Particle measurement equipment

Abstract

The purpose of the present invention is to provide a particle measuring apparatus capable of discriminating the type of a particle. The particle measuring apparatus according to the present invention causes a focal position of light to scan repeatedly along an optical axis direction in a three-dimensional region in a sample, and calculates two or more from among (a) one or more parameters representing a classification of a particle, obtained from the maximum intensity of reflected light from the particle, acquired from each focal position over the course of scanning; and (b) one or more parameters representing a classification of the particle, obtained on the basis of a particle position acquired by continuously tracking the particle, and the particle measuring apparatus outputs the result of said calculation (see fig. 1).

Description

[Technical Field]

[0001] The present invention relates to a particle measuring device. [Background technology]

[0002] Biopharmaceuticals offer superior efficacy compared to small molecule drugs, with glycosylated antibody molecules exerting effects against specific targets, such as cancer and rare diseases. While small molecule drugs are synthesized through chemical reactions, biopharmaceuticals are produced using the biological functions of cells. A representative biopharmaceutical, immunoglobulin G (IgG), is a large molecule with a complex structure and a molecular weight of approximately 150,000, making structural heterogeneity nearly impossible to prevent. Therefore, testing techniques for confirming the safety and efficacy of biopharmaceutical formulations play an even more important role. While there are many different types of testing for biopharmaceuticals, aggregation is one of the most important. Because biopharmaceuticals are polymers, they are prone to aggregation, which can cause toxicity. Therefore, measuring the size and number of aggregates in formulations and properly managing them is essential.

[0003] In addition to aggregates, biopharmaceutical samples to be tested may contain various types of particles, such as silicone oil droplets, air bubbles, and fragments of stainless steel, glass, and rubber (in this specification, these particulate-like substances will be collectively referred to as particles). Because the impact on pharmaceutical safety and efficacy varies depending on the type of particle, and measures to prevent particle generation vary depending on the type of particle, it is important to not only measure the size of particles but also to identify the type of particle during testing.

[0004] Patent Document 1 discloses a conventional particle measurement technique called Nano Particle Tracking Analysis (NTA), which calculates the hydrodynamic size of a particle based on the diffusion constant of the particle obtained by observing the Brownian motion of the particle for a certain period of time.

[0005] Patent Document 2 discloses a method for measuring particle size from the settling velocity of particles due to gravity.

[0006] Patent Document 3 discloses a method for measuring the size of particles based on the amount of light reflected from the particles.

[0007] Non-Patent Document 1 discloses a resonant mass measurement (RMM) method that calculates the mass of a particle based on a change in the resonance frequency of a cantilever that occurs when the particle flows through a channel provided in the cantilever. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Publication No. 2020-109419 [Patent Document 2] Patent No. 4339924 [Patent Document 3] Patent No. 6559555 [Non-patent literature]

[0009] [Non-Patent Document 1] TP Burg and SR Manalis, Appl. Phys. Lett., 2003, 83,2698-2700. Summary of the Invention [Problem to be solved by the invention]

[0010] The particle diffusion constant evaluated by NTA (Patent Document 1) is related only to particle size, so information other than size cannot be obtained. In the sedimentation method (Patent Document 2), only particles with a density greater than the solvent are detected, and the only information obtained is the particle's sedimentation velocity, making it difficult to distinguish between particles. In RMM (Non-Patent Document 1), the sign of the resonant frequency change differs depending on the particle's density relative to the solvent, making it possible to narrow down the particle type to a certain extent based on the sign. However, it is not possible to distinguish between different types of particles that have the same size relationship relative to the solvent. As such, conventional particle measurement devices detect only one of the particle-related information, such as diffusion constant or mass, making it difficult to distinguish between particle types.

[0011] The present invention has been made in view of the above-mentioned problems, and has an object to provide a particle measuring device that can distinguish the type of particle. [Means for solving the problem]

[0012] The particle measuring device according to the present invention repeatedly scans the focal position of light along the optical axis in a three-dimensional region within a sample, and calculates and outputs the results of at least two of the following: (a) one or more parameters representing particle type obtained from the maximum intensity of reflected light from particles acquired from each focal position during the scanning process; and (b) one or more parameters representing particle type obtained based on particle positions acquired by continuously tracking particles. This allows for accurate particle type determination based on multiple determination parameters.

[0013] As an example, we calculated the particle size according to the relationship between the maximum light intensity, the refractive index of the particle, the refractive index of the sample solvent, and the particle size, which allows us to determine the particle type based on the particle size.

[0014] As an example, we calculated the sphericity of the maximum light intensity of a particle from the maximum light intensity of multiple particles corresponding to each 3D image, which makes it possible to identify particles based on their maximum light intensity and sphericity.

[0015] As an example, the time required for one scan of a three-dimensional area is set to a predetermined value or less depending on the concentration of the particles to be measured and the diffusion constant of the smallest particle. This makes it possible to obtain multiple pieces of information about particles with high accuracy and to identify particles with high reproducibility.

[0016] As an example, by repeating the steps of scanning the focal position of light on a two-dimensional plane perpendicular to the optical axis and moving the position of the focal point relative to the sample in the optical axis direction at predetermined intervals, the focal position of light is repeatedly scanned over a specific three-dimensional region of the sample, and the time required to scan the two-dimensional plane once is set to a predetermined value or less depending on the width of the three-dimensional region in the optical axis direction, the maximum particle concentration to be measured, the diffusion constant of the smallest particle to be measured, and the distance the focal point is moved in the optical axis direction. This makes it possible to obtain multiple pieces of information about particles with high accuracy and to identify particles with high reproducibility.

[0017] As an example, the widths of the plane perpendicular to the optical axis of the three-dimensional area in the vertical and horizontal directions and the width in the optical axis direction are set to predetermined values ​​or greater depending on the time required for one scan of the three-dimensional area, the number of repeated scans of the three-dimensional area, the diffusion constant of the smallest particle to be measured, the particle density, the solvent density, the solvent viscosity, the gravitational acceleration, and the particle size. This makes it possible to repeatedly detect reflected light from a finite number of particles with a high probability, enabling particle identification with high reproducibility.

