonly used glass lens that focuses the laser light emitted from the laser light source 14 and focuses it on the reaction region 4.
[0083]
Others are the same as FIG.
[0084]
According to such a configuration, since the lens 20 is arranged on the optical path of the light emitted from the laser light source 14 as the condensing laser light source unit 19, the gold fine particle 7d labeled on the sample 7 is placed. A focused beam can be applied to the surface. Thus, for example, if a lens 20 having a large NA of 0.9 or more is used, the lens 20 has a very small area on the surface on which the labeled gold fine particle 7d is placed, for example, a range of about 1 μm or less in diameter. Light can be condensed and irradiated, and light irradiation in a specific extremely narrow range can be performed. Further, since the light from the laser light source 14 becomes a condensed beam and the area of the irradiation cross section is reduced, the surface of the reaction region 4 other than the surface on which the gold fine particle 7d labeled on the sample 7 is placed is placed. It is possible to prevent the laser beam from irradiating the part and becoming noise light and becoming a factor of being mixed into the light from the sample 4b. Furthermore, if the direction of each laser light source 14 is adjusted so that the light from the laser light source 14 is condensed at one point on the surface on which the gold fine particle 7d labeled on the sample 7 is placed, the light from the laser light source 14 is obtained. Can be irradiated only to a desired specific portion in the surface on which the gold fine particle 7d labeled on the sample 7 is placed. Thereby, it is possible to efficiently irradiate only the desired portion with the light from the laser light source 14 or to scan the light spot. Further, since light is not diffused, it is possible to prevent erroneous irradiation of light including a part in the reaction region 4 other than the surface on which the gold fine particle 7d labeled on the sample 7 is placed, for example, a side wall. Can do.
[0085]
In this modified example 1, as another modified example, the distance between the laser light source 14 and the lens 20 is adjusted, and the sample 4b is irradiated with the laser light emitted from the laser light source 14 as a diffused beam. Also good.
[0086]
(Modification 2)
FIG. 6 shows a schematic configuration of the second modification of the first embodiment, and the same parts as those in FIG. In the second modification, the block diagram of the entire optical inspection apparatus described with reference to FIG. 4 is used.
[0087]
In this case, the laser light source unit 11 is disposed at a symmetrical position with the objective lens 5 interposed therebetween. The sample stage 23 is formed of a hollow or transparent glass at the center, and has a structure in which light is irradiated to a reaction region (not shown) with little attenuation.
[0088]
On the other hand, laser light source units 11 (two in the figure) are also arranged substantially symmetrically with respect to the optical axis below the sample stage 23.
[0089]
These laser light source units 11 are held such that the center of the light beam emitted from each laser light source 14 coincides with the center of the reaction area 4 surface. Further, a laser light source unit position adjusting mechanism 15 is attached to these laser light source units 11, and the tilt angle, vertical position, horizontal position, etc. of the laser light source unit 11 can be adjusted by the laser light source unit position adjusting mechanism 15. The axial direction, vertical position, horizontal position, etc. of the light beam can be adjusted. These laser light sources 14 are arranged such that those having the same spectral characteristics come to positions facing each other with the objective lens 5 interposed therebetween.
[0090]
By adopting such a configuration, the substrate (latex particle) 4a portion of the sample 7 in the reaction region 4 can be simultaneously irradiated with light from above and below, and forward scattered light from various carriers can be captured, Scattered light can be detected more efficiently.
[0091]
(Modification 3)
FIG. 7 shows a schematic configuration of the third modification of the first embodiment, and the same parts as those in FIG. 1 are denoted by the same reference numerals. In the third modification, the block diagram of the optical inspection apparatus described with reference to FIG. 4 is also used.
[0092]
In this case, there are a plurality of laser light sources 14 and they are arranged at symmetrical positions with the objective lens 5 interposed therebetween. Then, the reaction region 4 on the sample stage 1 is directly irradiated with a light beam emitted from the laser light source 14. These laser light sources 14 are held so that the centers of the light beams emitted from the respective laser light sources 14 coincide with the centers of the surfaces of the reaction region 4. Further, a laser light source unit position adjusting mechanism 15 is attached to these laser light sources 14, and the laser light source unit position adjusting mechanism 15 can adjust the tilt angle, vertical position, etc. of the laser light source 14, and the axial direction of the light beam. Etc. can be adjusted.
