WO2016024429A1

WO2016024429A1 - Fine particle detection device

Info

Publication number: WO2016024429A1
Application number: PCT/JP2015/065302
Authority: WO
Inventors: 優紀田中
Original assignee: シャープ株式会社
Priority date: 2014-08-11
Filing date: 2015-05-27
Publication date: 2016-02-18
Also published as: JP2016038360A

Abstract

A fine particle detection device of the present invention comprises a specimen chip (7) having a specimen injection part into which a specimen including fine particles is injected; a light source (8); an irradiation optical system (1) that irradiates the specimen with light from the light source (8); a photodetection optical system (2, 13 to 27) that detects light emitted from the fine particles due to the irradiation of light; and a detector that detects the fine particles on the basis of the strength of the light from the fine particles. The photodetection optical system (2, 13 to 27) has an objective lens element (2) that transmits light from the light source (8) toward the specimen while condensing light emitted from the fine particles. The objective lens element (2) has a beam light transmission part having a shape that condenses light from the light source (8) toward the specimen. When the thickness of the specimen injection part is D, the wavelength of irradiation light from the light source (8) is λ, and a rate of decrease in beam radius of irradiation light in the beam light transmission part is R, a relational expression D≤λ/R² is satisfied.

Description

Fine particle detector

The present invention relates to a microparticle detection apparatus.

Conventionally, as a microparticle detection device, light is irradiated to microparticles developed in a liquid or on a membrane or slide glass, and fluorescence or scattered light generated from the microparticles is detected, and particle counting or property inspection is performed. There is something to do. Here, the fine particles include inorganic particles, microorganisms, cells, erythrocytes in blood, leukocytes, platelets, vascular endothelial cells, fine cell fragments of the tissue, and the like. The microparticles become a microparticle suspension when in the liquid.

Several detection methods are known as methods for detecting the fine particles.

In the flow cytometer, the microparticle suspension is flowed to the capillary together with the sheath liquid. Then, the type of particle and the size of the particle are classified by irradiating a part of the capillary with laser light and detecting scattered light or fluorescence generated when the fine particle is irradiated with light. For example, by labeling particles with a fluorescent reagent that binds to specific particles, the number of fluorescent particles can be counted to count only the specific particles.

By using this flow cytometer, it is possible to measure while suspending a suspension of microparticles at high speed, so that a large amount of specimens can be processed in a short time, and fluorescence emitted from microparticles, scattered light, etc. The intensity of can be measured quantitatively.

However, a flow cytometer capable of quantitatively measuring the intensity of scattered light emitted from the sub-micron particles has a large apparatus and is an expensive system.

In general, the flow cytometer requires a complicated mechanism such as a flow mechanism, which causes deterioration in maintainability and cost increase.

As a method for detecting particles other than the flow cytometer, without using the flow mechanism, image a predetermined range in which microparticles are two-dimensionally distributed, and coefficient the number of microparticles from information of the captured image, Furthermore, there is a method for determining the type and size. In this method, since detection and analysis of particles are performed using a captured image, the flow cytometer is referred to as an image cytometer.

As an imaging method in the image cytometer, a method of imaging a two-dimensional region of a visual field range with a light source and the imaging device fixed using an imaging device composed of a microscope and a digital camera having a certain visual field range In addition, there is a method in which scattered light or fluorescence is detected while scanning laser light two-dimensionally to image particles in a scan region.

In the case of imaging with the above-mentioned microscope and digital camera, high-precision image measurement is possible with particles of 1 μm or more, but when measuring submicron particles, a microscope with a high-magnification objective lens and a high-power lens are used. A sensitive digital camera (that is, low noise and wide dynamic range) is required, which makes the system very expensive. In the case of submicron particles, the wavelength of light and the size of the particles are equivalent, so that the imaging performance is degraded due to the diffraction limit, making it difficult to accurately determine the particle size.

Furthermore, if a fluorescence microscope system is used as the microscope, particles can be easily detected. However, similarly, in the case of submicron particles, the wavelength of light is equal to the size of the particles, and thus the particle size cannot be accurately determined. Also, when measuring fine particles, a high-magnification objective lens must be used, and the field of view is inevitably reduced, so the number of measurements increases. That is, in a high magnification microscope, the measurement time becomes enormous and the quantitativeness becomes a problem.

On the other hand, in the system that detects scattered light or fluorescence while scanning the laser beam, the laser beam is mounted while the laser beam is focused on the particle and irradiated, and the scattered light or fluorescence generated from the particle is detected. The obtained optical head is scanned two-dimensionally to form an image. In this case, laser light is condensed and irradiated on the particles, and the optical head is scanned two-dimensionally to form an image while detecting scattered light or fluorescence generated from the particles.

Thus, in a system that detects light while scanning the optical head relative to the sample, when detecting submicron particles, the laser spot diameter is equal to or smaller than the particle size. More than that. For this reason, the image obtained as a result of the two-dimensional scan is not an image in which each particle is resolved, and it is difficult to directly measure the size of the particle from the image. However, even if the laser beam irradiation spot is larger than the particle size, the intensity of the scattered light generated from the particle varies depending on the particle size, so that the particle diameter can be determined from the intensity of the scattered light. The reason is that there is a correlation between the particle diameter and the scattered light intensity.

In that case, a detector that detects scattered light with high sensitivity (a detector with low noise and a wide dynamic range) and a laser light source are required, but a microscope with a high-magnification objective lens and a high-sensitivity digital camera are required. Compared to the system to be used, an inexpensive system configuration is possible.

As a system for detecting light while scanning the optical head relative to the sample, there is a scanning laser microscope disclosed in Japanese Patent Application Laid-Open No. 2012-83621 (Patent Document 1). In this scanning laser microscope, a scanning unit for two-dimensionally scanning laser light on a specimen, an objective lens for condensing the laser light scanned by the scanning part on the specimen, and light from the specimen are detected. And a light detection unit. Then, based on the intensity of the light detected by the light detection unit and the scanning position information of the scanning unit by the enlarged partial image generation unit, the partial image generation unit includes a partial image of the adjacent sample without overlapping each other, and Then, an enlarged partial image larger than this image is generated with the same pixel resolution as the partial image. Further, the plurality of enlarged partial images generated by the enlarged partial image generating unit are accumulated by the enlarged partial image accumulating unit, and the plurality of enlarged partial images accumulated in the enlarged partial image accumulating unit by the image synthesizing unit. Are combined so as to partially overlap each other to generate an entire image of the specimen.

However, the conventional scanning laser microscope disclosed in Patent Document 1 has the following problems. That is, when detecting scattered light or fluorescence emitted from microparticles while scanning laser light two-dimensionally, the measurement time is correspondingly increased as the scanning area is increased. Therefore, in order to shorten the measurement time when detecting the same amount of specimen, it is effective to increase the sample amount (microparticle amount) per unit scanning area.

However, in the above-described conventional scanning laser microscope, no consideration has been made to increase the sample amount per unit scanning area. Therefore, when the conventional scanning laser microscope is used for detecting fine particles, there is a problem that the measurement time becomes longer as the scanning area becomes larger.

JP 2012-83621 A

Therefore, an object of the present invention is to provide a microparticle detection apparatus capable of detecting microparticles in a specimen at high speed and efficiently.

