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

Abstract

The purpose of the present invention is to provide a technology that makes it possible to easily perform individual identification on a reference sample that is used to calibrate a result of measuring the size of particles in a sample by using light. A reference sample according to the present invention is provided with a transparent flat plate having a window. The transparent flat plate is provided with a boundary surface having a reflectance that is substantially equal to the reflectance of a sample. One surface of the transparent flat plate has formed thereon a shape pattern that represents ID information for identifying the reference sample (see fig. 12).

Description

Particle measurement device, particle measurement method, reflectance standard sample

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

[0002] In recent years, pharmaceutical development has been shifting from small molecule drugs to biopharmaceuticals. Because biopharmaceuticals are polymers, they are prone to aggregation, which can lead to toxicity. For example, the U.S. Food and Drug Administration and other organizations are seeking to strengthen regulations on aggregate concentration control. Therefore, a technology is needed to quantitatively measure the size distribution of aggregates at a desired density in the submicron range of 0.1 to 1 μm. Protein aggregates float in a solvent, and their position changes over time due to Brownian motion. 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 technology for detecting particles using optical measurement. The document discloses an optical measurement method for focusing light to generate a light spot and measuring a test object approximately three times the size of the light spot, the method comprising: a signal acquisition step for detecting reflected light from the test object by irradiating the test object with the light while moving at least the focal position of the light in the optical axis direction; a step for 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 for obtaining the size of the test object by referencing the correspondence data using the intensity of the reflected light (claim 1). The technology described in the document achieves high-resolution measurement without preprocessing by causing interference between the reflected light and a reference light to enhance the signal.

[0004] Patent Document 2 discloses a technology 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 technology based on the technology of Patent Document 1 that speeds up scanning of a light spot so as not to be affected by the movement of particles undergoing Brownian motion in a liquid.

[0005] JP 2017-102032 A

[0006] In conventional technologies such as those disclosed in Patent Documents 1 and 2, interference between signal light and reference light is used to measure the size of protein aggregates. The interference efficiency changes due to factors such as the laser temperature, changes over time, and dust accumulation on the objective lens. In order to ensure consistently accurate measurements in the user's environment, Patent Document 2 uses a reference sample to calibrate the sensitivity of the interference efficiency. To expand the scope of measurement to include objects other than protein aggregates, various reference samples will be prepared depending on the optical properties of the measurement object. Therefore, it is necessary to avoid mixing up reference samples. In conventional technologies, such mixing up of reference samples has not been 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.

[0008] The reference sample of the present invention comprises a transparent flat plate having a window, the transparent flat plate having an interface surface with a reflectance approximately equal to the reflectance of the 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.

[0009] The reference sample according to the present invention can easily identify individual reference samples used to calibrate the results of measuring the size of particles in a sample using light. Problems, configurations, and effects other than those described above will become clear from the description of the following embodiments.

[0010] FIG. 1 is a schematic diagram illustrating the relationship between particle size and the magnitude of the reflected light amount in Patent Documents 1 and 2. FIG. 2 is a configuration diagram of a particle measuring device according to a first embodiment. FIG. 3 is a diagram illustrating a frame and a metal plate on which a reflectance reference sample is placed. FIG. 4 is a diagram illustrating a frame and a metal plate on which a reflectance reference sample is placed. FIG. 5 is a side cross-sectional view illustrating a reflectance reference sample 303 and a focal position. FIG. 6 is a schematic diagram illustrating a method for quantifying sensitivity correction. FIG. 7 is a schematic diagram illustrating a method for quantifying sensitivity correction. ０ 1 is a diagram showing an example of a measurement flow. １ 1 is a diagram showing an example of a measurement flow. １ FIG. 10 is a diagram showing another example of the measurement flow. １11A is a diagram showing an example of a measurement flow in which a real sample is subsequently measured after measuring the reflectance reference sample 303. FIG. 11B is a diagram showing the arrangement of the reflectance reference sample 303. FIG. 11C is a calculation result showing the specifications for the thickness variation of the transparent flat plate 402 for performing sensitivity calibration to achieve high-precision size measurement. As with FIG. 11A, this shows the calculation result of the relationship between the refractive index of the transparent flat plate 402 and the Strehl intensity. FIG. 11D is a schematic diagram showing the ID information of the reflectance reference sample 303. FIG. 11E is a diagram showing an example of the data configuration of ID information 1201. FIG. 11F is a diagram showing an example of an ID information pattern. FIG. 14A is an enlarged view of the ID information pattern in FIG. 14A. FIG. 14B is another example of an ID information pattern. FIG. 15A is an enlarged view of the ID information pattern in FIG. 15A. FIG. 11C is another example of an ID information pattern. FIG. 11D is a diagram showing an example of a schematic diagram showing the shape of the reflectance reference sample 303 in the xy plane. FIG. 11F is another example of a schematic diagram showing the shape of the reflectance reference sample 303 in the xy plane. The structure and reflectivity of the high reflectivity portion of the ID information mark for ID-A are shown. Figures 19A to 19C show the specific structure and reflectivity of the high reflectivity portion and low reflectivity portion of the ID information mark for ID-B. Figures 19A to 19C show the specific structure and reflectivity of the high reflectivity portion and low reflectivity portion of the ID information mark for ID-B. Figures 19A to 19C show the specific structure and reflectivity of the high reflectivity portion and low reflectivity portion of the ID information mark for ID-B. Figures 20A to 20C show the specific structure and reflectivity of the high reflectivity portion and low reflectivity portion of the ID information mark for ID-C. Figures 20A to 20C show the specific structure and reflectivity of the high reflectivity portion and low reflectivity portion of the ID information mark for ID-C. Figures 20A to 20C show the specific structure and reflectivity of the high reflectivity portion and low reflectivity portion of the ID information mark for ID-C. When the ID information is ID-A, ID-B, or a mixed pattern of these in FIG. 12, A linked to the ID information ０ 12 is a diagram showing an example of a measurement flow of the ID information. ０12. When the ID information is ID-A, ID-B or a mixed pattern of these in FIG. 12, after reproducing the ID information, A is １ 12. When the ID information is ID-C in FIG. 12, after reproducing the ID information, A is measured. １ 12. When the ID information is ID-A, ID-B or a mixed pattern of these in FIG. 12, after reproducing the ID information, A is measured. １ 12 is a diagram showing an example of a measurement flow in which the ID information is measured and then the actual sample is measured. １ 27A is a diagram showing an example of a measurement flow in which a sample containing particles to be measured and a solvent is measured first, and then a real sample is measured. FIG. 27B is a schematic diagram showing the relationship between a container containing a real sample containing particles to be measured and a solvent, and the focal position of laser light when measuring the solvent refractive index of the real sample. Following FIG. 27A, the focal position is moved to a predetermined position within the sample to measure the size of particles contained in the sample. FIG. 27C shows the reflectance of a reflectance reference sample 303 in embodiment 2. FIG. 27D shows the reflectance of another example configuration of the reflectance reference sample 303 in embodiment 2. FIG. 27E shows another example configuration of the reflectance reference sample 303.