[0018] As an example, we calculated the diffusion constant of a particle based on the time change of the particle's position, or calculated the particle size based on the diffusion constant, which makes it possible to identify particles based on the maximum light intensity and the diffusion constant.

[0019] As an example, we calculated the sedimentation velocity of particles based on the time change in the particle's position in the direction of gravity, which makes it possible to identify particles based on their maximum light intensity and sedimentation velocity.

[0020] As an example, an interference optical system is provided that splits light from a light source to generate signal light and reference light, and combines the signal light reflected from the sample with the reference light to generate three or more interference lights with different phase relationships. This configuration makes it possible to detect reflected light from weak particles with high sensitivity, allowing for the measurement of smaller particles.

[0021] As an example, at least two or more pieces of information among the following four items are output: (a) the maximum light intensity of the reflected light from the particle for each focal position of the light along the optical axis, (b) the particle's diffusion constant calculated based on the change in particle position over time or the particle size calculated from the diffusion constant, (c) the particle's settling velocity calculated based on the change in particle position over time in the direction of gravity, and (d) the particle's sphericity calculated from the maximum light intensity of multiple particles corresponding to each of multiple 3D images. This allows for more accurate identification of particle types. [Effects of the Invention]

[0022] According to the present invention, it is possible to provide a particle measuring device that can measure the size of particles and distinguish the type of particles. Problems, configurations, and effects other than those described above will become clear from the description of the following embodiments. [Brief explanation of the drawings]

[0023] [Figure 1] 1 is a diagram illustrating an example of the configuration of a particle measuring device according to a first embodiment. [Figure 2] 10A and 10B are diagrams illustrating the relationship between particle size and the amount of reflected light when particles smaller than the spot size are present at the focal position of light. [Figure 3] 10 shows the results of a simulation of the relationship between particle size (diameter of a particle) and the amplitude of a detection signal. [Figure 4] This shows the results of measuring a mixed sample of polystyrene beads with a diameter of 0.2 μm and polystyrene beads with a diameter of 1.0 μm using the particle measuring device of the present invention. [Figure 5] FIG. 1 is a schematic diagram showing how non-spherical particles are measured. [Figure 6] 10A and 10B are diagrams illustrating the operation of the Z stage 115 during repeated scanning. [Figure 7A] FIG. 10 is a schematic diagram showing the results expected when the particle measuring device of the present invention is used to repeatedly acquire reflected light from particles with high sphericity and particles with low sphericity. [Figure 7B] FIG. 7B is a schematic diagram showing the same predicted results as FIG. 7A. [Figure 8] This is an example of the results expected when a mixed sample of aggregates and silicone oil droplets is measured using the particle measurement device of the present invention and the particles are plotted against the average signal amplitude Aave (or particle size) and the sphericity parameter ε. [Figure 9] The dependence of tc on concentration is shown. [Figure 10] FIG. 1 is a schematic diagram of XY images obtained in the mth and (m+1)th scans at a certain z position. [Figure 11] FIG. 1 is a schematic diagram showing a three-dimensional region within a sample. [Figure 12] The dependence of ΔZ and particle size d for aggregates (positive settling velocity) and silicone oil (negative settling velocity) is shown. [Figure 13] 1 shows an example of the expected results when a mixed sample of protein aggregates and gas bubbles is measured using the particle measurement device of the present invention and the particles are plotted against the signal amplitude A and the particle size dB calculated based on Brownian motion. [Figure 14] This is an example of the results expected when measuring a mixed sample of protein aggregates, air bubbles, and silicone oil droplets using the particle measurement device of the present invention and plotting particles against signal amplitude A and sedimentation velocity v. [Figure 15] FIG. 10 is a schematic diagram showing an example of the configuration of a particle measuring device according to a fourth embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0024] <Embodiment 1: Device Configuration> 1 is a diagram showing an example of the configuration of a particle measuring device according to a first embodiment of the present invention. Light emitted from a light source 100, the light emission state of which is controlled by a laser driver 101, is converted into parallel light by a collimating lens 102, and the polarization direction is adjusted by a λ / 2 plate 103, the optical axis direction of which is adjustable. The light is then separated into signal light (reflected light component) and reference light (transmitted light component) by a polarizing beam splitter 104. The branching ratio between the signal light and the reference light can be freely set by adjusting the optical axis direction of the λ / 2 plate 103.

[0025] The polarization of the reference light is converted to a circularly polarized state by the λ / 4 plate 105, and then reflected by the reference light mirror 106. The λ / 4 plate 105 rotates the polarization of the reference light by 90 degrees from that of the outward path, and the signal light is reflected by the polarizing beam splitter 104. The signal light has its optical axis direction deflected in the X and Y directions by the two-dimensional scanner 107, and then the polarization is converted to a circularly polarized state by the action of the λ / 4 plate 108, and the signal light is focused by the objective lens 109 inside the sample 114 held in the sample container 110.

[0026] The two-dimensional scanner 107 plays a role of two-dimensionally scanning the focal position of the light by the objective lens 109 in the XY plane (in the plane perpendicular to the optical axis), and specifically, a combination of a resonant type and a non-resonant type galvanometer mirror can be used. The Z stage 115, which moves the sample in the Z-axis direction (optical axis direction), plays a role of scanning the focal position of the signal light along the Z-axis relative to the sample 114. The two-dimensional scanner 107 performs the step of scanning the focal position of the light in the XY plane perpendicular to the optical axis, and the step of moving the Z position of the focal point relative to the sample 114 by the Z stage 115 at a predetermined interval p z By repeating the steps of moving with , the focal position of the light can be scanned over a particular three-dimensional region within the sample 114.

