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

Abstract

To provide a technique capable of easily identifying individuals of standard sample used for calibrating the results of light-based particle size measurements in a sample.SOLUTION: A standard sample includes a transparent plate with a window. The transparent plate includes an interface with a reflectance approximately equal to that of the sample. On either side of the transparent plate, a shape pattern that represents ID information for identifying the standard sample is formed.SELECTED DRAWING: Figure 12

Description

[Technical field]

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

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

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

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

[0005] [Patent Document 1] JP 2017-102032 A [Patent Document 2] WO2020 / 144754 publication Summary of the Invention [Problem to be solved by the invention]

[0006] In the conventional techniques such as Patent Documents 1 and 2, the interference between the signal light and the reference light is used to measure the size of the protein aggregate. The interference efficiency changes due to the temperature of the laser, changes over time, dust on the objective lens, and the like. In order to always achieve accurate measurements in the user environment, Patent Document 2 uses a reference sample to calibrate the sensitivity of the interference efficiency. In order to expand the measurement target to other than protein aggregates, various reference samples will be prepared according to the optical properties of the measurement target. Therefore, it is necessary to avoid mixing up the reference samples. In the conventional techniques, such mixing up of the reference samples is not sufficiently considered.

[0007] The present invention has been made in consideration of the above-mentioned problems, and aims to provide a technology that can easily identify individual reference samples used to calibrate the results of measuring the size of particles in a sample using light. [Means for solving the problem]

[0008] The reference sample of the present invention comprises a transparent flat plate having a window, the transparent flat plate having an interface surface having a reflectance approximately equal to the reflectance of a sample, and a shape pattern representing ID information for identifying the reference sample is formed on one of the surfaces of the transparent flat plate. Effect of the Invention

[0009] According to the reference sample of the present invention, it is possible to easily identify individual reference samples used for calibrating the results of measuring the size of particles in a sample using light. Problems, configurations, and effects other than those described above will become apparent from the description of the following embodiments. [Brief description of the drawings]

[0010] [Figure 1] FIG. 2 is a schematic diagram for explaining the relationship between particle size and the amount of reflected light in Patent Documents 1 and 2. [Diagram 2] FIG. 1 is a configuration diagram of a particle measuring device according to a first embodiment. [Figure 3A] FIG. 2 is a diagram illustrating a frame and a metal plate on which a reflectance reference sample is placed. [Figure 3B] FIG. 2 is a diagram illustrating a frame and a metal plate on which a reflectance reference sample is placed. [Figure 4] FIG. 11 is a side cross-sectional view illustrating a reflectance reference sample 303 and a focal position. [Figure 5A] FIG. 13 is a schematic diagram showing a method for quantifying sensitivity correction. [Figure 5B] FIG. 13 is a schematic diagram showing a method for quantifying sensitivity correction. [Figure 6] FIG. 13 is a diagram showing an example of a measurement flow of A0. [Figure 7] FIG. 13 is a diagram showing an example of a measurement flow of A1. [Figure 8] FIG. 13 is a diagram showing another example of the measurement flow of A1. [Figure 9] FIG. 13 is a diagram showing an example of a measurement flow in which A1 is measured and then an actual sample is measured. [Figure 10] FIG. 3 is a diagram showing the arrangement of a reflectance reference sample 303. [Figure 11A] 13 is a calculation result showing the specification of the thickness variation of the transparent plate 402 for implementing sensitivity calibration to realize highly accurate size measurement. [Figure 11B] Similar to FIG. 11A, the results of calculation of the relationship between the refractive index of the transparent plate 402 and the Strehl intensity are shown. [Figure 12] 13 is a schematic diagram showing ID information of a reflectance reference sample 303. FIG. [Figure 13] 12 illustrates an example of the data configuration of ID information 1201. [Figure 14A] This is an example of an ID information pattern. [Figure 14B] FIG. 14B is an enlarged view of the ID information pattern in FIG. 14A. [Figure 15A] This is another example of an ID information pattern. [Figure 15B] FIG. 15B is an enlarged view of the ID information pattern in FIG. 15A. [Figure 16A] This is another example of an ID information pattern. [Figure 16B] This is another example of an ID information pattern. [Figure 17A] 1 is a schematic diagram showing an example of the shape of a reflectance reference sample 303 in the xy plane. [Figure 17B] 13 is a schematic diagram showing another example of the shape of the reflectance reference sample 303 in the xy plane. [Figure 17C] 13 is a schematic diagram showing another example of the shape of the reflectance reference sample 303 in the xy plane. [Figure 18] The structure and reflectance of the highly reflective portion of the ID information mark in ID-A are shown. [Figure 19A] 19A to 19C show the specific structures and reflectances of the high reflectance portion and the low reflectance portion of the ID information mark of ID-B. [Figure 19B] 19A to 19C show the specific structures and reflectances of the high reflectance portion and the low reflectance portion of the ID information mark of ID-B. [Figure 19C]19A to 19C show the specific structures and reflectances of the high reflectance portion and the low reflectance portion of the ID information mark of ID-B. [Figure 20A] 20A to 20C show the specific structures and reflectances of the high reflectance portion and the low reflectance portion of the ID information mark ID-C. [Figure 20B] 20A to 20C show the specific structures and reflectances of the high reflectance portion and the low reflectance portion of the ID information mark ID-C. [Figure 20C] 20A to 20C show the specific structures and reflectances of the high reflectance portion and the low reflectance portion of the ID information mark ID-C. [Figure 21] 13 is a diagram showing an example of a measurement flow of A0 linked to ID information when the ID information is ID-A, ID-B, or a mixed pattern of these in FIG. 12. [Figure 22] 13 is a diagram showing an example of a measurement flow of A0 linked to ID information when the ID information is ID-C in FIG. 12. [Figure 23] FIG. 13 is a diagram showing an example of a flow for measuring A1 after reproducing ID information when the ID information is ID-A, ID-B or a mixed pattern of these in FIG. [Figure 24] FIG. 13 is a diagram showing an example of a flow for measuring A1 after reproducing ID information when the ID information is ID-C in FIG. [Diagram 25] FIG. 13 is a diagram showing an example of a measurement flow in which, when the ID information is ID-A, ID-B, or a mixed pattern of these in FIG. 12, after reproducing the ID information, A1 is measured and then the actual sample is measured. [Figure 26] FIG. 13 is a diagram showing an example of a measurement flow in which, when the ID information is ID-C in FIG. 12, A1 is measured after reproducing the ID information, and then the real sample is measured. [Figure 27A] 1 is a schematic diagram showing the relationship between a container containing an actual sample containing particles to be measured and a solvent, and the focal position of laser light when measuring the solvent refractive index of the actual sample. FIG. [Figure 27B]FIG. 27A illustrates how the focal position is moved to a predetermined position within the sample to measure the size of particles contained within the sample. [Figure 28A] 13 shows the reflectance of a reflectance reference sample 303 in the second embodiment. [Figure 28B] 13 shows the reflectance in another configuration example of the reflectance reference sample 303 of the second embodiment. [Figure 29] Another configuration example of the reflectance reference sample 303 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] <Problems with conventional technology> FIG. 1 is a schematic diagram explaining the relationship between particle size and the amount of reflected light in Patent Document 1 and Patent Document 2. The techniques in Patent Document 1 and Patent Document 2 use the interference between reflected signal light from the particles and a reference light to measure the size of particles dispersed in a solvent. The reflected light detected changes according to the particle size and the refractive index. In the techniques described in Patent Document 1 and Patent Document 2, the electric field amplitude of the reflected light can be converted into a voltage by utilizing the interference of light to obtain a detection signal. At this time, the magnitude E of the detection signal sig is the refractive index of the particle, n p , the refractive index of the solvent, n m Using Fresnel's law, if the coherence length of the light source is sufficiently long, it can be expressed by the following equation: where S is the electric field amplitude of the light irradiated to the sample, R is the electric field amplitude of the reference light, σ is a coefficient according to the particle size shown above, and η is a constant indicating the interference efficiency between the reflected light and the reference light and the efficiency of the high-electric conversion of the photodetector.