In order to solve the above problems, a microparticle detection apparatus according to the present invention includes:
A sample chip having a sample injection part into which a sample containing fine particles is injected;
A light source;
An irradiation optical system for irradiating light emitted from the light source to the specimen in the specimen chip;
A light detection optical system for detecting light emitted from the microparticles in the specimen by the light irradiation;
A detection unit that detects the microparticles based on the intensity of light from the microparticles detected by the photodetection optical system;
The optical detection optical system includes an objective lens element that transmits light emitted from the light source toward the specimen, and collects light emitted from the microparticles in the specimen by irradiation with the light. ,
The objective lens element includes a beam light transmitting portion having a shape for transmitting the light from the light source and condensing the transmitted light toward the specimen,
The thickness of the sample chip in the sample injection portion in the light irradiation direction is D, the wavelength of the light irradiated from the light source is λ, and the rate of decrease in the beam radius of the irradiated light due to the condensing in the beam light transmitting portion is R. In this case, the relational expression D ≦ λ / R ²
It is characterized by meeting.

Moreover, in the microparticle detection apparatus of one embodiment,
The following relational expression D ≦ λ / R ² ≦ 3D
It comes to satisfy.

Moreover, in the microparticle detection apparatus of one embodiment,
The surface of the beam light transmitting portion in the objective lens element has a curvature for condensing light by refraction,
The thickness of the specimen chip in the specimen injection portion in the direction of light irradiation is D, the wavelength of the irradiation light from the light source is λ, the beam radius of the irradiation light transmitted through the beam light transmission section is r0, and the beam light transmission section Where f is the focal length of the light after passing through the light, the relational expression D ≦ λ (f / r0) ²
It comes to satisfy.

Moreover, in the microparticle detection apparatus of one embodiment,
The beam light transmitting portion in the objective lens element is a region along the optical axis, and has a shape such that the refraction angle of light after transmission is smaller than other regions excluding the beam light transmitting portion. And has a function of narrowing the irradiation light from the light source toward the specimen,
The other region of the objective lens element excluding the beam light transmission part has a shape such that the refraction angle of light from the fine particles after transmission increases from the beam light transmission part side toward the outer peripheral edge side. Thus, it has a function of condensing light emitted from the fine particles at a wide angle.

Moreover, in the microparticle detection apparatus of one embodiment,
An aperture member provided between the objective lens element and the detection unit, and having a hole through which light from the microparticles transmitted through the objective lens element is transmitted;
The diameter of the hole in the aperture member is taken in from all the light from the microparticles distributed in the range of the thickness D in the specimen injection part, and from the microparticles existing in the range of the thickness D of the specimen injection part. The variation rate of the light detection efficiency of the emitted light by the detection unit is set to be 10% or less.

Here, the fluctuation rate of the light detection efficiency is within 10%, assuming that a state in which the point light sources having the same light intensity are distributed in the range of the thickness D, the detection intensity variation is 10%. It means less than%.

As is clear from the above, in the microparticle detection apparatus of the present invention, the thickness D in the light irradiation direction of the sample injection portion of the sample chip satisfies the relational expression “D ≦ λ / R ² ”. Yes. Further, the reduction rate R of the beam radius of the irradiation light at the beam light transmitting portion of the objective lens element is substantially the same as one half of the reciprocal of the F value, and R = 1 / (2F) = NA. There is a relationship. Therefore, the relational expression “D ≦ λ / R ² ” can be modified as follows.
D ≦ λ / R ² = λ / NA ²

Then, using the depth of focus d = λ / NA ² , the above relational expression can be further modified as follows.
D ≦ λ / R ² = d

That is, in the present invention, the focal depth d of the irradiation light in the objective lens element is set to be equal to or greater than the thickness D in the light irradiation direction in the specimen injection portion. Therefore, it is possible to uniformly irradiate the irradiation region onto the sample region having a thickness that is injected into the sample injection unit. In other words, even if microparticles exist at different positions in the thickness direction of the specimen region, the microparticles can be detected.

Further, when the test chip rotates or scans in one direction, even if the test chip causes surface blurring in the light irradiation direction, the focal depth d is equal to or greater than the thickness D of the specimen injection part. The influence of the surface blur can be reduced by the amount of the focal depth d larger than the thickness D.

Furthermore, since the specimen having the thickness D can be measured at a time, it becomes possible to detect fine particles in the specimen at high speed and efficiently, and to shorten the measurement time.

It is a figure which shows schematic structure in the microparticle detection apparatus of this invention. It is the perspective view and sectional drawing which show schematic structure of the test | inspection chip in FIG. FIG. 3 is a diagram in which fine particles to be detected exist in the center of the specimen injection layer in FIG. 2. FIG. 3 is a diagram in which particles to be detected are present on the specimen injection layer. It is a figure at the time of assuming that scattered light generate | occur | produces on the surface of the said test | inspection chip. It is a figure which shows schematic structure of the microparticle detection apparatus different from FIG. It is a figure which shows schematic structure of the microparticle detection apparatus different from FIG. 1 and FIG. It is a figure which shows the scanning state of the sample by the optical module in FIG.

Hereinafter, the present invention will be described in detail with reference to embodiments shown in the drawings.

-1st Embodiment FIG. 1: is a figure which shows schematic structure of the microparticle detection apparatus of this Embodiment. This microparticle detection apparatus includes a disk-shaped inspection chip into which a specimen is injected, a rotational drive system that rotates the inspection chip, a light detection optical system that detects scattered light and fluorescence, and the detection optical system in the radial direction. A driving mechanism for driving and a detection unit that receives the signal from the light detection optical system and detects the microparticles are roughly configured.
In FIG. 1, 1 is a light source device, 2 is an objective lens, and 3 to 5 are first to third detection devices. The light source device 1, the objective lens 2, and the first to third detection devices 3 to 5 constitute an optical module 6 housed in a frame. A circular disk-shaped inspection chip 7 is arranged above the optical module 6 so as to face the objective lens 2. In the inspection chip 7, for example, a suspension in which fine particles labeled with a fluorescent substance are distributed. A transfer support such as a gel support or a membrane is injected as a specimen.

The light source device 1 of the optical module 6 is provided with a semiconductor laser 8 as a light source. On the optical axis of the semiconductor laser 8, a collimator lens 9, a first band pass filter 10, and a first ND (dimming). The filter 11 and the first aperture member 12 are arranged in this order. Here, the semiconductor laser 8, the collimator lens 9, the first band-pass filter 10, the first ND filter 11, and the first aperture member 12 are housed in one case, and the light source device 1 which is an example of the above-described irradiation optical system. It is composed.

Further, on the optical axis of the semiconductor laser 8, a prism mirror 13 that reflects the light transmitted through the first aperture member 12 toward the objective lens 2 is disposed. Further, below the prism mirror 13 in FIG. 1 on the optical axis of the objective lens 2, a first dichroic mirror 14 that transmits fluorescence from the objective lens 2 and reflects scattered light in order from the prism mirror 13 side. Among the fluorescence transmitted through the first dichroic mirror 14, the second fluorescence that transmits the first fluorescence having the first wavelength and reflects the second fluorescence having the second wavelength shorter than the first wavelength is reflected. A dichroic mirror 15 is arranged.

The “scattered light” referred to in the present invention is light in which the light emitted from the semiconductor laser 8 is isotropically or anisotropically scattered from the irradiated portion of the specimen to the surroundings. It has the same wavelength as the incident light. On the other hand, “fluorescence” means that the light emitted from the semiconductor laser 8 irradiates the sample to excite the fluorescent substance that labels the microparticles, and isotropically passes from the irradiated position of the sample to the surroundings. The scattered fluorescence is light having a wavelength different from that of the outgoing light.

Here, although not described in detail, the objective lens 2 is stored in a lens holder (not shown), and is moved in the optical axis direction by a drive unit (not shown) such as a stepping motor, so that the focal position is adjusted. It can be changed.