[0027] The signal light reflected from the particles contained in the sample 114 is converted back into parallel light by the objective lens 109, and the polarization state of the light is rotated 90 degrees from that of the outward path by the action of the λ / 4 plate 108. After the optical axis direction is deflected in the same direction as that of the outward path by the two-dimensional scanner 107, the light passes through the polarizing beam splitter 104. The sample container 110 has a transparent window 112 that transmits the signal light, a resin member 113 that forms a well, and a base plate 111 that contacts the transparent window 112 to mechanically hold the sample container 110 and stabilize the temperature of the sample 114.

[0028] The signal light and the reference light are combined by polarizing beam splitter 104 to form combined light, which is then guided to detection optical system 116. After pinhole 117 removes a portion of unnecessary light, such as reflected light from transparent window 112, the combined light is split into transmitted light and reflected light by half beam splitter 118. The transmitted combined light passes through λ / 2 plate 119, whose optical axis is set at approximately 22.5 degrees with respect to the horizontal direction, and is then focused by condenser lens 120 and split into two by polarizing beam splitter 121. These are converted into electrical signals by photodetectors 126 and 127, and then differentially amplified by current differential amplifier 130 to become detection signal 132, which is input to 3D image generator 134. The reflected light of the combined light passes through a λ / 4 plate 122, whose optical axis is set at approximately 0 degrees with respect to the horizontal direction, and a λ / 2 plate 123, whose optical axis is also set at approximately 0 degrees with respect to the horizontal direction, and is then collected by a collecting lens 124 and split into two by a polarizing beam splitter 125. These are converted into electrical signals by photodetectors 128 and 129, respectively, and then differentially amplified by a current differential amplifier 131 to become a detection signal 133, which is input to a three-dimensional image generator 134. Here, the optical axes of the λ / 2 plates 119 and 123 are adjustable, which makes it possible to adjust the light splitting ratio by the polarizing beam splitters 121 and 125.

[0029] If the detection signal 132 is I and the detection signal 133 is Q, they are expressed by the following equation: E sig , E refare the electric field amplitudes of the signal light and the reference light, respectively, and θ is the phase difference between the signal light and the reference light. Constants such as the photoelectric conversion efficiency of the photodetector are omitted for simplicity.

[0030]

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[0031] By performing the square root sum calculation on I and Q as follows, a signal A proportional to the amplitude of the signal light can be obtained.

[0032]

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[0033] The three-dimensional image generation unit 134 calculates a signal A expressed by Equation 2 for each focal position of the light, and generates a three-dimensional image of the sample 114. The analysis unit 135 analyzes the generated three-dimensional images to derive particle information, which is output to the display unit 136 and presented to the user.

[0034] <Embodiment 1: Principle of size measurement based on the amount of reflected light> FIG. 2 is a diagram illustrating the relationship between particle size and the amount of reflected light when a particle smaller than the spot size is present at the focal position of the light. The principle of particle size measurement using the particle measuring device of the present invention will be explained using FIG. 2. In the range where the particle size is smaller than the spot size, the larger the particle, the greater the ratio of the particle's projected area within the light spot, and the greater the amount of reflected light. That is, the amount of reflected light from the relatively large particle shown on the right of FIG. 2 is greater than the amount of reflected light from the relatively small particle shown on the left of FIG. 2. Therefore, by analyzing a three-dimensional image and extracting the amount of reflected light when the particle is in focus (the maximum amount of reflected light relative to the particle position), it is possible to detect differences in particle size as differences in the amount of reflected light. The reflection signal amplitude A obtained from a particle placed at the focal position is calculated as a function of the particle size d, the refractive index n of the particle, and particle , the refractive index of the solvent n solvantis given by the following equation using β, where β is a constant determined by the detector sensitivity, spot size, etc.

[0035]

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[0036] By measuring a sample with a known size and refractive index, such as polystyrene beads, the proportionality coefficient β can be determined. Substituting the refractive indices of the particles and solvent into Equation 3 allows for the calculation of particle size. The light spot size can be adjusted by the wavelength and numerical aperture of the objective lens, and can be set according to the particle size range to be measured using this principle. For example, when the light source wavelength is 785 nm and the objective lens numerical aperture is 0.45, the spot size is approximately 1.74 μm. In this case, particles with a diameter of 1.74 μm or less can be measured using this principle. For particles larger than the spot size, the particle size can be calculated using an approach similar to microscopy based on the obtained 3D image.

[0037] Figure 3 shows the results of a simulation of the relationship between particle size (particle diameter) and detection signal amplitude. The simulation was performed using the "wave dynamic ray tracing method" described in Patent Document 3, with the light source wavelength set to 785 nm and the objective lens numerical aperture set to 0.45. As shown in Equation 3, it can be seen that the detection signal amplitude is proportional to the particle diameter. By storing data on this correspondence in advance, it is possible to calculate particle size from the magnitude of the detection signal using the particle measuring device of the present invention.

[0038] Figure 4 shows the results of measuring a mixed sample of polystyrene beads with a diameter of 0.2 μm and polystyrene beads with a diameter of 1.0 μm using the particle measuring device of the present invention. It can be seen that the two types of particles with different sizes are detected separately based on the difference in the amount of reflected light.

[0039] <Embodiment 1: Measurement of sphericity> Next, we will explain the principle of particle sphericity measurement in the particle measuring device of the present invention. When the particles to be measured are silicone oil droplets or air bubbles, their shape is a perfect sphere (perfect sphere), but when they are protein aggregates, their shape generally differs from a sphere. Therefore, by evaluating the sphericity of the particles (how close the shape is to a sphere), it is possible to distinguish the type of particle.

[0040] Figure 5 is a schematic diagram showing how to measure particles that are not perfectly spherical. As shown in Figure 5, when a particle is not perfectly spherical, the amount of light reflected from the particle varies depending on the particle's orientation. Specifically, the particle shown on the right in Figure 5 has a larger proportion of its projected area within the light spot than the particle shown on the left in Figure 5, and therefore a larger amount of light is reflected.

[0041] Because the orientation of particles in a liquid is constantly changing, if the amount of reflected light from the same particle is repeatedly detected at different times, the amount of reflected light from a non-spherical particle will not be a constant value but will have a certain degree of variability. In other words, differences in particle sphericity can be detected as differences in the magnitude of variability in the amount of reflected light repeatedly obtained. Therefore, in this invention, in order to repeatedly detect reflected light from the same particle, the focal position of the light is repeatedly scanned over a specific three-dimensional area.