[0012]

number

[0013] Particle size can be measured by measuring the detection signal expressed by formula 1. In actual measurements, the detection signal may fluctuate due to changes in the environmental temperature, aging, or dust on the objective lens, which can cause the wavelength shift of the semiconductor laser and fluctuations in the laser power. To suppress this, sensitivity calibration is performed using the following procedure.

[0014] (1) Prepare a standard sample by diluting polystyrene standard beads of known particle size with pure water to an appropriate concentration. (2) Measure the standard sample immediately before measuring the sample you want to actually measure. (3) From the measurement results of the standard sample, a sensitivity correction coefficient is calculated so that the measured size does not differ from the true size, and this value is manually entered.

[0015] The above calibration procedure requires a lot of time and labor to prepare the standard beads, dilute them accurately, and measure them, which is problematic in terms of user convenience. There is also a problem that the size of the standard beads may vary depending on the lot, and the beads may be damaged during the bead dilution and mixing, making it difficult to perform accurate measurements. Furthermore, the standard beads made of polystyrene have a large refractive index difference with the pure water solvent, so the reflectance is large and the measurement laser power needs to be set relatively low, whereas when measuring protein particles, for example, the refractive index difference between the protein particles and the solvent is small, so the reflectance is small and the measurement laser power needs to be set relatively high. The difference in laser power between the two may cause a difference in laser temperature, which may result in different laser wavelengths for the two. This wavelength shift may cause the sensitivity coefficients to differ between the two, which is a problem in accurate sensitivity correction.

[0016] <Basic principle of the present invention> (Sensitivity quantification) In order to solve the above problems, the present invention calibrates the sensitivity using a reflectance reference sample with a flat surface that has a reflected signal equivalent to the signal light obtained from the particle to be measured. Specifically, particles are measured using the following equation, which introduces a sensitivity coefficient K into equation 1.

[0017]

number

[0018] 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 when the reflectance reference sample is measured immediately before measuring the sample to be actually measured.

[0019]

number

[0020] If this reflectance reference sample is measured for each particle measurement or for each specified condition, it becomes possible to correct the sensitivity in a simpler manner using the sensitivity coefficient K.

[0021] (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 a sample container for measuring particles, and has a structure in which a substance having a reflectance substantially equal to the reflectance of the measurement sample is formed at the boundary surface in contact with the glass window. This makes it possible to make the measurement conditions of the reflectance reference sample and the measurement sample the same. In particular, by making the laser power during sample measurement and during reference sample measurement the same, the interference efficiency between the reflected light and the reference light can be made the same, enabling more accurate sensitivity correction.

[0022] 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 makes the refractive index difference between the transparent plate and the resin comparable to the refractive index difference between the protein particles and the solvent, and the reflectance of the reflectance reference sample and the reflectance of the protein particles in the solvent are approximately equal, so that the reflectance reference sample can be used as a reflectance reference sample for measuring protein particles. "Approximately equal" here means that the error of the measurement result falls within an allowable range. In other words, it is necessary that the difference between the reflectance of the material (here, the resin) in contact with the transparent plate and the reflectance of the sample falls within an allowable range from the viewpoint of measurement error.

[0023] As another example of a reflectance standard sample, a dielectric film was formed in contact with a transparent flat plate, as described below. 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 of the metal particles. By selecting the material and thickness of the dielectric film, and the material and thickness of the metal film to be formed on the dielectric film, it is possible to design a specified reflectance and to accommodate a variety of particles.

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

[0025] 2 is a configuration diagram of a particle measuring device according to embodiment 1 of the present invention. Laser light emitted from a light source 100, whose light emission state is controlled by a laser driver 101 that performs high-frequency superposition and output power control, is converted into parallel light by a collimating lens 102, and 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. Then, the light is separated by a polarizing beam splitter 104 into a signal light and a reference light.

[0026] The reference light is converted to a circularly polarized state by the λ / 4 plate 105, then reflected by the reference light mirror 106, and its polarization state is rotated by 90 degrees from that of the outward path by the λ / 4 plate 105, and reflected by the polarizing beam splitter 104. The signal light is deflected in its traveling direction by the XY-direction composite deflection element 107, then converted to a circularly polarized state by the action of the built-in λ / 4 plate, and focused within the sample 204 by the objective lens 108.

[0027] A driving mechanism 109 that moves the sample in the Z-axis direction has a function of scanning the focal position of the signal light along the Z-axis (optical axis) direction. The component of the signal light reflected from the sample 204 is deflected in the same direction as the outward path by the XY-direction composite deflection element 107, and is made circular by the action of the built-in λ / 4 plate, and the polarization state is rotated 90 degrees from the outward path, and then it passes through the polarizing beam splitter 104.

[0028] The sample container 200 holds a sample 204 in a well and guides signal light into the sample through a transparent window 202. 203 is a resin member that forms the well of the sample container. A metal plate 201 contacts the transparent window 202 and mechanically holds the sample container 200 while stabilizing the temperature of the sample.

[0029] The signal light and the reference light are combined by the polarizing beam splitter 104 and guided to the detection optical system 112 , and are split by the half beam splitter 113 via the pinhole 111 into transmitted light and reflected light.

[0030] The reflected light passes through a λ / 4 plate 114, whose optical axis is set at approximately 45 degrees with respect to the horizontal direction, and is then focused by a focusing lens 115 and split into two by a polarizing beam splitter 116. The two beams are then photoelectrically converted by photodetectors 150 and 151, and differentially amplified by a current differential amplifier 152 to become a detection signal 123.

[0031] The transmitted light passes through a λ / 2 plate 118, whose optical axis is set at approximately 22.5 degrees with respect to the horizontal direction, and is then focused by a focusing lens 119 and split into two by a polarizing beam splitter 120. The two beams are then photoelectrically converted by photodetectors 153 and 154, and differentially amplified by a current differential amplifier 155 to become a detection signal 122.

[0032] The detection optical system 112 implements 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 information stored in an information storage unit 125, and displays the calculation results including the correction on a display unit 126 to present them to a user. The information stored in the information storage unit 125 and the sensitivity correction based on it will be described later.

