Particle measurement device, particle measurement method, and reflectance reference sample

Abstract

The purpose of the present invention is to provide a technique that, when measuring the size of particles contained in a liquid sample using light, can cope with sensitivity that changes due to various reasons. This particle measurement device corrects the size of particles contained in a sample using a reference sample that is provided with a reference sample window having the same material and thickness as a sample container window, and that is provided with a substance having the same reflectance as the sample. The sample size is corrected using the reflectance of the substance measured at two different points in time (referring to Figure 9).

Description

Technical Field

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

[0002] In recent years, the development of drugs has shifted from low-molecular drugs to biopharmaceuticals. Since biopharmaceuticals are macromolecules, they are prone to aggregation, and sometimes become toxic when aggregated. For example, the U.S. Food and Drug Administration and others have strengthened the concentration management restrictions on aggregates. Therefore, for aggregates in the submicron region of 0.1~1μm, a technology for quantitatively measuring the size distribution of the required density is required. Protein aggregates are suspended in a solvent, and their positions change over time due to Brownian motion. Below, in the present invention, the measurement technology of the size and density of protein aggregates and standard particles such as polystyrene beads is described. These test objects are collectively referred to as "particles" for description.

[0003] Patent document 1 describes a technique for detecting particles using optical measurement. The document discloses "an optical measurement method, which focuses light to generate a light spot, and measures a test object that is approximately 3 times smaller than the size of the light spot, characterized in that it includes: a signal acquisition step, irradiating the test object while at least moving the focal position of the light in the direction of the optical axis, thereby detecting reflected light reflected from the test object; a step of acquiring correspondence data describing the correspondence between the intensity of the reflected light and the size of the test object; and a size calculation step of obtaining the size of the test object by comparing the intensity of the reflected light with the correspondence data" (claim 1). The technology described in the document enhances the signal by interfering the reflected light with the reference light, does not require pre-processing, and can achieve high-resolution measurement.

[0004] Patent document 2 discloses the following technology: physically scanning the objective lens and receiving the interference between the signal light and the interference light through four detectors with different phase conditions, thereby eliminating the need for phase adjustment of the reference light based on the scanning of the reflector in time domain OCT (Optical Coherence Tomography). Furthermore, Patent document 2 discloses a technology based on the technology of Patent document 1 that speeds up the scanning of the light spot in a manner that is not affected by the movement of particles performing Brownian motion in the liquid.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Publication No. 2017-102032

[0008] Patent Document 2: WO2020 / 144754 Summary of the invention

[0009] Problems to be solved by the invention

[0010] The technology described in patent documents 1 and 2 uses the interference signal generated by the interference of the signal light reflected from the particles in the liquid sample and the reference light to measure the particle size. In actual measurement, the wavelength shift of the semiconductor laser and the change of the laser power are generated due to the change of ambient temperature, the change over time, the dust attached to the objective lens, etc., so that the detection signal sometimes changes. In order to suppress this situation, the sensitivity correction is implemented using standard beads with known particle size.

[0011] However, this calibration procedure requires the preparation of standard beads, etc. In addition, due to the difference between the light reflectance from the standard beads and the light reflectance from the particles, it is necessary to change the laser power between the laser irradiation of the standard beads and the laser irradiation of the particles, and as a result, the sensitivity coefficient may be different between the two.

[0012] The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a technology capable of coping with sensitivity that varies due to various reasons when measuring the size of particles contained in a liquid sample using light.

[0013] Means for solving problems

[0014] The particle counting device of the present invention uses a reference sample to calibrate the size of particles contained in the sample. The reference sample has a reference sample window having the same material and thickness as the sample container window, and has a substance having the same reflectivity as the sample. The reflectivity of the substance measured at two different time points is used to calibrate the sample size.

[0015] Effects of the Invention

[0016] According to the particle counting method of the present invention, when measuring the size of particles contained in a liquid sample using light, it is possible to cope with sensitivity that changes due to various reasons. Other problems, structures, and effects than those described above will become clear from the following description of the embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram for explaining the relationship between the particle size and the amount of reflected light in Patent Documents 1 and 2.

[0018] Figure 2 This is a configuration diagram of the particle counting device according to the first embodiment.

[0019] Figure 3A It is a diagram for explaining a frame and a metal plate for configuring a reflectance reference sample.

[0020] Figure 3B It is a diagram for explaining a frame and a metal plate for configuring a reflectance reference sample.

[0021] Figure 4 3 is a side cross-sectional view illustrating the reflectivity reference sample 303 and the focal position.

[0022] Figure 5A It is a schematic diagram showing the quantitative method of sensitivity correction.

[0023] Figure 5B It is a schematic diagram showing the quantitative method of sensitivity correction.

[0024] Figure 6 This is a diagram showing an example of the measurement flow of A0.

[0025] Figure 7 This is a diagram showing an example of the measurement flow of A1.

[0026] Figure 8 This is a diagram showing another example of the measurement flow of A1.