Further, in FIG. 1, the second dichroic mirror 15 on the optical axis of the objective lens 2 is condensed by the objective lens 2 and converted into parallel light in order from the second dichroic mirror 15 side in FIG. In addition, the first fluorescence having the first wavelength that has passed through the second band-pass filter 16 and the second band-pass filter 16 for dimming light from the specimen (light having a wavelength different from the first wavelength) is collected. The 1st lens 17 and the 2nd aperture member 18 which cuts the 1st fluorescence stray light which passed the 1st lens 17 are arranged. Furthermore, a detection element such as a photomultiplier tube (PMT) that detects the first fluorescence that has passed through the second aperture member 18 is provided below the second aperture member 18 on the optical axis of the objective lens 2. One detector 19 is arranged. Here, the 2nd aperture member 18 and the 1st detector 19 are stored in one case, and constitute the 1st detection device 3 which is an example of the above-mentioned optical detection optical system.

A third bandpass filter 20, a second lens 21, a third aperture member 22, and a second detector 23 are arranged in this order from the second dichroic mirror 15 side on the left side of the second dichroic mirror 15 in FIG. The third aperture member 22 and the second detector 23 are housed in one case and constitute a second detection device 4 that is an example of the light detection optical system. The third band pass filter 20, the second lens 21, the third aperture member 22, and the second detector 23 are basically composed of the second band pass filter 16, the first lens 17, and the second aperture member 18. The first detector 19 has the same configuration. However, it is different in that the processing for the second fluorescence of the second wavelength is performed.

Further, a second ND filter 24, a third lens 25, a fourth aperture member 26, and a third detector 27 are arranged in this order from the first dichroic mirror 14 side on the right side of the first dichroic mirror 14 in FIG. The fourth aperture member 26 and the third detector 27 are housed in one case and constitute the third detection device 5 which is an example of the light detection optical system. The second ND filter 24, the third lens 25, the fourth aperture member 26, and the third detector 27 are basically composed of the second bandpass filter 16, the first lens 17, the second aperture member 18, and the first detection. It has the same configuration as the container 19. However, it is different in that processing is performed on scattered light from the specimen.

That is, in the present embodiment, the light detection optical system includes the objective lens 2, the dichroic mirrors 14 and 15, the

filters

16, 20, and 24, the

lenses

17, 21, and 25, the

aperture members

18, 22, and the like. 26 and the

detectors

19, 23 and 27.

The inspection chip 7 is configured to be transparent and circular, and is placed on a transparent and circular table 29 fixed to the central shaft 28 and fixed to the central shaft 28. The central shaft 28 can be rotated by a motor 30 as an example of the rotational drive system. On the other hand, the optical module 6 can be moved by the drive mechanism in the radial direction of the disk formed by the inspection chip 7. The drive mechanism of the optical module 6 is not particularly limited. For example, the frame body of the optical module 6 is configured to be movable by being guided by the guide rail disposed in the radial direction by a timing belt or the like reciprocated in the radial direction by a stepping motor or the like.

FIG. 2 shows a schematic configuration of the inspection chip 7. 2A is an external perspective view, and FIG. 2B is a cross-sectional view taken along the line A-A ′ in FIG. The inspection chip 7 has a structure in which two

transparent substrates

31 and 32 each provided with a center hole 33 for fixing are bonded to each other via a spacer 34 provided with a center hole 35 for fixing. Have.

The spacer 34 is formed with a circular groove 36 concentric with the center hole 35. Then, by closing both side surfaces of the spacer 34 with the two

substrates

31 and 32, the circular groove 36 becomes a donut-shaped space and becomes a specimen injection layer into which the specimen is injected. The specimen injection layer 36 has a thickness D corresponding to the thickness of the spacer 34. The thickness D of the specimen injection layer 36 is preferably about several tens of micrometers. In addition, a sample injection port 37 for injecting the sample into the sample injection layer 36 is formed in one substrate 31 (for example, located on the upper side in FIG. 2). The sample injection port 37 is provided at a plurality of locations in the circumferential direction (4 locations in FIG. 2A) and a plurality of locations in the radial direction (2 locations in FIG. 2A) and sealed after the sample is injected. Is done.

The test chip 7 into which the sample has been injected is placed on the table 29, and the optical module 6 is moved in the radial direction while being rotated by the motor 30 to detect fluorescence and scattered light from the microparticles in the sample. Is done.

Hereinafter, as an example, the operation and function of the microparticle detection apparatus will be described by taking as an example the case where the two types of fluorescence and the scattered light are detected simultaneously.

Laser light is emitted from the semiconductor laser 8 of the light source device 1. The emitted laser light is collimated by the collimator lens 9 and passes through the first bandpass filter 10. The first band pass filter 10 cuts light having an unnecessary wavelength. Next, the laser light passes through the first ND filter 11, a part of the irradiation light intensity is reduced, passes through the first aperture member 12 for shaping the incident light beam diameter, and is guided to the prism mirror 13. The laser light is reflected by the prism mirror 13, passes through the objective lens 2 and the table 29, and is collected on the specimen injection layer 36 of the test chip 7.

In this case, the length of the prism mirror 13 in the longitudinal direction (horizontal direction) is short, the width in the direction perpendicular to the longitudinal direction is narrow, and the laser light from the semiconductor laser 8 is near the optical axis of the objective lens 2. It passes through only the (beam light transmission part). In this way, when the microscopic particles in the specimen injection layer 36 are irradiated with focused light, fluorescence and scattered light that is isotropically scattered from the portion irradiated with the focused light are generated.

At this time, the beam diameter of the laser beam transmitted through the first aperture member 12 is smaller than the aperture diameter of the objective lens 2. That is, the effective NA of the laser beam is made smaller than the NA (numerical aperture) of the objective lens 2. Therefore, the depth of focus of the laser light is increased, and the laser light can be uniformly irradiated onto the thick specimen region injected into the thick specimen injection layer 36. Thereby, even if microparticles exist at different positions in the thickness direction of the specimen region, the microparticles can be detected.

Even if surface blurring occurs in the extending direction of the central axis 28 when the test chip 7 is rotated, if the depth of focus is larger than the thickness D of the specimen injection layer 36, the effect of the surface blurring is correspondingly increased. Can be reduced. Furthermore, since the thick specimen can be measured at a time, a large amount of specimen can be measured and processed at high speed, and the measurement time can be shortened. The depth of focus is generally defined as a distance to a position twice the root of the spot diameter at the focal position. The spot diameter is generally the diameter at a position where the intensity is 1 / e ² with respect to the light intensity at the center position of the spot.

The depth of focus is determined by the wavelength of irradiation light / (effective NA of irradiation light) ² , and the value is not less than the thickness of the specimen injection layer 36 in the test chip 7 and not more than three times the thickness of the specimen injection layer 36. Is set. Therefore, it is possible to keep the spot diameter of the irradiation light at a constant size and to secure the necessary depth of focus.

The effective NA may be different between the tangential direction and the radial direction of the inspection chip 7. For example, the effective NA in the tangential direction may be larger than the effective NA in the radial direction. In that case, the beam diameter in the radial direction is larger in the vicinity of the focal position than in the tangential direction. By doing this, even when the disk-shaped inspection chip 7 is rotated, even if eccentricity that is asynchronous with rotation occurs, there is a margin for the spread of the beam in the radial direction, and fine particles are skipped. Can be prevented. Further, the resolution in the tangential direction can be improved as compared with the case where the effective NA is the same in the tangential direction and the radial direction. Moreover, the irradiation light intensity per unit area in the vicinity of the focal position can be increased.

When the laser light focused by the objective lens 2 is irradiated onto the microparticles in the inspection chip 7, scattered light and fluorescence scattered by the microparticles are generated (hereinafter, the scattered light and fluorescence are combined). Called signal light). The generated signal light is focused by the first to

third lenses

17, 21, 25. Here, the first to

third lenses

17, 21 and 25 may be the same lens as the objective lens 2 used when condensing the laser light. In that case, it is possible to reduce the size of the microparticle detection apparatus by using the same lens. Further, when the same lens as the objective lens 2 is used for detecting the scattered light, so-called back scattered light is detected from the light scattered by the fine particles, and the classification of the fine particle system by the scattered light is easy. become.