[0011] <Problems with the Prior Art> Figure 1 is a schematic diagram illustrating the relationship between particle size and the amount of reflected light in Patent Documents 1 and 2. The techniques in Patent Documents 1 and 2 utilize the interference between reflected signal light from the particles and a reference light to measure the size of particles dispersed in a solvent. The detected reflected light changes depending on the particle size and its refractive index. The techniques described in Patent Documents 1 and 2 utilize the interference of light to convert the electric field amplitude of the reflected light into a voltage 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 ｍUsing Fresnel's law, when the coherence length of the light source is sufficiently long, it can be expressed by the following equation: where S is the electric field amplitude of the light irradiating 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-voltage conversion of the photodetector.

[0012]

[0013] Particle size can be measured by measuring the detection signal expressed by Equation 1. In actual measurements, fluctuations in the detection signal can occur due to wavelength shifts in the semiconductor laser or fluctuations in laser power caused by changes in environmental temperature, aging, or dust on the objective lens. 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 measure. (3) From the measurement results of the standard sample, determine the sensitivity correction coefficient that will eliminate the discrepancy between the measured size and the true size, and manually enter that value.

[0015] The above calibration procedure requires a significant amount of time and effort for the preparation, accurate dilution, and measurement of standard beads, posing challenges in terms of user convenience. Furthermore, there is a risk of size variation between standard bead lots and beads being lost during bead dilution and mixing, which can hinder accurate measurements. Furthermore, polystyrene standard beads have a large refractive index difference from the pure water solvent, resulting in a high reflectance and requiring a relatively low measurement laser power. In contrast, when measuring protein particles, for example, the refractive index difference between the protein particles and the solvent is small, resulting in a low reflectance and requiring a relatively high measurement laser power. The difference in laser power between these two types of beads can result in differences in laser temperature, potentially resulting in different laser wavelengths. This wavelength difference can result in different sensitivity coefficients, posing a challenge for accurate sensitivity correction.

[0016] <Basic Principle of the Present Invention> (Quantification of Sensitivity) In order to solve the above-mentioned problems, the present invention calibrates sensitivity using a reflectance reference sample having a flat surface that has a reflection 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]

[0018] The sensitivity coefficient K is defined by the following equation: ０ is the reflectance of a reflectance standard sample measured at the time of shipment of the particle measuring device of the present invention. １ is the reflectance when the reflectance reference sample is measured immediately before the measurement of the sample to be actually measured.

[0019]

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

[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 a substance is formed in contact with the glass window, with the boundary surface having a reflectance substantially equal to that of the measurement sample. This allows the measurement conditions for the reflectance reference sample and the measurement sample to be the same. In particular, by using the same laser power during sample measurement and during reference sample measurement, 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 flat plate. This makes the difference in refractive index between the transparent flat plate and the resin comparable to the difference in refractive index between the protein particles and the solvent, making the reflectance of the reflectance reference sample and the reflectance of the protein particles in the solvent approximately equal, allowing it to be used as a reflectance reference sample for measuring protein particles. "Almost equal" here means that the error in the measurement results falls within an acceptable range. In other words, the difference between the reflectance of the material (here, the resin) in contact with the transparent flat plate and the reflectance of the sample must fall within an acceptable range in terms of measurement error.

[0023] As another example of a reflectance reference 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 particles. By selecting the material and thickness of the dielectric film, as well as the material and thickness of the metal film formed on the dielectric film, it is possible to design a sample to achieve a specified reflectance, making it possible to accommodate a variety of particles.

[0024] In the following description of the embodiments of the present invention, the explanation will be made in a unified coordinate system in which the z-axis is set to the optical axis direction, 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, and then the light is split into signal light and reference light by a polarizing beam splitter 104.