[0027] Fig. 9 This is a diagram showing an example of a measurement flow in which an actual sample is measured after A1 is measured.

[0028] Fig.10 It is a diagram showing the arrangement of the reflectance reference sample 303 .

[0029] Fig.11A This is a calculation result showing the specification of the thickness deviation of the transparent flat plate 402 for implementing sensitivity correction to achieve high-precision dimension measurement.

[0030] Fig. 11B Representation and Fig.11A Similarly, the relationship between the refractive index of the transparent plate 402 and the Strehl intensity is calculated.

[0031] Fig.12 It is a schematic diagram showing the ID information of the reflectance reference sample 303 .

[0032] Fig.13 This is a diagram showing an example of the measurement flow of playback ID information.

[0033] Fig.14A This is a schematic diagram showing the relationship between a container containing an actual sample including particles to be measured and a solvent and the focal position of a laser beam when measuring the refractive index of the solvent of the actual sample.

[0034] Fig. 14B then Fig.14A , shows a situation where the focal position is moved to a predetermined position in a sample to measure the size of particles contained in the sample.

[0035] Fig.15AThe reflectance of the reflectance reference sample 303 in Embodiment 2 is shown.

[0036] Fig. 15B The reflectance in another structural example of the reflectance reference sample 303 according to the second embodiment is shown.

[0037] Fig.16 Another structural example of the reflectance reference sample 303 is shown. DETAILED DESCRIPTION

[0038] <Issues of the Prior Art>

[0039] Figure 1 This is a schematic diagram illustrating the relationship between the particle size and the amount of reflected light in Patent Documents 1 and 2. The techniques of Patent Documents 1 and 2 use the interference between the reflected signal light from the particles and the reference light to measure the size of the particles dispersed in a solvent. The detected reflected light varies with the size of the particles according to their refractive index. In the techniques described in Patent Documents 1 and 2, the electric field amplitude of the reflected light can be converted into a voltage to obtain a detection signal by using the interference of light. At this time, the refractive index n of the particles is used. p , the refractive index of the solvent n m According to the Fresnel law, when the coherence length of the light source is long enough, the size of the detection signal E sig It can be expressed by the following formula: 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 size of the particle as shown above, and η is a constant representing the interference efficiency of the reflected light and the reference light and the efficiency of the high electrical conversion of the photodetector.

[0040] [Formula 1]

[0041]

[0042] The particle size can be measured by measuring the detection signal shown in formula 1. In actual measurement, the wavelength shift of the semiconductor laser and the change of the laser power are generated due to changes in ambient temperature, changes over time, or dust attached to the objective lens, so the detection signal sometimes changes. In order to suppress this situation, sensitivity correction is implemented in the following order.

[0043] (1) Prepare a standard sample by diluting polystyrene standard beads of known particle size with pure water.

[0044] (2) Measure the standard sample immediately before measuring the sample you want to actually measure.

[0045] (3) Based on the measurement results of the standard sample, calculate the sensitivity correction factor that eliminates the deviation between the measured dimension and the true dimension, and manually input the value.

[0046] The above-mentioned calibration sequence requires complicated work and a lot of time for the preparation of standard beads, accurate dilution, measurement, etc., and there are problems from the perspective of user convenience. In addition, depending on the batch of standard beads, there are sometimes deviations in size. In addition, beads are sometimes damaged during bead dilution and stirring, so there is a problem that accurate measurement cannot be performed sometimes. Furthermore, the standard beads formed by polystyrene have a large difference in refractive index with pure water as a solvent, so the reflectivity is large, and it is necessary to set the measuring laser power to a relatively low level. On the contrary, for example, in the case of measuring protein particles, the refractive index difference between the protein particles and the solvent is small, so the reflectivity is small, and the measuring laser power must be set to a relatively high level. Due to the difference in laser power between the two, the difference in laser temperature occurs, and the laser wavelength may be different in the two. Due to the deviation in this wavelength, the sensitivity coefficient may be different in the two, which becomes a problem when performing accurate sensitivity correction.

[0047] <Basic Principle of the Invention>

[0048] (Sensitivity quantification)

[0049] To solve the above problems, the present invention performs sensitivity calibration using a reflectance reference sample having a plane having a reflection signal equivalent to the signal light obtained from the particle to be measured. Specifically, the particle is measured by the following formula in which the sensitivity coefficient K is introduced into Formula 1.

[0050] [Formula 2]

[0051]

[0052] The sensitivity coefficient K is defined by the following formula: A0 is the reflectance of the reflectance reference sample measured when the particle counting device of the present invention is shipped. A1 is the reflectance of the reflectance reference sample measured immediately before measuring a sample to be actually measured.

[0053] [Formula 3]

[0054]

[0055] If the reflectance reference sample is measured for each particle measurement or for each predetermined condition, the sensitivity can be corrected using the sensitivity coefficient K in a simpler method.