[0093]
With such a configuration, the laser light from the laser light source 14 can be directly applied to the surface on which the gold fine particles 7d labeled on the sample 7 are placed. Therefore, it is necessary to use a lens or the like for the configuration of the light source. The optical axis can be easily adjusted, and light measurement can be easily performed.
[0094]
Further, since the laser light source 14 can freely change the direction of the light beam by operating the laser light source unit position adjusting mechanism 15, not only the reaction region 4 having various shapes but also the uneven sample 4b can be changed. Can respond.
[0095]
In the above description, the case where a plurality of laser light source units 11 are arranged around the objective lens 5 has been described. However, when the sample 7 is extremely small, only one laser light source 14 is arranged around the objective lens 5. You can also Even in this case, the same effect as described above can be expected.
[0096]
(Modification 4)
By the way, in order to illuminate the surface on which the gold fine particle 7d labeled on the sample 7 is placed with light of uniform brightness without unevenness, it is necessary to confirm this state in advance.
[0097]
FIG. 8 shows a schematic configuration of the modified example 4, and the same parts in FIG. In the modification, the block diagram of the optical inspection apparatus described with reference to FIG. 4 is used.
[0098]
In this case, a light intensity detector 31 is arranged as a light intensity detection means on the sample stage 1 irradiated with light from a plurality of (two in the illustrated example) laser light source units 11 instead of the reaction region 4. Yes. Here, as the light intensity detector 31, a solid-state imaging device (CCD camera or CMOS sensor), an imaging tube, or the like is used. The light intensity detector 31 individually detects the intensity of light from each laser light source unit 11. The light detection output of the light intensity detector 31 is taken into the computer 10 described with reference to FIG. 4 as an electric signal.
[0099]
The computer 10 analyzes the variation in light intensity from the output of the light intensity detector 31 corresponding to the light intensity from each laser light source unit 11, sends a signal to the laser light source unit driving device 18, and sends each laser light source unit 11. The current supplied to the laser light source 14 is individually controlled. Alternatively, at the same time, a command is also input from the computer 10 to the laser light source unit driving device 18 to control the inclination, vertical position, and horizontal position of each laser light source 14, and on the surface on which the gold fine particle 7d labeled on the sample 7 is placed. Uniform light can be irradiated.
[0100]
FIG. 9 shows a specific circuit configuration of the laser light source unit 11 and a power supply device 53 for supplying a current to the laser light source 14 of the laser light source unit 11.
[0101]
In this case, the power supply device 53 conducts voltage adjustment by guiding the output from the AC power supply 57 to the transformer circuit 58 including a transformer. The output from the transformer circuit 58 is sent to a bridge rectifier circuit 59 composed of four diodes, subjected to full-wave rectification, and then smoothed by a smoothing capacitor 60. The smoothed output is led to a constant voltage circuit 64 composed of two power transistors 61 and 62 and an OP amplifier 63 connected in Darlington, and output as a constant voltage. In this case, the terminal voltage of the Zener diode 65 is used as a reference voltage, and the variable reference voltage generation circuit 67 configured by circuit elements such as an OP amplifier 66 and a resistor generates the variable reference voltage. Then, by changing the value of the variable reference resistor 68 connected to the OP amplifier 63, the voltage output of the constant voltage circuit 64 is adjusted from the relationship with the variable reference voltage of the variable reference voltage generation circuit 67, and the laser light source unit drive A drive current is supplied to the device 18. That is, by changing the value of the variable reference resistor 68, the drive current to the laser light source unit drive device 18 can be adjusted simultaneously. Here, an FET (Field Effect Transistor) 70 plays a role of preventing an unexpected situation in which a sudden current flows to the OP amplifier 63 when the current is short-circuited and the OP amplifier 63 is damaged. . The power switch 69 can be used to turn on / off the power. The power switch 69 is connected to the computer 10, and the computer 10 also adjusts the power input. The power switch 69 may be switched manually.
