Light scattering detection device

Abstract

The present invention provides a light scattering detection device, which can set the vertical solid angle of the incident side slit of the imaging lens to be larger without setting the tilt angle α of the incident light to be larger, and can perform measurement with a high S / N ratio and high detection accuracy. The light scattering detection device (1) comprises: a transparent sample pool (10) for holding a liquid sample; a light source (20) for irradiating the sample pool (10) with coherent light; an imaging optical system (50) for focusing light scattered from the sample pool (10) to the surroundings at different scattering angles; a slit plate (40) for limiting the scattering angle range incident on the imaging optical system (50); and a detector (70) for receiving the focused light from the imaging optical system (50), wherein the central axis (41S) of the slit (41) is configured to be eccentric to one side in the vertical direction from the central axis (50S) of the imaging optical system (50).

Description

Technical Field

[0001] The present invention relates to a light scattering detection device used in a microscopic particle detection device for measuring the molecular weight, gyration radius (size), etc. of microparticles dispersed in a liquid sample. Background Art

[0002] Size exclusion chromatography (SEC) is known as a method for separating microparticles such as proteins dispersed in a liquid sample. In recent years, multi-angle light scattering (MALS) detection devices have been used as chromatographic detection devices in addition to ultraviolet (UV) absorbance detection devices and differential refractive index detection devices. MALS detection devices have the advantage of being able to calculate the molecular weight and particle size of the measured sample (see Patent Documents 1 and 2).

[0003] Figure 7 The coordinate system of the scattered light radiation direction when the scattered light generating light source is arranged at the origin is shown. Figure 7 As shown, on the XY plane, light is incident in the positive direction of the X direction, the scattering angle from the traveling direction of the light on the XY plane is defined as θ, and the angle from the XY plane is defined as φ.

[0004] Next, Figure 8 A top view showing a basic configuration example of a MALS detection device, Fig. 9 A side view is shown. Figure 8 as well as Fig. 9 In FIG. 1 , 110 is a sample pool, 111 is a liquid sample, 120 is a light source, 121 is a focusing lens, 140 is a slit plate, 150 is an imaging lens, 160 is an aperture plate, and 170 is a detector. Figure 8 as well as Fig. 9 As shown, a liquid sample 111 is passed into a cylindrical sample cell 110, and light is irradiated from a light source 120 in a manner that passes through the sample cell 110 and the center of the flow path. Visible laser light is generally used as a light source. An angle θ from the direction of travel of the light is defined as a scattering angle on a horizontal plane (on an XY plane), and a plurality of detectors 170 are arranged on a horizontal plane (on an XY plane) passing through the sample cell 110 and the center of the flow path to detect different scattering angles. Figure 8 This is an example in which two detectors 170 are arranged at arrangement angles of θ1 and θ2.

[0005] In the case of the cylindrical sample cell 110, there is a technical problem that reflected light at the interface between glass and air and at the interface between glass and the flow path enters the detector 170 as stray light, which deteriorates the detection accuracy of the detector 170. As a solution, Fig. 9As shown, the inventors have found a method for reducing the above-mentioned stray light by tilting the incident light with respect to the sample cell 110 (angle α) (see Non-Patent Document 1).

[0006] Next, Fig.10 as well as Fig.11 The relationship between the scattered light intensity and the scattering angle is shown. That is, Fig.10 as well as Fig.11 The scattering pattern of particles with a refractive index of 1.59 in a solvent with a refractive index of 1.33 and an incident wavelength of 660 nm is calculated according to the Mie scattering theory. The particle sizes are 1 nm, 100 nm, and 500 nm. Fig.10 is the scattering angle pattern in the horizontal direction (θ direction), Fig.11 This is the scattering angle pattern in the vertical direction (φ direction). When the sample is sufficiently small compared to the wavelength of the incident light, scattered light in the θ direction is generated isotropically, and the scattered light intensity has no dependence on the scattering angle. As the particle size of the sample increases, the scattered light scatters more strongly in the forward direction (in the direction where θ is smaller). In the φ direction, the scattered light intensity tends to decrease as φ increases.