[0056] (Benchmark sample)

[0057] The reflectivity reference sample has the following structure: a glass window having the same material and thickness as the glass window of the light incident surface of the sample container for measuring particles, and a material having a reflectivity substantially equal to the reflectivity of the measured sample at the boundary surface in contact with the glass window. Thus, the measuring conditions of the reflectivity reference sample and the measured sample can be made the same. In particular, by making the laser power the same when measuring the sample and the reference sample, the interference efficiency of the reflected light and the reference light can be made the same, so that more accurate sensitivity correction can be performed.

[0058] As an example of a reflectivity reference sample, a resin having a refractive index of 1.47±0.03 is formed by contacting a transparent plate. As a result, the refractive index difference between the transparent plate and the resin becomes the same as the refractive index difference between the protein particles and the solvent, and the reflectivity of the reflectivity reference sample is roughly equal to the reflectivity of the protein particles in the solvent, so it can be used as a reflectivity reference sample for protein particle measurement. The roughly equal here refers to the degree to which the error of the measurement result converges within the allowable range. In other words, from the perspective of measurement error, it is necessary to make the difference between the reflectivity of the substance in contact with the transparent plate (here, the resin) and the reflectivity of the sample converge within the allowable range.

[0059] As another example of a reflectivity reference sample, a dielectric film is formed by contacting with a transparent plate as described later. When the measured particle is a metal particle, the reflectivity of the metal particle in the solvent can vary from 0.01% to a value of several % depending on its material, texture, etc. By selecting the material and film thickness of the dielectric film, and the material and film thickness of the metal film formed on the dielectric film, it can be designed to a specified reflectivity, and can cope with various particles.

[0060] <Implementation Method 1>

[0061] Hereinafter, in the description of the embodiments of the present invention, as shown in the drawings, the coordinate system is unified for description, with the optical axis direction being the z-axis. In addition, the size of the particle to be measured is handled as the diameter of a sphere of equal volume.

[0062] Figure 2 This is a block diagram of a particle counting device according to Embodiment 1 of the present invention. The laser light emitted from the light source 100 whose light emission state is controlled by the laser driver 101 that implements high frequency superposition and emission power control is converted into parallel light by the collimating lens 102, and after the polarization direction is adjusted by the λ / 2 plate 103 whose optical axis is set to about 22.5 degrees relative to the horizontal direction, it is separated into signal light and reference light by the polarization beam splitter 104.

[0063] The reference light is converted into a circular polarization state by the λ / 4 plate 105, reflected by the reference light reflector 106, and becomes a polarization state rotated 90 degrees relative to the outgoing path by the λ / 4 plate 105, and is reflected by the polarization beam splitter 104. The signal light is deflected in the direction of travel by the XY-direction composite deflection element 107, and then converted into a circular polarization state by the action of the built-in λ / 4 plate, and is focused in the sample 204 by the objective lens 108.

[0064] The driving mechanism 109 that moves the sample in the Z-axis direction has the function of scanning the focal position of the signal light along the Z-axis direction (optical axis direction). The component of the signal light reflected from the sample 204 is deflected in the same direction as the outgoing path by the XY-direction composite deflection element 107, and becomes a polarization state rotated 90 degrees relative to the outgoing path by the action of the built-in λ / 4 plate, and passes through the polarization beam splitter 104.

[0065] The sample container 200 holds a sample 204 in a hole and guides signal light into the sample via a transparent window 202. Reference numeral 203 denotes a resin member forming the hole of the sample container. A metal plate 201 contacts the transparent window 202, mechanically holds the sample container 200, and is responsible for stabilizing the temperature of the sample.

[0066] The signal light and the reference light are combined by the polarization beam splitter 104 , guided to the detection optical system 112 , and branched into transmitted light and reflected light by the half beam splitter 113 via the pinhole 111 .

[0067] After passing through the λ / 4 plate 114 whose optical axis is set at approximately 45 degrees relative to the horizontal direction, the reflected light is focused by the focusing lens 115 and branched into two parts by the polarization beam splitter 116. The reflected light is photoelectrically converted by the photodetectors 150 and 151 respectively, and differentially amplified by the current differential amplifier 152 to become the detection signal 123.

[0068] After passing through the λ / 2 plate 118 whose optical axis is set at approximately 22.5 degrees relative to the horizontal direction, the transmitted light is focused by the focusing lens 119 and branched into two parts by the polarization beam splitter 120, and is photoelectrically converted by the photodetectors 153 and 154 respectively, and differentially amplified by the current differential amplifier 155 to become the detection signal 122.

[0069] The detection optical system 112 forms a homodyne phase diversity method, and the detection signals 122 and 123 are processed by a signal processing unit 124. The signal processing unit 124 (processing unit) performs sensitivity correction based on the information stored in the information storage unit 125, and displays the calculation result including the correction on the display unit 126, and presents the calculation result to the user. The information stored in the information storage unit 125 and the sensitivity correction based on the information will be described later.

