Particle measuring device, particle measuring method, sample container

Abstract

The purpose of the present invention is to reduce, when measuring the size or density, etc., of particles by light irradiation, the burden on a user of inputting the refractive index of the particles or the refractive index of a solvent into a measurement device, and improve refractive-index-dependent measurement accuracy. The particle measurement device according to the present invention specifies a boundary position along the optical axis direction between a light transmission window and a sample, and calculates the refractive index of the sample using the refractive index of the light transmission window and the refractive index of a known sample at the boundary position (see fig. 6).

Description

[Technical field]

[0001] The present invention relates to a technique for measuring the size distribution of particles suspended in a sample containing a solvent and the particles. [Background technology]

[0002] In recent years, the focus of pharmaceutical development has been shifting from small molecule drugs to biopharmaceuticals. Biopharmaceuticals are polymeric and therefore prone to aggregation, which may cause toxicity. For example, the U.S. Food and Drug Administration and others are trying to strengthen regulations on aggregate concentration management. Therefore, a technology is needed to quantitatively measure the size distribution of desired density for aggregates in the submicron region of 0.1 to 1 um. Protein aggregates float in a solvent, and their position changes over time due to Brownian motion. In the following, this invention describes a technology for measuring the size and density of protein aggregates and standard particles such as polystyrene beads. These test objects are collectively referred to as "particles."

[0003] Patent Document 1 describes a technique for detecting particles using optical measurement. The document discloses "an optical measurement method for generating an optical spot by converging light and measuring an object having a size approximately three times or less than the size of the optical spot, the optical measurement method comprising a signal acquisition step of detecting reflected light reflected from the object by irradiating the object with the light while moving at least the focal position of the light in the optical axis direction, a step of acquiring correspondence data describing the correspondence between the intensity of the reflected light and the size of the object, and a size calculation step of acquiring the size of the object by consulting the correspondence data using the intensity of the reflected light" (Claim 1). The technology described in the document can achieve high-resolution measurement without pre-processing by causing the reflected light to interfere with a reference light to enhance the signal.

[0004] Patent Document 2 discloses a technique that eliminates the need for phase adjustment of a reference light by mirror scanning in time domain OCT (Optical Coherence Tomography) by physically scanning an objective lens and receiving the interference between signal light and interference light with four detectors with different phase conditions. Furthermore, Patent Document 2 discloses a technique based on the technique of Patent Document 1 for speeding up scanning of a light spot so as not to be affected by the movement of particles undergoing Brownian motion in a liquid.

[0005] Patent Document 3 discloses a technology for measuring tomographic images of a living body using a semiconductor laser as a light source, in which a high frequency is superimposed on a driving current to control the coherence length within a predetermined range, thereby reducing the effect of noise contained in the acquired tomographic images.

[0006] Patent Document 4 discloses a technique for measuring the refractive index of a solvent using a special container and an optical system.

[0007] Non-Patent Document 1 discloses standard information regarding the functions and shapes of microplates, which are representative containers used in the analysis of biopharmaceuticals. These standards will be mentioned in the embodiment of the sample container described later. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2017-102032 A [Patent Document 2] WO2020 / 144754 [Patent Document 3] JP 2015-049204 A [Patent Document 4] Japanese Patent Application Publication No. 7-055700 [Non-patent literature]

[0009] [Non-Patent Document 1] URL:https: / / www.slas.org / education / ansi-slas-microplate-standards / (retrieved January 18, 2022) Summary of the Invention [Problem to be solved by the invention]

[0010] Consider measuring the size and density of particles such as protein aggregates using the technology described in Patent Document 1 or 2. The reflected light detected using the technology described in the same document changes depending on the particle size and its refractive index. The technology described in the same document can obtain a detection signal by converting the electric field amplitude of the reflected light into a voltage using optical interference. The magnitude of this detection signal changes depending on not only the particle size but also the refractive index of the particle and the refractive index of the solvent. On the other hand, in biopharmaceuticals, for example, pH, salt, sugar, surfactant, etc. are added in appropriate amounts to prevent protein aggregation as much as possible. Therefore, the refractive index of the solvent changes depending on the conditions of the additive.

[0011] In the case of protein aggregates with a small difference in refractive index from the solvent, the magnitude of the detection signal changes greatly with respect to changes in the refractive index of the solvent. Therefore, when measuring the size and density of protein aggregates using the techniques described in Patent Documents 1 and 2, the user needs to input the refractive indexes of the protein aggregates and the solvent to the measurement device with high accuracy.

[0012] Instead of the user inputting the refractive index, it is possible to use a device for measuring the refractive index as described in Patent Document 4. However, in commercially available refractive index measuring devices including the method described in the same document, the wavelength of the light source used is often a discrete value. Therefore, from the viewpoint of user convenience, it is difficult to accurately determine the refractive index of the solvent and the particles according to the difference in wavelength and the difference in additive conditions.

[0013] The present invention has been made to solve the above-mentioned problems, and aims to reduce the burden on a user of inputting the refractive index of a particle or a solvent into a measurement device when measuring particle size, density, etc. by light irradiation, and to improve the measurement accuracy that depends on the refractive index. [Means for solving the problem]

[0014] The particle measuring device of the present invention identifies the boundary position along the optical axis direction between the light transmission window and the sample, and calculates the refractive index of the sample using the refractive index of a known sample at that boundary position and the refractive index of the light transmission window. Effect of the Invention

[0015] According to the present invention, it is possible to improve the convenience and measurement accuracy when measuring the size and density of particles in a medium using light. Problems, configurations, effects, etc. other than those described above will become clear from the description of the following embodiments. [Brief description of the drawings]