[0042] 6 is a diagram illustrating the operation of the Z stage 115 during repeated scanning. z The region is repeatedly scanned N times at time intervals t (the time required to scan a specific three-dimensional region once).

[0043] Figure 7A is a schematic diagram of the results expected when the particle measuring device of the present invention is used to repeatedly acquire reflected light from particles with high and low sphericity. The signal amplitude of particles with high sphericity is expected to be almost constant, whereas the signal amplitude of particles with low sphericity is expected to vary as shown in Figure 7. In the present invention, the parameter ε representing the sphericity of a particle is defined by the following equation. A in the equation min , A maxare the minimum and maximum values ​​of the amplitude A of the reflected signal from the particle acquired repeatedly, respectively.

[0044]

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[0045] ε corresponds to the ratio of the maximum and minimum projected areas of particles in the light spot, and when ε = 1, the particle is a perfect sphere. The particle size of a non-spherical particle can be calculated by, for example, the average signal amplitude A ave The particle size and sphericity of particles larger than the spot size can be evaluated directly from the image, similar to microscopy.

[0046] FIG. 7B is a schematic diagram showing predicted results similar to those of FIG. 7A. To measure sphericity with high accuracy, it is desirable to acquire the signal amplitude from a particle several tens of times or more. FIG. 7A shows an example in which the reflected signal amplitude from a particle is acquired 31 consecutive times, from the 0th scan to the 30th scan, but it is not necessary to acquire the signal amplitude continuously from the first (0th) scan to the last scan in this manner. For example, a total of 300 scans may be performed, and the signal amplitude of particles present in the scan range may be acquired continuously from the 100th scan to the 131st scan. FIG. 7B shows one such example.

[0047] FIG. 8 shows the average signal amplitude A ave This is an example of the expected results when particles are plotted against the particle size (or particle diameter) and the sphericity parameter ε. Protein aggregates are distributed in the low sphericity region, while silicone oil droplets are distributed in the high sphericity region, making it possible to distinguish between the two while measuring particle size.

[0048] <Embodiment 1: Conditions necessary for measuring sphericity> Next, we will explain the conditions necessary to acquire reflected light from the same particle multiple times. First, the identity of the particle must be maintained between the mth scan and the m+1th scan (m is a natural number less than or equal to N). Here, particle identity means a state in which it can be confirmed that each particle detected in the mth scan is the same particle as each particle detected in the m+1th scan. Particles in liquid undergo Brownian motion, and the typical particle movement amount Δr due to Brownian motion over a time interval t is given by the following equation, where D is the particle diffusion constant. α is a constant greater than or equal to 1.

[0049]

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[0050] The particle movement due to Brownian motion follows a Gaussian distribution. For example, when α = 1, there is a probability of approximately 68% that the particle movement is less than Δr, and when α = 2, there is a probability of approximately 95% that the particle movement is less than Δr. The particle diffusion constant D is expressed by the Boltzmann constant k B It is expressed by the following formula using the absolute temperature T of the solvent, the viscosity η of the solvent, and the particle size d.

[0051]

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[0052] For example, α=2, t=5 seconds, d=0.2 μm, k B =1.38×10 -23 J / K, T = 25°C (298.15K), η = 0.00089 Pa*s, Δr is approximately 5.0 μm. On the other hand, if the particle density is n, the average particle spacing l ave is given by the following formula:

[0053]

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[0054] For example, if the particle concentration is 10 7 In the case of pieces / mL, l aveis approximately 46 μm. In order for the particle identity to be maintained between the mth and m+1th scans, the average particle interval must be greater than twice the particle movement distance due to Brownian motion during the time interval t. This can be expressed as the following formula: D max is the diffusion constant for the particle with the largest Brownian motion, i.e., the smallest particle, within the particle size range to be measured.

[0055]

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[0056] Regarding the value of α, for example, when α = 1, particle identity is maintained for approximately 68% or more, and when α = 2, particle identity is maintained for approximately 95% or more. By solving Equation 8 for t, the following condition for the scanning time t per scan of the three-dimensional region required to maintain particle identity is obtained:

[0057]

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[0058] t c Since depends on the particle concentration and particle size (through the diffusion constant), t c must be set to an appropriate value.

[0059] Figure 9 shows the c The dependence of the particle concentration on the particle size is shown in Fig. 1. The parameters used in the calculation are d = 0.2 μm, T = 25°C (298.15 K), η = 0.00089 Pa*s, and α = 3. As the particle concentration increases, the average particle spacing decreases, so the scanning time t required to maintain particle identity is c becomes shorter. For example, n=10 7 When the concentration is high, it is difficult to satisfy the formula 9, so the tc is about 9.1 seconds. If the concentration is high and it is difficult to satisfy the formula 9, the condition of the formula 9 can be satisfied by diluting the sample 114 by an appropriate factor.

[0060] The scanning time per scan for the three-dimensional area is t, where Δt is the scanning time per scan for the XY plane, and p is the amount of sample movement in the Z direction by the Z stage 115. z Then, it can be expressed by the following formula.

[0061]

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[0062] Substituting Equation 10 into Equation 9 leads to the following conditions that Δt, Lz, and pz must satisfy in order to maintain particle identity:

[0063]

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[0064] In the present invention, L is set to satisfy Equation 11. z , p z By setting Δt, the particle identity is maintained during N repeated scans. The smaller the particle, the greater the t c Therefore, it is necessary to satisfy Equation 11 for the smallest particle size among the particles to be measured (the smallest particle size the user wants to measure). For example, pz = 1.0 μm, Lz = 100 μm, n = 10 7 When the parameters are particles / mL, d = 0.2 μm, T = 25°C (298.15 K), η = 0.00089 Pa*s, and α = 2 (this parameter condition will be referred to as condition I for later reference in relation to Figure 12), Equation 11 becomes Δt < 0.09, and a scanning speed that satisfies this condition can be easily achieved with a galvanometer mirror.