[0070] (Measurement of reflectivity of boundary surface)

[0071] Figure 3A~Figure 3B 303 is a diagram for explaining a frame and a metal plate for arranging a reflectance reference sample. In the figure, a reflectance reference sample 303 is inserted into a frame 302 and arranged in a manner of contacting the surface of a metal plate 201. In this case, the reflectance reference sample 303 may also be arranged on the frame 302 together with other sample containers 304. When the reflectance reference sample 303 is arranged on the frame 302 together with other sample containers 304, both can be measured together in a series of processes.

[0072] Figure 4 2 is a side cross-sectional view illustrating a reflectivity reference sample 303 and a focal position. The reflectivity reference sample 303 is composed of a transparent plate 402, a container 403, and a substance 404 in contact with the transparent plate 402, and is arranged in contact with the metal plate 201. The light 400 focused by the object lens 108 passes through a window 405 for measurement and irradiates the boundary surface between the transparent plate 402 and the substance 404. By adjusting the relative position between the boundary surface and the object lens 108, the focal position of the object lens 108 is made consistent with the boundary surface, and the reflectivity from the boundary surface is obtained. Furthermore, the position of the reference light reflector 106 is adjusted, and the optical path length is adjusted so that the reflectivity from the boundary surface becomes the maximum.

[0073] (Sensitivity quantification)

[0074] Figure 5A~Figure 5B : is a schematic diagram showing a quantitative method of sensitivity correction. First, the reflectance (A0) of the boundary surface between the transparent plate 402 of the reflectance reference sample 303 and the substance 404 in contact with the transparent plate 402 is measured by the above method when the device is shipped, and the information is stored in the information storage unit 125. Next, when measuring the actual sample, the reflectance (A1) of the boundary surface between the transparent plate 402 of the reflectance reference sample 303 and the substance 404 in contact with the transparent plate 402 is similarly measured, and the information is stored in the information storage unit 125. The sensitivity coefficient K can be calculated from the values ​​of A0 and A1, and the detection signal can be corrected by Formula 2.

[0075] FIG5 shows the case where A0>A1. The main causes of this are the wavelength shift of the semiconductor laser caused by the change in ambient temperature and the change over time, the change in laser power, the dust attached to the optical element including the objective lens, the change over time of the optical element itself, etc., which lead to the change in optical characteristics and the reduction of output signals. By using the quantitative method of sensitivity correction described above, the influence of these factors on the measurement results can be greatly reduced.

[0076] (Measurement process)

[0077] Figure 6 1 is a diagram showing an example of the measurement process of A0. This flowchart can be implemented by the signal processing unit 124. The same applies to the following flowcharts. The focusing position of the signal light is moved to the plane position of the reflectivity reference sample 303, and the light source 100 is made to emit light at the standard power P0. The Z-axis direction position Z0 of the boundary surface and the position R of the reference light reflector 106 are initialized respectively. The signal is measured while scanning Z0 around the transparent window 405, thereby determining Z0. By measuring the signal while scanning R at the determined Z0, the reflector position R0 at which the optical path lengths of the signal light and the reference light are consistent with each other is determined. The signal level obtained at this time point is stored in the information holding unit 125 as A0.

[0078] A0 and A1 are measured using the same light emission power, so the standard power is used here. The standard power is used so that the signal level of the measurement result is not saturated. Under the condition of obtaining this unsaturated signal level, the reflectivity of the sample needs to be roughly the same as the reflectivity of the reflectivity reference sample 303 (from the perspective of measurement accuracy, the difference between the two is within the allowable range).

[0079] Figure 7 is a diagram showing an example of the measurement process of A1. Figure 6 The signal processing unit 124 performs the measurement in the same manner. Figure 6 and Figure 7 The obtained A0 and A1 are used to calculate the sensitivity coefficient K.

[0080] Figure 8 FIG. 1 is another diagram showing another example of the measurement process of A1. Figure 7 Based on the process of FIG. 1 , a predetermined value is set for the sensitivity coefficient K, and when the sensitivity coefficient K calculated based on A0 and A1 is outside the predetermined value range, sensitivity abnormality processing can be performed. For example, the signal processing unit 124 can output a signal or message indicating that the sensitivity coefficient K is outside the predetermined range to notify the user of this.

[0081] Fig. 9 1 is a diagram showing an example of a measurement process of measuring an actual sample after measuring A1. When measuring an actual sample, Z0 and R0 can be determined in the same manner as the reflectance reference sample. The focus position is moved from Z0 to a predetermined measurement position Z, and the detection signal is processed while scanning the focus according to predetermined scanning conditions. The measurement result of the actual sample is corrected based on the sensitivity coefficient K. Through the above processing, the particle size and density distribution of the actual sample are calculated, and the results are displayed on the display unit 126.

[0082] (Configuration of reflectivity reference sample)

[0083] Fig.103 shows an example in which the reflectance reference sample 303 is arranged in the frame 302 together with other sample containers 304, but it can also be arranged in other positions. For example, a portion of the sample stage 1001 that holds the metal plate 201 and is larger than the metal plate 201 can be arranged with a reflectance reference sample 303. Figure 4 The reflectance reference sample 303 of the structure shown in the figure. At this time, the reflectance reference sample 303 can be arranged outside the frame 302 in a manner of contacting a metal plate 1002 different from the metal plate 201. The different metal plate 1002 can use a metal plate different from the metal plate 201, or a metal plate integrated with the metal plate 201.

