Particle measurement device, particle measurement method

Abstract

To provide a technique capable of coping with sensitivity that changes for various reasons when a size of particles contained in a liquid sample is measured using light.SOLUTION: A particles measurement device is configured to calibrate a size of particles included in a sample using a reference sample. The reference sample includes a reference sample window having the same material and thickness as those of a sample container window, and a substance having the same reflectance as that of the sample. A sample size is calibrated by using the reflectance of the substance measured at two different points of time.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] This invention relates to a technique for measuring the size of particles contained in a liquid sample. [Background technology]

[0002] In recent years, the focus of pharmaceutical development has been shifting from small molecule drugs to biopharmaceuticals. Because biopharmaceuticals are macromolecules, they are prone to aggregation, and aggregation can lead to toxicity. For example, the U.S. Food and Drug Administration and other organizations are strengthening regulations on the concentration of aggregates. Therefore, there is a need for a technology to quantitatively measure the size distribution of aggregates in the submicron region of 0.1 to 1 μm in terms of desired density. Protein aggregates are suspended 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 subjects will be 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 a light spot by focusing light and measuring an object that is approximately three times or less in size than the light spot, characterized by comprising: a signal acquisition step of detecting reflected light reflected from an object by irradiating the object while moving the focal position of the light in the direction of the optical axis; a step of acquiring correspondence relationship data describing the correspondence relationship between the intensity of the reflected light and the size of the object; and a size calculation step of obtaining the size of the object by querying the correspondence relationship data using the intensity of the reflected light (Claim 1). The technique described in the document enables high-resolution measurement without the need for preprocessing by enhancing the signal by interfering the reflected light with a reference light.

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

[0005] [Patent Document 1] Japanese Patent Publication No. 2017-102032 [Patent Document 2] WO2020 / 144754 publication [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The techniques described in Patent Documents 1 and 2 measure particle size using interference signals generated by the interference between signal light reflected from particles in a liquid sample and reference light. In actual measurements, changes in ambient temperature, changes over time, and dust adhering to the objective lens can cause wavelength shifts and fluctuations in laser power of the semiconductor laser, which can cause fluctuations in the detection signal. To suppress this, sensitivity calibration is performed using standard beads with known particle sizes.

[0007] However, this calibration procedure is time-consuming, requiring the preparation of standard beads. Furthermore, due to the difference between the light reflectance of the standard beads and the light reflectance of the particles, the laser power must be changed when irradiating the standard beads with the laser versus when irradiating the particles with the laser, which may result in different sensitivity coefficients between the two.

[0008] This invention has been made in view of the above problems, and aims to provide a technology that can accommodate sensitivity that changes for various reasons when measuring the size of particles contained in a liquid sample using light. [Means for solving the problem]

[0009] The particle measuring device according to the present invention calibrates the size of particles contained in a sample using a reference sample. The reference sample includes a reference sample window having the same material and thickness as the sample container window, and a material with a reflectivity similar to that of the sample. The sample size is calibrated using the reflectivity of the material measured at two different time points. [Effects of the Invention]

[0010] The particle measurement method according to the present invention can accommodate varying sensitivity for various reasons when measuring the size of particles contained in a liquid sample using light. Other issues, configurations, and effects will be clarified by the following description of the embodiments. [Brief explanation of the drawing]

[0011] [Figure 1] This is a schematic diagram illustrating the relationship between particle size and the magnitude of reflected light intensity as described in Patent Documents 1 and 2. [Figure 2] This is a diagram showing the configuration of the particle measuring device according to Embodiment 1. [Figure 3A] This diagram illustrates the frame and metal plate on which the reflectance reference sample is placed. [Figure 3B] This diagram illustrates the frame and metal plate on which the reflectance reference sample is placed. [Figure 4] This is a side cross-sectional view illustrating the reflectance reference sample 303 and its focal point. [Figure 5A] This is a schematic diagram illustrating a method for quantifying sensitivity correction. [Figure 5B] This is a schematic diagram illustrating a method for quantifying sensitivity correction. [Figure 6]It is a diagram showing an example of the measurement flow of A0. [Figure 7] It is a diagram showing an example of the measurement flow of A1. [Figure 8] It is a diagram showing another example of the measurement flow of A1. [Figure 9] It is a diagram showing an example of the measurement flow in which, after measuring A1, a real sample is subsequently measured. [Figure 10] It is a diagram showing the arrangement of the reflectance reference sample 303. [Figure 11A] It is a calculation result showing the specification of the thickness variation of the transparent flat plate 402 for realizing high-precision size measurement by performing sensitivity calibration. [Figure 11B] It shows the result of calculating the relationship between the refractive index of the transparent flat plate 402 and the Strehl intensity, similar to FIG. 11A. [Figure 12] It is a schematic diagram showing the ID information of the reflectance reference sample 303. [Figure 13] It is a diagram showing an example of the measurement flow for reproducing the ID information. [Figure 14A] It is a schematic diagram showing the relationship between the container containing the real sample including the particles to be measured and the solvent, and the focal position of the laser beam when measuring the refractive index of the solvent of the real sample. [Figure 14B] Following FIG. 14A, it shows the state of moving the focal position to a predetermined position within the sample and measuring the size of the particles encapsulated in the sample. [Figure 15A] It shows the reflectance of the reflectance reference sample 303 in Embodiment 2. [Figure 15B] It shows the reflectance in another configuration example of the reflectance reference sample 303 of Embodiment 2. [Figure 16] It shows another configuration example of the reflectance reference sample 303.