[0016] [Figure 1] 1 is a schematic diagram showing the relationship between defocus and the detection signal and particle size in the technology described in Patent Document 1. FIG. [Diagram 2] 1 is a simulation result showing the relationship between particle diameter and signal intensity. [Diagram 3] 1 is a calculation result showing the relationship between the refractive index of the solvent calculated based on Equation 1 and the magnitude of the detection signal. [Figure 4] FIG. 2 is a schematic diagram showing the relationship between a container containing a solvent containing particles to be measured and laser light to be measured. [Figure 5A] 13 is an experimental result showing a detection signal when the focal position is changed by moving the sample in the Z direction. [Figure 5B] This is an experimental result showing the change in the detection signal when the focal position is fixed at Z0 and the position of the reference beam mirror is changed. [Figure 6] 4 is a flowchart showing a measurement procedure in the first embodiment. [Figure 7] 11 is another flowchart showing the measurement procedure in the first embodiment. [Figure 8] 1 shows the results of measuring the refractive index of a solvent using an aqueous sucrose solution as the solvent. [Figure 9A] FIG. 2 is a schematic diagram showing the light path when the focus of the laser light is at the boundary between the transparent window and the solvent. [Figure 9B] FIG. 13 is a schematic diagram showing the amount of movement when the focus of laser light is moved into a sample. [Figure 10] 13 is a calculation result showing the relationship between the focal position Z in the sample and the detection signal. [Figure 11] 13 is a calculation result showing the relationship between the focal position Z in a sample and the detection signal when the coherence length L of the semiconductor laser light source is set to 210 μm, using the high frequency superposition technique described in Patent Document 3. [Figure 12] 13 is a calculation result showing the specification of the thickness variation of the transparent window for realizing the measurement of the refractive index of a solvent with high accuracy according to the present invention. [Figure 13] FIG. 1 is a schematic diagram showing a cross-sectional structure of a sample container described in Patent Document 2. [Figure 14A] FIG. 2 shows a plan view of a sample container suitable for use in the present invention. [Figure 14B] 14B is a cross-sectional view taken along the line AA′ of FIG. 14A. [Figure 15A] The following are experimental results showing the ratio of cases where measurements were performed normally without the influence of air bubbles occurring in the measurement area when the sample was dispensed, using the sample container described in Patent Document 2. [Figure 15B] The following are experimental results showing the ratio of cases where measurements were performed normally without the influence of air bubbles occurring in the measurement area when the sample was dispensed, using the sample container described in Patent Document 2. [Figure 16A] 13 is an experimental result showing the ratio of cases in which the measurement was performed normally without the influence of air bubbles occurring in the measurement area when the sample was dispensed using the sample container of embodiment 3. [Figure 16B]13 is an experimental result showing the ratio of cases in which the measurement was performed normally without the influence of air bubbles occurring in the measurement area when the sample was dispensed using the sample container of embodiment 3. [Figure 17A] FIG. 13 is a diagram showing the configuration of a sample container, a frame, and a base plate according to the third embodiment. [Figure 17B] FIG. 11 is a schematic diagram showing the relationship between a container and a base plate in embodiment 3. [Figure 18A] FIG. 2 is a schematic diagram showing the configuration of a container suitable as a reference for measuring the refractive index of a solvent. [Figure 18B] FIG. 2 is a schematic diagram showing how a solvent of a target sample is measured, similarly to the first embodiment. [Figure 19] 13 is a diagram showing an example of the configuration of a particle measuring device according to embodiment 4. [Figure 20A] 1 is a schematic diagram showing a particle size distribution measuring method of the present invention in which a detection signal is acquired while a sample container is moved in the Z direction. [Figure 20B] 1 is a schematic diagram showing a particle size distribution measuring method of the present invention in which a detection signal is acquired while a sample container is moved in the Z direction. [Figure 21A] 10 is a schematic diagram showing a particle size distribution measuring method of the present invention in which a detection signal is acquired while an objective lens is moved in the Z direction. [Figure 21B] 10 is a schematic diagram showing a particle size distribution measuring method of the present invention in which a detection signal is acquired while an objective lens is moved in the Z direction. [Figure 22A] 1A and 1B are schematic diagrams showing a case where a sample container of the present invention is attached to and detached from a particle measuring device of the present invention. [Figure 22B] 1 is a schematic cross-sectional view of a sample container according to the present invention attached to a particle measuring device. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] <First embodiment: Problems with the prior art> FIG. 1 is a schematic diagram showing the relationship between defocus and the detection signal and particle size in the technology described in Patent Document 1. The upper left of FIG. 1 is a geometrical diagram of an optical system when there is defocus for a relatively small particle. The geometric spot size at the z position (coordinate along the optical axis) of the particle is geometrically determined by the numerical aperture of the objective lens and the amount of defocus. The detection signal attenuates according to the ratio of the projected area of ​​the particle in the area of ​​the geometric beam. This ratio of the projected areas corresponds to the reaction cross-sectional area of ​​the reflected light. The upper right of FIG. 1 is a schematic diagram showing the case when there is defocus for a relatively large particle. Compared to the upper left of FIG. 1, the particle size is large, so the reaction cross-sectional area is large and the amount of reflected light is also large. Therefore, it is possible to detect the difference in particle size as the difference in the amount of reflected light (difference in the amount of detected signal). The lower part of FIG. 1 is an experimental result showing the relationship between the amount of detected signal at the focal position and the particle size.

[0018] Figure 2 shows the results of a simulation showing the relationship between particle diameter and signal intensity. The simulation method used was the "wave dynamic ray tracing method" described in Patent Document 1, with the wavelength of the light source set to 785 nm and the numerical aperture of the objective lens set to 0.45. As can be seen from the figure, the magnitude of the detection signal changes depending on the size of the particle. By storing this correspondence data in advance, it is possible to measure the particle size from the magnitude of the detection signal.

[0019] On the other hand, the reflected light detected varies depending on the particle size and the refractive index of the particle. In the technology described in Patent Documents 1 and 2, a detection signal can be obtained by converting the electric field amplitude of the reflected light into a voltage using optical interference. At this time, the magnitude of the detection signal |E sig | is the refractive index of the particle, n p , and the refractive index of the solvent, n m Using Fresnel's law, when the coherence length of the light source is sufficiently long, it can be expressed by the following equation: where S is the electric field amplitude of the light irradiated to the sample, R is the electric field amplitude of the reference light, σ is a coefficient that changes depending on the particle size, and η is a constant that indicates the interference efficiency between the reflected light and the reference light and the efficiency of the photoelectric conversion of the photodetector.

[0020]

number

[0021] As can be seen in Equation 1, the magnitude of the detection signal depends not only on the size of the particle but also on the refractive index of the particle, n p , and the refractive index of the solvent, n m On the other hand, in biopharmaceuticals, appropriate amounts of, for example, pH, salt, sugar, surfactant, etc. are added to prevent protein aggregation as much as possible. Therefore, the refractive index of the solvent changes depending on the additive conditions.

[0022] Figure 3 shows the calculation results showing the relationship between the refractive index of the solvent calculated based on Equation 1 and the magnitude of the detection signal. Here, the refractive index of polystyrene is set to 1.58, and the refractive index of the protein aggregate is set to 1.40. As can be seen from the figure, in the case of protein aggregates with a small refractive index difference from the solvent, the change in the magnitude of the detection signal is large with respect to the change in the refractive index of the solvent.

[0023] Because of this relationship, when measuring the size and density of protein aggregates using the techniques described in Patent Documents 1 and 2, the user needs to input the refractive indexes of the protein aggregates and the solvent into the measurement device with high accuracy. This input can be a burden for the user during measurement. In the first embodiment of the present invention, a method for reducing the burden on the user associated with inputting such refractive indexes is described.

[0024] In the following description, the explanation will be made in a unified coordinate system in which the optical axis direction is the z-axis, as shown in Figure 1. Furthermore, the size of the particle to be measured is treated as the diameter of a sphere with the same volume.

[0025] <Embodiment 1: Method for measuring the refractive index of a solvent and target particles> 4 is a schematic diagram showing the relationship between a container containing a solvent containing particles to be measured and a laser beam to be measured. In the figure, 108 is an objective lens, 300 is a laser beam, 204 is a sample, 203 is a container, and 202 is a transparent window provided on the bottom surface of the container. In this embodiment, in order to measure the refractive index of the solvent, the reflectance of the boundary between the solvent and the transparent window 202 depends on the refractive index of the solvent. By determining the condition for maximizing the detection signal while moving a sample stage (not shown) in the Z direction, the focus of the objective lens 108 can be positioned at the boundary between the sample 204 and the transparent window 202. At this time, the magnitude E of the detection signal can be calculated from the Fresnel equation. sig is the refractive index of the solvent, n m , the refractive index of the transparent window n g Using these, it is expressed by the following formula, where S is the electric field amplitude of the light irradiating the sample, R is the electric field amplitude of a reference light (not shown), and η is a constant indicating the interference efficiency between the reflected light and the reference light and the efficiency of photoelectric conversion of the photodetector.