Scattered light of the signal light is reflected by the first dichroic mirror 14, attenuated by the second ND filter 24, converged by the third lens 25, and guided to the third detector 27. A fourth aperture member 26 is disposed at the focal position of the third lens 25, and stray light is removed.

The aperture diameter of the fourth aperture member 26 allows light from a substance located within the focal depth of the laser light (irradiation light) to pass sufficiently, but can remove scattered light and stray light from other than the in-focus position. The diameter is set to a certain diameter. For example, since the reflected light generated on the surface of the substrate 32 and the surfaces of the

lenses

2 and 25 in the inspection chip 7 is greatly deviated from the focal position of the objective lens 2, the fourth aperture member is formed by the optical system following the objective lens 2. At position 26, the light spreads. Therefore, it is cut without being able to efficiently pass through the fourth aperture member 26.

Here, the method for determining the aperture diameter of the fourth aperture member 26 is as follows.

・ Aperture diameter determination method A
(1) As shown in FIG. 3, when the detected microparticle 38 exists at the center of the specimen injection layer 36 in the thickness direction, the scattered light is collected by the objective lens 2 and the third lens 25, and the fourth aperture is obtained. The light intensity transmitted through the member 26 and detected by the third detector 27 is 100.
(2) As shown in FIG. 4, when the detected particles 38 are present in the upper or lower part of the specimen injection layer 36 in the thickness direction, the scattered light is condensed by the objective lens 2 and the third lens 25, and the fourth The light intensity transmitted through the aperture member 26 and detected by the third detector 27 becomes 90 or more.

By determining the aperture diameter so that the above A- (1) and A- (2) are established, even if microparticles exist at different positions in the thickness direction in the specimen injection layer 36, the fourth aperture member 26 The variation rate of the light intensity that is transmitted through and detected by the third detector 27 is within 10%, and the error of the minute particle diameter when detecting scattered light can be within 10%.

・ Aperture diameter determination method B
(1) As shown in FIG. 3, when the detected microparticle 38 exists at the center of the specimen injection layer 36 in the thickness direction, the scattered light is collected by the objective lens 2 and the third lens 25, and the fourth aperture is obtained. The light intensity transmitted through the member 26 and detected by the third detector 27 is 100.
(2) When it is assumed that scattered light is generated at a position twice or above the thickness direction in the specimen injection layer 36 in the thickness direction, the scattered light is collected by the objective lens 2 and the third lens 25, The light intensity transmitted through the fourth aperture member 26 and detected by the third detector 27 becomes 70 or more.

By determining the aperture diameter so that the above B- (1) and B- (2) are established, the inspection chip 7 is moved in the thickness direction due to surface blurring in the direction of the central axis 28 (that is, the light irradiation direction). However, the fluctuation rate of the light intensity that is transmitted through the fourth aperture member 26 and detected by the third detector 27 is within 30%, and the error of the minute particle diameter at the time of detecting scattered light is also within 30%. it can.

・ Aperture diameter determination method C
(1) As shown in FIG. 3, when the detected microparticle 38 exists at the center of the specimen injection layer 36 in the thickness direction, the scattered light is collected by the objective lens 2 and the third lens 25, and the fourth aperture is obtained. The light intensity transmitted through the member 26 and detected by the third detector 27 is 100.
(2) As shown in FIG. 5, when it is assumed that scattered light is generated on the front surface or the back surface of the inspection chip 7, the scattered light is collected by the objective lens 2 and the third detection lens 25, and the fourth aperture member 26. And the light intensity detected by the third detector 27 becomes 0.01 or less.

By determining the aperture diameter so that the above C- (1) and C- (2) are established, scattered light is generated due to scratches and dust existing on the surface of the inspection chip 7 and the surface of the table 29. However, the light intensity transmitted through the fourth aperture member 26 and detected by the third detector 27 is 0.01% or less, and the influence of scattered light from an unnecessary surface is also 0.01% or less. Can do.

The aperture diameter that satisfies all the conditions of the aperture diameter determination methods A, B, and C is most suitable as the aperture diameter of the fourth aperture member 26. The reason is that unintended scattered light is assumed to be mixed due to foreign matters such as scratches and dust, and the setting of the aperture diameter when detecting scattered light is particularly important.

Of the signal light transmitted through the first dichroic mirror 14, the second fluorescence having the shorter second wavelength is reflected by the second dichroic mirror 15 and transmitted through the third bandpass filter 20. The light is converged by the second lens 21 and guided to the second detector 23. A third aperture member 22 is disposed at the focal position of the second lens 21, and stray light is removed by the third aperture member 22.

Similarly, the first fluorescence having the longer first wavelength transmitted through the second dichroic mirror 15 is transmitted through the second band-pass filter 16 and then converged by the first lens 17 to be the first detector. 19 leads. A second aperture member 18 is disposed at the focal position of the first lens 17 and stray light is removed.

In the case of fluorescence detection by the first detector 19 and the second detector 23, the possibility that fluorescence is generated due to scratches is low. Therefore, the aperture diameters of the second aperture member 18 and the third aperture member 22 are not necessarily the aperture diameter. It is not necessary to satisfy all the conditions of the diameter determination methods A, B, and C. It is sufficient that only the aperture diameter determining method A is satisfied.

As described above, when the first fluorescence is guided to the first detector 19, the second fluorescence is guided to the second detector 23, and the scattered light is guided to the third detector 27, In the first detector 19, the second detector 23, and the third detector 27, photoelectric conversion is performed on the signal light, and the obtained electric signal is guided to the signal processing unit. The signal processing unit AD-converts the electrical signal, which is an analog signal, into a digital signal, and further processes the digital signal to process an information processing apparatus such as a personal computer 80 that is an example of the detection unit. Forwarded to

Then, in the information processing apparatus, based on the processed digital signal, the type of minute particles that generate fluorescence or scattered light are quantitatively analyzed, and the analysis result is displayed. .

As described above, in the present embodiment, the laser light emitted from the semiconductor laser 8 is passed through the collimator lens 9 and the prism mirror 13 to the specimen injected into the specimen injection layer 36 of the test chip 7. Irradiate. The table 29 on which the inspection chip 7 is placed is rotated by a motor 30, while the optical module 6 in which the light source device 1, the objective lens 2, and the first to third detection devices 3 to 5 are housed is moved in the radial direction. Move to. Thus, the first to third detection devices 3 to 5 detect the fluorescence and scattered light that are isotropically scattered from the minute particles in the specimen irradiated with the focused laser beam. Detection is performed by means of the

devices

19, 23, 27.

Thus, when detecting scattered light or fluorescence emitted from a minute particle while scanning laser light two-dimensionally, it is necessary to shorten the measurement time because the scanning area becomes wider and the measurement time becomes longer. . For this purpose, it is effective to increase the amount of fine particles per unit scanning area.

For this purpose, the specimen injection layer 36 of the test chip 7 is given a thickness D, and the optical system is optimized as follows.

First, in order to improve the accuracy of detection of fine particles, it is desirable to irradiate the specimen with the diameter of the laser that is the irradiation light as narrow as possible. Therefore, the sample is irradiated with the laser beam with the objective lens 2 so as to be focused on the position of the sample. When the laser beam is squeezed using a lens, the diameter of the laser beam cannot be reduced to infinity due to the diffraction effect. The spot radius r, which is the minimum value of the diameter when the laser beam is narrowed, is r = 0.61λ / NA (NA: numerical aperture, λ: when considering a circular franphopha diffraction image without an intensity distribution). It is generally known that it is given by (wavelength of light). Therefore, the beam radius is set to a value close to the spot radius r in the entire range of the thickness D into which the specimen is injected (for example, the beam radius is within twice the root of the spot radius r). It is desirable to do.