Mode for Carrying Out the Invention

[0012] <Problems of the Prior Art> Figure 1 is a schematic diagram illustrating the relationship between particle size and the magnitude of reflected light as described in Patent Documents 1 and 2. The techniques described in Patent Documents 1 and 2 utilize the interference of reflected signal light from particles and a reference light to measure the size of particles dispersed in a solvent. The detected reflected light changes with the particle size and its refractive index. In the techniques 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 the interference of light. At this time, the magnitude of the detection signal E sig The refractive index n of the particle p , the refractive index n of the solvent m Using Fresnel's Law, when the coherence length of the light source is sufficiently long, it can be expressed by the following equation: S is the electric field amplitude of the light irradiating the sample, R is the electric field amplitude of the reference light, σ is a coefficient corresponding to the particle size shown above, and η is a constant indicating the interference efficiency of the reflected light and the reference light and the efficiency of the high-voltage conversion of the photodetector.

[0013]

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[0014] Particle size can be measured by measuring the detection signal represented by Equation 1. In actual measurements, the detection signal may fluctuate due to changes in ambient temperature, changes over time, or fluctuations in the wavelength shift and laser power of the semiconductor laser caused by dust adhering to the objective lens. To suppress this, sensitivity calibration is performed according to the following procedure.

[0015] (1) Prepare a standard sample by diluting polystyrene standard beads of known particle size with pure water to an appropriate concentration. (2) Immediately before measuring the sample to be measured, measure the standard sample. (3) From the measurement results of the standard sample, determine the sensitivity correction coefficient that eliminates the discrepancy between the measured size and the true size, and manually input that value.

[0016] The calibration procedure described above requires cumbersome effort and a great deal of time for preparing standard beads, accurate dilution, and measurement, posing challenges from a user convenience standpoint. Furthermore, there can be variations in size between standard bead lots, and beads may break during dilution and stirring, which can lead to inaccurate measurements. In addition, standard beads made of polystyrene have a large refractive index difference with the solvent (pure water), resulting in high reflectivity and requiring a relatively low laser power setting. In contrast, when measuring protein particles, for example, the refractive index difference between the protein particles and the solvent is small, resulting in low reflectivity and requiring a relatively high laser power setting. This difference in laser power between the two can lead to a difference in laser temperature, potentially resulting in different laser wavelengths. This wavelength shift can lead to different sensitivity coefficients, posing a challenge in accurate sensitivity correction.

[0017] <Basic Principles of the Invention> (Sensitivity Quantification) To solve the above problems, this invention performs sensitivity calibration using a reflectance reference sample having a plane that has a reflected signal equivalent to the signal light obtained from the particle being measured. Specifically, the particle is measured by the following equation, which is obtained by introducing a sensitivity coefficient K into Equation 1.

[0018]

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[0019] The sensitivity coefficient K is defined by the following formula. A0 is the reflectance of the reflectance reference sample measured at the time of shipment of the particle measuring device of the present invention. A1 is the reflectance of the reflectance reference sample measured immediately before measuring the sample to be actually measured.

[0020]

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[0021] By measuring this reflectance reference sample for each particle measurement or under predetermined conditions, it becomes possible to correct the sensitivity using a simpler method based on the sensitivity coefficient K.

[0022] (Reference sample) The reflectance reference sample has a glass window made of the same material and thickness as the glass window on the light incident surface of the sample container used for measuring particles, and a material is formed in contact with this glass window, having an interface with a reflectance approximately equal to that of the measurement sample. This allows the measurement conditions for the reflectance reference sample and the measurement sample to be identical. In particular, by making the laser power the same during sample measurement and reference sample measurement, the interference efficiency of the reflected light and the reference light can be made the same, enabling more accurate sensitivity correction.