[0026]

number

[0027] In this embodiment, by selecting the material of the transparent window 202, the refractive index can be set to a specified value. Similarly, the electric field amplitude S of the light irradiated to the sample 204 and the electric field amplitude R of the reference light can be treated as constant values ​​by setting the output power condition of the semiconductor laser (not shown) to be constant. Furthermore, the magnitude of the detection signal when a liquid with a known refractive index n0, such as pure water for a clean room, is used as the sample is measured in advance, and the value is expressed as |E0 sig |E0 sig | is expressed by the following formula.

[0028]

number

[0029] From equations 2 and 3, the following equation holds:

number

[0030] In Equation 4, the refractive index of the solvent, n m The rest are known or measured values. Therefore, using Equation 4, the refractive index of the solvent, n m In this case, since the wavelength of the light source is the measurement wavelength itself, there is no need to convert the value that varies depending on the wavelength as described above into a value equivalent to the measurement wavelength.

[0031] Figure 5A shows the experimental results of the detection signal when the sample is moved in the Z direction to change the focal position. The peak seen at a focal position of about 200 μm represents the reflected light from the interface between the transparent window and air, and the peak seen at a focal position of about 375 μm represents the reflected light from the interface between the transparent window and the sample (solvent). The distance between the two represents the thickness of the transparent window. In this way, by acquiring the detection signal while scanning the focal position so as to include the interface between the transparent window and air, and the interface between the transparent window and the solvent, the focal position Z0 of the boundary between the transparent window and the solvent can be determined by finding the position of the second peak from the left in the figure.

[0032] Figure 5B shows the experimental results showing the change in the detection signal when the focal position is fixed at Z0 and the position of the reference beam mirror is changed. As can be seen from the figure, when the position of the reference beam mirror is about 300 μm, the optical path length until the reflected beam reaches the detector becomes equal to the optical path length until the reference beam reaches the same detector, and the detection signal becomes maximum. The maximum detection signal at this time is E in Equation 4. sig Although a description of the reference light has been omitted, the details of the optical system will be described later.

[0033] 6 is a flowchart showing the measurement procedure in this embodiment. In step S600, the focal position Z0 and the reference beam mirror position R0 are initialized to predetermined initial values ​​Z00 and R00. In steps S601 and S602, while scanning the focal position Z0, a detection signal is acquired, and Z0 is updated so as to focus on the boundary between the transparent window and the solvent. In steps S603 and S604, the reference beam mirror position R0 is updated so that the optical path length of the detection signal and the optical path length of the reference beam match. In step S605, the refractive index n of the solvent is calculated using Equation 4. m In step S606, the focal position is moved into the sample to obtain the refractive index n m The size and density of particles contained in a sample are measured using this method. To measure particle size and density, the focal position is moved in a specified range in the X, Y, and Z directions while the reflected light from the particles is converted into an electrical signal as a detection signal, and the maximum detection signal at the focal position for each particle is obtained, and the refractive index n of the medium is calculated. m The size and density of each particle are measured and displayed in a form that can be visually recognized by the user. In this case, the refractive index of the solvent, n m The relationship in Figure 2 is used, corrected accordingly.

[0034] FIG. 7 is another flowchart showing the flow of measurement in this embodiment. In the present invention, the sample container can be a disposable type in order to improve the convenience of the user and the accuracy of the size distribution measurement. In this case, the condition for focusing on the boundary between the transparent window and the solvent may be significantly deviated (e.g., about 10 μm or more) from the predetermined initial value due to individual differences in the shape of the sample container or positional deviations that occur when the user installs the sample container in the device. FIG. 7 is a flowchart of this embodiment that is suitable for such a case. This can be achieved by repeatedly updating Z0 and R0 until the change from the previous value of the laser light spot position and the position of the reference light mirror becomes equal to or less than a predetermined threshold value Δ.

[0035] In step S700, the focal position Z0 and the reference beam mirror position R0 are initialized to predetermined initial values ​​Z00 and R00. Steps S701 to S704 are the same as S601 to S604. In step S705, it is determined whether the difference between the current value and the previous value of Z0 and the difference between the current value and the previous value of R0 are both smaller than the threshold value Δ. If this is not satisfied, the previous values ​​of Z0 and R0 are updated by the current values, and the process returns to S701 to repeat the trial. If the condition of S705 is satisfied, steps S706 to S707 are carried out. These are the same as steps S605 to S606. The threshold value Δ for Z0 and the threshold value Δ for R0 may be the same value or different values.

[0036] Through the experimental investigations of the inventors, it has been found that when a semiconductor laser with a wavelength of 785 nm is used as the light source, a microscope lens with a numerical aperture of 0.45 is used as the objective lens, and borosilicate glass with a thickness of 175 μm (actual refractive index = 1.520) is used as the transparent window, the threshold value Δ is preferably about 1 μm in the Z direction and about 5 μm in the R direction. In addition, it has been found that the movement range of the focal position in the size distribution measurement is preferably about 50 to 100 μm in the Z direction and about 300 to 500 μm in the X and Y directions. In order to measure the size of a particle in response to the change in the position of a particle that randomly moves Brownianally in a solvent, it is preferable to use a resonant galvanometer mirror that is widely used in scanning laser microscopes for either one of the focus scans in the X or Y direction. The numerical values ​​described here can be appropriately determined by an engineer familiar with general interference measurement according to the wavelength, beam diameter, and specifications of the objective lens.

[0037] FIG. 8 shows the measurement results of the refractive index of a solvent measured using a sucrose aqueous solution as the solvent. As is well known, the refractive index of a sucrose aqueous solution changes linearly depending on the concentration. The reference data in the figure shows the published data (http: / / www.shimadzu.co.jp / products / opt / products / ref / ref-app05.html) of the refractive index of a sucrose aqueous solution measured at the C line (656.27 nm) using a Kalnew precision refractometer. As described above, a general refractive index measurement method uses parallel light and is performed for discrete wavelengths, so it is difficult to directly correspond to the wavelength when a semiconductor laser with a wavelength of 785 nm is used as the light source. In general, it is known that the refractive index of a dielectric material tends to decrease as the wavelength becomes longer, and it is also known that these depend on temperature. The refractive index measured using this embodiment is the refractive index for a laser light focused by an objective lens using the same light source for measuring the size distribution, and the temperature conditions of the sample are the same as those for performing the size measurement, so that the user does not need to convert these factors and input the refractive index as described above, which improves convenience.

[0038] <Embodiment 2: Regarding the movement amount of the focal position into the sample and the movement amount of the reference light mirror> In the present invention, weak reflected light from particles suspended in a solvent is amplified and detected by the homodyne phase diversity method. The details of the optical system constituting the homodyne phase diversity method will be described later, but in order for this to achieve a predetermined amplification, it is required that the optical path lengths of the reflected light and the reference light match on the photodetector. In the second embodiment of the present invention, a specific method for matching the optical path lengths will be described.

[0039] FIG. 9A is a schematic diagram showing the optical path when the focus of the laser light is at the boundary between the transparent window and the solvent. The position of the sample held by the Z stage (not shown) is Z0, and the position of the reference light mirror held by the R stage (not shown) is R0. A part of the light irradiated from the semiconductor laser, which is the light source, proceeds toward the sample as signal light, is reflected, and is guided to the detection optical system. At the same time, another part of the light irradiated from the semiconductor laser proceeds toward the reference light mirror as reference light, is reflected, and is guided to the detection optical system. At Z0 and R0, the optical path lengths of the signal light and the reference light from the semiconductor laser to the detection optical system match on the order of wavelength.