[0084] If the reflectivity reference sample 303 is arranged outside the frame 302, the samples to be measured can be arranged at all positions in the frame 302, so the number of samples that can be measured can be maximized. In addition, through this arrangement, it can be configured so that the user cannot remove the reflectivity reference sample 303 arranged outside the frame 302. Thus, human errors such as forgetting to arrange the reflectivity reference sample 303 and mistaking the arrangement position can be avoided. For example, it can be considered to place the reflectivity reference sample 303 in a place that the user cannot see. The reflectivity reference sample 303 can be configured to be detachable or fixedly installed.

[0085] (Thickness of transparent plate)

[0086] Fig.11A The calculation result of the specification of the thickness deviation of the transparent plate 402 used for implementing sensitivity correction and realizing high-precision size measurement is shown. In order to measure the particle size distribution with high precision, the structure of the reflectivity reference sample 303 also needs to meet the specified specifications, similar to the composition specifications of the sample container. It is well known that when the thickness of the transparent plate 402 deviates from the specified value, spherical aberration is generated optically. The influence on the detection signal can be treated as a decrease in Strehl intensity based on wavefront aberration. The decrease in the detection signal (Strehl intensity) caused by the deviation of the thickness of the transparent plate 402 is calculated. Here, the results are shown when a semiconductor laser with a wavelength of 785nm is used as the light source 100, a microscope lens with a numerical aperture of 0.45 is used as the objective lens 108, and a borosilicate glass with a thickness of 175μm (the measured refractive index value = 1.520) is used as the transparent plate 402. As shown in the figure, it can be seen that in order to make the decrease in the detection signal less than 0.2%, the deviation of the thickness of the transparent plate 402 is required to be less than 70μm.

[0087] Fig. 11B and Fig.11ASimilarly, the result of calculating the relationship between the refractive index and the Strehl intensity of the transparent plate 402 is shown. As shown in the figure, the permissible range of the refractive index of the transparent plate 402 is ±0.22.

[0088] As an example of the present invention, if Figure 4 and the following Fig.12 and Fig.16 If the reflectivity reference sample 303 shown in FIG. 3 is not optically interchangeable with the sample container 304 shown in FIG. 3 , accurate sensitivity cannot be obtained. Specifically, the thickness of the transparent plate 402 formed on the bottom must be 175±70 μm, and the refractive index must be 1.520±0.22. As a material of the transparent plate 402 that can meet these conditions, an optical quality borosilicate glass substrate can be used.

[0089] (ID information)

[0090] Fig.12 Schematic diagram showing the ID information of the reflectivity reference sample 303. The reflectivity reference sample 303 may have ID information. Specifically, the information corresponding to the reflectivity of the reflectivity reference sample 303 is recorded in a manner that can be optically recognized, such as a bar code, on the surface of the transparent flat plate 402 on the side in contact with the metal plate 201 and on the inner side 1202 of the transparent window 405 at a position closer to the outside than the container opening 1203. The information corresponding to the reflectivity may be the reflectivity value itself or the level of the value (high, medium, low, etc.). In addition, it may be a mark, information, etc. associated with the value or level of the reflectivity. The recording method may be performed by known methods such as printing or engraving.

[0091] By aligning the focus of the laser light within the surface of the transparent flat plate 402 on the side in contact with the metal plate 201 and scanning in the xy direction of the figure, it is possible to reproduce the ID information 1201. By reproducing the ID information before measuring the reflectivity reference sample 303, it is known that the sample is reliably the reflectivity reference sample, thereby avoiding human errors such as mistaking the reference sample.

[0092] As the ID information, other information corresponding to the reflectivity may be recorded, for example, predetermined specific identification information, the serial number of the reference sample, etc. Thus, the quality of the reference sample can be ensured.

[0093] The surface of the transparent plate 402 on which the ID is recorded may not be the surface on the side in contact with the metal plate 201. The ID information may be formed in advance on the transparent plate 402, and the surface on which the ID information is formed may be bonded as the container side, thereby manufacturing the reflectance reference sample 303. In this case, the ID information is also formed on the inner side of the transparent window 405 and the outer side of the container opening 1203.

[0094] Fig.13 1 is a diagram showing 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 when measuring A1 is shown. For example, ID information can be reproduced before scanning the boundary surface position Z0 and the reflector position R.

[0095] The ID information can be used to check in advance whether the correct reflectivity reference sample 303 is used. Since the light emission power is predetermined for each sample type, when the light emission power is determined, the sample type corresponding to it is also determined. Therefore, it is preferred to perform the step of reproducing the ID information after determining the light emission power and before measuring the reflectivity.

[0096] (Mechanical error, etc.)