[0007] In addition, for the MALS detection device, a device capable of measuring low-concentration samples and having a high S / N ratio is desired. To this end, it is required to allow the scattered light generated from the sample to enter the optical system of the detector efficiently. In other words, it is necessary to make the solid angle of the scattered light entering each detector larger. When the solid angle of the detector is increased, if the solid angle in the horizontal direction (on the optical axis plane) is increased, the angular resolution of each detector deteriorates, so it is desired to increase the solid angle in the vertical direction. However, increasing the size of the detector in order to increase the solid angle will increase the dark current, so it is not preferred.

[0008] In order to increase the solid angle without increasing the size of the detector, a method of focusing the scattered light with a lens or the like is adopted (non-patent document 1). Specifically, the scattered light generated in the pool flow path is imaged on the detector via an imaging lens. Therefore, in order to reduce the solid angle in the horizontal direction and increase the solid angle in the vertical direction, a slit with a narrow opening width in the horizontal direction and a wide opening length in the vertical direction is provided on the incident side of the imaging lens. Thus, the scattered light can be efficiently detected without deteriorating the angular resolution.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent Application Laid-Open No. 07-72068

[0012] Patent Document 2: Japanese Patent Application Publication No. 2015-111163

[0013] Non-patent document 1: "Analysis of absolute molecular weight and complex formation of proteins using light scattering method", Otaka Masafumi, Biotechnology, Vol. 89 Summary of the invention

[0014] Technical problem to be solved by the invention

[0015] In order to increase the amount of light entering the detector, it is necessary to set the vertical opening length of the slit disposed on the incident side of the imaging lens to be larger. However, if the vertical opening length of the slit is set to be larger, the reflected light at the interface between the glass and air of the sample cell and at the interface between the glass and the flow path enters the detector as stray light. Since the intensity of the reflected light is greater than that of the scattered light, the dynamic range of the detector is reduced, and the detection accuracy of the detector is deteriorated due to the intensity fluctuation of the stray light. On the other hand, in order to remove the stray light, if the inclination angle α of the incident light is set to be larger, as shown in FIG. Fig.11 As shown, the scattering angle φ in the vertical direction becomes larger, the intensity of scattered light entering the slit decreases, and the S / N ratio decreases.

[0016] Therefore, an object of the present invention is to provide a light scattering detection device that can set the vertical solid angle of the incident side slit of the imaging lens to be larger without setting the inclination angle α of the incident light to be larger, and can perform measurement with a high S / N ratio and high detection accuracy.

[0017] Solutions for solving the above technical problems

[0018] A light scattering detection device according to one embodiment of the present invention is a light scattering detection device for detecting microparticles in a liquid sample, and is characterized in that it comprises: a transparent sample cell for holding a liquid sample; a light source for irradiating the sample cell with coherent light; an imaging optical system for focusing light scattered from the sample cell to the surroundings at different scattering angles; a slit plate for limiting the range of scattering angles incident on the imaging lens; a detector for receiving the focused light from the imaging optical system; and the central axis of the slit is configured to be eccentric in a vertical direction from the central axis of the imaging optical system.

[0019] In the configuration of the light scattering detection device, it is preferred that the slit is vertically elongated and at least a side along the vertical direction is linear.

[0020] Furthermore, it is preferable that the slit is arranged to be eccentrically disposed upward in the vertical direction from the central axis of the imaging optical system.

[0021] Furthermore, it is preferable that, when a is defined as a lower length of the slit in the vertical direction from the central axis of the imaging optical system and b is defined as an upper length, a<b is satisfied.

[0022] Furthermore, preferably, a plurality of detection optical systems from the sample pool to the detector are arranged around the sample pool at equal intervals from the center axis of the pool, and the length ratio of the lower length a and the upper length b of the slit is adjusted according to the configuration angle of each detector relative to the sample pool under the condition of a≤b.

[0023] In addition, it is preferable that the light source is arranged so that an optical axis of coherent light incident on the sample cell from the light source is inclined at a predetermined angle with respect to a plane including the sample cell and the detector.