[0033] (Reflectance measurement of the interface) 3A and 3B are diagrams illustrating a frame and a metal plate on which a reflectance reference sample is placed. In the figures, a reflectance reference sample 303 is inserted into a frame 302 and placed so as to contact the surface of a metal plate 201. At this time, the reflectance reference sample 303 may be placed on the frame 302 together with other sample containers 304. When the reflectance reference sample 303 is placed on the frame 302 together with other sample containers 304, it becomes possible to measure both of them together in a single flow.

[0034] FIG. 4 is a side cross-sectional view for explaining the reflectance reference sample 303 and the focal position. The reflectance reference sample 303 is composed of a transparent flat plate 402, a container 403, and a substance 404 in contact with the transparent flat plate 402, and is arranged in contact with the metal plate 201. The light 400 collected by the objective lens 108 passes through a measurement window 405 and is irradiated onto the interface between the transparent flat 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 and the interface are matched to obtain the reflectance from the interface. Furthermore, the position of the reference beam mirror 106 is adjusted to adjust the optical path length so that the reflectance from the interface is maximized.

[0035] (Sensitivity quantification) 5A and 5B are schematic diagrams showing a method for quantifying sensitivity correction. First, the reflectance (A0) from the interface between the transparent flat plate 402 of the reflectance reference sample 303 installed in the device and the material 404 in contact with the transparent flat plate 402 is measured by the above-mentioned method at the time of shipment of the device, and the information is stored in the information storage unit 125. Next, when measuring an actual sample, the reflectance (A1) from the interface between the transparent flat plate 402 of the reflectance reference sample 303 and the material 404 in contact with the transparent flat plate 402 is measured in the same manner, and the information is stored in the information storage unit 125. The sensitivity coefficient K can be found from the values ​​of A0 and A1, and the detection signal can be corrected by Equation 2.

[0036] Figure 5 shows the case where A0>A1. Possible factors that cause this include wavelength shifts and laser power fluctuations of semiconductor lasers due to changes in environmental temperature and aging, dust on optical elements including the objective lens, and aging of the optical elements themselves, which changes the optical characteristics and reduces the output signal. By using the above method of quantifying sensitivity correction, it is possible to greatly reduce the impact of these factors on the measurement results.

[0037] (Measurement flow) FIG. 6 is a diagram showing an example of the measurement flow of A0. This flowchart can be implemented 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 caused to emit light at the 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 are the same is determined by measuring the signal while scanning R at the determined Z0. The signal level obtained at this point is stored in the information storage unit 125 as A0.

[0038] Since A0 and A1 are measured using the same optical output power, the standard power is used here. The reason for using the standard power is to ensure that the signal level of the measurement result does not saturate. Under the condition that this non-saturated signal level is 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 allowable range from the viewpoint of measurement accuracy).

[0039] Fig. 7 is a diagram showing an example of a measurement flow of A1. A1 can be measured in the same manner as in Fig. 6. The signal processing unit 124 calculates the sensitivity coefficient K from A0 and A1 obtained from Figs.

[0040] Fig. 8 is a diagram showing another example of the measurement flow of A1. In addition to the flow of Fig. 7, a predetermined value can be set for the sensitivity coefficient K, and when the sensitivity coefficient K calculated from A0 and A1 falls outside the range of predetermined values, sensitivity abnormality processing can be executed. 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 to that effect.

[0041] FIG. 9 is a diagram showing an example of a measurement flow in which A1 is measured and then a real sample is measured. When measuring a real sample, Z0 and R0 can be determined in the same way as for 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 result of the real sample is 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.

[0042] (Arrangement of reflectance standard samples) FIG. 10 is a diagram showing the arrangement of the reflectance reference sample 303. Although FIG. 3 shows an example in which the reflectance reference sample 303 is arranged in the frame 302 together with other sample containers 304, the reflectance reference sample 303 may be arranged in other positions. For example, the reflectance reference sample 303 having the structure shown in FIG. 4 may be arranged in a part of a sample stage 1001 that holds the metal plate 201 and is larger than the metal plate 201. In this case, the reflectance reference sample 303 may be arranged outside the frame 302 so as to be in contact with a metal plate 1002 that is separate from the metal plate 201. The separate metal plate 1002 may be separate from the metal plate 201, or may be integrated with the metal plate 201.

[0043] By arranging the reflectance reference sample 303 outside the frame 302, samples to be measured can be arranged at all positions within the frame 302, thereby maximizing the number of samples that can be measured. This arrangement also makes it possible to configure the reflectance reference sample 303 arranged outside the frame 302 so that the user cannot remove it. This makes it possible to avoid human error, such as forgetting to place the reflectance reference sample 303 or placing it in the wrong position. For example, it is possible to place the reflectance reference sample 303 in a place that is not visible to the user. The reflectance reference sample 303 may be configured to be removable, or may be attached in a fixed manner.

[0044] (Thickness of transparent plate) FIG. 11A shows the calculation results of the thickness variation of the transparent plate 402 for performing sensitivity calibration to achieve high-precision size measurement. In order to measure the particle size distribution with high precision, the configuration of the reflectance reference sample 303 must satisfy a certain specification, similar to the configuration specification 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 based on 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 for a case where a semiconductor laser with a wavelength of 785 nm 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 borosilicate glass with a thickness of 175 μm (actual refractive index = 1.520) is used as the transparent plate 402. As can be seen from the figure, in order to keep the decrease in the detection signal to 0.2% or less, the variation in thickness of the transparent flat plate 402 must be 70 μm or less.

[0045] 11A, Fig. 11B shows the results of calculating the relationship between the refractive index and the Strehl strength of the transparent plate 402. As can be seen from the figure, the tolerance of the refractive index of the transparent plate 402 is ±0.22.

[0046] As an example of the present invention, the reflectance reference sample 303 shown in Fig. 4 and Fig. 12 and Fig. 29 described later must be optically compatible with the sample container 304 shown in Fig. 3 in order to obtain accurate sensitivity. Specifically, the transparent flat plate 402 formed at the bottom must have a thickness of 175±70 μm and a refractive index of 1.520±0.22. An optical quality borosilicate glass substrate can be used as the material for the transparent flat plate 402 that satisfies this requirement.

[0047] (ID information) 12 is a schematic diagram showing ID information of the reflectance reference sample 303. The reflectance reference sample 303 can have ID information 1201. Specifically, as shown as ID-A, information corresponding to the reflectance of the reflectance reference sample 303 can be formed in an optically identifiable form at a location inside an outer diameter 1202 of the transparent window 405 and outside a container opening 1203, within the surface of the transparent flat plate 402 on the side opposite to the side in contact with the metal plate 201.

[0048] Instead of or in combination with ID-A, a configuration such as ID-B or ID-C is also possible. ID-B can be formed in an optically identifiable form in a location inside the container opening 1203 within the surface of the transparent flat plate 402 opposite to the side in contact with the metal plate 201. ID-C can be formed in an optically identifiable form in a location inside the outer diameter 1202 of the transparent window 405 and outside the container opening 1203 within the surface of the transparent flat plate 402 in contact with the metal plate 201. As an optically identifiable form, an information mark with a reflectance higher than the reflectance of a portion without information (background reflectance) can be recorded (white information mark), or an information mark with a reflectance lower than the background reflectance can be recorded (black information mark).