[0040] FIG. 9B is a schematic diagram showing the amount of movement when the focus of the laser light is moved into the sample. As shown in the figure, in order to move the focus of the laser light into the sample, the Z stage holding the sample is moved by dz, and the R stage holding the reference light mirror is moved by dr. The particles to be measured by the present invention move randomly in the medium with Brownian motion, and the position is not fixed, so it is difficult to determine the optical path length so that it matches while scanning Z and R as described above. If this is carried out, the particle size distribution shown above is measured while changing the reference light mirror position several tens of times at intervals of about 5 μm, and the position of the reference light mirror is determined as the condition where the average value of the detection signal from the detected multiple particles is maximized. However, such a procedure requires extra time for measurement, greatly impairing the convenience of the user. Therefore, in this embodiment, the relationship between dz and dr is determined from an optical viewpoint as follows.

[0041] The change in the focal position dFP when the sample position is moved by dz toward the objective lens is the refractive index n of the solvent. m and can be approximated by the following equation using Snell's law or the paraxial theory in optics:

[0042]

number

[0043] The change in the optical path length dL from the focal point until the reflected light reaches the detection optical system can be expressed by the following equation, since the optical path length in air becomes smaller by dz and the optical path length in a medium becomes larger by dFP.

[0044]

number

[0045] Therefore, the amount of movement dr of the reference mirror for matching the optical path length of the signal light with that of the reference mirror can be expressed by the following equation, where α is the correction term of the above approximation.

[0046]

number

[0047] The correction term α is mainly affected by the change in the spherical aberration of the objective lens, and can be obtained by ray tracing if the structure of the objective lens is known. Through the inventors' experimental study, it has been found that when a semiconductor laser with a wavelength of 785 nm is used as the light source, a microscope lens with a numerical aperture of 0.45 is used as the objective lens, and borosilicate glass with a thickness of 175 μm (measured refractive index = 1.520) is used as the transparent window, good correction is possible with α = 1.029 in the range of dz from 100 to 900 μm. When changing the wavelength of the light source or the objective lens, the value of α can be appropriately determined by calculation using ray tracing or experimental study as described above. The value of α changes depending on the selection of the optical system, but its range is 0.8 to 1.2.

[0048] By using Equations 5 and 7, it is possible to uniquely determine the focal position relative to the sample movement and the movement of the reference beam mirror without having to comprehensively scan the reference beam mirror position as described above. This makes it possible to simultaneously ensure measurement accuracy and improve user convenience by suppressing extension of measurement time.

[0049] Here, the relationship between the amount of movement of the sample position toward the objective lens and the amount of movement of the reference beam mirror is shown, but it is also possible to fix the sample position and move the objective lens. In this case, if the objective lens position is moved by the same amount dz toward the sample, the amount of movement dr of the reference beam mirror can be determined by directly using the relationships of Equations 5 to 7. A specific embodiment will be described later.

[0050] Next, we will discuss the suitable value of the sample movement amount dz in the particle size distribution measurement of the present invention. In the present invention, weak reflected light from particles suspended in a solvent is amplified by the homodyne phase diversity method, converted into an electrical signal, and detected. Considering the effect of the coherence length of the semiconductor laser, which is the light source, on Equation 1, the magnitude of the detection signal from a particle at the focus of the laser light |E sig | is the electric field of the reflected light E s and the electric field E of the reference beam r is proportional to the magnitude of the coherence length, L, and the difference in optical path length between the signal light and the reference light, ΔL, and is expressed as follows:

[0051]

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[0052] The coherence length is the square of the detected signal |E sig | 2 is the FWHM range of the optical path length difference where is 1 / 2 of the maximum value. The other terms overlap with the explanation of Equation 1, so the explanation will be omitted here.

[0053] In the present invention, the reflected light from the target particles is detected by the homodyne phase diversity method, but as with other optical measurement methods, the reflected light from the boundary between the transparent window and the solvent is also received by the detection optical system. The reflected light from the transparent window and the solvent is about 10 to 100 times larger than the reflected light from the target particles, and acts as unnecessary noise when measuring the particle size distribution, so the effect of this will be described next.

[0054] When the focal point of the laser light is within the sample, the reflected light from the boundary between the transparent window and the solvent is detected with defocus aberration as reflected light from a surface that is out of focus. The magnitude of the detection signal in this case is |E sig_boundary | is expressed by the following formula.

[0055]

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[0056] In Equation 9, the effect of defocus aberration is a term of the sinc function, Z is the focal position in the sample (corresponding to the amount of defocus), λ is the wavelength of the light source, and NA is the numerical aperture of the objective lens.

[0057] FIG. 10 shows the calculation results showing the relationship between the focal position Z in the sample and the detection signal. Here, assuming that the coherence length of the laser light is sufficiently large, the detection signal obtained from the target particle and the noise obtained from the boundary between the transparent window and the solvent were calculated using Equation 8 and Equation 9. In the figure, Z=0 is the boundary position between the transparent window and the solvent. Here, the refractive index of the solvent is 1.333, the refractive index of the particle is 1.400, the size is 0.1 μm, and the position is Z=500 μm. As can be seen in the figure, the signal obtained from the boundary between the transparent window and the solvent attenuates with increasing Z, and if the position of the target particle is 500 μm, it can be seen that the detection signal from the particle is about 10 times larger than the noise from the boundary. The condition for both sizes to be the same is Z>about 50 μm, as shown in the figure. From this result, it can be seen that in the size distribution measurement of the present invention, it is preferable that the focal position of the laser light in the sample is at least 50 μm away from the transparent window.

[0058] 11 shows the calculation results showing the relationship between the focal position Z in the sample and the detection signal when the coherence length L of the semiconductor laser light source is set to 210 μm by the high frequency superposition technique described in Patent Document 3. Through experimental studies by the inventors, it has been found that when a semiconductor laser with a wavelength of 785 nm is used as the light source and a microscope lens with a numerical aperture of 0.45 is used as the objective lens, the high frequency superposition frequency is set to about 300 MHz and the coherence length is set to about 210 μm, which is the high frequency superposition condition suitable for the present invention from the viewpoint of laser noise and noise from the boundary between the transparent window and the solvent. The calculation results are based on this.

[0059] As can be seen from the figure, by controlling the coherence length of the semiconductor laser, which is the light source, by high frequency superposition, when the target particle is located at 500 μm, the detection signal from the particle becomes about 100 times larger than the noise from the boundary, and the effect of high frequency superposition reduces the influence of noise to about 1 / 10. From the figure, it can be seen that in order to perform measurement with an S / N ratio of 10 or more by using high frequency superposition, the focal position in the sample should be separated from the boundary of the transparent window by 130 μm or more. Generally, it can be considered that an S / N ratio of about 3 is sufficient to perform measurement, so it can be said that the effect of this embodiment can be obtained by measuring the sample at a distance of 100 μm or more from the boundary of the transparent window.

[0060] As for the control of the coherence length by high frequency superposition, the suitable conditions change depending on the circuit type and impedance matching conditions used. While observing the time change of the emission power and the spread of the oscillation wavelength from the viewpoint of laser noise, and taking into consideration the noise effect on the measurement according to Equation 9, the technique of this embodiment can be applied to other optical systems by maximizing the S / N ratio of the detection signal for the target particle. This is easy for general engineers in the relevant technical field.

[0061] The reflected light from the boundary between the top of the sample liquid surface and the air also affects the measurement as noise. As can be seen from the figure, the effect of noise can be sufficiently reduced by moving the measurement focal position about 500 μm away from the transparent window, so in order to effectively realize the effects of this embodiment, the top of the sample liquid surface must be at least 1 mm away from the boundary of the transparent window, which is twice 500 μm.