[0023] As an example of a reflectance reference sample, a resin with a refractive index of 1.47 ± 0.03 was formed in contact with a transparent plate. This resulted in a refractive index difference between the transparent plate and the resin being approximately the same as the refractive index difference between the protein particles and the solvent. As a result, the reflectance of the reflectance reference sample and the reflectance of the protein particles in the solvent became approximately equal, making it usable as a reflectance reference sample for protein particle measurement. Here, "approximately equal" means that the error in the measurement result falls within an acceptable range. In other words, the difference between the reflectance of the substance in contact with the transparent plate (in this case, the resin) and the reflectance of the sample must fall within an acceptable range from the standpoint of measurement error.

[0024] As another example of a reflectance reference sample, a dielectric film was formed in contact with a transparent plate, as described later. When the particles to be measured are metal particles, the reflectance of the metal particles in the solvent can vary from 0.01% to several percent depending on the material and composition. By selecting the material and thickness of the dielectric film, as well as the material and thickness of the metal film formed on top of the dielectric film, it is possible to design a system that achieves a predetermined reflectance, making it possible to accommodate various types of particles.

[0025] <Embodiment 1> In the following description of embodiments of the present invention, we will proceed using a coordinate system in which the optical axis direction is taken as the z-axis, as shown in the figure. Furthermore, the size of the particles to be measured will be treated as the diameter of spheres with equal volume.

[0026] Figure 2 is a diagram of the configuration of a particle measuring device according to Embodiment 1 of the present invention. Laser light emitted from a light source 100, whose emission state is controlled by a laser driver 101 that performs high-frequency superposition and emission power control, 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, it is separated into signal light and reference light by a polarizing beam splitter 104.

[0027] The reference light is converted to a circularly polarized state by the λ / 4 plate 105, reflected by the reference light mirror 106, and then reflected by the λ / 4 plate 105 with a polarization state rotated 90 degrees from the forward path, and then reflected by the polarizing beam splitter 104. The signal light is deflected in the direction of propagation by the XY direction composite deflection element 107, then 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.

[0028] The drive mechanism 109, which 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 forward path by the XY-direction composite deflection element 107, and due to the action of the built-in λ / 4 plate, it becomes a circular polarization state with the polarization rotated by 90 degrees from the forward path, and passes through the polarizing beam splitter 104.

[0029] The sample container 200 holds the sample 204 in its well and guides signal light into the sample through the transparent window 202. 203 is a resin component that forms the well of the sample container. The metal plate 201 contacts the transparent window 202 to mechanically hold the sample container 200 and to stabilize the sample temperature.

[0030] The signal light and the reference light are combined by a polarizing beam splitter 104 and guided to the detection optical system 112, where they are split into transmitted light and reflected light by a half-beam splitter 113 via a pinhole 111.

[0031] The reflected light passes through a λ / 4 plate 114 with its optical axis set at approximately 45 degrees to the horizontal, then is focused by a focusing lens 115 and split into two by a polarizing beam splitter 116. Each split is then photoelectrically converted by photodetectors 150 and 151, and differentially amplified by a current differential amplifier 152 to produce a detection signal 123.

[0032] 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. Each of these is then photoelectrically converted by photodetectors 153 and 154, and differentially amplified by a current differential amplifier 155 to produce a detection signal 122.

[0033] The detection optical system 112 constitutes a homodyne phase diversity method, and the detection signals 122 and 123 are processed by the signal processing unit 124. The signal processing unit 124 (processing unit) performs sensitivity correction based on the information held in the information holding unit 125, and displays the calculation result including the correction on the display unit 126 to present it to the user. The information held in the information holding unit 125 and the sensitivity correction based thereon will be described later.

[0034] (Measurement of reflectance at the interface) Figures 3A and 3B illustrate the frame and metal plate on which the reflectance reference sample is placed. In the figures, the reflectance reference sample 303 is inserted into the frame 302 and positioned so as to be in contact with the surface of the metal plate 201. At this time, the reflectance reference sample 303 may also be placed in the frame 302 together with other sample containers 304. When the reflectance reference sample 303 is placed in the frame 302 together with other sample containers 304, it becomes possible to measure both together in a single flow.

[0035] Figure 4 is a side cross-sectional view illustrating the reflectance reference sample 303 and its focal position. The reflectance reference sample 303 consists of a transparent plate 402, a container 403, and a substance 404 in contact with the transparent plate 402, and is positioned in contact with the metal plate 201. Light 400 focused by the objective lens 108 passes through the measurement window 405 and irradiates the interface between the transparent plate 402 and the substance 404. By adjusting the relative position between the interface and the objective lens 108, the focal position of the objective lens 108 is aligned with the interface, and the reflectance from the interface is obtained. Furthermore, the position of the reference light mirror 106 is adjusted to adjust the optical path length so that the reflectance from the interface is maximized.