[0049] In Fig. 12, ID information 1201 is arranged in four symmetrical positions in each of ID-A, ID-B, and ID-C, but the arrangement is not necessarily limited to this. For example, the arrangement may be in one to three positions, or in more than four positions. The arrangement does not need to be symmetrical, and arrangement is possible without particular restrictions. Furthermore, recording may be made in a pattern in which two or more of the three patterns of ID-A, ID-B, and ID-C are mixed.

[0050] In ID-A and ID-B, the ID information 1201 is formed inside the reflectance reference sample 303, so there is little risk that the ID information mark will be rubbed off and erased during sample handling. ID-A has the advantage of being able to widen the ID information area, and ID-B has the advantage that the actual measurement position and the ID information position are closer than in the cases of ID-A and ID-C, so that the movement distance of the sample stage can be shortened and the measurement time can be shortened. ID-C not only has the same advantage as ID-A in that the ID information area can be widened, but also has the advantage that the information can be formed after the reflectance reference sample 303 is assembled, making the sample fabrication process easier. However, there is a risk that the ID information mark will be rubbed off and erased during sample handling.

[0051] The ID information 1201 records information corresponding to the reflectance of the reflectance reference sample. Specifically, it may be the reflectance value itself, or the reflectance value divided into levels based on a certain criterion, for example, high reflectance, medium reflectance, low reflectance, etc. The ID information may be the refractive index value itself, or the refractive index value divided into levels based on a certain criterion, for example, high refractive index, medium refractive index, low refractive index, etc. The ID information may be the optimal power value of the laser light for reflectance measurement itself, or the optimal laser light power value divided into levels based on a certain criterion, for example, high power, medium power, low power, etc. Note that, as an example of each of the above criteria, three levels, high, medium, and low, are used, but the number of levels can be selected arbitrarily. The criteria can also be determined arbitrarily. The ID information may be information on the material used for the reflectance reference sample 303, for example, resin, dielectric, metal, pure water, oil, etc. The level-divided information based on a certain criterion and the information on the material are defined as the type information of the sample. By using the type information, it is possible to determine that the sample having that information is of a different type from other samples.

[0052] The ID information may be a serial number. If the serial number is linked to the reflectance, etc., it is possible to record information similar to that described above. When the serial number is recorded, it is advantageous in that the traceability of each reflectance reference sample 303 becomes easy, and it becomes easier to guarantee the quality.

[0053] The ID information can be recorded by a known method such as printing or engraving, for example, photolithography, inkjet printing, or laser marker.

[0054] The ID information pattern can be a one-dimensional barcode, a two-dimensional barcode, a combination of a specific dot size and density, etc. Note that the patterns that can be used are not limited to these, and other known patterns can be used as long as they are optically identifiable.

[0055] FIG. 13 shows an example of the data configuration of the ID information 1201. Here, the ID information is configured by adding 8 bytes of error correction code to 8 bytes of ID information. The ID information includes 4 bytes of the serial number of the reflectance reference sample 303, 1 byte of type information of the reflectance reference sample 303, 1 byte of the refractive index of the reflectance reference sample 303, 1 byte of the laser power to be irradiated to the reflectance reference sample 303, and 1 byte of other information, but the contents of the ID information are not limited to this. For example, reflectance information can also be recorded. Also, certain information can be recorded in multiple locations. The error correction code can be easily realized by applying the standards of optical discs such as Blu-ray (registered trademark) discs.

[0056] FIG. 14A shows an example of an ID information pattern. First, information on the orientation of ID information is recorded at the four corners of a 300 μm×300 μm area. Here, information indicating the upper left, upper right, lower left, and lower right is recorded at four locations: the upper left, upper right, lower left, and lower right. As this information, a one-dimensional barcode, a two-dimensional barcode, a combination of a specific dot size and density, or other known patterns can be used. Note that the information indicating the orientation of ID information does not need to be recorded at all four locations, and it is sufficient to record it at least at one location. However, if it is recorded at multiple locations, it becomes easier to identify the orientation of the ID information, and in particular, if different information is recorded at four locations, it is sufficient to read information at least at one of the four locations to identify the orientation of the ID information, and it becomes easier to identify the orientation of the ID information pattern, so that the start position of the ID information can be identified in a short time.

[0057] Any information different from the information of the top left, top right, bottom left, and bottom right can be recorded between the top left and top right, the top right and bottom right, the bottom right and bottom left, and the bottom left and top left. It may be a dummy pattern such as a meaningless repeated pattern, or information indicating the top side, right side, bottom side, and left side may be recorded. Information regarding the position, such as address information, may be recorded on each of these sides. By using the information represented by each side, for example, the top left pattern is identified and the position of the side connected to it is identified, and the position of the main part of the ID information can be identified based on these positions. In other words, the information represented by each side can suggest the position of the ID information.

[0058] FIG. 14B is an enlarged view of the ID information pattern in FIG. 14A. Each piece of information at the top left, top right, bottom left, and bottom right is recorded in a size of, for example, about 50 μm×50 μm, and ID information is recorded inside in a range of about 200 μm×200 μm. As shown in FIG. 14B, the ID information is recorded as a barcode row about 25 μm wide from the top left to the top right. The length of one unit of the barcode is about 5 μm, and white or black information marks are recorded in a predetermined pattern. 2 Bytes of information are recorded in one barcode row as 1 Byte of information for 20 barcode units. Since the width of the barcode row is 25 μm, 8 barcode rows can be recorded in an ID information range of about 200 μm×200 μm, and thus 16 Bytes of information can be recorded in total. A barcode row with a width of 25 μm and a length of 5 μm per unit can be produced by photolithography, but the production method is not limited thereto.

[0059] Fig. 15A shows another example of an ID information pattern. In this example, the area of ​​the ID information pattern is 1960 µm x 600 µm, and similarly to Fig. 14A, position-related information is recorded at the four corners, and a barcode of ID information is recorded in an area of ​​1860 µm x 500 µm.

[0060] FIG. 15B is an enlarged view of the ID information pattern in FIG. 15A. As shown in FIG. 15B, the width of the barcode is about 100 μm, and the length of one unit of information mark is about 22 μm. This allows four rows of barcodes, each row being 4 bytes, to be recorded, allowing a total of 16 bytes of information to be recorded. This barcode row can be created by inkjet printing at 2400 dpi, but the method of creation is not limited to this, and it can also be created by, for example, a laser marker. Inkjet printing allows for easier recording, and using a laser marker makes it semi-permanent and will not rub off.

[0061] 16A and 16B show another example of the ID information pattern. In this example, the ID information is recorded using a combination of a predetermined dot size and a predetermined dot density. If three types of dot sizes and three types of dot densities are specified, 3×3=9 types of information can be recorded as combinations of these (FIG. 16B). This method has the disadvantage that the amount of information as ID information is small, but since the same pattern is repeatedly recorded, it has the advantage that if it is recorded over a wider range than the scanning range of the laser beam, the ID information can be easily read even if the scanning range is shifted. Similarly, the same information may be repeatedly arranged in the barcode patterns described in FIGS. 14 and 15.