[0097] By using the reflectivity reference sample 303, even if there are mechanical errors in the particle counting device (such as slight differences in laser wavelength, optical system, and detector sensitivity), it is possible to obtain substantially the same results for the same sample. Furthermore, it can also be used for fault diagnosis of the device. Specifically, when the sensitivity coefficient K deviates from a certain numerical range, the signal processing unit 124 can, for example, issue an alarm on the display unit 126 to prompt maintenance and repair of the device, taking into account the degradation caused by the time-dependent change of the laser, the adhesion of dust to the optical element, the deviation of the optical system, etc.

[0098] (Solvent refractive index measurement)

[0099] Fig.14A Schematic diagram showing the relationship between a container containing an actual sample of particles and a solvent to be measured and the focal position of a laser when measuring the refractive index of the solvent of the actual sample. The reflectivity reference sample 303 can also be used to measure the refractive index of the solvent of the actual sample. In the figure, 1401 is a sample and 1402 is a sealing tape. In order to measure the refractive index of the solvent, the present invention utilizes the fact that the reflectivity of the boundary between the transparent plate 402 and the sample depends on the refractive index of the solvent. By moving the sample stage (not shown) in the Z direction while determining the condition for the detection signal to be the maximum, the focus of the objective lens 108 can be positioned at the boundary between the sample 1401 and the transparent plate 402. At this time, according to the Fresnel formula, the size of the detection signal can be expressed by Formula 1.

[0100] In the present invention, the material of the transparent plate 402 can be selected so that its refractive index can be used as a predetermined value. Similarly, the electric field amplitude S of the light irradiating the sample and the electric field amplitude R of the reference light can be treated as constant values ​​by making the emission power condition of the semiconductor laser (not shown) constant.

[0101] Furthermore, as a material in contact with the interface of the reflectance reference sample 303, the refractive index n is measured in advance. o The size of the detection signal when the substance is used as a sample is set as E0 sig . E0 sig It is expressed by Formula 4. Formula 5 is derived from Formula 1 and Formula 4. In Formula 5, the refractive index n of the solvent is m The rest are known values ​​or measured values, so the refractive index n of the solvent can be measured using Formula 5. m .

[0102] [Formula 4]

[0103]

[0104] [Formula 5]

[0105]

[0106] Fig. 14B Shown next Fig.14A Then, the focal position is moved to a predetermined position in the sample to measure the size of the particles contained in the sample.

[0107] <Implementation Method 2>

[0108] (Refractive index: dielectric film)

[0109] Fig.15A : represents the reflectance of the reflectance reference sample 303 in the second embodiment of the present invention. Figure 4 A dielectric film is used as a material in contact with the transparent flat plate 402 of the reflectivity reference sample 303. ZnSSiO2 is used as the dielectric film and is formed by sputtering on the transparent flat plate 402 made of borosilicate glass having a refractive index of 1.52. The reflectivity of the reflectivity reference sample formed with the dielectric film varies according to the film thickness of the dielectric film due to interference between reflection from the surface of the dielectric film on the laser incident side and reflection from the surface on the deep side thereof. Fig.15A The relationship between the film thickness and reflectivity of ZnSSiO2 used in the second embodiment is shown.

[0110] Fig. 15B 1 shows the reflectivity of another structural example of the reflectivity reference sample 303 of Embodiment 2. In this structural example, Al 100 nm and Ag 20 nm are further formed as metal films by sputtering on the dielectric film ZnSSiO2 formed on the transparent flat plate 402. The reflectivity of the reflectivity reference sample 303 with this structure is shown in FIG. Fig. 15B Change like that.

[0111] As described above, the reflectivity reference sample 303 is any one of (a) a dielectric film alone or a multilayer structure of two or more dielectric films, and (b) a structure in which a dielectric film and a metal film are stacked in sequence. By appropriately selecting the thickness of each film, the reflectivity of the reflectivity reference sample 303 can be set to a specified value. Fig. 15B The vicinity of C and D shown in means that there is a wide margin for the variation in film thickness. If this point is used when designing a film corresponding to a predetermined reflectance value, the margin for the film formation rate and the like becomes wide, which is preferable.

[0112] When the particle to be measured is larger than the spot size or is a metal particle, the reflectivity becomes higher due to the material, etc. In this case, the material and thickness of the dielectric film and the material and thickness of the metal film can be selected to achieve a structure corresponding to the reflectivity of the particle to be measured.

[0113] <Implementation Method 3>

[0114] In the third embodiment of the present invention, a specific example of the reflectance reference sample 303 will be described. The structure and measurement procedure of the particle counting device are the same as those of the first and second embodiments.

[0115] (Reflectivity reference sample 1)

[0116] As Figure 4 The material in contact with the transparent plate 402 of the reflectivity reference sample 303 is a UV curable resin (DVD310) manufactured by Nippon Kayaku Co., Ltd. If bubbles are mixed into the boundary surface, the reflectivity signal will be affected. Therefore, in order to avoid this, the UV curable resin is placed in a vacuum container and degassed by vacuum exhaust for 10 minutes. Then, the resin is injected into the container 403 and irradiated with about 300 mJ / cm using an ultraviolet irradiation device (ECS-201G1) manufactured by Eye Graphics. 2 The ultraviolet rays cure the resin.