[0036] (Sensitivity Quantification) Figures 5A and 5B are schematic diagrams illustrating the method for quantifying sensitivity correction. First, the reflectance (A0) from the interface between the transparent plate 402 of the reflectance reference sample 303 installed in the device and the substance 404 in contact with the transparent plate 402 is measured using the method described above at the time of shipment of the device, and this information is stored in the information storage unit 125. Next, when measuring an actual sample, the reflectance (A1) from the interface between the transparent plate 402 of the reflectance reference sample 303 and the substance 404 in contact with the transparent plate 402 is measured in the same manner, and this information is stored in the information storage unit 125. The sensitivity coefficient K can be determined from the values ​​of A0 and A1, and the detection signal can be corrected using Equation 2.

[0037] Figure 5 shows the case where A0 > A1. Possible causes of this include changes in the wavelength shift and laser power of the semiconductor laser due to changes in ambient temperature and time, dust adhering to optical elements including the objective lens, and changes in the optical elements themselves over time, which alters the optical properties and reduces the output signal. By using the above-described method for quantifying sensitivity correction, it is possible to significantly reduce the influence of these factors on the measurement results.

[0038] (Measurement flow) Figure 6 shows an example of the A0 measurement flow. This flowchart can be performed by the signal processing unit 124. The following flowcharts are similar. The focusing position of the signal light is moved to the planar position of the reflectance reference sample 303, and the light source 100 is made to emit light at standard power P0. The Z-axis position Z0 of the interface and the position R of the reference light mirror 106 are initialized. Z0 is determined by measuring the signal while scanning Z0 around the transparent window 405. The mirror position R0 where the optical path lengths of the signal light and the reference light match is determined by measuring the signal while scanning R at the determined Z0. The signal level obtained at this point is stored as A0 in the information holding unit 125.

[0039] Since A0 and A1 are measured using the same light emission power, we decided to use the standard power here. The reason for using the standard power is to ensure that the signal level of the measurement result does not saturate. Under the conditions under which this non-saturating signal level can be obtained, it is necessary that the reflectance of the sample and the reflectance of the reflectance reference sample 303 are approximately the same (the difference between the two is within an acceptable range from the standpoint of measurement accuracy).

[0040] Figure 7 shows an example of the measurement flow for A1. A1 can be measured in the same way as in Figure 6. The signal processing unit 124 calculates the sensitivity coefficient K from A0 and A1 obtained in Figures 6 and 7.

[0041] Figure 8 shows another example of the measurement flow for A1. In addition to the flow in Figure 7, a predetermined value can be set for the sensitivity coefficient K, and sensitivity abnormality processing can be performed when the sensitivity coefficient K obtained from A0 and A1 falls outside the range of the predetermined value. For example, the signal processing unit 124 can notify the user that the sensitivity coefficient K is outside the predetermined range by outputting a signal or message.

[0042] Figure 9 shows an example of a measurement flow in which A1 is measured, followed by the measurement of a real sample. When measuring a real sample, Z0 and R0 can be determined in the same way as the reflectance reference sample. The focal position is moved from Z0 to a predetermined measurement position Z, and the detection signal is processed while scanning the focal point according to predetermined scanning conditions. The measurement results of the real sample are calibrated based on the sensitivity coefficient K. Through the above processing, the particle size and density distribution of the real sample are calculated, and the results are displayed on the display unit 126.

[0043] (Placement of reflectance reference samples) Figure 10 shows the arrangement of the reflectance reference sample 303. In Figure 3, an example is shown in which the reflectance reference sample 303 is placed inside the frame 302 together with other sample containers 304, but it can also be placed in other locations. For example, the reflectance reference sample 303, which has the structure shown in Figure 4, can be placed in a part of the sample stage 1001 that is larger than the metal plate 201 and holds the metal plate 201. In this case, the reflectance reference sample 303 can be placed outside the frame 302 so as to be in contact with a metal plate 1002 separate from the metal plate 201. The other metal plate 1002 may be a different one from the metal plate 201, or it may be one that is integrated with the metal plate 201.

[0044] By placing the reflectance reference sample 303 outside the frame 302, it becomes possible to place the sample to be measured at all positions within the frame 302, thereby maximizing the number of samples that can be measured. Furthermore, this arrangement makes it possible to configure the system so that the reflectance reference sample 303 placed outside the frame 302 cannot be removed by the user. This helps to avoid human errors such as forgetting to place the reflectance reference sample 303 or placing it in the wrong position. For example, the reflectance reference sample 303 could be placed in a location that is not visible to the user. The reflectance reference sample 303 may be configured to be detachable or fixedly attached.