[0062] In the case of the ID information pattern shown in Fig. 14A, the scanning range of the laser light of the particle measuring device according to this embodiment is about 300 μm × 300 μm, so it is possible to read all the ID information with one scan, and reading can be completed in about 0.1 seconds. On the other hand, in the case of the ID information pattern shown in Fig. 15A, multiple scans are required to read all the ID information. Specifically, all the ID information can be read by scanning seven times in the horizontal direction and two times in the vertical direction, and the time required for this is about 15 seconds.

[0063] The size of the entire ID information pattern and the contents of the ID information can be changed as appropriate by setting the scanning range of the laser light and the time required to read the ID information. For example, if it is desired to set the laser light scanning range to be small or the reading time to be short, it is possible to make the size of the entire ID information pattern smaller or to reduce the amount of ID information. If it is not necessary to set the laser light scanning range to be large or the reading time to be so short, it is also possible to make the size of the entire ID information pattern larger or to increase the amount of ID information.

[0064] The dots are arranged so that no dots will overlap when any three of the nine patterns are combined. This allows any three of the nine patterns to be combined. When the ID information is read, the information content can be identified as long as it is known which patterns have been combined, so the orientation of the patterns does not matter.

[0065] FIG. 17A is an example of a schematic diagram showing the shape of the reflectance reference sample 303 in the xy plane. The reflectance reference sample 303 has four transparent windows and is shaped so that it can be directly installed on the frame 302 of FIG. 3A. This makes it possible to limit the positional deviation in the xy plane to a value according to the design dimensions and processing accuracy of the frame 302 and the reflectance reference sample 303. This makes it possible to shorten the access time of the optical head for reading the ID information, since the rough position can be known in advance when reading the ID information placed at the position shown in FIG. 12. In this case, if a notch is provided in a part of the reflectance reference sample 303 as shown in the figure, the orientation of the reflectance reference sample 303 when it is installed on the frame 302 will not be mistaken. Note that the reflectance reference sample 303 shown in this figure does not necessarily need to be directly installed on the frame 302, and may be installed outside the frame 302.

[0066] 17B and 17C are schematic diagrams showing another example of the shape of the reflectance reference sample 303 in the xy plane. In FIG. 17B, there is one transparent window, and in FIG. 17C, there are two transparent windows. These can be installed outside the frame 302. By installing them outside the frame 302, measurement samples can be installed at all locations within the frame 302, making it possible to maximize the number of measurement samples. The reflectance reference sample 303 shown in FIG. 17B and FIG. 17C may be installed inside the frame 302. It is possible to install them so that their short sides, long sides, corners, notches, etc. match the frame 302.

[0067] FIG. 18 shows the structure and reflectance of the high reflectance part of the ID information mark in ID-A. Specifically, the material of the high reflectance part (part where information is recorded) is Al, and the calculation results of the Al film thickness and reflectance are shown. The reflectance (background reflectance) of the part without ID information in ID-A is 1% or less when a black plastic container is used as the container 403. Therefore, if the Al film thickness is set to 2 nm or more, a reflectance about 10 times the background reflectance can be obtained, and the ID information mark can be read reliably. In order to clearly recognize the ID information, it is considered that the reflectance of the ID information mark should be at least twice the background reflectance (i.e., the reflectance of the side wall of the container 403). The same is true for ID-B and ID-C.

[0068] 19A to 19C show the specific structure and reflectance of the high reflectance portion and the low reflectance portion of the ID information mark of ID-B. The background reflectance of ID-B is about 5% when a resin material is used as the filling material. Therefore, in order to set the reflectance of the ID information mark higher than the background reflectance, it is desirable to set the reflectance of the high reflectance portion to 50% or more, which is 10 times or more. This makes it possible to read the ID information mark reliably. FIG. 19A shows the calculation result of the reflectance with respect to the Al film thickness when Al is used as the high reflectance portion. From these, it can be seen that if the Al film thickness is set to 7 nm or more, a reflectance about 10 times the background reflectance can be obtained, and the ID information mark can be read reliably.

[0069] On the other hand, when the reflectance of the ID information mark is set lower than the background reflectance in ID-B, the reflectance of the ID information mark should be set to 0.5% or less, since the background reflectance of ID-B is about 5%. FIG. 19B shows the calculation result of the reflectance versus W film thickness when the part with low reflectance has a four-layer structure of ZnS(1) / W / ZnS(2) / Al. Here, the film thickness of ZnS(1) is 20 nm, the film thickness of ZnS(2) is 60 nm, and the film thickness of Al is 50 nm. From these, when the W film thickness is set to 8.2 to 11.3 nm, the reflectance is less than one-tenth of the background reflectance, and the ID information mark can be read reliably.

[0070] Figure 19C shows another example of ID-B where the reflectance of the ID information mark is set lower than the background reflectance. Here, a multilayer structure with ZnS and SiO2 dielectric layers is used in the area with low reflectance. ZnS is provided on the surface in contact with the glass bottom plate, and SiO2 and ZnS are provided alternately on top of it. The layer furthest from the glass bottom plate is ZnS. In a certain multilayer structure, all SiO2 layers have the same thickness, all ZnS layers except the layer closest to the glass bottom plate and the layer furthest from the glass bottom plate have the same thickness, and the ZnS layer closest to the glass bottom plate and the ZnS layer furthest from the glass bottom plate are half the thickness of the other ZnS layers. The SiO2 and ZnS layers are different thicknesses. Under these conditions, the reflectance of a certain number of layers is calculated by changing the SiO2 and ZnS thicknesses independently, and the reflectance for that number of layers is calculated by taking the layer with the smallest reflectance as the reflectance for that number of layers, as shown in Figure 19C. From these results, it was found that if the number of dielectric layers is five or more, a reflectance of 0.5% or less, which is one-tenth of the background reflectance, can be obtained, and the ID information mark can be read reliably.

[0071] 19B and 19C, it has been explained that the reflectance of the ID information mark is 1 / 10 or less of the background reflectance (i.e., the reflectance of the substance 404), but it is believed that the ID information can be sufficiently identified if it is at least 1 / 2 or less. Similarly, when making the ID information mark dark in ID-C, the reflectance of the ID information mark should be 1 / 2 or less of the background reflectance (i.e., the reflectance of the side wall of the container 403).

[0072] 20A to 20C show the specific structure and reflectance of the high reflectance and low reflectance parts of the ID information mark of ID-C. The background reflectance of ID-C is the reflectance of air and the glass bottom plate, which is about 4%. Therefore, in order to set the reflectance of the ID information mark high, the reflectance of the high reflectance part should be set to 40% or more, which is 10 times higher, so that the ID information mark can be read reliably. FIG. 20A shows the calculation result of the reflectance with respect to the Al film thickness when Al is used as the high reflectance part (white). From these, it can be seen that if the Al film thickness is set to 5 nm or more, the reflectance is about 10 times higher than the background reflectance, and the ID information mark can be read reliably.

[0073] On the other hand, in ID-C, when the reflectance of the ID information mark is set lower than the background reflectance, the background reflectance of ID-C is about 4%, so the reflectance of the ID information mark should be set to 0.4% or less. Fig. 20B shows the calculation result of the reflectance against the ZnS film thickness when the part with low reflectance has a four-layer structure of SiO2 / W / ZnS / Al. Here, the SiO2 film thickness is 95 nm, the W film thickness is 10 nm, and the Al film thickness is 50 nm. From these, when the ZnS film thickness is 50 to 60 nm, a reflectance of 1 / 10 or less of the background reflectance can be obtained, and the ID information mark can be read reliably.