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

[0027] The driving mechanism 109, which moves the sample in the Z-axis direction, has the function of scanning the focal position of the signal light along the Z-axis (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-axis composite deflection element 107, and is made circular by the action of the built-in λ / 4 plate, with the polarization rotated 90 degrees from the outward path, and then 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. Reference numeral 203 denotes 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, guided to the detection optical system 112, and split into transmitted light and reflected light by the half beam splitter 113 via the pinhole 111.

[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 relative 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 the information stored in an information storage unit 125, and displays the calculation results including the correction on a display unit 126 to present to the user. The information stored in the information storage unit 125 and the sensitivity correction based on the information will be described later.

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

[0034] FIG. 4 is a side cross-sectional view illustrating the reflectance reference sample 303 and the focal position. The reflectance reference sample 303 comprises a transparent flat plate 402, a container 403, and a substance 404 in contact with the transparent flat plate 402, and is placed in contact with the metal plate 201. 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 aligned, and the reflectance from the interface is obtained. 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] 5A and 5B are schematic diagrams showing a method for quantifying sensitivity correction. First, the reflectance (A ０ ) is measured by the above method before shipping the device, and the information is stored in the information storage unit 125. Next, when measuring the actual sample, the reflectance (A １ ) and stores the information in the information storage unit 125. ０ and A １ The sensitivity coefficient K can be calculated from the value of , and the detection signal can be corrected using Equation 2.

[0036] In FIG. ０ >A １ This is shown for the case of . Possible causes of this include wavelength shifts and fluctuations in laser power of semiconductor lasers due to changes in environmental temperature and aging, dust on optical elements including the objective lens, and changes in the optical elements themselves over time, which change the optical characteristics and reduce the output signal. By using the above-mentioned method for quantifying sensitivity correction, it is possible to greatly reduce the impact of these factors on the measurement results.

[0037] (Measurement flow) FIG. ０ This flowchart can be implemented by the signal processing unit 124. The same applies to the following flowcharts. The focus position of the signal light is moved to the plane position of the reflectance reference sample 303, and the light source 100 is set to the standard power P ０ The Z-axis position Z0 of the boundary surface and the position R of the reference beam mirror 106 are initialized. Z0 is determined by measuring the signal while scanning Z0 around the transparent window 405. The signal is measured while scanning R at the determined Z0, and the mirror position R0 where the optical path lengths of the signal beam and the reference beam match is determined. The signal level obtained at this point is called A. ０ and stores the result in the information storage unit 125 as the above.

[0038] A ０and A １ Since measurements are performed using the same optical output power, the standard power is used here. The reason for using the standard power is to prevent the signal level of the measurement result from saturating. Under the condition that this non-saturated signal level is obtained, the reflectance of the sample and the reflectance of the reflectance reference sample 303 must be approximately the same (the difference between the two must be within an acceptable range from the viewpoint of measurement accuracy).

[0039] FIG. 7 shows A １ 1 is a diagram showing an example of a measurement flow. １ 6 and 7. The signal processing unit 124 measures the A obtained by ０ and A １ Calculate the sensitivity coefficient K from

[0040] FIG. 8 shows A １ 7 is a diagram showing another example of the measurement flow of . In addition to the flow of FIG. 7, a predetermined value is set for the sensitivity coefficient K, and A ０ and A １ If the sensitivity coefficient K calculated from is outside a predetermined range, the signal processor 124 can execute sensitivity abnormality processing. For example, the signal processor 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 shows A １ This figure shows an example of a measurement flow in which a real sample is subsequently measured after measuring the reflectance reference sample. 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 results of the real sample are calibrated based on the sensitivity coefficient K. Through the above processing, the particle size and density distribution of the real sample are calculated, and the results are displayed on the display unit 126.

[0042] (Arrangement of Reflectance Reference Sample) FIG. 10 is a diagram showing the arrangement of the reflectance reference sample 303. While FIG. 3 shows an example in which the reflectance reference sample 303 is arranged together with other sample containers 304 in the frame 302, it may also 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 the 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 placing the reflectance reference sample 303 outside the frame 302, samples to be measured can be placed at all positions within the frame 302, thereby maximizing the number of samples that can be measured. This placement also makes it possible to configure the reflectance reference sample 303 placed outside the frame 302 so that it cannot be removed by the user. 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 detachable or may be fixedly attached.

[0044] (Thickness of Transparent Plate) Figure 11A shows the calculation results for the thickness variation specifications of the transparent plate 402 required to perform sensitivity calibration and achieve high-precision size measurement. To measure particle size distribution with high precision, the configuration of the reflectance reference sample 303 must also satisfy certain specifications, similar to the configuration specifications of the sample container. As is well known, deviations in the thickness of the transparent plate 402 from a predetermined value optically result in spherical aberration. The effect on the detection signal can be treated as a decrease in Strehl intensity due to wavefront aberration. The decrease in the detection signal (Strehl intensity) due to deviations in the thickness of the transparent plate 402 was calculated. The results shown here were obtained using a semiconductor laser with a wavelength of 785 nm as the light source 100, a microscope lens with a numerical aperture of 0.45 as the objective lens 108, and borosilicate glass with a thickness of 175 μm (measured refractive index = 1.520) 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 the 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 Strehl intensity of the transparent plate 402. As can be seen from the figure, the tolerance range for 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 in FIGS. 12 and 29 described below must be optically compatible with the sample container 304 shown in FIG. 3 in order to obtain accurate sensitivity. Specifically, the transparent plate 402 formed at the bottom must have a thickness of 175±70 μm and a refractive index of 1.520±0.22. An optical-quality borosilicate glass substrate can be used as the material for the transparent plate 402 that satisfies these requirements.