Here, the condition for the beam radius to be less than or equal to twice the route of the spot radius r is given by D ≦ d using the focal depth d = λ / NA ² . Further, the reduction rate R of the beam radius of the laser light due to the focusing of the objective lens 2 is generally synonymous with half of the reciprocal of the F value, and has a relationship of R = 1 / (2F) = NA. Therefore, the above conditions can be modified as follows.
D ≦ d = λ / NA ² = λ / R ²

In Patent Document 1, as described above, “the beam radius is set to be less than twice the route radius of the spot radius r” or “the thickness D into which the specimen is injected is set to a depth of focus d or less. There is no description regarding consideration. Therefore, when the above-mentioned conditions are not satisfied in Patent Document 1, even if the specimen injection portion has a thickness D, a portion where the beam diameter of the laser beam is large and a portion within the thickness D are formed. . In particular, since the light density of the laser beam is small at a location where the beam diameter is large, the intensity of light emitted from the microparticles is weak, and the detection sensitivity of the microparticles in the region of the thickness D is lowered.

Therefore, it cannot be effectively used for detecting all the fine particles in the specimen injection part having the thickness D. Alternatively, since the distribution of the intensity of light emitted from the microparticles in the specimen injection portion having the thickness D is made, the variation in the intensity of the light from the microparticles to be detected becomes large.

Further, the focal depth d (= λ / R ² ) is set to 3 times or less the thickness D of the specimen injection layer 36. If the depth of focus d (= λ / R ² ) is too large, the following problem occurs.

That is, the spot radius r of the laser beam is proportional to the beam radius reduction rate R (as described above, r = 0.61λ / NA = 0.61λ / R (NA: numerical aperture, λ: wavelength of light)). . For this reason, when the decrease rate R is small (that is, the laser beam condensing rate is small), the beam radius of the laser beam irradiated onto the specimen is increased, and the detection accuracy of microparticles is decreased. End up.

That is, when the reduction rate R is small, the depth of focus d can be increased while the beam radius of the laser beam cannot be reduced.

Therefore, in order to make the beam radius as small as possible while satisfying the relationship of D ≦ λ / R ² (= d), the focal depth d (= λ / R ² ) is set to It is set to 3 times or less of the thickness D. Here, “3D” which is the upper limit value of the focal depth d is the accuracy of the irradiation optical system and the light detection optical system, and the extension direction of the central axis 28 during scanning of the optical module 6 in the radial direction. In consideration of blur and the like, the first detector 19, the second detector 23, and the third detector 27 are set so that light can be detected with optimum accuracy.

In the present embodiment, since the light emitted from the microparticles is emitted in a wide angle range, the light is efficiently guided to the first to

third detectors

19, 23, 27. A wide objective lens 2 is used. Furthermore, the objective lens 2 is configured so that part of the objective lens 2 along the optical axis can transmit laser light. That is, in the present embodiment, the objective lens 2 has a function for transmitting laser light and guiding it to the specimen, and a function for condensing light from the microparticles.

Then, the laser beam from the semiconductor laser 8 passes through only a very narrow region near the optical axis of the objective lens 2, and after passing through the objective lens 2, satisfies the condition of D ≦ λ / R ² and is irradiated to the specimen. It has become so. Further, the entire region of the objective lens 2 guides the light emitted from the fine particles to the first to

third detectors

19, 23, 27.

In the present embodiment, in order to realize the above, the shape of the objective lens 2 is set as follows.

That is, as described above, the beam light transmitting portion that is a part of the vicinity of the optical axis of the objective lens 2 and transmits the laser light from the semiconductor laser 8 that is the light source has a gentle curvature and is a semiconductor. The laser beam from the laser 8 (hereinafter simply referred to as a beam) has a function of focusing toward the inspection chip 7. Here, if the radius of the beam incident on the objective lens 2 is r0 and the focal length of the beam after passing through the objective lens 2 is f, an effective F value that is a value obtained by dividing the focal length f by the effective aperture (2 · r0). , The definition of the reduction rate R is given by R = 1 / (2F) = 1/2 (f / (2 · r0)) = r0 / f. Therefore, the condition of “D ≦ λ / R ² ” described above can be modified as D ≦ λ / R ² = λ (f / r 0) ² . Since the laser beam is incident only on a part of the objective lens 2, it is not an ordinary F value having the lens diameter as r0, but an effective F value having r0 as the diameter of the incident beam.

The entire objective lens 2 including the beam light transmitting portion in the vicinity of the optical axis of the objective lens 2 and the other peripheral portions, the first to third detectors 19 emit scattered light and fluorescence emitted from the specimen at a wide angle. , 23 and 27, it is a specimen light transmitting portion that condenses toward the light. Then, the shape is such that the angle of refraction of scattered light and fluorescence after transmission gradually increases from the region of the beam light transmitting portion toward the outside.

That is, in the present embodiment, the beam light transmitting portion along the optical axis in the objective lens 2 squeezes the laser light loosely (however, if the aperture is too narrow, the focal length f becomes too small, so the reduction rate R is (Because the above condition “D ≦ λ / R ² ” is not satisfied), the laser beam after passing through the objective lens 2 has a refraction angle smaller than that of the peripheral portion. Yes. On the other hand, in the other regions, in order to collect the laser light emitted from the specimen at a wide angle, the refraction angles of the scattered light and the fluorescence after passing through the objective lens 2 are changed from the beam light transmitting part side to the outer peripheral side. It has a shape that gradually becomes larger toward.

In the present embodiment, the fluorescence and scattered light collected by the objective lens 2 are converged by the first to

third lenses

17, 21, 25, and the second to

fourth aperture members

18, 22, 26 are collected. To the first to

third detectors

19, 23, 27. In this case, the aperture diameters of the

aperture members

18, 22, and 26 are determined so that all the fluorescence and the scattered light from the fine particles distributed in the range of the thickness D in the specimen injection layer 36 can be taken in. In that case, even if microparticles exist at different positions in the thickness direction in the specimen injection layer 36, detection of light transmitted through the

aperture members

18, 22, 26 and detected by the

detectors

19, 23, 27. The variation rate of efficiency is within 10%. Here, the fluctuation rate of the light detection efficiency is within 10%, assuming that the point light sources having the same light intensity are distributed in the range of the thickness D, the variation in the detection intensity is less than 10%. It means that.

Normally, the aperture member is designed to transmit only the target light and shield other light. For example, the light emitted from a specific depth in the optical axis direction is transmitted, and the light from other depths is designed to be shielded. Here, when the range of the depth at the position where the fluorescence and the scattered light transmitted through the

aperture members

18, 22, and 26 are emitted is smaller than the thickness D of the specimen injection layer 36, the distribution is in the range of the thickness D. It becomes impossible to detect all the fine particles. Therefore, in the present invention, not only the focal depth d of the laser beam is set to be larger than the thickness D of the specimen injection layer 36 as described above, but also the position where the light transmitted through each

aperture member

18, 22, 26 is emitted. The depth range is also set larger than the thickness D of the specimen injection layer 36.

By doing so, even if microparticles exist at different positions in the thickness direction in the specimen injection layer 36, they pass through the

aperture members

18, 22, and 26 and are detected by the

detectors

19, 23, and 27. The fluctuation rate of the light intensity can be made within 10%, and the error of the fine particle diameter when detecting fluorescence and scattered light can be made within 10%.

Second Embodiment This embodiment relates to an irradiation optical system different from the irradiation optical system in the first embodiment, and has a configuration in which irradiation laser light is reflected by a dichroic mirror.