[0074] Figure 20C shows another example of ID-C where the reflectance of the ID information mark is set lower than the background reflectance. Here, a multilayer structure with ZnS and SiO2 dielectric layers is used in the area with low reflectance. ZnS is provided on the surface in contact with the glass bottom plate, and SiO2 and ZnS are provided alternately on top of it. The layer furthest from the glass bottom plate is ZnS. In a certain multilayer structure, all SiO2 layers have the same thickness, all ZnS layers except for the layer closest to the glass bottom plate and the layer furthest from the glass bottom plate have the same thickness, and the ZnS layer closest to the glass bottom plate and the ZnS layer furthest from the glass bottom plate are half the thickness of the other ZnS layers. Note that the SiO2 and ZnS layers have different thicknesses. Under these conditions, the reflectance of a certain number of layers is calculated by changing the SiO2 and ZnS thicknesses independently, and the reflectance for that number of layers is calculated by taking the layer with the smallest reflectance as the reflectance for that number of layers, as shown in Figure 20C. From these results, it was found that if the number of dielectric layers is five or more, a reflectance of 0.4% or less, which is one-tenth of the background reflectance, can be obtained, and the ID information mark can be read reliably.

[0075] The structures shown in FIGS. 18 to 20 can be formed by photolithography and thin film processes.

[0076] FIG. 21 is a diagram showing an example of a measurement flow of A0 linked to ID information when the ID information is ID-A, ID-B, or a mixed pattern of ID-A, ID-B, or ID-B in FIG. 12. The light collection position of the signal light is moved to the planar position of the reflectance reference sample 303, and the light source 100 is caused 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 mirror 106 are initialized. Z0 is determined by measuring the signal while scanning in the Z direction around the transparent window 405. The signal is measured while scanning R at the determined Z0, and the mirror position R0 at which the optical path lengths of the signal light and the reference light match is determined. Next, while keeping the Z position at Z0, the light collection position is scanned in the XY direction and moved to the ID information position, and the ID information 1201 of the reflectance reference sample 303 is read. Light is emitted at the power P1 for measuring the reflectance reference sample based on the acquired ID information, and the signal level obtained at this point is set as A0. This signal level A0 is stored in the information storage unit 125 in association with the ID information. When there are multiple types of reflectance reference samples, the above flow is repeated and the signal level A0 corresponding to each ID information is stored in the information storage unit 125.

[0077] Figure 22 is a diagram showing an example of the measurement flow of A0 linked to ID information when the ID information is ID-C in Figure 12. It is the same as Figure 21, except that the Z position before reading the ID information is moved in the negative Z direction from Z0 by the thickness of the glass bottom plate.

[0078] FIG. 23 is a diagram showing an example of a flow for measuring A1 after reproducing ID information when the ID information is ID-A, ID-B, or a mixed pattern of these in FIG. 12. When the user selects a measurement object, the ID information of the reflectance reference sample suitable for the measurement object is first read. From the contents of the ID information, it is judged whether the reflectance reference sample for the measurement object selected by the user is normal, and if it is normal, the next flow is carried out, and if it is abnormal, abnormality processing such as issuing an alarm is carried out. Since the reflectance of the reflectance reference sample 303 is the same as that of the measurement object, by reading the reflectance information of the reflectance reference sample 303 described in the ID information, it is automatically known which measurement object it corresponds to. This makes it possible to prevent human errors such as mistaking the reflectance reference sample. After it is judged that the reflectance reference sample is normal, A1 is measured by emitting light with a laser light power for measuring the signal level of the reflectance reference sample based on the contents of the ID information, and the sensitivity coefficient K is calculated from A0 and A1. A predetermined value is set for the sensitivity coefficient K, and when the sensitivity coefficient K calculated from A0 and A1 falls outside the range of predetermined values, sensitivity abnormality processing can be executed. 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 to that effect.

[0079] Fig. 24 is a diagram showing an example of a flow for measuring A1 after reproducing ID information when the ID information is ID-C in Fig. 12. It is the same as Fig. 23 except that the Z position before reading the ID information is moved in the negative Z direction from Z0 by the thickness of the glass bottom plate.

[0080] FIG. 25 is a diagram showing an example of a measurement flow in which A1 is measured after reproducing the ID information in the case where the ID information is ID-A, ID-B, or a mixed pattern of these in FIG. 12, and then an actual sample is measured. When a user selects a measurement object, the ID information of the reflectance reference sample for that object is first read. From the contents of the ID information, it is determined whether the reflectance reference sample is normal for the measurement object selected by the user, and if it is normal, the next flow is performed, and if it is abnormal, an abnormality process such as issuing an alarm is performed. This makes it possible to prevent human errors such as mixing up the reflectance reference sample. After it is determined that the reflectance reference sample is normal, A1 is measured by emitting light with a laser light power for measuring the signal level of the reflectance reference sample based on the contents of the ID information, and a sensitivity coefficient K is calculated from A0 and A1. A predetermined value is set for the sensitivity coefficient K, and when the sensitivity coefficient K calculated from A0 and A1 falls outside the range of a predetermined value, a sensitivity abnormality process can be performed. For example, the signal processing unit 124 can notify the user of the fact by outputting a signal or message indicating that the sensitivity coefficient K is outside the predetermined range. The flow after obtaining the sensitivity coefficient K is the same as that in FIG.

[0081] Figure 26 is a diagram showing an example of a measurement flow in which A1 is measured after reproducing the ID information and then the actual sample is measured when the ID information is ID-C in Figure 12. It is the same as Figure 25, except that the Z position before reading the ID information is moved from Z0 in the negative Z direction by the thickness of the glass bottom plate.

[0082] 21 to 26, the important thing is to use the same P1 laser light power for the measurement of A0 linked to the ID information, the measurement of A1 before the actual measurement, and the actual measurement. This makes it possible to prevent sensitivity deviations due to differences in laser light power.

[0083] (machine difference etc.) By using the reflectance reference sample 303, even if there is an instrumental difference in the particle measuring device (such as minute differences in the laser wavelength, optical system, detector sensitivity, etc.), it is possible to obtain almost the same result for the same sample. Furthermore, it can also be used for diagnosing malfunctions in the device. Specifically, if the sensitivity coefficient K deviates from a certain numerical range, it is possible that the laser has deteriorated due to aging, dust has adhered to the optical element, the optical system has shifted, etc., and therefore the signal processing unit 124 can issue an alert on the display unit 126, for example, to prompt maintenance or repair of the device.

[0084] (Solvent refractive index measurement) FIG. 27A is a schematic diagram showing the relationship between a vessel containing a real sample containing a particle to be measured and a solvent, and the focal position of a laser beam when measuring the solvent refractive index of the real sample. The reflectance reference sample 303 can also be used to measure the refractive index of the solvent of the real 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 reflectance of the boundary between the transparent plate 402 and the sample depends on the refractive index of the solvent. By determining the condition that maximizes the detection signal while moving the sample stage (not shown) in the Z direction, 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, the magnitude of the detection signal can be expressed by Equation 1 from the Fresnel equation.