[0102]
In the laser light source unit driving device 18, series circuits of variable resistors 71 a (˜71 b) and switches 72 a (˜72 b) are separately prepared as drive circuits for the laser light source unit 11.
[0103]
In the laser light source unit driving device 18, by adjusting the resistance values of the variable resistors 71a and 71b, the current supplied to the laser light source unit 11 can be adjusted, and the output light intensity can be controlled. ing. The adjustment of the output light intensity may be performed manually or may be automatically adjusted electrically by the computer 10.
[0104]
Further, in the laser light source unit driving device 18, the laser light source unit 11 can be turned on and off independently by controlling on and off of the switches 72a and 72b. These on / off controls may be automatically performed by computer control or may be manually operated.
[0105]
When adjusting the resistance values of the variable resistors 71a and 71b under the control of the computer 10, the intensity of the light from the laser light source 14 is detected, the detected signal is guided to the computer 10, and the computer 10 analyzes the light intensity. Then, the resistance values of the variable resistors 71a and 71b are adjusted. Alternatively, the resistance values of the variable resistors 71a and 71b may be manually adjusted based on the intensity of light from each laser light source 14 obtained by this detection.
[0106]
As a result, the magnitude of the current supplied to each laser light source 14 can be controlled based on the light intensity obtained in the vicinity of the surface on which the gold fine particle 7d labeled on the sample 7 is placed. To the same brightness, and the surface on which the gold fine particles 7d labeled on the sample 7 are placed can be illuminated with uniform brightness. For this reason, uneven illumination on this surface can be suppressed, the light in the reaction region 4 can be uniformly irradiated, and light detection with high reliability can be performed.
[0107]
In the above description, the computer 10 is used. However, the laser light source unit driving device 18 is manually operated in the manner described above based on the light intensity from the laser light source 14 of each laser light source unit 11 obtained by the light intensity detector 31. Adjust or adjust the laser light source unit position adjusting mechanism 15 to align the light from the laser light source 14 to the same brightness, and then remove the photodetector 31 and re-install the reaction region 4 at this position. You may do it.
[0108]
On the other hand, an imaging means (for example, a CCD camera or a CMOS sensor (both not shown)) is used as the light intensity detector 31, and the reaction region 4 is placed on the sample stage 1 as it is (without introducing a sample). The light from the laser light source 14 of each laser light source unit 11 is individually applied to the reaction region 4, the light from the reaction region 4 is passed through the objective lens 5 and the lens 8, respectively, and is imaged by the imaging means. Then, the brightness of each pixel of the light image of the monitor 17 is analyzed by the computer 10 to place the gold fine particle 7d labeled on the sample 7 of the light from each laser light source 14. The intensity variation in the surface is determined, and the laser light source unit driving device 18 or the laser light source unit position adjusting mechanism 1 is determined based on the result. The adjusted, it is also to current supplied to each of the laser light source 14, or the spatial position of the laser light source is controlled individually.
[0109]
Further, in the laser light source unit driving device 18, all the laser light sources 14 of the laser light source unit 11 can be turned on / off independently by controlling on / off of the shutter 13. These on / off operations may be automatically performed by computer control, or the shutter 13 may be manually operated.
[0110]
Thereby, it can adjust so that the light from the laser light source 14 may be irradiated into the reaction region 4 substantially uniformly. Further, the light intensity can be partially adjusted, for example, by concentrating the light beam at a desired location in the reaction region 4. Furthermore, since the magnitude of the drive current flowing through all the laser light sources can be controlled, the intensity of the laser light can be increased or decreased all at once. Thereby, it is possible to irradiate each sample with laser light having an appropriate light intensity, and to detect light efficiently.
[0111]
In addition, the individual laser light sources constituting the laser light source unit 11 can be easily turned on and off, and the output light intensity can be individually adjusted, so that laser light of an appropriate intensity is irradiated. It is possible to detect light efficiently.