[0047] 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 on the surface of the transparent flat plate 402 opposite to the side that contacts the metal plate 201, at a location inside the outer diameter 1202 of the transparent window 405 and outside the container opening 1203.

[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 on the surface of the transparent flat plate 402 opposite the side that contacts the metal plate 201, at a location inside the container opening 1203. ID-C can be formed in an optically identifiable form on the surface of the transparent flat plate 402 on the side that contacts the metal plate 201, at a location inside the outer diameter 1202 of the transparent window 405 and outside the container opening 1203. 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, it is also possible to record in a pattern that mixes two or more of the three patterns of ID-A, ID-B, and ID-C.

[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, thereby shortening the movement distance of the sample stage and enabling shorter measurement time. ID-C not only has the same advantage as ID-A in that it can widen the ID information area, but also has the advantage that the information can be formed after the reflectance reference sample 303 is assembled, simplifying the sample fabrication process. 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, the ID information may be the reflectance value itself, or the reflectance value classified into levels based on a certain standard, such as high reflectance, medium reflectance, and low reflectance. The ID information may also be the refractive index value itself, or the refractive index value classified into levels based on a certain standard, such as high refractive index, medium refractive index, and low refractive index. The ID information may also be the optimal power value of the laser beam for reflectance measurement, or the optimal laser beam power value classified into levels based on a certain standard, such as high power, medium power, and low power. While the above standards are exemplified as three levels, high, medium, and low, the number of levels can be selected arbitrarily. The standard can also be determined arbitrarily. The ID information may also be information about the material used in the reflectance reference sample 303, such as resin, dielectric, metal, pure water, or oil. The information classified into levels based on a certain standard and the information about the material are defined as the type information of the sample. The type information makes it possible to determine that a 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 or the like, it is possible to record information similar to that described above. Recording a serial number has the advantage of facilitating traceability of each reflectance reference sample 303 and making it easier to guarantee quality.

[0053] The ID information can be recorded by known methods 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. However, the patterns that can be used are not limited to these, and any other known pattern can be used as long as it is optically identifiable.

[0055] FIG. 13 shows an example of the data structure of ID information 1201. Here, the ID information is configured to include 8 bytes of ID information plus 8 bytes of error correction code. 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 laser power to be irradiated onto 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 implemented 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 regarding the orientation of the 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. This information can be a one-dimensional barcode, a two-dimensional barcode, a combination of a specific dot size and density, or other known patterns. Note that the information indicating the orientation of the ID information does not need to be recorded in all four locations; it only needs to be recorded in at least one location. However, recording information in multiple locations makes it easier to identify the orientation of the ID information. In particular, if different information is recorded in four locations, it is only necessary to read information from at least one of the four locations to identify the orientation of the ID information. This makes it easier to identify the orientation of the ID information pattern, and therefore 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, top right and bottom right, bottom right and bottom left, and bottom left and top left. It may be a dummy pattern such as a meaningless repeated pattern, or information indicating the top edge, right edge, bottom edge, and left edge, respectively, may be recorded. Information regarding the position, such as address information, may be recorded on each of these edges. By using the information represented by each edge, for example, the top left pattern can be identified and the position of the edge connected to it can be 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 edge can suggest the position of the ID information.

[0058] Figure 14B is an enlarged view of the ID information pattern in Figure 14A. Each of the information in the upper left, upper right, lower left, and lower right areas is recorded, for example, in a size of approximately 50 μm x 50 μm, with ID information recorded within an area of ​​approximately 200 μm x 200 μm. As shown in Figure 14B, the ID information is recorded as a barcode string approximately 25 μm wide from the upper left to the upper right. Each barcode unit is approximately 5 μm long, and white or black information marks are recorded in a predetermined pattern. With 20 barcode units representing 1 byte of information, 2 bytes of information are recorded per barcode string. Since the barcode string width is 25 μm, eight barcode strings can be recorded in an ID information area of ​​approximately 200 μm x 200 μm, allowing a total of 16 bytes of information to be recorded. A barcode string with a width of 25 μm and a length of 5 μm per unit can be fabricated by photolithography, but this fabrication method is not limited to this.

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

[0061] Figures 16A and 16B show another example of an ID information pattern. In this example, ID information is recorded using a combination of a predetermined dot size and a predetermined dot density. By specifying three types of dot size and three types of dot densities, it is possible to record 3 x 3 = 9 different types of information by combining these (Figure 16B). This method has the disadvantage of a small amount of ID information, but has the advantage that the same pattern is recorded repeatedly, so if it is recorded over a wider area than the scanning range of the laser beam, the ID information can be easily read even if the scanning range is shifted. Similarly, the barcode patterns described in Figures 14 and 15 may also have the same information repeatedly arranged.

[0062] In the case of the ID information pattern shown in Figure 14A, the scanning range of the laser light of the particle measuring device according to this embodiment is approximately 300 µm x 300 µm, so it is possible to read all of the ID information with a single scan, which takes approximately 0.1 seconds. On the other hand, in the case of the ID information pattern shown in Figure 15A, multiple scans are required to read all of the ID information. Specifically, all of the ID information can be read by scanning seven times horizontally and two times vertically, which takes approximately 15 seconds.