[0062] Next, a method for measuring the refractive index of the target particle will be described. After measuring the refractive index of the solvent in advance according to this embodiment, the focal position of the laser light is moved within the sample according to Equations 5 to 7 to measure the detection signal from the target particle, thereby making it possible to measure detection signals corresponding to the respective refractive indices of the solvent and the target particle. When the size of the target particle is known, such as polystyrene standard particles, the refractive index n of the target particle can be calculated using the obtained detection signal and Equation 8. p On the other hand, in the case of a target with an unknown size, such as a protein aggregate formed under specific conditions, a medium with a known refractive index, such as the sucrose aqueous solution shown in Figure 8, can be used. By measuring samples diluted with at least two or more concentrations of sucrose aqueous solutions, two average values ​​of the detection signal corresponding to the refractive index of the solvent and the size distribution (it is better to use the average value because there is a size distribution), it is possible to use Equation 8 to calculate the quantity σ related to the size distribution and the refractive index n of the target particle. p It is possible to calculate both by solving these two unknowns simultaneously.

[0063] <Embodiment 3: Sample Container> In order to measure the particle size distribution with high accuracy according to the first and second embodiments, the sample container including the transparent window, which is a part of the measurement optical system, needs to satisfy certain specifications. As described above, a liquid level height of 1 mm or more is one of the conditions. In the third embodiment of the present invention, a specific example of a sample container that satisfies these specifications will be described. The measurement method is the same as that of the first and second embodiments.

[0064] FIG. 12 is a calculation result showing the specification of the thickness variation of the transparent window for realizing the measurement of the refractive index of the solvent with high accuracy according to the present invention. As is well known, when the thickness of the transparent window deviates from a predetermined value, spherical aberration occurs optically. The influence on the detection signal shown in Equation 1 to Equation 2 can be treated as a decrease in Strehl intensity due to wavefront aberration. FIG. 12 is a calculation result showing the state of the decrease in the detection signal (Strehl intensity) due to the deviation in the thickness of the transparent window. Here, the result is shown when a semiconductor laser with a wavelength of 785 nm is used as the light source, a microscope lens with a numerical aperture of 0.45 is used as the objective lens, and borosilicate glass with a thickness of 175 μm (actual refractive index value = 1.520) is used as the transparent window. As can be seen from the figure, in order to keep the decrease in the detection signal to 0.2% or less, it is found that the variation in the thickness of the transparent window (thickness difference between the thickest and thinnest points) is required to be 70 μm or less.

[0065] As shown in the first embodiment, in order to measure the refractive index and particle size distribution of a solvent, it is preferable to convert reflected light into a detection signal and obtain it while moving the sample container in the Z direction. In principle, the measurement can be performed while fixing the position of the sample container and moving the entire optical system, but since the weight and volume of the entire optical system are very large compared to the sample container, it is not preferable in terms of power consumption and miniaturization of the device. As described above, when the sample container is moved in the Z direction by the motorized stage, it is inevitable that the liquid surface vibration occurs in the sample due to the influence of the acceleration and deceleration. In order to make this liquid surface vibration sufficiently small compared to the amount of Brownian motion, it is necessary to increase the viscous resistance by forming a narrowed portion on the wall of the container.

[0066] FIG. 13 is a schematic diagram showing the cross-sectional structure of the sample container described in Patent Document 2. Here, the state in which the liquid level vibrates due to the effect of the sample container moving in the Z direction by the motorized stage as described above is shown. As can be seen in the figure, the sample container described in Patent Document 2 has a structure in which two regions are connected so that air bubbles can escape through the air bubble flow path to prevent air bubbles from being mixed into the measurement section. As a result, a mode in which the liquid level heights of the two regions vibrate up and down is likely to occur as an effect of the movement in the Z direction. This tends to be larger than the Brownian motion of the particles being measured, and is likely to lead to a decrease in measurement accuracy.

[0067] FIG. 14A shows a plan view of a sample container suitable for the present invention. In the figure, 1401 is a container, 1402 is a narrowed portion formed on the outer periphery of the well, 1403 is a sample injected into the well, and 1404 is a well. As can be seen in the figure, in this embodiment, wells 1404 into which samples are injected are integrally molded and arranged in an array. A feature of this embodiment is that narrowed portion 1402 is formed on the outer periphery of well 1404. Narrowed portion 1402 is formed so as to protrude from the side wall of well 1404 toward the outside of well 1404. The cross-sectional area of ​​narrowed portion 1402 on the plane of FIG. 14A is smaller than the cross-sectional area of ​​well 1404.

[0068] FIG. 14B is a cross-sectional view taken along the line AA' in FIG. 14A. 1405 denotes a transparent window. In order to reduce the vibration of the liquid surface caused by the movement of the container in the Z direction, the container 1401 does not use a bubble passage as in Patent Document 2, but has a narrowed portion 1402 instead. The narrowed portion 1402 has a function of trapping the bubbles on the outer periphery of the well 1404, rather than allowing the bubbles to escape, thereby suppressing their influence on the measurement. Furthermore, by forming the narrowed portion 1402, the flow resistance between the liquid and the wall surface is effectively increased by utilizing the capillary phenomenon, thereby reducing the vibration of the liquid surface.

[0069] The container 1401 of this embodiment solves the above-mentioned problem of liquid surface vibration, making it possible to realize stable particle size distribution measurement. In addition, in order to reduce the amount of valuable biopharmaceutical sample used, the diameter of the well 1404 is set to 4.0 mm or less so that the liquid surface height is 1 mm or more for a 20 μL sample. The material of the transparent window 1405 is borosilicate glass (measured refractive index = 1.520) with a thickness of 175 μm. The thickness accuracy was controlled to an accuracy of ±15 μm so as to satisfy the above specification (variation ≦ 70 μm). The outer shape of the container 1401 was made by molding black polypropylene resin while controlling so that the transparent window 1405 would not tilt.

[0070] 15A and 15B show experimental results showing the ratio of cases where the measurement was performed normally without the influence of air bubbles in the measurement area when the sample was dispensed using the sample container described in Patent Document 2. FIG. 15A shows the cross-sectional structure of the container. Here, a container was created in which the inner diameter of the well was changed in the range of 2 to 3 mm as the range of conditions where the liquid level height was 1 mm or more with a sample volume of 20 μL according to the above specifications. FIG. 15B shows experimental results in a table summarizing the ratio of cases where size distribution measurement was performed normally after 50 dispensing tests. As can be seen in the figure, when the inner diameter of the well was 3 mm, no air bubbles were mixed in and size distribution measurement was performed. However, a measurement waiting time of 1 minute was required for the influence of vibration of the liquid surface to become smaller than Brownian motion. It was found that this was long compared to the size distribution measurement time of 20 seconds.

[0071] 16A and 16B are experimental results showing the ratio of measurements performed normally without the influence of air bubbles in the measurement area when the sample was dispensed by the sample container of this embodiment. As shown in FIG. 16A, a container was created in which the number of constrictions formed was changed in the range of 0 to 4, and the inner diameter of the well was changed in the range of 2 to 4 mm. FIG. 16B is an experimental result in which the ratio of normal size distribution measurements was performed after 50 dispensing tests was performed and summarized in a table. As shown in the figure, when the number of constrictions is 0, there is no condition that allows normal measurement of all samples. On the other hand, when a constriction is formed as in the container of this embodiment, if there are two or more constrictions or one constriction and the inner diameter of the well is in the range of 3.5 mm to 4.0 mm, there is a condition that allows normal measurement of all samples. At this time, the measurement waiting time for the influence of vibration of the liquid surface to become smaller than Brownian motion was about 5 seconds.