[0112]
The number of laser light source units is not particularly limited, and a plurality of laser light source units can be individually controlled by a plurality of drive circuits.
[0113]
In this way, since the light from each laser light source 14 can be made to have the same brightness, irradiation unevenness on the surface on which the gold fine particle 7d labeled on the sample 7 is placed can be suppressed, and the reaction The gold fine particles in the region can be irradiated with light almost uniformly.
[0114]
(Second Embodiment)
Next, a second embodiment of the present invention will be described.
[0115]
FIG. 10 shows a schematic configuration of the second embodiment. The same parts as those in FIG. 1 are denoted by the same reference numerals. In the second embodiment, the block diagram of the optical inspection apparatus described with reference to FIG. 4 is also used.
[0116]
In this case, a laser light source 14 is disposed in the laser light source unit 11, and a polarizing filter 21 as a first polarizing element is disposed on the optical path of light emitted from the laser light source 14. Here, the polarization filter 21 converts the polarization of light emitted from the laser light source 14 into linearly polarized light. Further, the lens 12 is the same as that shown in FIG. 1, and after the laser light emitted from the laser light source is enlarged, it is converted into parallel light and emitted. In this case, the position of the polarizing plate 22 may be between the laser light source 14 and the lens 12.
[0117]
The laser light source unit 11 is disposed at a symmetrical position with the objective lens 5 interposed therebetween. Each laser light source unit 11 is provided with a laser light source unit position adjusting mechanism 15. By this laser light source unit position adjusting mechanism 15, the tilt angle, vertical position, horizontal position, etc. of the laser light source unit 11 can be adjusted. The axial direction, vertical position, horizontal position, etc. of the light beam can be adjusted. That is, the light intensity of the irradiation light on the surface on which the gold fine particle 7d labeled on the sample 7 is placed is adjusted, and the light irradiation angle is finely adjusted. As described in FIG. By setting the central axis of the light emitted from the laser light source unit 11 to be approximately at the center of the reaction region 4, the surface on which the gold fine particle 7d labeled on the sample 7 is placed is evenly uniform and uniform. It can be illuminated with. Further, the laser light source unit 11 can control the output light intensity by adjusting the drive current supplied to each laser light source 14. The adjustment of the output light intensity may be performed manually or may be automatically adjusted electrically by a computer. Thereby, the drive current of each laser light source 14 of the laser light source unit 11 can be individually controlled.
[0118]
Then, after passing through the objective lens 5 in the reflected light path from the sample, a polarizing plate 22 is disposed as a second polarizing element in front of the photodetector 9. The polarizing plate 22 defines the polarization direction of the light passing through the polarizing plate 22. The polarizing plate 22 allows only polarized light in a certain direction to pass through and is received by the photodetector 9 through the lens. For example, a CCD camera is used as the photodetector 9.
[0119]
According to such a configuration, it is possible to reduce the background light using polarized light, that is, to improve the S / N ratio of the signal. For example, when latex particles are used as the carrier, spherical particles are preferable. When such particles are irradiated with linearly polarized light, the polarization plane of the scattered light becomes the same as the polarization plane of the incident light. That is, the polarization plane of the scattered light keeps the polarization plane of the incident light as it is. However, the scattered light or reflected light from the wall surface or bottom surface of the reaction region or dust mixed in the sample becomes light having a polarization plane different from the polarization plane of the incident light. Therefore, these background light and noise light are shut out by the polarizing plate 22 placed behind the objective lens 5 and at a position from the photodetector 9, and the light passing through the polarizing plate 22 is the signal light and polarized light. Only a small amount of background light passes through the plate 22. In this way, the S / N ratio of the signal light can be improved.
[0120]
Next, a specific operation of light detection using such a light detector will be described. First, target DNA is set for one sample, and then a sample solution containing DNA labeled with a carrier is dropped onto the reaction region 4 to perform hybridization. By this reaction, the probe and the DNA to be examined are bound. At this time, the unreacted DNA is washed with a buffer solution (PBS (phosphate buffer solution), EDTA (ethylenediaminetetraacetate disodium salt), NaCl mixed solution: PH 7.4).