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

[0064] The dots are arranged so that no dots 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 by knowing which patterns are combined, so the orientation of the patterns is not important.

[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 in the frame 302 of FIG. 3A. This allows for positional deviation in the xy plane to be limited to a value corresponding to the design dimensions and processing accuracy of the frame 302 and the reflectance reference sample 303. This allows for the approximate position to be known in advance when reading ID information placed in the position shown in FIG. 12, thereby shortening the access time of the optical head for reading the ID information. In this case, by providing a notch in part of the reflectance reference sample 303 as shown in the figure, it is possible to prevent incorrect orientation when installing the reflectance reference sample 303 in the frame 302. Note that the reflectance reference sample 303 shown in this figure does not necessarily need to be directly installed in the frame 302; it may be installed outside the frame 302.

[0066] 17B and 17C are schematic diagrams showing other examples of the shape of the reflectance reference sample 303 in the xy plane. FIG. 17B has one transparent window, while FIG. 17C has 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, maximizing the number of measurement samples. Note that the reflectance reference samples 303 shown in FIGS. 17B and 17C may also be installed inside the frame 302. They can be installed so that their short sides, long sides, corners, or notches match the frame 302.

[0067] FIG. 18 shows the structure and reflectance of the highly reflective portion of the ID information mark in ID-A. Specifically, the material of the highly reflective portion (the portion where information is recorded) is Al, and the calculation results for Al film thickness and reflectance are shown. The reflectance of the portion of ID-A without ID information (background reflectance) 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 approximately 10 times the background reflectance can be obtained, allowing the ID information mark to be reliably read. To clearly recognize the ID information, it is considered sufficient for the reflectance of the ID information mark to be at least twice the background reflectance (i.e., the reflectance of the sidewall of the container 403). The same is true for ID-B and ID-C.

[0068] Figures 19A to 19C show the specific structure and reflectance of the high-reflectance and low-reflectance portions of the ID information mark of ID-B. The background reflectance of ID-B is approximately 5% when a resin material is used as the filler material. Therefore, 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 at least 10 times that of the background reflectance. This enables reliable reading of the ID information mark. Figure 19A shows the calculation results of the reflectance versus Al film thickness when Al is used for the high-reflectance portion. From these results, it can be seen that setting the Al film thickness to 7 nm or more achieves a reflectance approximately 10 times the background reflectance, allowing reliable reading of the ID information mark.

[0069] On the other hand, when the reflectance of the ID information mark is set lower than the background reflectance in ID-B, the background reflectance of ID-B is approximately 5%, so the reflectance of the ID information mark should be set to 0.5% or less. Figure 19B shows the calculation results for the reflectance versus W film thickness when the low-reflectance portion has a four-layer structure of ZnS(1) / W / ZnS(2) / Al. Here, the ZnS(1) film thickness is 20 nm, the ZnS(2) film thickness is 60 nm, and the Al film thickness is 50 nm. Therefore, if the W film thickness is 8.2 to 11.3 nm, a reflectance of less than one-tenth of the background reflectance can be obtained, allowing the ID information mark to be reliably read.

[0070] 19C shows another example of ID-B where the reflectance of the ID information mark is set lower than the background reflectance. In this example, ZnS and SiO ２ A multilayer structure with a dielectric layer of ZnS was used. ２ and ZnS were alternately provided. The layer furthest from the glass bottom plate was ZnS. In one multilayer structure, SiO ２ All the ZnS layers except for the layer closest to the glass bottom plate and the layer furthest from the glass bottom plate had the same thickness, and the ZnS layer closest to the glass bottom plate and the ZnS layer furthest from the glass bottom plate had half the thickness of the other ZnS layers. ２ The thicknesses of the SiO and ZnS layers were set to different values. ２ The reflectance versus number of layers was calculated by changing the thickness of the ZnS and ZnS layers independently, and the reflectance with the smallest reflectance was taken as the reflectance for that number of layers, as shown in Figure 19C. From these results, it can be seen that if the number of dielectric layers is five or more, a reflectance of 0.5% or less, which is one-tenth or less of the background reflectance, can be obtained, and the ID information mark can be reliably read.

[0071] 19B and 19C, it has been explained that the reflectance of the ID information mark is one-tenth 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 one-half or less. Similarly, when making the ID information mark darker in ID-C, it is sufficient to make the reflectance of the ID information mark one-half or less of the background reflectance (i.e., the reflectance of the side wall of the container 403).

[0072] Figures 20A to 20C show the specific structure and reflectance of the high-reflectance and low-reflectance portions of the ID-C ID information mark. The background reflectance of the ID-C is the reflectance of air and the glass bottom plate, which is approximately 4%. Therefore, to set a high reflectance for the ID information mark, the reflectance of the high-reflectance portion must be set to 40% or more, which is 10 times that of the high-reflectance portion, thereby enabling reliable reading of the ID information mark. Figure 20A shows the calculation results for reflectance versus Al film thickness when Al is used for the high-reflectance portion (white). From these results, setting the Al film thickness to 5 nm or more results in a reflectance approximately 10 times the background reflectance, allowing reliable reading of the ID information mark.