[0085] In the present invention, the refractive index can be set to a specified value by selecting the material of the transparent plate 402. 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 keeping the output power condition of the semiconductor laser (not shown) constant.

[0086] Furthermore, a material having a refractive index n o The magnitude of the detection signal when the substance is used as a sample is measured and the value is called E0 sig Let E0 sigis expressed by Equation 4. Equation 5 is derived from Equation 1 and Equation 4. In Equation 5, the refractive index of the solvent, n m Since all values ​​except for the above are known or measured values, the refractive index of the solvent, n m It is now possible to measure the

[0087]

number

[0088]

number

[0089] FIG. 27B shows, following FIG. 27A, how the focal position is moved to a predetermined position within the sample to measure the size of particles contained in the sample.

[0090] <Embodiment 2> (Refractive index: dielectric film) FIG. 28A shows the reflectance of the reflectance reference sample 303 in the embodiment 2 of the present invention. In the embodiment 2, a dielectric film was used as a material in contact with the transparent flat plate 402 of the reflectance reference sample 303 in FIG. 4. ZnS was used as the dielectric film, and it was formed by sputtering on the transparent flat 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 was formed changes depending on the film thickness of the dielectric film due to interference between reflection from the surface of the dielectric film on the laser light incident side and reflection from the surface on the back side. FIG. 28A shows the relationship between the film thickness of ZnS used in the embodiment 2 and the reflectance.

[0091] Fig. 28B shows the reflectance in another configuration example of reflectance reference sample 303 of embodiment 2. In this configuration example, a dielectric film SiO2 is formed by sputtering on a dielectric film ZnS formed on a transparent flat plate 402, and a dielectric film ZnS is further formed by sputtering. The reflectance of reflectance reference sample 303 with this structure changes with the thickness of the dielectric film ZnS when the SiO2 film thickness is 133 nm, as shown in Fig. 28B.

[0092] From the above, the reflectance reference sample 303 can be either (a) a single dielectric film or (b) a multilayer structure made of two or more types of dielectric films, and the reflectance of the reflectance reference sample 303 can be set to a predetermined value by appropriately selecting the film thickness of each.

[0093] In addition to proteins, particles that can be measured in the present invention can also be metal particles, plastic particles, etc. Reflectance varies depending on the type of particle, but in any case, the actual measurement sample is a sample in which submicron-sized particles are dispersed in a solvent, so the absolute value of the reflectance does not become large. From the viewpoint that the laser light power needs to be the same in the actual measurement and the reflectance reference sample measurement for correct sensitivity adjustment, it is preferable that the absolute value of the reflectance of the reflectance reference sample is not set to a large value.

[0094] In the structure (a), a reflectance between a minimum reflectance of about 4% and a maximum reflectance of about 23% can be obtained. When a reflectance of 4% or more is required as a reflectance reference sample, the structure (a) is preferable because it is simple and easy to fabricate, but when a reflectance lower than 4% is required, this structure cannot be realized. The structure (b) can obtain a reflectance between a minimum reflectance of about 2% and a maximum reflectance of about 49%. In particular, when a reflectance of 3% or less is required as a reflectance reference sample, the structure is preferable because the ZnS film thickness range where the reflectance is 3% is wide, 0 to 20 nm and 160 to 200 nm, and the structure is highly robust.

[0095] In addition, when the particle to be measured is larger than the light spot size, the reflectance may be high depending on the material, composition, etc. In that case, the material and film thickness of the dielectric film can be selected to provide a configuration that corresponds to the target particle reflectance.

[0096] <Embodiment 3> In the third embodiment of the present invention, a specific example of the reflectance reference sample 303 will be described. The configuration of the particle measuring device and the measurement procedure are the same as those in the first and second embodiments.

[0097] (Reflectance standard sample) An ultraviolet curing resin (DVD310) manufactured by Nippon Kayaku Co., Ltd. was used as the material in contact with the transparent flat plate 402 of the reflectance reference sample 303 in Fig. 4. Since bubbles in the interface would affect the reflected signal, to avoid this, the ultraviolet curing resin was placed in a vacuum container and degassed by evacuating for 10 minutes. After that, the resin was poured into the container 403 and ultraviolet rays were applied at approximately 300 mJ / cm using an ultraviolet irradiation device (ECS-201G1) manufactured by Eye Graphics. 2 The resin was cured by irradiation.

[0098] (Refractive index: for proteins) The reflectance from the interface is proportional to the difference in refractive index between the two substances. For example, when protein particles are used as the sample for measuring the size distribution, the difference in refractive index between the solvent and the protein particles is approximately 0.06. In the reflectance standard sample using the above UV-curable resin, the refractive index of the UV-curable resin DVD310 after curing is 1.50. If borosilicate glass with a refractive index of 1.52 is used for the transparent plate, the difference in refractive index between the two is 0.02, so the reflectance from the protein particles and the interface reflectance are also approximately the same.

[0099] This makes it possible to measure the reflectance reference sample 303 under the same laser power conditions as those for measuring the size distribution of protein particles, and to obtain the sensitivity coefficient K under these conditions. Although 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, making it possible to accurately measure particle sizes.

[0100] It is desirable for the reflectance reference sample 303 to have little optical change over time and be usable for a long period of time.

[0101] Samples with different refractive indices were prepared by using a different ultraviolet curing resin instead of the ultraviolet curing resin (DVD310) manufactured by Nippon Kayaku Co., Ltd. as the material in contact with the transparent flat plate 402 of the reflectance reference sample 303. Measurements were carried out for each sample to determine the sensitivity coefficient K, and the laser power conditions were as follows:

[0102] 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 equal power to protein particle sample measurement 1.50: Approximately equal power to protein particle sample measurement 1.52: Higher power than protein particle sample measurement

[0103] From these results, it is preferable that the refractive index of the material in contact with transparent plate 402 of reflectance reference sample 303 is 1.43 or more and 1.50 or less.

[0104] Fig. 29 shows another example of the configuration of the reflectance reference sample 303. In the above embodiment, an ultraviolet curing resin (DVD310) manufactured by Nippon Kayaku Co., Ltd. is used as the material in contact with the transparent flat plate 402, but a liquid resin (such as immersion oil for microscope objective lenses) can be used instead. In Fig. 16, 1601 is the liquid resin. In this case, it is sealed with sealing tape or the like to prevent the liquid from leaking.

[0105] (How to use the reflectance standard sample) When measuring a plurality of real samples, 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 measurement of the plurality of real samples.

[0106] When the measurement time required to measure a real sample is relatively short, the changes in the environmental temperature and the laser temperature during the measurement of the real sample are relatively small, so that it is sufficient to measure the reflectance reference sample once at the start of the measurement and perform sensitivity correction, thereby shortening the overall time required for measurement.

[0107] On the other hand, when the measurement time required for measuring a real sample is relatively long, there is a possibility that the environmental temperature or the laser temperature may change during the measurement, so that more accurate measurements can be performed by measuring a reflectance reference sample for each measurement of a plurality of real samples and redoing the sensitivity correction each time. In this case, it is also possible to measure a reflectance reference sample and redo the sensitivity correction each time after a certain number of real sample measurements, rather than after each measurement.