[0045] (Thickness of the transparent sheet) Figure 11A shows the calculation results for the specification of thickness variation of the transparent plate 402 to achieve high-precision size measurement by performing sensitivity calibration. In order to measure the particle size distribution with high precision, the configuration of the reflectance reference sample 303 must satisfy predetermined specifications, similar to the configuration specifications of the sample container. As is well known, if the thickness of the transparent plate 402 deviates from a predetermined value, spherical aberration occurs optically. The effect on the detection signal can be treated as a decrease in Strehl intensity due to wavefront aberration. The decrease in the detection signal (Strehl intensity) due to the deviation in the thickness of the transparent plate 402 was calculated. Here, the results are shown when a semiconductor laser with a wavelength of 785 nm is used as the light source 100, a microscope lens with an numerical aperture of 0.45 is used as the objective lens 108, and borosilicate glass with a thickness of 175 μm (measured refractive index = 1.520) is used as the transparent plate 402. As can be seen in the figure, in order to reduce the decrease in the detection signal to 0.2% or less, the variation in the thickness of the transparent plate 402 must be 70 μm or less.

[0046] Figure 11B shows the results of calculating the relationship between the refractive index and Strehl intensity of the transparent plate 402, similar to Figure 11A. As can be seen in the figure, the allowable range for the refractive index of the transparent plate 402 is ±0.22.

[0047] As an example of the present invention, the reflectance reference sample 303 shown in Figure 4 and later in Figures 12 and 16 cannot obtain accurate sensitivity unless it is optically compatible with the sample container 304 shown in Figure 3. Specifically, the transparent plate 402 formed at the bottom must have a thickness of 175 ± 70 μm and a refractive index of 1.520 ± 0.22. A borosilicate glass substrate of optical quality can be used as the material for the transparent plate 402 that satisfies these requirements.

[0048] (ID information) Figure 12 is a schematic diagram showing the ID information of the reflectance reference sample 303. The reflectance reference sample 303 can be given ID information. Specifically, information corresponding to the reflectance of the reflectance reference sample 303 is recorded in an optically identifiable form such as a barcode on the surface of the transparent flat plate 402 that is in contact with the metal plate 201 and on the inside 1202 of the transparent window 405, outside the container opening 1203. The information corresponding to the reflectance may be the reflectance value itself, or the level of the value (high, medium, low, etc.). It may also be a symbol or information linked to the reflectance value or level. The recording method can be a known method such as printing or engraving.

[0049] By focusing the laser beam onto the surface of the transparent flat plate 402 that is in contact with the metal plate 201 and scanning it in the x and y directions as shown in the figure, the ID information 1201 can be reconstructed. By reconstructing the ID information before measuring the reflectance reference sample 303, it can be confirmed that the sample is indeed the reflectance reference sample, thus avoiding human errors such as mistaking the reference sample.

[0050] ID information can also include information other than that corresponding to reflectance. For example, it could include predetermined specific identification information or the serial number of the reference sample. This makes it possible to guarantee the quality of the reference sample.

[0051] The surface of the transparent plate 402 on which the ID is recorded does not have to be the surface in contact with the metal plate 201. The reflectance reference sample 303 can be manufactured by pre-forming the ID information on the transparent plate 402 and then bonding it with the surface on which the ID information is formed facing the container. In this case as well, the ID information is formed on the inside of the transparent window 405 and on the outside of the container opening 1203.

[0052] Figure 13 shows an example of a measurement flow for reproducing ID information. ID information can be reproduced before measuring A0 or A1. Here, an example of reproduction during the measurement of A1 is shown. For example, ID information can be reproduced before scanning the interface position Z0 and the mirror position R.

[0053] The ID information can be used to pre-check whether the correct reflectance reference sample 303 is being used. Since the light emission power is predetermined for each sample type, determining the light emission power also determines the corresponding sample type. Therefore, it is desirable to perform the step of reproducing the ID information after determining the light emission power but before measuring the reflectance.

[0054] (Machine differences and other factors) By using the reflectance reference sample 303, even if there are slight differences in the particle measurement device (such as minute differences in laser wavelength, optical system, and detector sensitivity), nearly identical results can be obtained for the same sample. Furthermore, it can also be used for diagnosing device malfunctions. Specifically, if the sensitivity coefficient K deviates from a certain numerical range, it may indicate deterioration due to the aging of the laser, adhesion of dust to optical elements, or misalignment of the optical system. In such cases, the signal processing unit 124 can issue an alert, for example, on the display unit 126, prompting device maintenance or repair.

[0055] (Solvent refractive index measurement) Figure 14A is a schematic diagram showing the relationship between a container holding a real sample containing the particles to be measured and a solvent, and the focal position of the laser beam when measuring the refractive index of the solvent in the real sample. The reflectance reference sample 303 can also be used to measure the refractive index of the solvent in the real sample. In the figure, 1401 is the sample and 1402 is the sealing tape. The present invention utilizes the fact that the reflectance of the boundary between the transparent plate 402 and the sample depends on the refractive index of the solvent in order to measure the refractive index of the solvent. By moving a sample stage (not shown) in the Z direction and determining the condition under which the detection signal is maximized, the focal point of the objective lens 108 can be positioned at the boundary between the sample 1401 and the transparent plate 402. At this time, from Fresnel's equation, the magnitude of the detection signal can be expressed by Equation 1.