[0073] On the other hand, in the case of ID-C, when the reflectance of the ID information mark is set lower than the background reflectance, the reflectance of the ID information mark should be set to 0.4% or less, since the background reflectance of ID-C is about 4%. ２ This shows the calculation results of the reflectance versus the ZnS film thickness in the case of a four-layer structure of SiO / W / ZnS / Al. ２ The thickness of the ZnS film was 95 nm, the thickness of the W film was 10 nm, and the thickness of the Al film was 50 nm. From these results, if the thickness of the ZnS film is set to 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] 20C shows another example of ID-C where the reflectance of the ID information mark is set lower than the background reflectance. Here, ZnS and SiO ２ A multilayer structure with a dielectric layer of ZnS was used. ２ and ZnS were alternately provided. The layer furthest from the glass bottom plate was ZnS. In one multilayer structure, SiO ２ All the ZnS layers except for the layer closest to the glass bottom plate and the layer furthest from the glass bottom plate had the same thickness, and the ZnS layer closest to the glass bottom plate and the ZnS layer furthest from the glass bottom plate had half the thickness of the other ZnS layers. ２ The thicknesses of the SiO and ZnS layers were set to different values. ２The reflectance versus number of layers was calculated by changing the thickness of the ZnS and ZnS layers independently, and the reflectance with the smallest reflectance was taken as the reflectance for that number of layers, as shown in Figure 20C. From these results, it can be seen that if the number of dielectric layers is five or more, a reflectance of 0.4% or less, which is one-tenth or less of the background reflectance, can be obtained, and the ID information mark can be reliably read.

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

[0076] FIG. 21 shows the A associated with the ID information when the ID information is ID-A, ID-B, or a mixed pattern of these in FIG. 12. ０ 1 is a diagram showing an example of a measurement flow of the signal light condensing position being moved to a plane position of the reflectance reference sample 303, and the light source 100 is set to the standard power P ０ The Z-axis position Z0 of the interface and the position R of the reference beam mirror 106 are initialized. Z0 is determined by measuring the signal while scanning in the Z direction around the transparent window 405. At the determined Z0, the signal is measured while scanning R, and the mirror position R0 at which the optical path lengths of the signal beam and the reference beam match is determined. Next, while keeping the Z position at Z0, the focusing position is scanned in the X and Y directions to move to the ID information position, and the ID information 1201 of the reflectance reference sample 303 is read. Based on the acquired ID information, light is emitted at power P1 for measuring the reflectance reference sample, and the signal level obtained at this point is expressed as A. ０ This signal level A ０ The signal level A is linked to the ID information and stored in the information storage unit 125. If there are multiple types of reflectance reference samples, the above flow is repeated to store the signal level A for each ID information. ０ is stored in the information storage unit 125.

[0077] FIG. 22 shows the A associated with the ID information when the ID information is ID-C in FIG. ０ 21 is a diagram showing an example of a measurement flow of the above-mentioned measurement method, which is the same as that of FIG. 21 except that the Z position before reading the ID information is moved in the minus Z direction from Z0 by the thickness of the glass bottom plate.

[0078] FIG. 23 shows the state of the ID information when it is ID-A, ID-B or a mixed pattern of these in FIG. 12, after reproducing the ID information. １ 1 is a diagram showing an example of a flow for measuring a reflectance reference sample. When a user selects a measurement object, the ID information of the reflectance reference sample suitable for that measurement object is first read. Based on the contents of the ID information, it is determined whether the reflectance reference sample for the measurement object selected by the user is normal, and if normal, the next flow is carried out, and if 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 mixing up reflectance reference samples. After it is determined that the reflectance reference sample is normal, a laser beam is emitted with a power for measuring the signal level of the reflectance reference sample based on the contents of the ID information, and A is measured. １ Measure and A ０ and A １ The sensitivity coefficient K is calculated from the following equation. A predetermined value is set for the sensitivity coefficient K, and A ０ and A １ If the sensitivity coefficient K calculated from is outside a predetermined range, the signal processor 124 can execute sensitivity abnormality processing. For example, the signal processor 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 shows a case where the ID information is ID-C in FIG. 12, and then after the ID information is reproduced, A １ 23 is a diagram showing an example of a flow for measuring the Z position before reading the ID information. This is the same as FIG. 23 except that the Z position before reading the ID information is moved in the minus Z direction from Z0 by the thickness of the glass bottom plate.

[0080] FIG. 25 shows the state of the ID information when it is ID-A, ID-B or a mixed pattern of these in FIG. 12, after reproducing the ID information. １This is a diagram showing an example of a measurement flow in which a reflectance reference sample is measured and then an actual sample is measured. When the 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 normal, the next flow is carried out, and if abnormal, abnormal processing such as issuing an alarm is carried out. This makes it possible to prevent human errors such as mixing up reflectance reference samples. After it is determined that the reflectance reference sample is normal, a laser beam is emitted with a power to measure the signal level of the reflectance reference sample based on the contents of the ID information, and A １ Measure and A ０ and A １ The sensitivity coefficient K is calculated from the following equation. A predetermined value is set for the sensitivity coefficient K, and A ０ and A １ If the sensitivity coefficient K calculated from is outside a predetermined range, 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. The flow after obtaining the sensitivity coefficient K is the same as that shown in FIG. 9.

[0081] FIG. 26 shows the state after reproducing the ID information when the ID information is ID-C in FIG. 12. １ 25 is a diagram showing an example of a measurement flow in which the Z position before reading the ID information is moved in the minus Z direction from Z0 by the thickness of the glass bottom plate.

[0082] The important thing in Figures 21 to 26 is the A linked to ID information. ０ Measurement of A, which is performed before the actual measurement １ The laser light power for measuring the sensitivity and for actually measuring the sensitivity should be set to the same value P1. This makes it possible to prevent sensitivity deviations due to differences in laser light power.