FIG. 6 is a diagram showing a schematic configuration in the microparticle detection apparatus of the present embodiment. Reference numeral 41 denotes a light source device, 42 denotes an objective lens, 43 denotes a first detection device, and 44 denotes a second detection device. The light source device 41, the objective lens 42, the first detection device 43, and the second detection device 44 are housed in a frame to constitute an optical module 45. A circular disk-shaped inspection chip 46 is disposed above the optical module 45 so as to face the objective lens 42. In the inspection chip 46, for example, a suspension in which fine particles labeled with a fluorescent substance are distributed. A sample injection layer 47 is provided in which a transfer support such as a gel support or a membrane is sealed as a sample.

The light source device 41 of the optical module 45 is provided with a first semiconductor laser 48 as a light source. On the optical axis of the first semiconductor laser 48, a first collimator lens 49, a spot size adjustment lens 50, and a first semiconductor laser 48 are provided. The one aperture member 51 is arranged in this order. Further, in addition to the first semiconductor laser 48, a second semiconductor laser 52 that emits a laser beam having a second wavelength different from the first wavelength of the laser beam emitted from the first semiconductor laser 48 is disposed. Further, a second collimator lens 53 for collimating the laser beam from the second semiconductor laser 52 is disposed. A first dichroic mirror that transmits the laser light having the first wavelength and reflects the laser light having the second wavelength is transmitted to the intersection between the optical axis of the first semiconductor laser 48 and the optical axis of the second semiconductor laser 52. 54 is arranged. Here, the first semiconductor laser 48, the first collimator lens 49, the spot size adjusting lens 50, the first aperture member 51, the second semiconductor laser 52, the second collimator lens 53, and the first dichroic mirror 54 are included in one case. The light source device 41 that is an example of the irradiation optical system is configured.

Further, a prism 55 is disposed on the optical axis of the first semiconductor laser 48 to reflect the light transmitted through the first dichroic mirror 54 toward the objective lens 42 side. A second dichroic mirror 56 that reflects the light from the prism 55 so as to enter the objective lens 42 is disposed at the intersection of the light reflected by the prism 55 and the optical axis of the objective lens 42. Here, the second dichroic mirror 56 transmits the fluorescence from the microparticles in the specimen injection layer 47 and reflects the scattered light.

In the above configuration, a plurality of light sources of the first semiconductor laser 48 and the second semiconductor laser 52 are mounted, but a plurality of light sources are not necessarily required.

Also, 57 is a bandpass filter, 58 is a first lens, 59 is a second aperture member, and 60 is a first detector, which detects fluorescence. On the other hand, 61 is an ND filter, 62 is a second lens, 63 is a third aperture member, and 64 is a second detector, which detects scattered light. That is, in the present embodiment, the optical detection optical system includes the objective lens 42, the second dichroic mirror 56, the

filters

57 and 61, the

lenses

58 and 62, the aperture members 59 and 63, and the detectors. 60, 64.

The inspection chip 46 is configured to be transparent and circular, and is accommodated in a circular dish-shaped holder 66 fixed to the central shaft 65 and fixed to the central shaft 65. The central shaft 65 can be rotated by a spindle motor 67 as an example of the rotational drive system. On the other hand, the optical module 45 is movable by the drive mechanism in the radial direction of the disk formed by the inspection chip 46. Reference numeral 66a denotes an outer peripheral surface of the holder 66 provided with the encoder ring, and 68 denotes a head on which a pair of light emitting elements and light receiving elements for detecting the encoder ring are mounted.

Hereinafter, as an example, the case of scattered light detection for detecting the scattered light will be described as an example.

Of the first semiconductor laser 48 and the second semiconductor laser 52 in the light source device 41, a semiconductor laser emitting a laser beam having a wavelength reflected by the second dichroic mirror 56, for example, a first semiconductor laser 48 to a first one. A laser beam having a wavelength is emitted.

The laser light emitted from the first semiconductor laser 48 is converged by the first collimator lens 49, the spot size adjusting lens 50, and the first aperture member 51, and passes through the first dichroic mirror 54. Next, the light is reflected by the prism 55 and the second dichroic mirror 56, passes through the objective lens 42 and the inspection chip 46, and is collected on the specimen injection layer 47. In this case, the length of the prism 55 in the longitudinal direction (horizontal direction) is short, the width in the direction orthogonal to the longitudinal direction is narrow, and the laser light from the first semiconductor laser 48 is near the optical axis of the objective lens 42. It passes through only the (laser light transmission part). In this way, when the microscopic particles in the specimen injection layer 47 are irradiated with focused light, scattered light isotropically scattered from the irradiated portion to the surroundings.

The scattered light is isotropically emitted from the portion of the specimen injection layer 47 irradiated with the focused light to the surroundings. The component of the emitted scattered light that has passed through the inspection chip 46 and entered the objective lens 42 passes through the objective lens 42, is reflected by the second dichroic mirror 56, and is ND filter 61 and second lens 62. Then, it passes through the third aperture member 63 and is detected by the second detector 64. The signal detected by the second detector 64 is subjected to processing such as AD conversion by a built-in AD converter or the like, and then sent to a personal computer (PC) 80 or the like as an example of the detection unit. The In this way, the distribution of scattered light intensity at each measurement point on the specimen injection layer 47 is recorded in the internal memory or the like. Further, when the particle count is performed based on the detection signal, the particle count data is recorded in the internal memory or the like.

The third aperture member 63 is arranged to cut spatial stray light. It also functions as a confocal aperture member, and removes unnecessary reflected light and stray light from other than the surface where the specimen injection layer 47 exists. For example, since the reflected light generated on the surface of the inspection chip 46 and the lens surface is deviated from the focal position of the objective lens 42, it becomes light spread at the position of the third aperture member 63 by the optical system following the objective lens 42. The third aperture member 63 cannot be efficiently transmitted.

Thus, by performing the detection as described above while rotating the inspection chip 46, the scattered light intensity at each measurement point is recorded in the internal memory of the PC 80 or the like.

In the above description, the scattered light detection by the first wavelength laser beam from the first semiconductor laser 48 has been described. However, the second wavelength laser beam from the second semiconductor laser 52 can also be detected by the first wavelength. Except for being reflected by the one dichroic mirror 54, it is exactly the same. The fluorescence detection for detecting the fluorescence is exactly the same except that the fluorescence transmitted through the second dichroic mirror 56 is detected by the first detection device 43.

According to the present embodiment, the fluorescence generated in the fine particles is not blocked by the prism 55 and its support (not shown), and the fluorescence can be detected with high sensitivity.

Also in this embodiment, the focal depth d of the objective lens 42 is the wavelength λ / (effective NA of the irradiation light) ² , that is, the wavelength λ / (beam radius reduction rate R) ² of the irradiation light. The value is set to be not less than the thickness D of the specimen injection layer 47 in the test chip 46 and not more than three times the thickness D of the specimen injection layer 47. In this way, the beam radius of the irradiation light is set to be equal to or less than twice the route of the spot radius r so as to ensure the necessary depth of focus d.

Further, the depth range of the position where the light transmitted through each of the aperture members 59 and 63 is emitted is set larger than the thickness D of the specimen injection layer 47.

In addition, the shape of the objective lens 42 is a region along the optical axis, and the beam light transmitting portion has a shape in which the refraction angle of the light after transmission becomes smaller than other regions, and each semiconductor The irradiation light from the

lasers

48 and 52 has a function of narrowing toward the specimen. On the other hand, the other region excluding the beam light transmission part has a shape such that the refraction angle of the light from the fine particles after transmission increases from the beam light transmission part side toward the outer peripheral edge side, A function of condensing light emitted from a minute particle at a wide angle is provided.

By doing this, even if microparticles exist at different positions in the thickness direction in the specimen injection layer 47, the fluctuations in the light intensity that are transmitted through the aperture members 59 and 63 and detected by the

detectors

60 and 64 are detected. The rate can be within 10%, and the error of the fine particle diameter when detecting fluorescence and scattered light can be within 10%.