[0056] In this invention, the refractive index of the transparent plate 402 can be used as a predetermined value by selecting the material. Similarly, the electric field amplitude S of the light irradiated onto the sample and the electric field amplitude R of the reference light can be treated as constant values ​​by keeping the output power conditions of the semiconductor laser (not shown) constant.

[0057] Furthermore, the refractive index n is the material in contact with the interface of the reflectance reference sample 303. o Measure the magnitude of the detection signal when the substance is used as a sample, and set that value to E0 sig Let's assume that E0 sig This is represented by Equation 4. From Equations 1 and 4, Equation 5 is derived. In Equation 5, the refractive index n of the solvent is expressed. m Since the values ​​other than those mentioned are known or measured, we can use Equation 5 to determine the refractive index n of the solvent. m It becomes possible to measure this.

[0058]

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[0059]

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[0060] Figure 14B shows the process of moving the focal point to a predetermined location within the sample, following Figure 14A, to measure the size of the particles contained within the sample.

[0061] <Embodiment 2> (Refractive index: Dielectric film) Figure 15A shows the reflectance of the reflectance reference sample 303 in Embodiment 2 of the present invention. In Embodiment 2, a dielectric film was used as the material in contact with the transparent plate 402 of the reflectance reference sample 303 in Figure 4. ZnSSiO2 was used as the dielectric film and was formed by sputtering on a transparent plate 402 made of borosilicate glass with a refractive index of 1.52. The reflectance of the reflectance reference sample on which the dielectric film is formed changes according to the thickness of the dielectric film due to the interference of reflection from the side of the dielectric film on the laser light incident side and reflection from the side on the far side. Figure 15A shows the relationship between the thickness of the ZnSSiO2 used in Embodiment 2 and the reflectance.

[0062] Figure 15B shows the reflectance of the reflectance reference sample 303 in another configuration example of Embodiment 2. In this configuration example, Al 100 nm and Ag 20 nm were further formed as metal films by sputtering on the dielectric film ZnSSiO2 formed on the transparent flat plate 402. The reflectance of the reflectance reference sample 303 in this structure changes with respect to the thickness of the dielectric film ZnSSiO2 as shown in Figure 15B.

[0063] Based on the above, the reflectance of the reflectance reference sample 303 can be set to a predetermined value by selecting either (a) a dielectric film alone or a multilayer structure of two or more dielectric films, or (b) a structure in which a dielectric film and a metal film are sequentially stacked, and by appropriately selecting the film thickness of each. The areas around C and D shown in Figure 15B indicate a wide margin for variations in film thickness. It is preferable to use these points when designing a film corresponding to a predetermined reflectance value, as this provides a wide margin for fabrication, such as the film deposition rate.

[0064] When the measurement target particle is larger than the light spot size or is a metal particle, the reflectivity increases depending on its material, texture, etc. In that case, by selecting the material and thickness of the dielectric film and the material and thickness of the metal film, a configuration corresponding to the reflectivity of the target particle may be adopted.

[0065] <Embodiment 3> In Embodiment 3 of the present invention, a specific example of the reflectivity reference sample 303 will be described. The configuration and measurement procedure of the particle measurement device are the same as those in Embodiments 1 and 2.

[0066] (Reflectivity reference sample 1) As the substance contacting the transparent flat plate 402 of the reflectivity reference sample 303 in FIG. 4, an ultraviolet curable resin (DVD310) manufactured by Nippon Kayaku Co., Ltd. was used. Since bubbles mixed into the interface affect the reflectivity signal, in order to avoid that, the ultraviolet curable resin was put into a vacuum container and degassing treatment was carried out by evacuating the vacuum for 10 minutes. Then, the resin was injected into the container 403, and ultraviolet rays were irradiated at about 300 mJ / cm 2 to cure the resin using an ultraviolet irradiation device (ECS-201G1) manufactured by Eye Graphic.

[0067] (Refractive index: for protein) The reflectivity from the interface is proportional to the difference in refractive indices of the two substances. For example, when protein particles are targeted as the sample for measuring the size distribution, the difference in refractive indices between the solvent and the protein particles is about 0.06. In the reflectivity reference sample using the above ultraviolet curable resin, the refractive index after curing of the ultraviolet curable resin DVD310 is 1.50, and when borosilicate glass with a refractive index of 1.52 is used for the transparent flat plate, the difference in refractive indices between the two is 0.02. Therefore, the reflectivity from the protein particles and the interface reflectivity are also of the same degree.