[0117] (Refractive index: for protein)

[0118] The reflectivity from the boundary surface is proportional to the difference in refractive index between the two substances. For example, in the case of protein particles as the sample for measuring the size distribution, the difference in refractive index between the solvent and the protein particles is about 0.06. In the reflectivity reference sample using the above-mentioned ultraviolet curing resin, the refractive index of the ultraviolet curing resin DVD310 after curing is 1.50, and in the case of using borosilicate glass with a refractive index of 1.52 as the transparent plate, the difference in refractive index between the two is 0.02, so the reflectivity from the protein particles and the interface reflectivity are also at the same level.

[0119] Thus, the reflectivity reference sample 303 can be measured under the same laser power conditions as those for measuring the size distribution of protein particles, and the sensitivity coefficient K under these conditions can be obtained. Differences in laser power conditions may cause differences in sensitivity coefficients, but according to the method of the present invention, this possibility can be eliminated and a more accurate sensitivity correction can be performed, so that the particle size can be accurately measured.

[0120] As the reflectance reference sample 303, a sample which has little optical change over time and can be used for a long period of time is preferred.

[0121] As the reflectivity reference sample 303 in contact with the transparent plate 402, a sample having a different refractive index was prepared by using another ultraviolet curing resin instead of the ultraviolet curing resin (DVD310) manufactured by Nippon Kayaku Co., Ltd. The measurement for obtaining the sensitivity coefficient K was performed under the following laser power conditions.

[0122] n: Laser power condition

[0123] 1.41: Lower power than protein particle sample measurement

[0124] 1.43: Roughly equivalent to the power measured for protein particle samples

[0125] 1.48: Roughly equivalent to the power measured for protein particle samples

[0126] 1.50: Roughly equivalent to the power used for protein particle sample measurement

[0127] 1.52: Higher power than protein particle sample measurement

[0128] Based on these results, the refractive index of the substance 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.

[0129] Fig.16 Another structural example of the reflectivity reference sample 303 is shown. In the above embodiment, as the material in contact with the transparent flat plate 402, a UV curable resin (DVD310) manufactured by Nippon Kayaku Co., Ltd. is used, but a liquid resin (silicone oil for microscopes, etc.) may be used instead. Fig.16 In the figure, 1601 is the liquid resin. In this case, sealing is performed with a sealing tape or the like so as not to leak the liquid.

[0130] (How to use the reflectance reference sample)

[0131] When a plurality of actual samples are measured, the reflectance reference sample 303 may be measured only once at the start of the measurement, or the reflectance reference sample 303 may be measured for each of the plurality of actual sample measurements.

[0132] When the measurement time required for measuring the actual sample is relatively short, the ambient temperature and laser temperature changes during the actual sample measurement are relatively small. Therefore, it is sufficient to measure the reflectance reference sample once at the beginning of the measurement and perform sensitivity correction, thereby shortening the overall measurement time.

[0133] On the other hand, when the measurement time required for the actual sample measurement is relatively long, the ambient temperature and the temperature of the laser may change during the measurement. Therefore, for each measurement of a plurality of actual samples, the reflectivity reference sample is measured and the sensitivity correction is re-performed each time, so that more accurate measurement can be performed. In this case, instead of each measurement, the reflectivity reference sample may be measured for a certain number of actual samples and the sensitivity correction may be re-performed each time.

[0134] The length of the measurement time of the actual sample can be determined by the measurer. In this case, the measurement time when similar samples were measured in the past can also be referred to. However, in the case of measuring multiple unknown samples, it is preferred to perform sensitivity correction again each time.

[0135] <Variations of the present invention>

[0136] The present invention is not limited to the aforementioned embodiments, and includes various variations. For example, the aforementioned embodiments are described in detail in order to explain the present invention in an easy-to-understand manner, and are not limited to all the structures described. In addition, a part of the structure of a certain embodiment can be replaced with the structure of other embodiments, and in addition, the structure of other embodiments can be added to the structure of a certain embodiment. In addition, for a part of the structure of each embodiment, other structures can be added, deleted, or replaced.

[0137] In the above embodiment, the reflectance reference sample 303 may be configured as a component of the particle counting device, or may be configured as another component that can be attached to and detached from the particle counting device, for example.

[0138] In the above embodiment, it is mentioned that the objective lens 108 and the drive mechanism 109 function as an irradiation unit that focuses the signal light and irradiates the sample, and the detection optical system 112 functions as a detection unit that detects an interference signal generated by interference between the signal light and the interference light.

[0139] Description of reference numerals:

[0140] 100: Light source

[0141] 101: Laser Driver

[0142] 108: Objective lens

[0143] 109: Driving mechanism

[0144] 112: Detection optical system

[0145] 124: Signal Processing Department

[0146] 200: Sample container

[0147] 201: Metal Plate

[0148] 202: Transparent Window

[0149] 303: Reflectivity reference sample

[0150] 402: Transparent tablet

[0151] 404: Matter

[0152] 1201: ID information.