[0072] As described above, by using the sample container of this embodiment, it is possible to obtain the same performance in suppressing the effects of air bubbles as the container described in Patent Document 1, while reducing the waiting time associated with moving the sample container in the Z direction, thereby improving user convenience by enabling measurement to be completed in a short period of time.

[0073] FIG. 17A is a diagram showing the configuration of the sample container, frame, and base plate of this embodiment. In FIG. 17A, 1701 indicates the base plate, 1702 indicates the frame, and 1703 indicates the sample container. The sample container 1703 is positioned and set on the base plate 1701 along the frame 1702. At this time, the transparent window, which is the bottom surface of the sample container, is configured to be in contact with the base plate. It is preferable to use a metal material with high thermal conductivity as the material of the base plate 1701. Here, a graphite-based aluminum composite material ACM-io, which has high thermal conductivity and excellent processing accuracy, is used. With this structure, the sample container can be disposable after each measurement, and it is possible to improve user convenience by avoiding the effects of adhesions on the container cleaning and measurement.

[0074] In this embodiment, the sample container has four wells in a set, taking into consideration ease of handling by the user, but the number of wells in the sample container is arbitrary. For example, a container with 32 wells in a set can be created, and the number of sets can be appropriately selected taking into consideration the cost and yield of the container.

[0075] 17B is a schematic diagram showing the relationship between the container and the base plate of this embodiment. In the figure, 1701 indicates the base plate, 1703 indicates the sample container, 1704 indicates the transparent window, 1705 indicates the sample, 1706 indicates a seal for preventing evaporation of the sample, 1710 indicates a gap for improving the shape accuracy of the container by injection molding, and 1711 indicates a heat flow from the sample to the base plate for temperature stabilization.

[0076] This structure allows the transparent window 1704 to come into direct contact with the base plate 1701. The base plate 1701 has a hole corresponding to the transparent window 1704, and the diameter of the hole is larger than the diameter of the well. In the particle size measurement method of the present invention, if there is a difference between the temperature of the sample and the temperature of the measurement device, a flow of particles due to convection occurs, and the measurement accuracy decreases. In this embodiment, the transparent window 1704 is made of a glass material with high thermal conductivity compared to a plastic material, and the transparent window 1704 is made of a metal and has a high thermal conductivity. This configuration allows the temperature of the sample to be made the same as the temperature inside the device in a short time, shortening the waiting time for temperature stabilization and improving user convenience. The waiting time for temperature stabilization, which the inventors experimentally confirmed, could be shortened from 30 minutes to 10 minutes compared to the sample container described in Patent Document 2. In addition, in this embodiment, 96 samples can be measured continuously in one operation. It took about 4.8 hours to complete the measurement of 96 samples under the condition that the above standard measurement was repeated 10 times for each sample.

[0077] As described in the first embodiment, the present invention uses a transparent window of a sample container as part of the optical system to measure the refractive index of a solvent. In the above-mentioned embodiment, a method of pre-storing a reference signal at the time of shipment of the device has been disclosed. On the other hand, it is known that the quantum efficiency of a semiconductor laser, which is a light source, decreases with long-term use, and it is also known that the emission wavelength distribution changes with temperature. Furthermore, dust or the like adhering to the surface of the objective lens may reduce the transmittance and reduce the magnitude of the detection signal. In such a case, if the signal level pre-stored at the time of shipment of the device is used as a reference value, the measurement error of the refractive index will be large.

[0078] Fig. 18A is a schematic diagram showing the configuration of a container suitable as a reference for measuring the refractive index of a solvent. Fig. 18A shows a method for measuring a reference signal using a sample container in which pure water for a clean room or ultraviolet-curable or thermosetting resin with a known refractive index (reference sample) is sealed instead of a sample. The focal point of the laser light is positioned at the interface between the transparent window and the pure water or ultraviolet-curable or thermosetting resin, and the position of the reference light mirror is adjusted so that the optical path lengths are equal, and then the reference signal is acquired, thereby obtaining the reference signal required in Equations 2 to 4.

[0079] FIG. 18B is a schematic diagram showing the state of measuring the solvent of the target sample as in the first embodiment. By using a sample container in which a substance with a known refractive index is sealed as in this embodiment, it is possible to eliminate the influence of the change in quantum efficiency of the semiconductor laser and the dust attached to the objective lens as a common term using formula 4, and it is possible to maintain the measurement accuracy of the refractive index of the solvent according to the present invention with high accuracy. Such a container may be installed on the frame described above when needed by the user, or may be fixed to a specific location in the device in advance. In the case of using pure water as a sealing material, there is an advantage that the user can easily prepare it as needed. In addition, in the case of sealing UV-curable resin or thermosetting resin, there is an advantage that there is no influence of the liquid volume or evaporation, making it easy to handle and improving the convenience of the user.

[0080] <Fourth embodiment: Particle measuring device> 19 is an example of a configuration diagram of a particle measuring device according to embodiment 4 of the present invention. Laser light emitted from a light source 100, whose light emission state is controlled by a laser driver 101 that controls high frequency superposition and emission power, is converted into parallel light by a collimating lens 102, and after the polarization direction is adjusted by a λ / 2 plate 103 whose optical axis is set at approximately 22.5 degrees with respect to the horizontal direction, the light is separated by a polarizing beam splitter 104 into a signal light and a reference light.

[0081] The reference light is converted to a circularly polarized state by the λ / 4 plate 105, reflected by the reference light mirror 106, and the polarization state is rotated by 90 degrees from the outward path by the λ / 4 plate 105, and reflected by the polarizing beam splitter 104. The signal light is deflected in its traveling direction by the XY-direction composite deflection element 107, converted to a circularly polarized state by the action of the built-in λ / 4 plate, and focused in the sample 204 by the objective lens 108. The driving mechanism 109 that moves the sample in the Z-axis direction has the function of scanning the focal position of the signal light along the Z-axis direction (optical axis direction). The component of the signal light reflected from the sample 204 is deflected in the same direction as the outward path by the XY-direction composite deflection element 107, and is circular by the action of the built-in λ / 4 plate, and the polarization state is rotated by 90 degrees from the outward path, and passes through the polarizing beam splitter 104. Here, the sample container 200 holds a sample 204 in a well, and guides signal light into the sample through a transparent window 202. 203 is a resin member that forms the well of the sample container. The base plate 201 contacts the transparent window 202 to mechanically hold the sample container 200 and stabilize the temperature of the sample.

[0082] The signal light and the reference light are combined by the polarizing beam splitter 104 and guided to the detection optical system 112 , and are split by the half beam splitter 113 via the pinhole 111 into transmitted light and reflected light.

[0083] The reflected light passes through a λ / 4 plate 114, whose optical axis is set at approximately 45 degrees with respect to the horizontal direction, and is then focused by a focusing lens 115 and split into two by a polarizing beam splitter 116. The two beams are then photoelectrically converted by photodetectors 150 and 151, and differentially amplified by a current differential amplifier 152 to become a detection signal 123.