[0083] (Instrumental Differences, etc.) By using the reflectance reference sample 303, even if there are instrumental differences in the particle measurement device (such as minute differences in laser wavelength, optical system, detector sensitivity, etc.), it is possible to obtain nearly identical results for the same sample. Furthermore, it can also be used to diagnose equipment malfunctions. Specifically, if the sensitivity coefficient K falls outside a certain numerical range, possible causes include deterioration of the laser due to aging, adhesion of dust or the like to the optical elements, or misalignment of the optical system. Therefore, the signal processing unit 124 can issue an alert on the display unit 126, for example, to prompt equipment maintenance or repair.

[0084] (Solvent Refractive Index Measurement) Figure 27A is a schematic diagram showing the relationship between a container containing a real sample containing particles 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 in the real sample. In the figure, 1401 is the sample, and 1402 is sealing tape. 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 for maximizing 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. In this case, using Fresnel's equation, the magnitude of the detection signal can be expressed by Equation 1.

[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 taken as E0 ｓｉｇ Let E0 ｓｉｇ is expressed by Equation 4. Equation 5 is derived from Equation 1 and Equation 4. In Equation 5, the refractive index of the solvent, n ｍSince the values ​​other than are known or measured values, the refractive index n of the solvent can be calculated using Equation 5. ｍ It is possible to measure the following.

[0087]

[0088]

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

[0090] <Embodiment 2> (Refractive Index: Dielectric Film) Figure 28A shows the reflectance of a reflectance reference sample 303 according to embodiment 2 of the present invention. In embodiment 2, a dielectric film was used as the material in contact with the transparent flat plate 402 of the reflectance reference sample 303 shown in Figure 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 varies depending on the film thickness of the dielectric film due to interference between reflection from the laser light incident surface of the dielectric film and reflection from the surface further back. Figure 28A shows the relationship between the film thickness of the ZnS film used in embodiment 2 and the reflectance.

[0091] 28B shows the reflectance of another example of the configuration of the reflectance reference sample 303 of the second embodiment. In this example, a dielectric film SiO is formed by sputtering on a dielectric film ZnS formed on a transparent flat plate 402. ２ Then, a dielectric film ZnS was formed by sputtering. The reflectance of the reflectance reference sample 303 having this structure was ２ At a film thickness of 133 nm, the film thickness changes as shown in FIG. 28B with respect to the film thickness of the ZnS dielectric film.

[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 film.

[0093] In addition to proteins, particles that can be measured in the present invention include metal particles, plastic particles, and the like. Reflectance varies depending on the type of particle, but in any case, the actual measurement sample is a dispersion of submicron-sized particles in a solvent, so the absolute value of the reflectance does not become large. From the viewpoint that the laser light power must be the same in the actual measurement and the reflectance reference sample measurement in order to adjust the sensitivity correctly, it is preferable that the absolute value of the reflectance of the reflectance reference sample also not be large.

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

[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, etc. In that case, the material and film thickness of the dielectric film can be selected to create a configuration that corresponds to the reflectance of the target particle.

[0096] Third Embodiment In a third embodiment of the present invention, a specific example of a 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 Reference 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 Figure 4. Since bubbles mixed in the interface affect the reflected signal, to avoid this, the ultraviolet curing resin was placed in a vacuum container and degassed by evacuating for 10 minutes. Thereafter, the resin was poured into the container 403, and ultraviolet light of approximately 300 mJ / cm was applied 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, if protein particles are used as the sample for measuring size distribution, the difference in refractive index between the solvent and the protein particles is approximately 0.06. In the reflectance reference 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 as the transparent flat 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 used to measure 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 that the reflectance reference sample 303 be one that undergoes little optical change over time and can be used 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 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 conditions 1.41: Lower power than that in the protein particle sample measurement 1.43: Power approximately equal to that in the protein particle sample measurement 1.48: Power approximately equal to that in the protein particle sample measurement 1.50: Power approximately equal to that in the protein particle sample measurement 1.52: Higher power than that in the protein particle sample measurement

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

[0104] Figure 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. was 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 Figure 16, 1601 is the liquid resin. In this case, the liquid is sealed with sealing tape or the like to prevent leakage.

[0105] (How to use the reflectance reference sample) When measuring multiple real samples, the reflectance reference sample 303 may be measured only once at the start of measurement, or the reflectance reference sample 303 may be measured for each individual measurement of multiple real samples.

[0106] When the measurement time required to measure the actual sample is relatively short, the changes in the environmental temperature and laser temperature during the measurement of the actual sample are relatively small, so 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, if the measurement time required to measure a real sample is relatively long, there is a possibility that the environmental temperature or the laser temperature may change during the measurement, and therefore, more accurate measurements can be achieved 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 measuring a certain number of real samples, rather than for each measurement.

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

[0109] <Regarding Modifications of the Present Invention> The present invention is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those including all of the described configurations. Furthermore, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace part of the configuration of each embodiment with other configurations.

[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.