[0108] The operator can decide how long or short the measurement time is for the actual sample. In this case, the operator may refer to the measurement time taken to measure a similar sample in the past. However, when measuring multiple unknown samples, it is preferable to redo the sensitivity correction each time.

[0109] <Modifications of the present invention> The present invention is not limited to the above-described embodiment, and includes various modified examples. For example, the above-described embodiment has been described in detail to clearly explain the present invention, and is not necessarily limited to those having all of the configurations described. In addition, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. In addition, it is possible to add, delete, or replace a part of the configuration of each embodiment with another configuration.

[0110] In the above embodiment, the reflectance reference sample 303 can be configured as a component of the particle measuring device, or can be configured as a separate member that is detachable from the particle measuring device, for example.

[0111] It should be noted that in the above embodiment, the objective lens 108 and the driving mechanism 109 serve as an irradiation unit that focuses and irradiates the signal light onto the sample, and the detection optical system 112 serves as a detection unit that detects an interference signal generated by interference between the signal light and the interference light. [Explanation of symbols]

[0112] 100: Light source 101: Laser driver 102: Collimating lens 103:λ / 2 plate 104: Polarizing beam splitter 105:λ / 4 plate 106: Reference mirror 107: Composite deflection element 108: Objective lens 109: Drive mechanism 111: Pinhole 112: Detection optical system 113: Half beam splitter 114:λ / 4 plate 115: Condenser lens 116: Polarizing beam splitter 118:λ / 2 plate 119: Condenser lens 120: Polarizing beam splitter 122: Detection signal 123: Detection signal 124: Signal processing unit 125: Information holding unit 126: Display section 150: Photodetector 151: Photodetector 152: Current Differential Amplifier 153: Photodetector 154: Photodetector 155: Current Differential Amplifier 200: Sample container 201: Metal plate 202: Transparent window 203: Resin member forming a well of a sample container 204: Sample 302: Frame 303: Reflectance standard sample 304: Sample container 400: Concentrated light 402: Transparent flat plate 403: Container 404: Substance 405: Transparent window for measurement 1001: Sample stage 1002: Metal plate 1201:ID information 1202: Outer diameter of transparent window 405 1203: Container opening

Claims

1. A reference sample used to calibrate a measurement result of a particle size in a particle measuring device that measures the size of particles contained in a liquid sample by irradiating the sample with light, comprising: The reference sample has a reference sample window having the same material and thickness as a sample container window of a sample container that contains the sample, The reference sample has a material having a reflectance that is within a tolerance range relative to the reflectance of the sample, the material being disposed in contact with the reference sample window; 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, a shape pattern representing ID information for identifying the reference sample is formed on any one surface of the transparent flat plate; The shape pattern is configured to be optically readable. A reference sample characterized by:

2. The ID information includes information indicating the orientation of the shape pattern.

2. The reference sample according to claim 1 .

3. The shape pattern has a rectangular outer periphery, The shape pattern has a portion representing information suggesting an orientation of the shape pattern at at least one of the four corners of the rectangle.

3. The reference sample according to claim 2.

4. The shape pattern has a rectangular outer periphery, The shape pattern has a portion representing information suggesting a position of the shape pattern on at least one of the four sides of the rectangle.

3. The reference sample according to claim 2.

5. The shape pattern is formed by a dot pattern, the dot pattern is configured to represent the ID information by a combination of dot size and dot plane density; The dot pattern is arranged such that the dots do not overlap each other for any combination of the sizes and the planar densities.

2. The reference sample according to claim 1 .

6. The reference sample includes a notch that indicates the orientation of the reference sample.

2. The reference sample according to claim 1 .

7. The planar size of the shape pattern is approximately 300 μm × approximately 300 μm, The ID information is represented by arranging a plurality of shape patterns representing the ID information in a planar area smaller than the planar size.

2. The reference sample according to claim 1 .

8. The planar size of the shape pattern is approximately 600 μm × approximately 2000 μm, The ID information is represented by arranging a plurality of shape patterns representing the ID information in a planar area smaller than the planar size.

2. The reference sample according to claim 1 .

9. The ID information includes at least information regarding the type of the reference sample, The ID information further comprises: The serial number of said reference sample; The type of the reference sample; the refractive index of the reference sample; the laser power of the light to be irradiated onto the reference sample; At least one of the following is included: The ID information includes information of 4 bytes or more.

2. The reference sample according to claim 1 .

10. the shape pattern is formed on a surface of the transparent flat plate that contacts the substance, The geometric pattern is disposed below a sidewall of a container in which the reference sample contains the substance; The reflectance of the shape pattern is at least twice as high as the reflectance of the sidewall.

2. The reference sample according to claim 1 .

11. the shape pattern is formed on a surface of the transparent flat plate that contacts the substance, the shape pattern is disposed between the substance and the transparent plate; The reflectance of the shape pattern is at least twice the reflectance of the material or is at most half the reflectance of the material.

2. The reference sample according to claim 1 .

12. the shape pattern is formed on a surface of the transparent flat plate that does not contact the substance, The geometric pattern is disposed below a sidewall of a container in which the reference sample contains the substance; The reflectance of the shape pattern is at least twice the reflectance of the sidewall or is at most half the reflectance of the sidewall.

2. The reference sample according to claim 1 .

13. 1. A particle measurement device for measuring the size of particles contained in a liquid sample, comprising: A light source that 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 condenses the signal light and irradiates the sample with the collected light; a detection unit for detecting an interference signal obtained by causing the reflected light from the particle to interfere with the reference light; a processing unit that measures a size of the sample using the interference signal detected by the detection unit, and calibrates the size using a measurement result of a reference sample; Equipped with The reference sample has a reference sample window having the same material and thickness as a sample container window of a sample container that contains the sample, The reference sample has a material having a reflectance that is within a tolerance range relative to the reflectance of the sample, the material being disposed in contact with the reference sample window; 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, a shape pattern representing ID information for identifying the reference sample is formed on any one surface of the transparent flat plate; The shape pattern is configured to be optically readable, The processing unit reads the ID information based on a signal obtained by irradiating the reference sample with the light. A particle measuring device characterized by:

14. 1. A method for measuring the size of particles contained in a liquid sample, comprising: Emitting light from a light source; splitting light from the light source into a signal light and a reference light; collecting the signal light and irradiating it onto the sample; detecting an interference signal obtained by interfering the reflected light from the particle with the reference light; measuring a size of the sample using the detected interference signal; calibrating said size using a reference sample; having The reference sample has a reference sample window having the same material and thickness as a sample container window of a sample container that contains the sample, The reference sample has a material having a reflectance that is within a tolerance range relative to the reflectance of the sample, the material being disposed in contact with the reference sample window; 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, a shape pattern representing ID information for identifying the reference sample is formed on any one surface of the transparent flat plate; The shape pattern is configured to be optically readable, In the step of calibrating, the ID information is read based on a signal obtained by irradiating the reference sample with the light. A particle measuring method comprising:

15. The particle measurement method further includes a step of determining whether the reference sample is of a type suitable for calibrating the size, and if not, performing the above process; In the determining step, it is determined whether the reference sample is suitable based on whether information describing the type of the reference sample described in the ID information corresponds to the sample.

15. The particle measuring method according to claim 14.

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