[0084] The transmitted light passes through a λ / 2 plate 118, whose optical axis is set at approximately 22.5 degrees with respect to the horizontal direction, and is then focused by a focusing lens 119 and split into two by a polarizing beam splitter 120. The two beams are then photoelectrically converted by photodetectors 153 and 154, and differentially amplified by a current differential amplifier 155 to become a detection signal 122.

[0085] The detection optical system 112 shown here constitutes a homodyne phase diversity method, and detection signals 122 and 123 are the real part and the imaginary part of the detection signal shown in (Equation 8) and (Equation 9), respectively. The magnitude of the detection signal is processed by the signal processing unit 124 as an absolute value calculated from these. The display unit 125 displays the calculation results by the signal processing unit 124 and presents them to the user. The signal processing unit 124 calculates the refractive index of the solvent, the refractive index of the particles, the particle size, etc. by implementing the method described in the above embodiments (for example, the flowcharts in Figures 6 to 7). The signal processing unit 124 also controls each unit equipped in the particle measuring device.

[0086] Using this device, continuous automatic measurements can be easily performed on the array of multiple sample containers shown in Figure 17A by adding a sample movement function in the X and Y directions as well as the Z direction to the driving mechanism 109.

[0087] <Fifth embodiment: Method for moving focal position in size distribution measurement> As shown in the first embodiment, in order to measure the particle size distribution by the present invention, it is necessary to obtain a detection signal while moving the focal position in the Z direction in the sample. At this time, it is preferable to obtain a detection signal while moving the sample container in the Z direction in the sense that the optical path length condition of the optical system is kept constant. On the other hand, even if the position of the sample container is fixed and the measurement is performed while moving the entire optical system, the effect of keeping the optical path length condition of the optical system constant is the same, but the weight and volume of the entire optical system are very large compared to the sample container, so it cannot be said to be preferable in terms of power consumption and miniaturization of the device. However, if the power fluctuation of the light transmitted through the objective lens is within a certain standard, it is possible to measure the particle size distribution by moving only the objective lens in the direction of the sample, not the entire optical system. In the fifth embodiment of the present invention, a specific example will be described. It is the same as the above embodiments except that the objective lens is moved instead of the sample container.

[0088] Figures 20A and 20B are schematic diagrams showing the particle size distribution measurement method of the present invention, in which a detection signal is acquired while a sample container is moved in the Z direction. Figure 20A is a schematic diagram showing a case where the focal position is at the start point. Figure 20B is a schematic diagram showing a case where the focal position is at the end point. In the figure, Δz indicates the difference in the positions of the two sample containers. As mentioned above, it is known that Δz is preferably 50 to 100 μm. The movement of the sample container is controlled by a Z stage (not shown).

[0089] 21A and 21B are schematic diagrams showing the particle size distribution measurement method of the present invention, in which a detection signal is obtained while moving the objective lens in the Z direction. FIG. 21A is a schematic diagram showing the case where the focal position is at the start point. FIG. 21B is a schematic diagram showing the case where the focal position is at the end point. In the figure, Δz indicates the difference in the positions of the two objective lenses. The movement of the objective lens is controlled by an objective lens driving unit 2101 (e.g., a Z stage, which corresponds to a "scanning unit" that scans the position of the objective lens). In the configuration shown in this figure, the position of the sample container does not move during measurement, so there is an advantage that vibrations of the sample liquid surface due to inertial force and propagation of vibrations from the stage are suppressed. On the other hand, there is a disadvantage that the device configuration becomes complicated because a Z stage that moves the objective lens is added. Since the amount of movement of the objective lens during size distribution measurement is about 50 to 100 μm, if the beam diameter is about 5 mm, it is easy for a general optical engineer to configure an optical system so that the power fluctuation of the light transmitted through the objective lens is sufficiently small, even if the focal position is scanned with a galvanometer mirror. Since the weight of the objective lens is sufficiently small compared to the entire optical system, the power consumption of this method is equivalent to that of the method of moving the sample container.

[0090] As described above, the technique of the present invention is applicable to both the method of moving a sample container and the method of moving an objective lens.

[0091] <Embodiment 6: Sample Container> FIG. 22A is a schematic diagram showing the state when the sample container according to the sixth embodiment of the present invention is attached to and detached from the particle measuring device of the present invention. In the figure, 1701 is a base plate, 1702 is a frame, and 1703 is a sample container. Here, the base plate 1701 and the frame 1702 are attached and fixed to the particle measuring device to form a sample holder. With this configuration, the sample container 1703 can be attached to and detached from the sample holder. The user can dispose of the sample container without cleaning the well into which the sample is dispensed or managing foreign matter, and rapid and stable measurement is possible. In addition, when all the sample containers are attached to the sample holder, the dimensions are the same as the 96-well standard (127.76 mm wide x 85.48 mm deep) of the microplate mentioned above. At the same time, the pitch between the wells is the same as the same standard (9 mm). This provides user convenience, such as shared use of a storage cabinet.

[0092] FIG. 22B is a cross-sectional view showing the state when the sample container of the present invention is attached to a particle measuring device. In the figure, 1701 is a base plate, 1702 is a frame, 1703 is a sample container, 1704 is a transparent window, 1705 is a sample, and 108 is an objective lens. In the present invention, it is necessary for the light emitted from the objective lens to be irradiated onto the sample container through the transparent window. For this purpose, the sample container needs to be positioned so that the well does not interfere with the measurement while being accommodated in the folder. The present invention is structurally designed to make this possible. As can be seen in the figure, the positioning of the well in the XY direction is realized by the dimensional accuracy of the outer shapes of the frame 1702 and the sample container 1703. The gap between the two is preferably about 30 to 300 μm. The positioning of the well in the Z direction is realized by contacting the transparent window 1704 with the base plate 1701.

[0093] The sample container of this embodiment incorporates four wells. Compared to a sample container incorporating one well, the container can be made approximately 17.5 mm wide in the XY direction and approximately 11 mm high, making it easy to pick up in the hand and allowing it to stand on its own and not tip over during dispensing, improving the user's workability. In addition, the width of the container of the present invention is set to 18 mm or less, which is twice the well pitch of 9 mm in the 96-well standard of the above-mentioned microplate, making it possible to arrange 96 wells without overlapping.

[0094] <Modifications of the present invention> The present invention is not limited to the above-described embodiment, and includes various modified examples. For example, the above-described embodiment has been described in detail to clearly explain the present invention, and is not necessarily limited to those having all of the configurations described. In addition, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. In addition, it is possible to add, delete, or replace a part of the configuration of each embodiment with another configuration.