[0121]
The test sample obtained in this way is set on the sample stage 1. Then, the laser light source 14 of each laser light source unit 11 is turned on, and the test sample on the sample stage 1 is irradiated with light emitted from the laser light source 14. However, the emission spectrum of the light emitted from the laser light source 14 is the same for all. By light irradiation, scattered light is emitted from the carrier, which passes through the objective lens 5 and reaches the polarizing plate 22. The linearly polarized light transmitted here passes through the lens 12, enters the photodetector 9, and is guided to the computer 10. The computer 10 synthesizes the light image by the light detector 9 and outputs it on the monitor 17 or displays the light intensity by the scattered light on the monitor 17 as a numerical value or a graph.
[0122]
These laser light source units 11 can be used properly according to the sample to be examined and the size and structure of the reaction region 4. That is, since the laser light source unit 11 is configured integrally by combining the laser light source 14 with optical elements such as the polarizing filter 21 and the lens 12, the laser light source unit 11 collects light or collimates light in units of each laser light source unit. It is possible to control the beam pattern of the light on the surface on which the carrier labeled on the test sample is placed, and various kinds of reaction regions 4 as well as various shapes can be controlled. It is possible to deal with a test sample of a size. The lens 12 may be a glass lens such as BK7, which is used as a material for a normal lens, but is condensed with respect to normal visible light such as quartz glass, a plastic lens, a diffractive optical element, or a liquid crystal lens. An element or a material that can act can be used.
[0123]
In addition, this invention is not limited to the said embodiment, In the implementation stage, it can change variously in the range which does not change the summary. For example, in the above-described embodiment, a laser light source is used as a light source. However, an incoherent light source that is a white light source such as a halogen lamp, a metal halide lamp, or a tungsten lamp is used without being limited to a coherent light source such as a laser beam. be able to. In addition, although the DNA microarray has been described as a sample to be applied in each of the above-described embodiments, it can be applied to all reaction containers called so-called DNA microarrays. Further, the present invention is not limited to DNA, and can be widely applied to inspections and measurements that handle various other biological substances. In this embodiment, the DNA microarray has been described as an example. However, various probes for testing various biological substances are immobilized on a substrate to test various biological substances such as immune reactions. It is also possible to do. Furthermore, the substrate on which various specific binding substances are immobilized can be applied in various forms, such as a two-dimensional substrate such as a silicon wafer or glass, various beads, and various porous materials. It is possible to apply a quality substrate and various gels. Further, when a specific binding substance is solid-phased on a two-dimensional substrate, the present invention can be applied to an ellipsometer that measures the change in the polarization plane of reflected light by illuminating with polarized light. In this case, the sensitivity is very high, which is preferable. In addition, when a specific binding substance is immobilized on various beads, the present invention can be applied to a bead array or a flow cytometer.
[0124]
When a specific binding substance is solid-phased on a two-dimensional plane such as a slide glass or a silicon wafer as a substrate, since the reaction region has a two-dimensional extent, the carrier is placed on a substantially flat plane. When a specific binding substance is solid-phased on a porous substrate having a general extent, the reaction region has a three-dimensional extent, so that the surface on which the carrier is placed has a three-dimensional extent. become.
[0125]
Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent requirements are deleted from all the constituent requirements shown in the embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and is described in the column of the effect of the invention. If the above effect is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.
[0126]
【The invention's effect】
As described above, according to the present invention, it is possible to provide a photodetector that can irradiate sufficient light efficiently and perform highly reliable light detection.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of an optical inspection apparatus based on a microscope structure according to a first embodiment of the present invention.
FIG. 2 is a diagram showing a schematic configuration of an example of a DNA reaction vessel used in the first embodiment.
FIG. 3 is a diagram showing a state of DNA hybridization and light scattering by gold fine particles in the DNA reaction vessel used in the first embodiment.
FIG. 4 is a block diagram showing a laser light source unit driving device and an image processing device used in the first embodiment.