[0068] This allows the reflectance reference sample 303 to be measured under the same laser power conditions as those used to measure the size distribution of protein particles, and the sensitivity coefficient K can be obtained under these conditions. While differences in laser power conditions can cause differences in the sensitivity coefficient, the method of the present invention eliminates this possibility and enables more accurate sensitivity correction, thus allowing for accurate measurement of particle size.

[0069] For the reflectance reference sample 303, it is desirable to have a sample that exhibits minimal optical changes over time and can be used for extended periods.

[0070] For the transparent flat plate 402 of the reflectance reference sample 303, samples were prepared with varying refractive indices by using a different UV-curing resin instead of the UV-curing resin (DVD310) manufactured by Nippon Kayaku. Measurements were performed to determine the sensitivity coefficient K for each sample, and the laser power conditions were as follows.

[0071] n: Laser power condition 1.41: Lower power than protein particle sample measurement 1.43: Approximately equal power to protein particle sample measurement. 1.48: Approximately equivalent power to protein particle sample measurement. 1.50: Approximately equivalent power to protein particle sample measurement. 1.52: Higher power than protein particle sample measurement.

[0072] Based on these results, the refractive index of the material in contact with the transparent flat plate 402 of the reflectance reference sample 303 is preferably 1.43 or more and 1.50 or less.

[0073] Figure 16 shows another configuration example of the reflectance reference sample 303. In the embodiments described above, a UV-curing resin (DVD310) manufactured by Nippon Kayaku was used as the material in contact with the transparent plate 402, but a liquid resin (such as microscope silicone oil) can be used instead. In Figure 16, 1601 is the liquid resin. In this case, the container should be sealed with sealing tape or the like to prevent leakage of the liquid.

[0074] (How to use the reflectance reference sample) When measuring multiple real samples, the reflectance reference sample 303 may be measured only once at the start of the measurement, or it may be measured for each individual measurement in the measurement of multiple real samples.

[0075] When the measurement time required for measuring a real sample is relatively short, the ambient temperature and laser temperature changes during the measurement are relatively small. Therefore, it is sufficient to perform a sensitivity correction by measuring a reflectance reference sample once at the start of the measurement, which can shorten the overall measurement time.

[0076] On the other hand, if the measurement time required for measuring actual samples is relatively long, there is a possibility of changes in ambient temperature and laser temperature during measurement. In such cases, it is more accurate to measure a reflectance reference sample and re-correct the sensitivity each time for each individual measurement of multiple actual samples. In this case, instead of measuring each individual measurement, it is also acceptable to measure a reflectance reference sample and re-correct the sensitivity each time for every certain number of actual sample measurements.

[0077] The length of measurement time for actual samples can be determined by the person performing the measurement. In this case, the measurement time used when measuring similar samples in the past may be used as a reference. However, when measuring multiple unknown samples, it is preferable to repeat the sensitivity correction each time.

[0078] <Regarding variations of the present invention> The present invention is not limited to the embodiments described above, and includes various modifications. For example, the embodiments described above are described in detail to make the present invention easier to understand, and are not necessarily limited to those having all the configurations described. Furthermore, it is possible to replace parts of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add configurations from other embodiments to the configuration of one embodiment. In addition, it is possible to add, delete, or replace parts of the configuration of each embodiment with other configurations.

[0079] In the embodiments described above, the reflectance reference sample 303 can be configured as a component of the particle measuring device, or it can be configured as a separate component that is detachable from the particle measuring device, for example.

[0080] In the embodiments described above, the objective lens 108 and the drive mechanism 109 serve as illumination units that focus and irradiate the sample with signal light, and the detection optical system 112 serve as a detection unit that detects interference signals generated by the interference between signal light and interference light. [Explanation of Symbols]

[0081] 100: Light source 101: Laser Driver 108: Objective lens 109: Drive mechanism 112: Detection Optics 124: Signal Processing Unit 200: Sample container 201: Metal plate 202: Transparent window 303: Reflectance Reference Sample 402: Transparent flat plate 404: Substance 1201: ID Information

Claims

1. A particle measuring device for measuring the size of particles contained in a liquid sample, A light source that emits light, A branching unit that splits the light from the aforementioned light source into signal light and reference light, An illumination unit that focuses the signal light and irradiates it onto the sample. A detection unit that detects an interference signal obtained by interfering the reflected light from the particles with the reference light. A processing unit measures the size of the sample using the interference signal detected by the detection unit and calibrates the size using the measurement result of a reference sample. Equipped with, The aforementioned reference sample is provided with a reference sample window having the same material and thickness as the sample container window of the sample container that contains the aforementioned sample, The reference sample includes a material having a reflectance such that the difference between it and the reflectance of the sample is within an acceptable range, positioned in contact with the reference sample window. The processing unit obtains the result of measuring the reflectance of the substance at the first time point as the first reflectance, The processing unit obtains the result of measuring the reflectance of the substance at a second time point, which is closer to the time point in time when the sample is measured than the first time point, as the second reflectance. The processing unit calibrates the size of the sample using the first reflectance and the second reflectance. A particle measuring device characterized by the following features.