[0065] Figure 10 is a schematic diagram of XY images obtained at a certain z position during the mth and m+1th scans. The crosses in the figure represent the positions of each particle during the mth scan, and the dotted circles are circles with a radius of Δr centered on the crosses. As shown in the upper part of Figure 10, when the condition of Equation 11 is not satisfied, the circles overlap (the particle movement amount is greater than the average particle spacing), making it impossible to ensure the identity of the particles. For example, particles 1 and 2 in the image obtained during the (m+1)th scan are located in an area where the circles overlap, making it impossible to distinguish which particle is which. On the other hand, as shown in the lower part of Figure 10, when the condition of Equation 11 is satisfied, the circles do not overlap, so the particle closest to the particle position detected during the mth scan can be determined to be the original particle.

[0066] Figure 11 is a schematic diagram showing a three-dimensional region within a sample. Another condition for repeatedly acquiring reflected light from the same particle N times is that the particle does not move outside the three-dimensional region of the scanning target due to Brownian motion or sedimentation during N scans. As shown in Figure 11, particles that exist on the periphery of the scanning region V at the start of scanning may move outside the scanning region V during N scans (Figure 11 shows an xz cross section). Let the width of the scanning region in the xyz directions be L. x ,L y ,L z Then, the scanning area V is expressed by the following formula.

[0067]

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[0068] Considering the Brownian motion and sedimentation of particles, consider region V' (the internal region of three-dimensional region V) where particles exist that can detect reflected light N times. Assuming that convection is sufficiently suppressed and that particle movement in the x and y directions occurs only through Brownian motion, the particles with the smallest size will have the largest movement in the x and y directions. The movement ΔL in both the x and y directions that occurs during time Nt for the smallest particle size among the particles to be measured isx , ΔL y is expressed by the following formula:

[0069]

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[0070]

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[0071] Therefore, in order to detect the light repeatedly reflected N times, the particle must be present in the following xy region, which is the range of region V' in the xy directions.

[0072]

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[0073] The movement of particles in the z direction occurs due to Brownian motion and sedimentation, and the amount of movement ΔZ of particles in the z direction during time Nt is expressed as the sum of both as follows:

[0074]

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[0075] Considering that the direction of gravity is the positive direction of the z axis, and that the sedimentation velocity can be either positive or negative depending on the density relationship between the solvent and the particles, the maximum particle movement amount ΔL that occurs in both the positive and negative z directions is z+ ,ΔL z- is expressed by the following formula:

[0076]

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[0077]

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[0078] Here, for example, the right-hand side of Equation 17 means that for particles with positive settling velocity, the value in the parentheses is maximized with respect to particle size. To repeatedly acquire reflected light from particles in the target particle size range N times, the particles must be in the following z region, which is the z-direction range of region V'.

[0079]

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[0080] From Equation 15 and Equation 19, in order for the volume of region V' to be equal to or greater than 0, the following conditions must be satisfied:

[0081]

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[0082]

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[0083]

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[0084] For example, α=2, N=10, t=5 seconds, d=0.2 μm, k B =1.38×10 -23 J / K, T=25℃ (298.15K), η=0.00089Pa*s. Equations 20 and 21 are L x ,L y >62.7 μm, which is a condition that can be easily achieved with a combination of a normal galvanometer scanner and an objective lens.

[0085] Next, ΔL z+ ,ΔL z- The specific values ​​of are explained below. The settling velocity of particles, v, is given by the following formula: d is the particle diameter, ρ particle is the particle density, c is the particle resistance coefficient, ρ solvent is the density of the solvent, and g is the acceleration due to gravity.

[0086]

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[0087] When a particle settles quietly, that is, when its inertia is very small compared to its viscosity, the resistance coefficient c of the particle is given by the following equation, where R is the Reynolds number:

[0088]

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[0089] The Reynolds number R is expressed by the following formula:

[0090]

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[0091] Substituting Equation 24 and Equation 25 into Equation 23 and rearranging, we obtain the following equation.

[0092]

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[0093] Figure 12 shows the dependence of ΔZ and particle size d for aggregates (positive settling velocity) and silicone oil (negative settling velocity), calculated by substituting Equations 6 and 26 into Equation 16. The parameters used in the calculation were N = 10, t = 5 seconds, k B =1.38×10 -23 J / K, T=25℃(298.15K), η=0.00089Pa*s, g=9.8m / s 2 , ρ solvent =1.0g / cm 3 , ρ particle is 1.2 g / cm for aggregates 3 , and 0.818 g / cm for silicone oil. 3When the particle size is small (approximately 1.0 μm or less), the contribution of Brownian motion is large, and when measuring particles of 1.0 μm or less, the effect of sedimentation can be almost ignored.

[0094] The particle size d to be measured is set to a range of 0.2 μm to 2.0 μm. As explained in Equations 16 to 18, ΔL z+ and ΔL z- is the maximum value of ΔZ in the variable range of d. Therefore, as shown in FIG. 12, when d=0.2 μm, the value of ΔZ is ΔL z+ and ΔL z- In this case, the condition of Equation 22 is L z >63.1 μm, which is satisfied by condition I, which is an example of the condition for satisfying equation 11 mentioned above.

[0095] If the measurement target is limited to small particles of 1.0 μm or less and particle settling can be virtually ignored, the condition in Equation 22 can be simplified. Substituting v = 0 into Equations 17 and 18, the following equation is obtained:

[0096]

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[0097] Substituting Equation 27 into Equation 22 gives the following equation.

[0098]

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[0099] Furthermore, substituting Equation 10 into Equation 28 and rearranging it, we obtain the following condition: This is the same condition as Equation 22 when particle settling can be ignored.

[0100]

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[0101] As described above, in the first embodiment, by repeatedly scanning a three-dimensional area to satisfy the conditions shown in Equation 11 and Equations 20 to 22 and repeatedly detecting the light reflected from the particles, it is possible to evaluate not only the particle size but also the sphericity of the particles, thereby making it possible to identify the type of particle.