Claims

1. A particle counting device for measuring the size of particles contained in a liquid sample, characterized in that: The particle counting device comprises: a light source, which emits light; a branching unit that branches the light from the light source into a signal light and a reference light; an irradiation unit that focuses the signal light and irradiates the sample; a detection unit that detects an interference signal obtained by interfering the reflected light from the particle with the reference light; and a processing unit that measures the size of the sample using the interference signal detected by the detection unit and corrects the size using the measurement result of a reference sample, The reference sample has a reference sample window having the same material and thickness as a sample container window of a sample container containing the sample. The reference sample has a substance at a position in contact with the reference sample window, the substance having a reflectivity whose difference with the reflectivity of the sample is within an allowable range, the processing unit acquires a result of measuring the reflectance of the substance at a first time point as a first reflectance, the processing unit acquires, as a second reflectance, a result of measuring the reflectance of the substance at a second time point closer to the time point when the sample is measured than the first time point, The processing section corrects a size of the sample using the first reflectivity and the second reflectivity.

2. The particle counting device according to claim 1, characterized in that The irradiation unit irradiates the light so that a focal position of the light becomes an interface between the reference sample window and the substance. The processing unit obtains the first reflectivity and the second reflectivity using the interference signal obtained from the boundary surface between the reference sample window and the substance.

3. The particle counting device according to claim 1, characterized in that The processing unit calculates a ratio of the second reflectivity to the first reflectivity as a sensitivity coefficient, The processing unit corrects the size of the sample by multiplying the size of the sample measured using the interference signal meter by the sensitivity coefficient.

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

5. The particle counting device according to claim 1, wherein: The light source emits the light having the same emission power both when measuring the size of the sample and when measuring the reflectivity of the substance. The processing unit obtains the size of the sample, the first reflectivity, and the second reflectivity using the interference signal obtained from the light having the same emission power.

6. The particle counting device according to claim 1, wherein: The particle counting device further includes a plate on which the sample container is placed and a frame covering the sample container placed on the plate. The reference sample is configured to be fixable relative to the plate at a position outside the frame on the plate.

7. The particle counting device according to claim 2, characterized in that: The irradiation unit irradiates the light so that a focal position of the light becomes a boundary surface between the sample container window and the liquid. The processing unit obtains the magnitude of the interference signal obtained from the boundary surface between the sample container window and the liquid as a sample interference signal value, The processing unit obtains the magnitude of the interference signal obtained from the boundary surface between the reference sample window and the substance as a reference sample interference signal value. The processing unit calculates a refractive index of the liquid using the sample interference signal value and the reference sample interference signal value.

8. The particle counting device according to claim 1, wherein: The substance is an ultraviolet curing resin.

9. The particle counting device according to claim 1, wherein: The substance is at least one of the following structures: A dielectric film or a multilayer structure of two or more dielectric films; and A structure in which a metal film is stacked on a dielectric film.

10. The particle counting device according to claim 1, wherein: The particle counting device includes the reference sample.

11. The particle counting device according to claim 10, wherein: The refractive index of the substance is 1.47±0.

03.

12. A particle measuring method for measuring the size of particles contained in a liquid sample, characterized in that: The particle counting method comprises: Steps of emitting light from a light source; a step of branching the light from the light source into a signal light and a reference light; A step of focusing the signal light and irradiating 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; and The step of correcting said dimensions using a reference sample, The reference sample has a reference sample window having the same material and thickness as a sample container window of a sample container containing the sample. The reference sample has a substance at a position in contact with the reference sample window, the substance having a reflectivity whose difference with the reflectivity of the sample is within an allowable range, In the calibration step, a result of measuring the reflectance of the substance at a first time point is obtained as a first reflectance. In the calibration step, a result of measuring the reflectance of the substance at a second time point closer to the time point when the sample is measured than the first time point is obtained as a second reflectance, In the correcting step, the size of the sample is corrected using the first reflectivity and the second reflectivity.

13. A reference sample used for correcting the measurement result of the size in a particle counting device that measures the size of particles contained in a liquid sample by irradiating the sample with light, characterized in that: The reference sample is configured to be attachable to and detachable from a sample holder, the sample holder being formed by a bottom plate provided in the particle counting device and holding a sample container containing the sample. The reference sample has a reference sample window having the same material and thickness as the sample container window of the sample container. The reference sample has a substance at a position in contact with the reference sample window, the substance having a reflectivity whose difference with the reflectivity of the sample is within an allowable range, The reference sample window is formed on a transparent plate disposed 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.

14. The reference sample according to claim 13, characterized in that: A shape pattern representing ID information for identifying the reference sample is formed on any surface of the transparent flat plate.

15. The reference sample according to claim 13, characterized in that: The refractive index of the substance is 1.47±0.03.

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