[0024] Effects of the Invention

[0025] According to the present invention, a light scattering detection device can be provided, which can set the vertical solid angle of the incident side slit of the imaging lens to be large without setting the inclination angle α of the incident light to be large, and can perform measurement with a high S / N ratio and high detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a side view of one embodiment of the light scattering detection device of the present invention.

[0027] Figure 2 It is a partially enlarged side view of the light scattering detection device according to this embodiment.

[0028] Figure 3 It is a top view of the light scattering detection device according to this embodiment.

[0029] Figure 4 This is a side view for explaining the configuration and component sizes in ray tracing simulation.

[0030] Figure 5 This is a plan view for explaining the results of a ray tracing simulation when the detectors are arranged at θ=15 degrees.

[0031] Figure 6 This is a side view for explaining the results of a ray tracing simulation when the detectors are arranged at θ=15 degrees.

[0032] Figure 7 It is a coordinate system of the scattered light radiation direction when the scattered light generating light source is arranged at the origin.

[0033] Figure 8 It is a top view of a basic configuration example of a MALS detection device.

[0034] Fig. 9 It is a side view of a basic configuration example of a MALS detection device.

[0035] Fig.10It is an explanatory diagram of the relationship between scattered light intensity and scattering angle (scattering angle pattern in the horizontal direction).

[0036] Fig.11 It is an explanatory diagram of the relationship between scattered light intensity and scattering angle (scattering angle pattern in the vertical direction). DETAILED DESCRIPTION

[0037] Hereinafter, one embodiment of the light scattering detection device of the present invention will be described with reference to the drawings. In addition, components denoted by the same reference numerals in the respective drawings have the same or similar configurations.

[0038] [Constitution of light scattering detection device]

[0039] First, refer to Figure 1 to Figure 3 The configuration of one embodiment of the light scattering detection device of the present invention will be described. Figure 1 It is a side view of one embodiment of the light scattering detection device of the present invention. Figure 2 FIG. 2 is a partially enlarged side view of the light scattering detection device of this embodiment. Figure 1 as well as Figure 2 As shown, the light scattering detection device 1 of this embodiment is a device for detecting the molecular weight and rotation radius (size) of microparticles such as proteins dispersed in a liquid sample. The light scattering detection device 1 includes a sample cell 10, a light source 20, a slit plate 40, an imaging optical system 50, an aperture plate 60, and a detector 70. Each component is described below.

[0040] The sample cell 10 is a transparent cylindrical cell that holds a liquid sample in an internal flow path. The sample cell 10 is formed of, for example, colorless and transparent quartz glass.

[0041] The light source 20 irradiates the sample cell 10 with coherent light. "Coherent light" refers to the following light: the phase relationship between the light waves of any two points in the light beam does not change with time and remains constant, and even if a large optical path difference is applied after the light beam is split by any method and overlapped again, it still shows complete coherence. As the light source 20, for example, a laser light source for irradiating visible light laser is used. There is no completely coherent light in nature, and laser oscillating in a single mode is light close to a coherent state.

[0042] A focusing optical system 21 is arranged on the optical path L1 of the incident light from the light source 20 to the sample cell 10. As the focusing optical system 21, for example, a single focusing lens is used. The focusing lens is a plano-convex lens, and the incident side of the light from the light source 20 is formed as a convex surface, and the exit side is formed as a flat surface. In the present embodiment, a single focusing lens is used as the focusing optical system 21, but the focusing optical system 21 can also be composed of a combination of multiple compound lenses and focusing lenses.

[0043] The light source 20 and the focusing optical system 21 are arranged in such a manner that the optical axis of the coherent light incident from the light source 20 to the sample cell 10 is tilted at a predetermined angle (tilt angle α) from a plane (XY plane) including the sample cell 10 and the detector 50. Specifically, the light source 20 and the focusing optical system 21 are arranged so that the incident light is incident from obliquely above relative to the sample cell 10. By tilting the incident light relative to the sample cell 10 (angle α), stray light caused by reflected light at the interface between the glass and air of the sample cell 10 and the interface between the glass and the flow path (hereinafter collectively referred to as the "cell interface") can be reduced. The laser irradiated from the light source 20 is focused near the central axis of the sample cell 10 after passing through the focusing optical system 21.