Third Embodiment In this embodiment, the microparticle detection method in the first embodiment and the second embodiment is two-dimensionally scanned in two directions orthogonal to each other on the inspection chip or the optical module. The present invention relates to a fine particle detection apparatus applied to a two-dimensional scanning method.

FIG. 7 shows, as an example, a microparticle detection apparatus (PC80 is omitted) that scans in the X direction while moving the inspection chip in the Y direction.

7, 71 is a light source device, 72 is an objective lens, 73 is a band pass filter, and 74 is a detection device. The light source device 71, the objective lens 72, the band pass filter 73, and the detection device 74, which are an example of the irradiation optical system, are housed in a frame and constitute the optical module 75. A glass stage 76 is disposed above the optical module 75 so as to face the objective lens 72. On the glass stage 76, for example, a suspension, a gel support, or a membrane in which fine particles labeled with a fluorescent substance are distributed. A test chip 77 in which a transfer support such as a sample is injected as a specimen is set.

Here, the glass stage 76 has a rectangular shape, and scans in a two-dimensional direction of a first scanning direction in the long side direction and a second scanning direction in the short side direction orthogonal to the first scanning direction. It is configured. The scanning method in that case is not particularly limited. In short, it is only necessary to include a first operation unit that reciprocates the glass stage 76 in the first scanning direction and a second operation unit that reciprocates in the second scanning direction. Alternatively, the optical module 75 side may be scanned in a two-dimensional direction, or the optical module 75 may be reciprocated in the first scanning direction while moving the glass stage 76 in the second scanning direction.

The light source device 71 has the same function as the light source device 1 shown in FIG. 1 in the first embodiment or the light source device 41 shown in FIG. 6 in the second embodiment.

In addition, the light detection optical system has a configuration that functions in the same manner as the light detection optical system shown in FIG. 1 in the first embodiment or the light detection optical system shown in FIG. 6 in the second embodiment. ing. The bandpass filter 73 is disposed in the rotary folder 79 and can be replaced with a filter having another wavelength according to the wavelength of the fluorescence.

As described above, in the microparticle detection apparatus according to the present embodiment, the intensity of scattered light or fluorescence is obtained by relatively scanning the glass stage 76 on which the optical module 75 or the inspection chip 77 is placed in a two-dimensional direction. The distribution image is read.

For example, as shown in FIG. 8, the microparticle detection apparatus scans the optical module 75 in the X direction that is the first scanning direction while moving the glass stage 76 in the Y direction that is the second scanning direction. In this way, a distribution image of the intensity of scattered light or fluorescence is generated.

Also in this embodiment, the focal depth d of the objective lens 72 is the wavelength λ / (effective NA of irradiation light) ² , that is, the wavelength λ / (beam radius reduction rate R) ² of irradiation light. The value is set to be not less than the thickness D of the specimen injection layer 78 in the test chip 77 and not more than three times the thickness D of the specimen injection layer 78. In this way, the beam radius of the irradiation light is set to be equal to or less than twice the route of the spot radius r, and the necessary depth of focus d is secured.

Further, the depth range of the position where the light transmitted through the aperture member is emitted is set larger than the thickness D of the specimen injection layer 78.

The beam light transmitting portion, which is a region along the optical axis of the objective lens 72, has such a shape that the refraction angle of the light after transmission is smaller than other regions, and a semiconductor laser ( A function of narrowing the irradiation light from the light source toward the specimen. On the other hand, the other region excluding the beam light transmission part has a shape such that the refraction angle of the light from the fine particles after transmission increases from the beam light transmission part side toward the outer peripheral edge side, A function of condensing light emitted from a minute particle at a wide angle is provided.

By doing so, even if microparticles exist at different positions in the thickness direction in the specimen injection layer 78, the variation rate of the light intensity that is transmitted through the aperture member and detected by the detector is within 10%, and the fluorescence In addition, the error of the fine particle diameter when detecting scattered light can be made within 10%.

As described above, the microparticle detection apparatus of the present invention is

Test chips

7, 46, 77 having

specimen injection portions

36, 47, 78 into which specimens containing fine particles are injected;

Light sources

8, 48, 52;
Irradiation

optical systems

1, 41, 71 for irradiating the specimens in the

test chips

7, 46, 77 with the light emitted from the

light sources

8, 48, 52;
A light detection optical system for detecting light emitted from the microparticles in the specimen by the light irradiation;
A detection unit that detects the microparticles based on the intensity of light from the microparticles detected by the photodetection optical system;
The light detection optical system transmits light emitted from the

light sources

8, 48, and 52 toward the specimen, and condenses light emitted from the microparticles in the specimen by irradiation with the light. Including

objective lens elements

2, 42, 72;
The

objective lens elements

2, 42, 72 include a beam light transmitting portion having a shape for transmitting the light from the

light sources

8, 48, 52 and condensing the transmitted light toward the specimen. ,
The thickness of the

test chip

7, 46, 77 in the

specimen injection part

36, 47, 78 in the light irradiation direction is D, the wavelength of the irradiation light from the

light source

8, 48, 52 is λ, and the beam light transmission part is When the reduction rate of the beam radius of the irradiation light by the above-mentioned condensing is R, the relational expression D ≦ λ / R ²
It is characterized by meeting.

According to the above configuration, the thickness D in the light irradiation direction of the

specimen injection portions

36, 47, 78 of the

test chips

7, 46, 77 satisfies the relational expression “D ≦ λ / R ² ”. Yes. Further, the reduction rate R of the beam radius of the irradiation light due to the condensing at the beam light transmitting portion of the

objective lens element

2, 42, 72 is substantially the same as one half of the reciprocal of the F value, and the numerical aperture NA. And R = 1 / (2F) = NA.

Therefore, the relational expression “D ≦ λ / R ² ” can be modified as follows.
D ≦ λ / R ² = λ / NA ²

That is, according to the present invention, the focal depth d of the irradiation light in the

objective lens elements

2, 42, 72 is set to be equal to or greater than the thickness D in the light irradiation direction in the

specimen injection portions

36, 47, 78. Has been. Therefore, the irradiation light can be uniformly irradiated onto the specimen region having a thickness that is injected into the

specimen injection portions

36, 47, and 78. In other words, even if microparticles exist at different positions in the thickness direction of the specimen region, the microparticles can be detected.

Further, when the

test chips

7, 46, 77 are rotated or scanned in one direction, even if the

test chips

7, 46, 77 cause surface blurring in the light irradiation direction, the depth of focus d is the sample injection. Since the thickness is equal to or greater than the thickness D of the

portions

36, 47, and 78, the influence of the surface blur can be reduced by the amount of the focal depth d larger than the thickness D.

As described above, the focal depth d of the irradiation light is desirably larger than the thickness D in the light irradiation direction. However, if the depth of focus d (= λ / R ² ) is too large, the rate of decrease R by the beam light transmitting portion becomes small, and the beam radius of the irradiation light cannot be reduced.

According to this embodiment, in addition, the focal depth d represented by λ / R ² is set to be not more than three times the thickness D in the light irradiation direction in the

specimen injection sections

36, 47, 78. Accordingly, considering the accuracy of the irradiation

optical systems

1, 41, 71 and the light detection optical system and surface blurring in the light irradiation direction of the

inspection chips

7, 46, 77, the light detection optical system is optimal. Light detection can be performed with high accuracy.