[0112] 100: Light source 101: Laser driver 102: Collimating lens 103: λ / 2 plate 104: Polarizing beam splitter 105: λ / 4 plate 106: Reference beam mirror 107: Composite deflection element 108: Objective lens 109: Driving 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 storage unit 126: Display unit 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 well of sample container 204: Sample 302: Frame 303: Reflectance reference sample 304: Sample container 400: Focused light 402: Transparent flat plate 403: Container 404: Material 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 size measurement results in a particle measuring device that measures the size of particles contained in a liquid sample by irradiating the sample with light, wherein the reference sample has a reference sample window made of the same material and thickness as a sample container window of a sample container that contains the sample, the reference sample has a material at a position in contact with the reference sample window, the reference sample has a reflectance whose difference with the reflectance of the sample is within an acceptable range, the reference sample window is formed on a transparent flat plate placed at the bottom of the reference sample, the transparent flat plate has a thickness in the range of 175±70 μm and a refractive index of the transparent flat plate in the range of 1.520±0.22, a geometric pattern representing ID information that identifies the reference sample is formed on one surface of the transparent flat plate, and the geometric pattern is configured to be optically readable.

2. The reference sample according to claim 1, characterized in that the ID information includes information indicating the orientation of the shape pattern.

3. The reference sample according to claim 2, characterized in that the shape pattern has a rectangular periphery, and the shape pattern has a portion at at least one of the four corners of the rectangle that represents information suggesting an orientation of the shape pattern.

4. The reference sample according to claim 2, characterized in that the shape pattern has a rectangular periphery, and the shape pattern has a portion on at least one of the four sides of the rectangle that represents information suggesting the position of the shape pattern.

5. The reference sample according to claim 1, characterized in that 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 planar density of the dots, and the dot pattern is arranged such that the dots do not overlap each other, regardless of the combination of size and planar density.

6. The reference sample according to claim 1, characterized in that the reference sample has a notch that indicates the orientation of the reference sample.

7. The reference sample according to claim 1, characterized in that the planar size of the shape pattern is approximately 300 μm × approximately 300 μm, and the ID information is represented by arranging multiple shape patterns representing the ID information within a planar area smaller than the planar size.

8. The reference sample according to claim 1, characterized in that the planar size of the shape pattern is approximately 600 μm × approximately 2000 μm, and the ID information is represented by arranging multiple shape patterns representing the ID information within a planar area smaller than the planar size.

9. The reference sample according to claim 1, characterized in that the ID information includes at least information regarding the type of the reference sample, and the ID information further includes at least one of the following: a serial number of the reference sample, the type of the reference sample, the refractive index of the reference sample, and the laser power of the light to be irradiated onto the reference sample, and the ID information includes 4 bytes or more of information.

10. The reference sample according to claim 1, characterized in that the shape pattern is formed on the surface of the transparent flat plate that comes into contact with the substance, the shape pattern is positioned below the side wall of a container in which the reference sample holds the substance, and the reflectance of the shape pattern is at least twice the reflectance of the side wall.

11. The reference sample according to claim 1, characterized in that the shape pattern is formed on the surface of the transparent flat plate that contacts the substance, the shape pattern is disposed between the substance and the transparent flat plate, and the reflectance of the shape pattern is more than twice the reflectance of the substance or less than half the reflectance of the substance.

12. The reference sample according to claim 1, characterized in that the shape pattern is formed on the surface of the transparent flat plate that is not in contact with the substance, the shape pattern is positioned below the side wall of a container in which the reference sample holds the substance, and the reflectance of the shape pattern is more than twice the reflectance of the side wall or less than half the reflectance of the side wall.

13. A particle measuring device for measuring the size of particles contained in a liquid sample, comprising: a light source for emitting light; a branching section for branching the light from the light source into signal light and reference light; an irradiation section for collecting the signal light and irradiating it onto the sample; a detection section for detecting an interference signal obtained by interfering between the reflected light from the particle and the reference light; and a processing section for measuring the size of the sample using the interference signal detected by the detection section and calibrating the size using the measurement result of a reference sample, wherein the reference sample has a reference sample window having the same material and thickness as a sample container window provided in a sample container that contains the sample, wherein the reference sample has a material having a reflectance whose difference with the reflectance of the sample is within an allowable range, at a position in contact with the reference sample window, wherein the reference sample window is formed on a transparent flat plate arranged at the bottom of the reference sample, wherein the transparent flat plate has a thickness in the range of 175±70 μm and a refractive index of the transparent flat plate in the range of 1.520±0.22, a shape pattern representing ID information for identifying the reference sample is formed on either surface of the transparent flat plate, the shape pattern is configured to be optically readable, and the processing unit reads the ID information based on a signal obtained by irradiating the reference sample with the light.

14. A particle measurement method for measuring the size of particles contained in a liquid sample, comprising: a step of emitting light from a light source; a step of splitting the light from the light source into signal light and reference light; a step of concentrating 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 a step of calibrating the size using a reference sample, wherein the reference sample has a reference sample window having the same material and thickness as a sample container window provided in a sample container that contains the sample, and the reference sample has a substance having a reflectance whose difference with the reflectance of the sample is within an allowable range, at a position in contact with the reference sample window, the reference sample window is formed on a transparent flat plate arranged at the bottom of the reference sample, the transparent flat plate has a thickness in the range of 175±70 μm and a refractive index of the transparent flat plate in the range of 1.520±0.22, and a shape pattern representing ID information for identifying the reference sample is formed on any surface of the transparent flat plate, the shape pattern is configured to be optically readable, and in the calibrating step, the ID information is read based on a signal obtained by irradiating the reference sample with the light.

15. The particle measuring method according to claim 14, further comprising a step of determining whether the reference sample is of a type suitable for calibrating the size, and if not, carrying out the above process, wherein in the step of determining, 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.

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