[0044] The detection optical system 30 is arranged on the optical path L2 of the outgoing light from the sample cell 10. The detection optical system 30 of the present embodiment is composed of a slit plate 40, an imaging optical system 50, an aperture plate 60, and a detector 70.

[0045] The imaging optical system 50 focuses light scattered from the sample cell 10 to the surroundings at different scattering angles. For example, a single imaging lens is used as the imaging optical system 50. The imaging lens is a plano-convex lens, and the incident side of the scattered light from the sample cell 10 is formed as a flat surface, and the exit side is formed as a convex surface. In the present embodiment, a single imaging lens is used as the imaging optical system 50, but the imaging optical system 50 can also be composed of a combination of multiple compound lenses and imaging mirrors.

[0046] The slit plate 40 is disposed between the sample cell 10 and the imaging optical system 50 on the optical path L2 of the outgoing light from the sample cell 10. The slit plate 40 limits the range of scattering angles incident on the imaging optical system 50. That is, the slit 41 opened in the slit plate 40 is vertically elongated in the vertical direction, and at least the side along the vertical direction is straight, in order to limit the scattering angle in the horizontal direction and capture more light beams in the vertical direction. Specifically, the slit 41 is in the shape of a rectangular or long hole that is vertically elongated.

[0047] The central axis 41S of the slit 41 is configured to be eccentric to one side in the vertical direction from the central axis 50S of the imaging optical system 50. Since the irradiation light of the light source 20 is incident from the oblique upper part of the sample cell 10, the reflected light RL generated at the interface between the air and the glass and the interface between the glass and the liquid (hereinafter referred to as the "cell interface") of the sample cell 10 is easily deviated downward as stray light. Therefore, in order to limit the reflected light (stray light) RL, the central axis 41S of the slit 41 of the present embodiment is configured to be eccentric to the upper part in the vertical direction from the central axis 50S of the imaging optical system 50.

[0048] Specifically, if Figure 2As shown, when the lower length of the slit 41 in the vertical direction from the central axis 50S of the imaging optical system 50 is set to a and the upper length is set to b, the relationship between the lower length a and the upper length b of the slit 41 is set to a<b. Incident light may also be incident from below to above, in which case, the relationship between the lower length a and the upper length b of the slit 41 is set to a>b.

[0049] The aperture plate 60 is disposed on the optical path L2 of the outgoing light from the sample cell 10 and on the imaging optical system side of the detector 70. The aperture plate 60 has a function of limiting stray light, and an aperture 61 opens in front of the light receiving surface of the detector 70.

[0050] The detector 70 receives the focused light from the imaging optical system 50. That is, the light receiving surface of the detector 70 is located at the focal point of the imaging optical system 50. As the detector 70 of this embodiment, for example, a photodiode (PD) is used, and an array detector such as a two-dimensional CMOS may also be used.

[0051] Figure 3 FIG. 1 is a top view of an embodiment of the light scattering detection device of this embodiment. Figure 3 As shown, the detection optical system 30 from the sample pool 10 to the detector 70 is arranged in multiple numbers around the sample pool 10 at equal intervals d from the pool center axis S. The angle θ from the direction of light travel is defined as the scattering angle on the horizontal plane (on the XY plane). Multiple detectors 70 are arranged on the horizontal plane (on the XY plane) passing through the sample pool 10 and the pool center axis S, so that different scattering angles can be detected. Figure 3 In the scheme, two detection optical systems 30 and 31 are arranged at the arrangement angles of θ1 and θ2.

[0052] When a plurality of detectors 70 are provided at equal intervals d around the sample cell 10, the length ratio of the lower length a to the upper length b of the slit 41 is adjusted under the condition of a ≤ b according to the arrangement angle of each detector 70 relative to the sample cell 10. Figure 3 In the above, the detection optical system 31 configured at the scattering angle θ2 is less affected by the reflected light at the cell interface than the detection optical system 30 configured at the scattering angle θ1. Since the detection optical system 31 with the scattering angle θ2 = 90 degrees is less affected by the reflected light at the cell interface, the vertical solid angle can be optimized by setting a larger value to make it closer to b.