Moreover, in the microparticle detection apparatus of one embodiment,
The surface of the beam light transmitting portion in the

objective lens element

2, 42, 72 has a curvature for condensing light by refraction,
The thickness of the

test chip

7, 46, 77 in the

specimen injection part

36, 47, 78 in the light irradiation direction is D, the wavelength of the light emitted from the

light source

8, 48, 52 is λ, and the beam light transmission part is The relation D ≦ λ (f / r0) ² , where r0 is the beam radius of the irradiated light to be transmitted and f is the focal length of the light transmitted through the beam light transmitting portion.
It comes to satisfy.

According to this embodiment, the surface of the beam light transmitting portion has a curvature for condensing light by refraction, and the thickness D of the

specimen injection portions

36, 47, 78 is expressed by the relational expression “D ≦ λ ( f / r0) ² "is satisfied. Therefore, it is desirable that the beam radius r0 of the irradiation light transmitted through the beam light transmitting portion is smaller within a range satisfying the relational expression “D ≦ λ / R ² ”.

Moreover, in the microparticle detection apparatus of one embodiment,
The beam light transmission part in the

objective lens element

2, 42, 72 is an area along the optical axis so that the refraction angle of the light after transmission is smaller than other areas excluding the beam light transmission part. And has a function of narrowing the irradiation light from the

light sources

8, 48, 52 toward the specimen,
In other regions of the

objective lens elements

2, 42 and 72 excluding the beam light transmission part, the refraction angle of the light from the fine particles after transmission increases from the beam light transmission part side toward the outer peripheral edge side. And has a function of condensing light emitted at a wide angle from the fine particles.

According to this embodiment, the surface of the beam transmitting portion having a curvature for condensing light by refraction and a small beam radius r0 of the transmitted irradiation light is used as the light in the

objective lens elements

2, 42, 72. A region along the axis is formed so that the refraction angle of light after transmission is smaller than that of other regions. Further, the other region (the region around the region along the optical axis) excluding the beam light transmission part is shaped so that the refraction angle of the light from the fine particles after transmission increases outward. Yes.

Therefore, the single

objective lens element

2, 42, 72 is focused on the light emitted from the

light sources

8, 48, 52 toward the specimen, and the light emitted from the fine particles at a wide angle is condensed. It is possible to have a function to perform.

Moreover, in the microparticle detection apparatus of one embodiment,

Aperture members

18, 22 provided between the

objective lens elements

2, 42, 72 and the detection unit and having holes for transmitting light from the fine particles that have passed through the

objective lens elements

2, 42, 72. 26, 59, 63,
The diameters of the holes in the

aperture members

18, 22, 26, 59, 63 are all taken in from the fine particles distributed in the range of the thickness D in the

sample injection sections

36, 47, 78, and the sample injection section The variation rate of the light detection efficiency of the light emitted from the fine particles existing in the range of the thickness D of 36, 47, 78 by the detection unit is set to 10% or less.

According to this embodiment, the

aperture members

18, 22, 26, 59, 63 are provided between the

objective lens elements

2, 42, 72 and the detection unit, and the

aperture members

18, 22, 26, 59 are provided. , 63 with a diameter of the hole in the range of the thickness D of the

specimen injection part

36, 47, 78, the variation rate of the light detection efficiency by the detection part of the light emitted from the fine particles is 10% or less. It is set as follows. Therefore, even if microparticles exist at different positions in the thickness direction in the

specimen injection portions

36, 47, and 78, the variation rate of the light intensity transmitted through the holes of the

aperture members

18, 22, 26, 59, and 63 is increased. It can be within 10%.

DESCRIPTION OF

SYMBOLS

1,41,71 ...

Light source device

2,42,72 ... Objective lens 3-5,43,44,74 ...

Detection device

6,45,75 ...

Optical module

7,46,77 ...

Inspection chip

8,48,52 ...

Semiconductor laser

9, 49, 53 ...

Collimator lens

10, 16, 20, 57, 73 ...

Band pass filter

11, 24, 61 ...

ND filter

12, 18, 22, 26, 51, 59, 63 ... Aperture member 13 ...

Prism Mirror

14, 15, 54, 56 ...

Dichroic mirror

17, 21, 25, 58, 62 ...

Lens

19, 23, 27, 60, 64 ...

Detector

28, 65 ... Center shaft 29 ... Table 30, 67 ...

Motor

31, 32 ... Substrate 34 ...

Spacers

36, 47, 78 ... Sample injection layer 37 ... Sample injection port 38 ... Detected fine particles 50 ... Spot size adjustment lens 55 ... Prism 66 ... Holder 76 ... Glass stage 80 ... Personal computer (Information processing device)

Claims

A sample chip (7, 46, 77) having a sample injection part (36, 47, 78) into which a sample containing fine particles is injected;
A light source (8, 48, 52);
An irradiation optical system (1, 41, 71) for irradiating the specimen in the specimen chip (7, 46, 77) with the light emitted from the light source (8, 48, 52);
A light detection optical system (3, 4, 5, 43, 44) for detecting light emitted from the microparticles in the specimen by the light irradiation;
A detection unit (80) for detecting the microparticles based on the intensity of light from the microparticles detected by the photodetection optical system (2, 13 to 27, 42, 55 to 64, 72, 74); With
The light detection optical system (2, 13 to 27, 42, 55 to 64, 72, 74) transmits the light emitted from the light source (8, 48, 52) toward the specimen, while the light. An objective lens element (2, 42, 72) for condensing light emitted from the microparticles in the specimen by irradiation of
The objective lens element (2, 42, 72) transmits the light from the light source (8, 48, 52) and has a shape for condensing the transmitted light toward the specimen. Contains
The thickness of the sample tip (7, 46, 77) in the sample injection part (36, 47, 78) in the light irradiation direction is D, the wavelength of the light emitted from the light source is λ, and the light beam transmission part is the above. When the reduction rate of the beam radius of the irradiated light due to condensing is R, the relational expression D ≦ λ / R 2
A fine particle detection device characterized by satisfying the above.
The fine particle detection apparatus according to claim 1,
The following relational expression D ≦ λ / R 2 ≦ 3D
A fine particle detection device characterized by satisfying the above.
In the microparticle detection apparatus according to claim 1 or 2,
The surface of the beam light transmitting portion in the objective lens element (2, 42, 72) has a curvature for condensing light by refraction,
The thickness in the light irradiation direction of the sample injection portion (36, 47, 78) of the sample chip (7, 46, 77) is D, the wavelength of the irradiation light from the light source (8, 48, 52) is λ, The relational expression D ≦ λ (f / r0) 2 , where r0 is the beam radius of the irradiating light transmitted through the beam light transmitting portion and f is the focal length of the light after passing through the beam light transmitting portion.
A fine particle detection device characterized by satisfying the above.
In the microparticle detection apparatus according to claim 3,
The beam light transmission part in the objective lens element (2, 42, 72) is an area along the optical axis, and the refraction angle of the light after transmission is smaller than other areas excluding the beam light transmission part. And has a function of narrowing the irradiation light from the light source (8, 48, 52) toward the specimen,
In the other area of the objective lens element (2, 42, 72) except for the beam light transmission part, the refraction angle of the light from the fine particles after transmission increases from the beam light transmission part side toward the outer peripheral edge side. A microparticle detection apparatus characterized by having a function of condensing light emitted from the microparticles at a wide angle.
In the microparticle detection apparatus according to any one of claims 1 to 4,
A hole provided between the objective lens element (2, 42, 72) and the detection unit (80) and transmitting light from the microparticles that have passed through the objective lens element (2, 42, 72). An aperture member (18, 22, 26, 59, 63) having
While taking in all the light from the said microparticle which distributes the diameter of the said hole in the said aperture member (18,22,26,59,63) in the range of the thickness D in the said specimen injection part (36,47,78). The fine particle detection is characterized in that the variation rate of the light detection efficiency by the detection part of the light emitted from the fine particles existing within the range of the thickness D of the specimen injection part is set to 10% or less. apparatus.