[0102] In the first embodiment, the case where particles present on the periphery of the scanning area V at the start of scanning are measured has been described, but for example, particles that move from outside the measurement area into the measurement area during repeated scanning may also be measured. In this case, it is not necessary to satisfy Equations 20 to 22. Furthermore, the number of repeated measurements N, the scanning area V, and the area V' do not need to be defined at the start of scanning until the final scan, and they can be redefined during scanning.

[0103] <Embodiment 2: Evaluation of Brownian motion> Particle coordinates are constantly changing due to Brownian motion, and the amount of particle movement due to Brownian motion varies depending on particle size. The particle measurement device of the present invention repeatedly scans a specific three-dimensional area, making it possible to continuously observe particle coordinates for a certain period of time. Therefore, in embodiment 2 of the present invention, the particle diffusion constant expressed by Equation 6 is calculated based on the amount of particle movement due to Brownian motion, and the particle size is then calculated from the value of the diffusion constant. The particle measurement device has the same configuration as embodiment 1. Because signal amplitude A depends on the particle diameter and refractive index, while Brownian motion depends only on the particle diameter as shown in Equation 6, the two provide different information about the particle.

[0104] In addition to Brownian motion, particle movement is also caused by solvent convection due to thermal non-uniformity within the sample 114, so suppressing the occurrence of convection or the effects of convection is important for ensuring stable measurement accuracy. In the present invention, the base plate 111 with high heat dissipation properties is used to suppress heat accumulation in the sample 114 and suppress convection. Furthermore, because particle movement caused by solvent convection is almost constant regardless of particle size, the effects of convection are removed when evaluating the amount of particle movement by subtracting the average movement of all particles.

[0105] FIG. 13 shows the particle size d calculated based on the signal amplitude A and Brownian motion when a mixed sample of protein aggregates and bubbles is measured using the particle measuring device of the present invention. B This is an example of the results expected when particles are plotted against . Bubbles have a larger difference in refractive index from the solvent than aggregates, and are distributed in areas with larger signal amplitudes than aggregates. This makes it possible to distinguish between aggregates and bubbles. In other words, in this invention, by repeatedly scanning a three-dimensional area and continuously observing the particle coordinates, it is possible to evaluate the amount of particle movement due to Brownian motion in addition to the signal amplitude, and to identify the type of particle.

[0106] <Embodiment 3: Evaluation of Sedimentation Velocity> Particles in a liquid settle due to the action of gravity, and so their position in the direction of gravity (Z coordinate) changes over time. In embodiment 3 of the present invention, a specific three-dimensional area is repeatedly scanned to continuously observe the Z coordinate of the particle for a certain period of time, and not only the amplitude of the reflected signal from the particle but also the settling velocity of the particle, expressed by Equation 23 or Equation 26, is evaluated. The configuration of the particle measuring device is the same as in embodiments 1 and 2.

[0107] The reflected signal amplitude A depends on the particle diameter and refractive index, and the settling velocity v depends on the particle diameter and density, so the reflected signal amplitude A and the moving velocity v give different information about the particle. For example, when d = 1.0 μm and ρ particle =1.2g / cm 3 , ρ solvent , =1.0g / cm3 , g=9.8m / s 2 Using Equation 26, the settling velocity is calculated as v = 0.12 μm / s.

[0108] Figure 14 shows an example of the expected results when a mixed sample of protein aggregates, air bubbles, and silicone oil droplets is measured using the particle measurement device of the present invention, and particles are plotted against signal amplitude A and sedimentation velocity v. Aggregates have a higher density than typical solvents, so they have a positive sedimentation velocity, while air bubbles and silicone oil have a lower density than solvents, so they have negative sedimentation velocities. Because air bubbles have a larger refractive index difference with the solvent than silicone oil and a lower density, they are thought to have a larger signal amplitude than silicone oil and to be distributed in a region with a smaller sedimentation velocity (larger absolute value). As a result, the three types of particles are detected separately, as shown in Figure 14.

[0109] In this way, in this embodiment 3, by repeatedly scanning a three-dimensional area and continuously observing the particle coordinates, it is possible to evaluate the particle movement amount due to the settling velocity in addition to the signal amplitude, and to identify the particle type. Furthermore, by simultaneously calculating not only the settling velocity but also the sphericity described in embodiment 1 and the particle size based on Brownian motion described in embodiment 2, it is also possible to identify the particle type with even higher accuracy.

[0110] <Embodiment 4: Application of stirring and heating stress> 15 is a schematic diagram showing an example of the configuration of a particle measuring device according to a fourth embodiment of the present invention. The fourth embodiment differs from the first to third embodiments in that it further includes a thermo-humidistat chamber 201 and a vibration stress applying unit 202. The other configurations are the same as those of the first to third embodiments.

[0111] It is known that aggregates form due to physical stress such as heating and vibration, and in formulation research, stress tests are conducted under various solvent conditions to explore conditions that are less likely to cause aggregate formation. Typically, samples are subjected to stress such as heating and vibration using a stress tester, and then measurements are performed using a particle measurement device. However, this process has issues such as the time required to transfer samples, a time lag between applying stress and measurement, and the inability to perform real-time measurements while applying stress.

[0112] In this fourth embodiment, the role of a stress tester is incorporated into the particle measuring device by providing a thermo-hygrostat 201 that keeps the sample at a constant temperature and a vibration stress applying unit 202 that applies vibration stress to the sample. This embodiment eliminates the need to transfer the sample during the stress test, and allows measurement immediately after applying stress. Another advantage is that real-time measurement is possible while applying heat stress.

[0113] <Modifications of the present invention> The present invention is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and are not necessarily limited to those including all of the described configurations. Furthermore, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, or to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace part of the configuration of each embodiment with other configurations.

[0114] In the above embodiments, the analysis unit 135 may calculate two or more parameters used to determine the particle type and output the results in some format, or may further determine the particle type using the parameters and output the results. For example, on a plot plane such as those shown in Figures 8, 13, and 14, each plot can be classified into one of the particle types by a method such as clustering the plots or identifying a dividing line that divides the plots.