[0053] [The role of light scattering detection device]

[0054] Next, refer to Figure 1 to Figure 6 The operation of the light scattering detection device of this embodiment will be described.

[0055] like Figure 1 as well as Figure 2 As shown, the liquid sample 11 is passed through the flow path of the cylindrical sample cell 10. When the passing of the liquid sample 11 is completed, visible laser light is irradiated as coherent light from the light source 20 via the focusing optical system 21. The visible laser light advances along the optical path L1, so that the laser light is incident on the liquid sample 11 in the flow path of the sample cell 10. When the laser light is incident on the liquid sample 11, the light is irradiated on the microparticles contained in the liquid sample 11 and scattered at a predetermined scattering angle. Then, the scattered light emitted from the sample cell 10 passes through the slit 41 of the slit plate 40, passes through the imaging optical system 50 and the aperture plate 60, and is received on the light receiving surface of the detector 70.

[0056] When the scattered light is emitted from the sample cell 10, reflected light RL as stray light is generated at the cell interface of the sample cell 10. Since the irradiation light of the light source 20 is incident from the oblique upper side of the sample cell 10 at an inclined angle α, the reflected light (stray light) generated at the cell interface of the sample cell 10 is easily deviated downward. Since the slit 41 of the present embodiment is configured to be eccentric upward in the vertical direction from the central axis 50S of the imaging optical system 50, the plate portion of the slit plate 40 can limit the reflected light (stray light) RL deviated downward.

[0057] In this way, by disposing the slit 41 of the slit plate 40 eccentrically upward in the vertical direction from the central axis 50S of the imaging optical system 50, scattered light useful for analysis can be positively incident on the imaging optical system 50. The imaging optical system 50 forms an image on the light receiving surface of the detector 70, and the aperture plate 60 is disposed in front of the light receiving surface. Thus, the aperture plate 60 can further limit stray light, so that the scattered light required for analysis is received on the light receiving surface of the detector 70.

[0058] Specifically, when the lower length of the slit 41 in the vertical direction from the central axis 50S of the imaging optical system 50 is set to a and the upper length is set to b, the relationship between the lower length a and the upper length b of the slit 41 is set to a<b. That is, by setting the lower length a of the slit 41 to be smaller, the reflected light RL at the cell interface can be blocked by the plate portion of the slit plate 40. As for the upper length b, by setting it larger with the effective diameter of the imaging optical system 50 as the upper limit, the solid angle in the vertical direction can be increased.

[0059] In addition, if Figure 3As shown, a plurality of detection optical systems 30 from the sample cell 10 to the detector 70 are arranged at equal intervals d around the sample cell 10 from the cell center axis S. In the case where the light scattering detection device 1 is provided with a plurality of detection optical systems 30 at equal intervals d around the sample cell 10, the length ratio of the lower length a and the upper length b of the slit 41 is adjusted under the condition of a≤b according to the arrangement angle of each detector 70 relative to the sample cell 10. That is, in the detection optical system 31 arranged at the scattering angle θ2=90 degrees where the influence of the reflected light RL at the cell interface is small, by setting a larger value to make it close to b, the vertical solid angle can be optimized.

[0060] 〔Ray tracing simulation〕

[0061] In order to confirm the effects of the above-described embodiment, a ray tracing simulation was performed. Figure 4 This is a side view for explaining the configuration and component sizes in ray tracing simulation. Figure 5 1 is a top view for explaining the results of a ray tracing simulation when a detector (PD) is arranged at θ=15 degrees. Figure 6 It is its side view.