[0095] In the above embodiments, the member constituting the transparent window (e.g., 202 or 1704) has been exemplified as a single plate-like member arranged below the member forming the well (e.g., 203 or 1703), but the structure of the member forming the transparent window is not limited to this. It is sufficient that the member can function as a transparent window by forming at least an area that transmits light between the member and the sample in the well. Furthermore, the member constituting the transparent window may be configured as a part of the sample container, or may be configured as a member that forms a transparent window by being attached to the sample container. [Explanation of symbols]

[0096] 100: Light source 101: Laser driver 108: Objective lens 109: Drive mechanism 112: Detection optical system 124: Signal processing unit 200: Sample container 201: Base plate 202: Transparent window 203: Resin member for forming a well

Claims

1. A particle measuring device for measuring a size of particles suspended in a sample containing a solvent and particles, comprising: A light source that emits light, an objective lens for illuminating the light onto the sample; a scanning unit that scans at least one of the position of the sample and the position of the objective lens along the optical axis direction of the light; a detector for detecting the intensity of the light reflected from the sample; A calculation unit that calculates a size of the particle based on the intensity and the refractive index of the solvent; Equipped with The light source irradiates the light through the light transmission window onto the sample in contact with the light transmission window; the calculation unit identifies a boundary position between the light transmission window and the sample along the optical axis direction by identifying a peak of the intensity in a process in which the scanning unit scans the position of the sample or the position of the objective lens; The calculation unit is a first intensity of the light reflected by irradiating the light at a boundary position along the optical axis between the light transmission window and the sample as a focal point of the objective lens; a second intensity of the light reflected by irradiating the light at a boundary position along the optical axis direction between the light transmission window and a known sample having a known refractive index with the objective lens as a focal point; the refractive index of the solvent; The refractive index of the known sample; the refractive index of the light-transmitting window; Calculate the refractive index of the solvent according to the relationship between A particle measuring device characterized by:

2. The scanning unit moves a focus of the light into the sample after the calculation unit acquires the refractive index of the solvent, The computing unit acquires data describing a relationship between a refractive index of the solvent, a size of the particle, and the first intensity; The calculation unit calculates the size of the particle by referencing the data using the refractive index of the solvent and the first intensity when the focus of the light is within the sample.

2. The particle measuring device according to claim 1,

3. the calculation unit calculates the boundary position candidates by repeating the process of identifying the intensity peak in the process of the scanning unit scanning the position of the sample or the position of the objective lens; the calculation unit calculates a difference between the candidate calculated in the process of the repetition and a previous value thereof; The calculation unit adopts the candidate at a point in time when the difference becomes less than a threshold as the boundary position.

2. The particle measuring device according to claim 1,

4. The particle measuring device further comprises: a light branching unit that branches the light emitted by the light source into a measurement light and a reference light; a light irradiation unit that condenses and irradiates the measurement light onto the sample; an optical path length adjustment unit that adjusts the optical path length of the reference light; an interference optical system that generates interference light by combining a signal light generated by reflection of the measurement light from the sample with the reference light; a light detection unit for detecting the interference light; Equipped with the calculation unit controls the optical path length adjustment unit to adjust the optical path length so that the interference light has a maximum intensity for each focal position of the light; The calculation unit calculates the size of the particle using the optical path length at which the maximum intensity is obtained.

2. The particle measuring device according to claim 1,

5. When the scanning unit moves the position of the sample in the dz direction toward the light irradiation unit or moves the position of the objective lens in the dz direction toward the sample along the optical axis direction, The optical path length adjustment unit increases the optical path length of the reference light by dr, d=a(. ２ -1) dz, n is the refractive index of the sample, α is a constant between 0.8 and 1.2, is 5. The particle measuring device according to claim 4.

6. The calculation unit is a signal value of the interference light detected by the light detection unit; the size of the particles; the electric field amplitude of the measurement light, the electric field amplitude of the reference beam, the refractive index of the solvent; the coherence length of the light; an optical path difference between the signal light and the reference light; Calculate the refractive index of the particle using 5. The particle measuring device according to claim 4.

7. The calculation unit, for each of the solvents having mutually different refractive indices, a signal value of the interference light detected by the light detection unit; the electric field amplitude of the measurement light, the electric field amplitude of the reference beam, the refractive index of the solvent; the coherence length of the light; an optical path difference between the signal light and the reference light; Get the combination of The calculation unit calculates the size of the particle and the refractive index of the particle using each of the combinations.

5. The particle measuring device according to claim 4.

8. The particle measuring apparatus further comprises a sample container; The sample container includes a well for receiving the sample; The sample container further includes a narrowed portion protruding from a side wall of the well toward an outside of the well, The cross-sectional area of ​​the narrowed portion on a plane perpendicular to the height direction of the sample container is smaller than the cross-sectional area of ​​the well on the plane.

2. The particle measuring device according to claim 1,

9. A particle measurement method for measuring a size of a particle suspended in a sample containing a solvent and particles, comprising: A step of irradiating light emitted from a light source through the light transmission window onto the sample in contact with the light transmission window by an objective lens; scanning at least one of the position of the sample and the position of the objective lens along the optical axis direction of the light; detecting the intensity of the light reflected from the sample; calculating a size of the particle based on the intensity and the refractive index of the solvent; having In the step of calculating the size of the particle, a peak of the intensity is identified during the process of scanning the position of the sample or the position of the objective lens by the scanning step, thereby identifying a boundary position between the light transmission window and the sample along the optical axis direction; In the step of calculating the size of the particles, a first intensity of the light reflected by irradiating the light at a boundary position along the optical axis between the light transmission window and the sample as a focal point of the objective lens; a second intensity of the light reflected by irradiating the light at a boundary position along the optical axis direction between the light transmission window and a known sample having a known refractive index with the objective lens as a focal point; the refractive index of the solvent; The refractive index of the known sample; the refractive index of the light-transmitting window; Calculate the refractive index of the solvent according to the relationship between A particle measuring method comprising:

10. the sample is held in a sample container having a well for receiving the sample; The sample container further includes a narrowed portion protruding from a side wall of the well toward an outside of the well, The cross-sectional area of ​​the narrowed portion on a plane perpendicular to the height direction of the sample container is smaller than the cross-sectional area of ​​the well on the plane.

10. The particle measuring method according to claim 9.

11. The inside diameter of the well ranges from 3.5 mm to 4.0 mm.

11. The particle measuring method according to claim 10.

12. The optically transparent window is configured as part of a sample vessel that contains the sample or by a member that can be attached to the sample vessel; The difference between the maximum and minimum thicknesses of the light-transmitting window is 70 μm or less.

10. The particle measuring method according to claim 9.

13. the sample is held in a sample container having a well for receiving the sample; The sample container is configured to come into contact with a metal base plate via a frame in which the light-transmitting window is formed, the base plate has a hole facing the light-transmitting window; The opening size of the hole is larger than the opening size of the light-transmitting window.

10. The particle measuring method according to claim 9.

14. The particle measurement method further includes measuring the refractive index of the solvent using a reference sample having a known refractive index provided separately from the sample. The particle measuring method according to claim 9.

15. A sample container that is detachable from a sample holder formed of a frame and a metal base plate provided in a size measurement device that irradiates a sample containing a solvent and particles with light to measure the size of the particles suspended in the solvent, and that holds the sample in a well formed therein, a shape feature that provides a positioning function of performing positioning in two directions perpendicular to the optical axis of the light by inserting the light into the frame and performing positioning in the optical axis direction of the light by contacting the base plate; a shape feature that provides a function of forming a light transmission window through which the light can be irradiated onto the sample by using a hole provided in the base plate and a transparent member disposed on the bottom of the sample container; A shape feature that provides a function of suppressing sample consumption to a minimum by setting the inner diameter of the well within a range of 3.5 mm to 4.0 mm and limiting the amount of sample used to approximately 20 μL; A shape feature that provides a function of facilitating handling during dispensing and detachment by forming four or more of the wells; the well has two or more and four or less constriction portions protruding outward from a side wall, and a cross-sectional area of ​​the constriction portions in a plane perpendicular to the optical axis of the light is smaller than a cross-sectional area of ​​the well, thereby providing a shape feature that provides a function of reducing the effect of bubbles generated in the sample; a shape feature that provides a function of enabling the refractive index of the solvent to be measured by the size measurement device by making the difference between the maximum value and the minimum value of the thickness of the transparent member within a range of 70 μm or less; A sample container comprising:

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