[0115] In the above embodiments, the three-dimensional image generation unit 134 and the analysis unit 135 can be configured by hardware such as a circuit device that implements these functions, or by a computing device such as a CPU (Central Processing Unit) that executes software that implements these functions. [Explanation of symbols]

[0116] 100: Light source 101: Laser driver 109: Objective lens 115: Z Stage 116: Detection optical system 134: 3D image generation unit 135:Analysis Department 136: Display section

Claims

1. A particle measuring device for measuring particles contained in a sample containing a solvent and particles, a light source that emits light; a light irradiation unit that condenses and irradiates the light onto the sample; a scanning unit that repeatedly scans the focal position of the light in a three-dimensional region within the sample; a light detection unit that detects reflected light from the particles; an image generating unit that generates a three-dimensional image of the sample based on the signal detected by the light detecting unit; an analysis unit that analyzes the three-dimensional image; Equipped with The analysis unit one or more parameters representing the type of the particle obtained from the maximum intensity of the reflected light obtained from each focal position in a process of repeatedly scanning the focal position within the three-dimensional region along the optical axis direction of the light; one or more parameters representative of the type of the particle obtained based on the position of the particle obtained by continuously tracking the particle; Calculate at least two of the above and output the results. The analysis unit calculates a numerical value representing the sphericity of the particle obtained from the variation in the reflected light as the parameter. A particle measuring device characterized by:

2. the scanning unit repeatedly moves the focal position back and forth along the optical axis direction within the three-dimensional area, the analysis unit acquires the maximum intensity obtained from each of the focal positions during the process of moving the focal position back and forth, for each of the repetitions; The analysis unit calculates a numerical value representing the sphericity of the particle as the parameter using the maximum and minimum values ​​of the maximum intensities in all the repetitions.

2. The particle measuring device according to claim 1.

3. A particle measuring device for measuring particles contained in a sample containing a solvent and particles, a light source that emits light; a light irradiation unit that condenses and irradiates the light onto the sample; a scanning unit that repeatedly scans the focal position of the light in a three-dimensional region within the sample; a light detection unit that detects reflected light from the particles; an image generating unit that generates a three-dimensional image of the sample based on the signal detected by the light detecting unit; an analysis unit that analyzes the three-dimensional image; Equipped with The analysis unit one or more parameters representing the type of the particle obtained from the maximum intensity of the reflected light obtained from each focal position in a process of repeatedly scanning the focal position within the three-dimensional region along the optical axis direction of the light; one or more parameters representative of the type of the particle obtained based on the position of the particle obtained by continuously tracking the particle; Calculate at least two of the above and output the results. When the time required for the scanning unit to scan the three-dimensional region once is t, the concentration of the maximum particle to be measured is n, the diffusion constant of the minimum particle to be measured is D, and a constant equal to or greater than 1 is α, [Equation 1] fulfill A particle measuring device characterized by:

4. A particle measuring device for measuring particles contained in a sample containing a solvent and particles, a light source that emits light; a light irradiation unit that condenses and irradiates the light onto the sample; a scanning unit that repeatedly scans the focal position of the light in a three-dimensional region within the sample; a light detection unit that detects reflected light from the particles; an image generating unit that generates a three-dimensional image of the sample based on the signal detected by the light detecting unit; an analysis unit that analyzes the three-dimensional image; Equipped with The analysis unit one or more parameters representing the type of the particle obtained from the maximum intensity of the reflected light obtained from each focal position in a process of repeatedly scanning the focal position within the three-dimensional region along the optical axis direction of the light; one or more parameters representative of the type of the particle obtained based on the position of the particle obtained by continuously tracking the particle; Calculate at least two of the above and output the results. the scanning unit repeatedly scans the focal position of the light in the three-dimensional region by repeating a step of scanning the focal position of the light in a two-dimensional plane perpendicular to the optical axis and a step of moving the position of the focal position of the light relative to the sample along the optical axis direction at intervals of p z ; When the width of the three-dimensional region along the optical axis direction is Lz, the time required for the scanning unit to scan the two-dimensional plane once is Δt, the concentration of the maximum particle to be measured is n, the diffusion constant of the minimum particle to be measured is D, and a constant of 1 or more is α, [Equation 2] fulfill A particle measuring device characterized by:

5. A particle measuring device for measuring particles contained in a sample containing a solvent and particles, a light source that emits light; a light irradiation unit that condenses and irradiates the light onto the sample; a scanning unit that repeatedly scans the focal position of the light in a three-dimensional region within the sample; a light detection unit that detects reflected light from the particles; an image generating unit that generates a three-dimensional image of the sample based on the signal detected by the light detecting unit; an analysis unit that analyzes the three-dimensional image; Equipped with The analysis unit one or more parameters representing the type of the particle obtained from the maximum intensity of the reflected light obtained from each focal position in a process of repeatedly scanning the focal position within the three-dimensional region along the optical axis direction of the light; one or more parameters representative of the type of the particle obtained based on the position of the particle obtained by continuously tracking the particle; Calculate at least two of the above and output the results. When the widths in the vertical and horizontal directions of the three-dimensional region in a plane perpendicular to the optical axis are L x and L y , respectively, the width of the three-dimensional region in the optical axis direction is L z , the time required for the scanning unit to scan the three-dimensional region once is t, the number of times the scanning unit repeatedly scans the three-dimensional region is N, the diffusion constant of the smallest particle to be measured is D, a constant of 1 or more is α, the density of the particle is ρ p , the density of the solvent is ρ s , the viscosity of the solvent is η, the gravitational acceleration is g, and the particle diameter is d, [Equation 3] fulfill A particle measuring device characterized by:

6. A particle measuring device for measuring particles contained in a sample containing a solvent and particles, a light source that emits light; a light irradiation unit that condenses and irradiates the light onto the sample; a scanning unit that repeatedly scans the focal position of the light in a three-dimensional region within the sample; a light detection unit that detects reflected light from the particles; an image generating unit that generates a three-dimensional image of the sample based on the signal detected by the light detecting unit; an analysis unit that analyzes the three-dimensional image; Equipped with The analysis unit one or more parameters representing the type of the particle obtained from the maximum intensity of the reflected light obtained from each focal position in a process of repeatedly scanning the focal position within the three-dimensional region along the optical axis direction of the light; one or more parameters representative of the type of the particle obtained based on the position of the particle obtained by continuously tracking the particle; Calculate at least two of the above and output the results. When the widths of the three-dimensional region in the vertical and horizontal directions in a plane perpendicular to the optical axis are L x and L y , respectively, the width of the three-dimensional region in the optical axis direction is L z , the time required for the scanning unit to scan the three-dimensional region once is t, the number of times the scanning unit repeatedly scans the three-dimensional region is N, the diffusion constant of the smallest particle to be measured is D, and a constant of 1 or more is α, [Equation 4] fulfill A particle measuring device characterized by:

7. A particle measuring device for measuring particles contained in a sample containing a solvent and particles, a light source that emits light; a light irradiation unit that condenses and irradiates the light onto the sample; a scanning unit that repeatedly scans the focal position of the light in a three-dimensional region within the sample; a light detection unit that detects reflected light from the particles; an image generating unit that generates a three-dimensional image of the sample based on the signal detected by the light detecting unit; an analysis unit that analyzes the three-dimensional image; Equipped with The analysis unit one or more parameters representing the type of the particle obtained from the maximum intensity of the reflected light obtained from each focal position in a process of repeatedly scanning the focal position within the three-dimensional region along the optical axis direction of the light; one or more parameters representative of the type of the particle obtained based on the position of the particle obtained by continuously tracking the particle; Calculate at least two of the above and output the results. the scanning unit repeatedly scans the focal position of the light in the three-dimensional region by repeating a step of scanning the focal position of the light in a two-dimensional plane perpendicular to the optical axis and a step of moving the position of the focal position of the light relative to the sample along the optical axis direction at intervals of p z ; When the widths of the three-dimensional region in the vertical and horizontal directions in a plane perpendicular to the optical axis are L x and L y , respectively, the width of the three-dimensional region in the optical axis direction is L z , the time required for the scanning unit to scan the two-dimensional plane once is Δt, the number of times the scanning unit repeatedly scans the three-dimensional region is N, the diffusion constant of the smallest particle to be measured is D, and a constant of 1 or more is α, [Equation 5] fulfill A particle measuring device characterized by:

8. A particle measuring device for measuring particles contained in a sample containing a solvent and particles, a light source that emits light; a light irradiation unit that condenses and irradiates the light onto the sample; a scanning unit that repeatedly scans the focal position of the light in a three-dimensional region within the sample; a light detection unit that detects reflected light from the particles; an image generating unit that generates a three-dimensional image of the sample based on the signal detected by the light detecting unit; an analysis unit that analyzes the three-dimensional image; Equipped with The analysis unit one or more parameters representing the type of the particle obtained from the maximum intensity of the reflected light obtained from each focal position in a process of repeatedly scanning the focal position within the three-dimensional region along the optical axis direction of the light; one or more parameters representative of the type of the particle obtained based on the position of the particle obtained by continuously tracking the particle; Calculate at least two of the above and output the results. the analysis unit calculates a settling velocity of the particle as the parameter based on a change over time in the position of the particle in the direction of gravity; The analysis unit classifies the particle types by using the signal amplitude of the reflected light and the sedimentation velocity as the parameters. A particle measuring device characterized by:

9. A particle measuring device for measuring particles contained in a sample containing a solvent and particles, a light source that emits light; a light irradiation unit that condenses and irradiates the light onto the sample; a scanning unit that repeatedly scans the focal position of the light in a three-dimensional region within the sample; a light detection unit that detects reflected light from the particles; an image generating unit that generates a three-dimensional image of the sample based on the signal detected by the light detecting unit; an analysis unit that analyzes the three-dimensional image; Equipped with The analysis unit one or more parameters representing the type of the particle obtained from the maximum intensity of the reflected light obtained from each focal position in a process of repeatedly scanning the focal position within the three-dimensional region along the optical axis direction of the light; one or more parameters representative of the type of the particle obtained based on the position of the particle obtained by continuously tracking the particle; Calculate at least two of the above and output the results. The analysis unit uses the two or more parameters to determine the type of the particle and outputs the result. A particle measuring device characterized by:

10. The analysis unit the maximum intensity, the refractive index of the particle, the refractive index of the solvent, and the size of the particle; Calculate the size of the particles according to the relationship between the parameters 10. The particle measuring device according to claim 1, wherein the particle measuring device is a particle measuring device.

11. The analysis unit calculates a diffusion coefficient of the particle as the parameter based on a change in the position of the particle over time.

10. The particle measuring device according to claim 1, wherein the particle measuring device is a particle measuring device.

12. The analysis unit calculates the size of the particle as the parameter based on the diffusion coefficient of the particle.

12. The particle measuring device according to claim 11.

13. The particle measuring device further comprises: an optical branching unit that branches the light emitted by the light source to generate signal light and reference light; an interference optical system that generates three or more interference lights having different phase relationships by combining the signal light reflected from the sample with the reference light; Equipped with The light detection unit detects the interference light and outputs it as an electrical signal.

10. The particle measuring device according to claim 1, wherein the particle measuring device is a particle measuring device.

14. The analysis unit the maximum strength, the sphericity of the particle calculated from the maximum intensity obtained by repeatedly moving the focal position back and forth along the optical axis within the three-dimensional region; a diffusion constant of the particle calculated based on a change in the position of the particle over time, or a size of the particle calculated from the diffusion constant; a settling velocity of the particle calculated based on a change in the position of the particle in the direction of gravity over time; At least two of the four parameters are output as the above.

10. The particle measuring device according to claim 1, wherein the particle measuring device is a particle measuring device.

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