[0062] like Figure 4 As shown, the sample pool 10 is, for example, a transparent cylindrical pool with an inner diameter of 1.6 mm and an outer diameter of 8.0 mm. An imaging lens (plano-convex lens) is arranged at a position S47 mm from the center axis of the sample pool 10. The imaging lens is formed, for example, with a convex diameter of φ12.7 mm and a focal length of 38 mm. An aperture plate 60 and a 2.4 mm PD are arranged at a position S140 mm from the center axis of the sample pool 10. The slit 41 of the slit plate 40 is formed, for example, as an opening with a horizontal width of 3 mm and a vertical length of 8.5 mm (Z direction). Regarding the vertical opening of the slit 41, for example, the lower length a from the XY plane containing the center axis 50S of the imaging optical system 50 is set to 2.5 mm along the -Z direction, and the upper length b is set to 6.0 mm along the +Z direction.

[0063] like Figure 5 as well as Figure 6 As shown, by setting the relationship between the lower length a and the upper length b to a<b, the reflected light RL generated at the cell interface of the sample cell 10 is blocked by the plate portion of the slit plate 40 and does not reach the detector 70, so high-precision measurement can be performed.

[0064] In addition, the stray light caused by the reflected light RL at the cell interface depends on the configuration angle θ of the detector 70. When θ is small, since the influence of the reflected light RL at the cell interface is large, a needs to be shortened, and when θ is close to 90 degrees, the reflected light RL at the cell interface is almost not generated. According to the configuration angle of the detector 70, the slit 41 is optimized under the condition of a≤b, thereby achieving the reduction of stray light and the improvement of the S / N ratio.

[0065] As described above, according to the light scattering detection device 1 of the present embodiment, it is possible to provide a light scattering detection device that can set the vertical solid angle of the slit 41 of the slit plate 40 disposed on the incident side of the imaging optical system 50 to be large without setting the inclination angle α of the incident light to be large, and can perform measurement with a high S / N ratio and high detection accuracy.

[0066] The above-mentioned embodiments are for easy understanding of the present invention and are not used to limit the interpretation of the present invention. The various elements and their configurations, materials, conditions, shapes and sizes of the embodiments are not limited to the examples, but can be appropriately changed. In addition, the components shown in different embodiments can be partially replaced or combined with each other.

[0067] Description of Reference Numerals

[0068] 1 Light scattering detection device

[0069] 10 Sample pool

[0070] 20 Light Source

[0071] 30 Detection optical system

[0072] 40 Slit Plate

[0073] 41 Slit

[0074] 41S Center axis of the slit

[0075] 50 Imaging Optical System

[0076] 51S The central axis of the imaging optical system

[0077] 70 detectors.

Claims

1. A light scattering detection device, which is a light scattering detection device for detecting microparticles in a liquid sample, It is characterized in that have: Transparent sample pool to hold liquid samples; a light source, irradiating the sample cell with coherent light; An imaging optical system for focusing light scattered from the sample cell to the surroundings at different scattering angles; A slit plate, used to limit the range of scattering angles incident on the imaging optical system, wherein the slit plate is arranged between the sample cell and the imaging optical system; a detector for receiving the focused light from the imaging optical system; The central axis of the slit of the slit plate is arranged to be vertically eccentric from the central axis of the imaging optical system.

2. The light scattering detection device according to claim 1, It is characterized in that The slit is longitudinally elongated in the vertical direction, and at least the side along the vertical direction is linear.

3. The light scattering detection device according to claim 1 or claim 2, It is characterized in that The slit is arranged to be eccentrically upward in the vertical direction from the central axis of the imaging optical system.

4. The light scattering detection device according to claim 3, It is characterized in that When a is defined as the lower length of the slit in the vertical direction from the central axis of the imaging optical system and b is defined as the upper length, a<b is established.

5. The light scattering detection device according to claim 4, It is characterized in that A plurality of detection optical systems from the sample cell to the detector are arranged around the sample cell at equal intervals from the cell center axis. The length ratio of the lower length a and the upper length b of the slit is adjusted according to the arrangement angle of each detector with respect to the sample cell under the condition of a≤b.

6. The light scattering detection device according to claim 1 or 2, It is characterized in that The light source is arranged so that the optical axis of the coherent light incident on the sample cell from the light source is inclined at a predetermined angle with respect to a plane including the sample cell and the detector.

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