FIG. 5 is a diagram showing a schematic configuration of a laser light source unit and an apparatus as a first modification of the first embodiment.
FIG. 6 is a diagram showing a schematic configuration of a laser light source unit and an apparatus as a second modification of the first embodiment.
FIG. 7 is a diagram showing a schematic configuration of a measurement optical system as a third modification of the first embodiment.
FIG. 8 is a diagram showing a schematic configuration of an optical state measurement optical system of a light source as a fourth modification of the first embodiment.
FIG. 9 is a diagram showing a specific circuit configuration of a power supply device that supplies a current to a laser light source of a laser light source unit used in Modification 4 of the first embodiment.
FIG. 10 is a diagram showing a schematic configuration of a measurement optical system used in a second embodiment.
[Explanation of symbols]
1 ... Sample stand 3 ... DNA slide glass reaction vessel
3a ... reaction vessel body, 4 ... reaction region
7a ... substrate, 7b ... target nucleic acid, 7c ... nucleic acid probe
7d: Gold fine particles, 5 ... Objective lens, 6 ... Objective lens holding mechanism
8 ... Lens, 9 ... Photo detector
10 ... computer, 11 ... laser light source unit
12 ... Lens, 13 ... Shutter, 14 ... Laser light source
15 ... Laser light source unit position adjustment mechanism
16 ... Shading box, 17 ... Monitor, 18 ... Laser light source unit drive device
19 ... Condensing laser source unit, 20 ... Lens
21 ... Polarizing filter, 22 ... Polarizing plate, 23 ... Sample stage,
31 ... Light intensity detector, 53 ... Power supply, 57 ... AC power supply
58 ... transformer circuit, 59 ... bridge rectifier circuit
60 ... smoothing capacitor, 61.62 ... power transistor
63 ... OP amplifier, 64 ... constant voltage circuit
65 ... Zener diode, 66 ... OP amplifier
67 ... Variable reference voltage generation circuit, 68 ... Variable reference resistor
69 ... Power switch, 71a. 71b ... Variable resistance
72a. 72b ... switch

Claims (10)

Light source means for emitting light for illuminating the carrier labeled on the sample;
A condensing lens for condensing the reflected light or scattered light emitted from the carrier by the illumination light from the light source means;
Photodetection means having a photodetector for detecting the light that has passed through the condenser lens,
An optical path of illumination light emitted from the light source and an optical path of the light detection means are separated from each other. 2. The light detection device according to claim 1, wherein a plurality of the light source means are arranged around the condenser lens. 3. The light detection device according to claim 2, wherein the light source means has substantially the same spectral characteristics of emission wavelengths. The light detection device according to claim 1, wherein the light source means is a white light source. 4. The light detection apparatus according to claim 1, wherein the light source means is a laser light source. A position adjusting means arranged around the light source means and capable of adjusting at least one of an inclination angle, a height position and a horizontal position of the light source means with respect to a surface on which a carrier labeled with the sample is placed; The photodetection device according to claim 1, further comprising: A first polarizing element that converts the polarized light of the light emitted from the light source means into linearly polarized light; and a second polarizing element that is disposed in front of the photodetector and transmits only polarized light in a certain direction. The photodetection device according to claim 1, wherein: 2. The light detection device according to claim 1, wherein the carrier is a metal particle or a dielectric particle. Furthermore, the light intensity detecting means for detecting the intensity of light emitted from the light source means in the vicinity of the surface on which the carrier labeled on the sample is placed, and a drive current for driving the light source means are supplied, and the drive current Drive means capable of controlling the magnitude of the light source, and the drive means can control the drive current to the light source means based on the intensity of light from the light source means detected by the light intensity detection means. The photodetection device according to claim 1. Furthermore, it has light intensity detecting means for detecting the intensity of light emitted from the light source means in the vicinity of the surface on which the carrier labeled on the sample is placed, and moving means for making the direction and position of the light source means movable. 2. The light detection apparatus according to claim 1, wherein the position and direction of the light source means can be controlled by the moving means based on the light intensity from the light source means detected by the light intensity detection means. .

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