2. The irradiation unit irradiates the light such that the focal position of the light is at the interface between the reference sample window and the substance. The processing unit uses the interference signal obtained from the interface between the reference sample window and the material to acquire the first reflectance and the second reflectance, respectively. The particle measuring device according to claim 1, characterized in that it is a particle measuring device.

3. The processing unit calculates the ratio of the second reflectance to the first reflectance as a sensitivity coefficient. The processing unit calibrates the size of the sample by multiplying the size of the sample measured using the interference signal by the sensitivity coefficient. The particle measuring device according to claim 1, characterized in that it is a particle measuring device.

4. The particle measuring device according to claim 3, characterized in that the processing unit outputs a signal indicating that the sensitivity coefficient is outside a predetermined range.

5. The light source emits light having the same emission power in both cases: when measuring the size of the sample and when measuring the reflectance of the material. The processing unit uses the interference signal obtained by the light having the same output power to acquire the size of the sample, the first reflectance, and the second reflectance, respectively. The particle measuring device according to claim 1, characterized in that it is a particle measuring device.

6. The particle measuring device further comprises a plate on which the sample container is placed, and a frame that covers the sample container placed on the plate. The reference sample is configured to be fixed to the plate at a position outside the frame on the plate. The particle measuring device according to claim 1, characterized in that it is a particle measuring device.

7. The irradiation unit irradiates the light such that the focal position of the light is at the interface between the sample container window and the liquid. The processing unit acquires the magnitude of the interference signal obtained from the interface between the sample container window and the liquid as the sample interference signal value. The processing unit acquires the magnitude of the interference signal obtained from the interface between the reference sample window and the material as the reference sample interference signal value. The processing unit calculates the refractive index of the liquid using the sample interference signal value and the reference sample interference signal value. The particle measuring device according to claim 2, characterized in that it is a particle measuring device.

8. The particle measuring device according to claim 1, characterized in that the substance is an ultraviolet-curing resin.

9. The aforementioned substance is A single dielectric film or a multilayer structure of two or more dielectric films, A structure in which a metal film is layered on top of a dielectric film. It is at least one of the following. The particle measuring device according to claim 1, characterized in that it is a particle measuring device.

10. The particle measuring apparatus according to claim 1, characterized in that it includes the aforementioned reference sample.

11. The substance has a refractive index of 1.47 ± 0.

03. The particle measuring device according to claim 10, characterized in that it is a particle measuring device.

12. A particle measurement method for measuring the size of particles contained in a liquid sample, Steps of emitting light from a light source, A step of splitting the light from the aforementioned light source into signal light and reference light, The steps of focusing the signal light and irradiating it onto the sample, A step of detecting an interference signal obtained by interfering the reflected light from the particle with the reference light, A step of measuring the size of the sample using the detected interference signal, A step of calibrating the size using a reference sample, It has, The aforementioned reference sample is provided with a reference sample window having the same material and thickness as the sample container window of the sample container that contains the aforementioned sample, The reference sample includes a material having a reflectance such that the difference between it and the reflectance of the sample is within an acceptable range, positioned in contact with the reference sample window. In the calibration step, the result of measuring the reflectance of the substance at the first time point is obtained as the first reflectance. In the calibration step, the result of measuring the reflectance of the substance at a second time point, which is closer to the time point in time when the sample is measured than the first time point, is obtained as the second reflectance. In the calibration step, the size of the sample is calibrated using the first reflectance and the second reflectance. A particle measurement method characterized by the following features.

13. The particle measuring device further comprises the reference sample and the sample container containing the sample, The aforementioned reference sample is provided with a reference sample window having the same material and thickness as the sample container window provided with the aforementioned sample container, The reference sample includes a material having a reflectance such that the difference between it and the reflectance of the sample is within an acceptable range, positioned in contact with the reference sample window. The reference sample window is formed on a transparent flat plate placed at the bottom of the reference sample. The thickness of the transparent plate is in the range of 175 ± 70 μm, and the refractive index of the transparent plate is in the range of 1.520 ± 0.

22. The particle measuring device according to claim 1, characterized in that it is a particle measuring device.

14. A shape pattern representing ID information for identifying the reference sample is formed on one of the surfaces of the transparent flat plate. The particle measuring device according to claim 13, characterized in that it is a particle measuring device.

15. The substance has a refractive index of 1.47 ± 0.

03. The particle measuring device according to claim 13, characterized in that it is a particle measuring device.

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