Particle measurement device

Abstract

The present invention provides a particle measurement device that measures the size of a particle by irradiating the particle in a liquid sample with light, and that suppresses variation in detection sensitivity caused by individual characteristics (machine difference) of the device. This particle measurement device measures the size of a particle in a liquid sample by irradiating the particle with light, and corrects the size of the particle using a correction function in which the irradiation position of a spot in at least one of the X, Y and Z directions is used as an input value.

Description

Technical Field

[0001] This invention relates to a technique for measuring particles in a solvent using light. Background Technology

[0002] In recent years, the focus of pharmaceutical development has shifted from low-molecular-weight drugs to biopharmaceuticals. Biopharmaceuticals, being high molecules, are prone to aggregation, which can sometimes lead to toxicity. For example, the U.S. Food and Drug Administration (FDA) and other regulatory bodies have tightened controls on the concentration of aggregates. Therefore, for aggregates in the submicron region (0.1–1 μm), techniques are needed to quantitatively measure the size distribution of desired concentrations. Protein aggregates float in solvents, and their positions change over time due to Brownian motion. Hereinafter, this invention describes techniques for measuring the size and concentration of protein aggregates and standard particles such as polystyrene beads. These analytes will be collectively referred to as "particles."

[0003] Patent Document 1 describes a technique for detecting particles using optical measurement. This document describes "an optical measurement method that focuses light to generate a light spot, and measures an object of inspection approximately three times the size of the light spot, characterized by comprising: a signal acquisition step in which the object of inspection is irradiated while at least the focal point of the light is moved in the direction of the optical axis, thereby detecting reflected light reflected from the object of inspection; a step in acquiring correspondence data describing the correspondence between the intensity of the reflected light and the size of the object of inspection; and a size calculation step in which the size of the object of inspection is obtained by querying the correspondence data using the intensity of the reflected light" (claim 1). The technique described in this document enhances the signal by interfering with a reference light, thereby eliminating the need for preprocessing and enabling high-resolution measurement.

[0004] Patent Document 2 discloses a technique that physically scans the objective lens and uses four detectors with different phase conditions to receive the interference between the signal light and the interference light, thereby eliminating the need for phase adjustment of the reference light in time-domain OCT (Optical Coherence Tomography) based on mirror scanning. Furthermore, based on the technique in Patent Document 1, a method is disclosed to accelerate the scanning of the light spot to avoid the influence of the motion of particles undergoing Brownian motion in the liquid.

[0005] Patent document 3 discloses the following technology: In the measurement of biological tomographic images using semiconductor lasers as light sources, the coherence length is controlled within a predetermined range by superimposing high frequency on the driving current, thereby reducing the influence of noise contained in the obtained tomographic images.

[0006] Patent document 4 discloses the following technology: an apparatus for measuring the three-dimensional shape of an electronic component by irradiating it with irradiation light that scans in mutually orthogonal main scanning direction and sub-scanning direction and receiving reflected light from the electronic component, wherein the positional offset of the irradiation light is corrected according to the light-receiving position of a pair of photodetectors disposed at a position offset from the optical axis of the irradiation light.

[0007] Patent document 5 discloses the following technology: irradiating an object with a laser, focusing the reflected light from the surface of the object onto an array of position detection elements, and measuring the signal of the position detection elements output according to the position of the reflected light by the principle of triangulation, thereby measuring the height or shape of the object in a non-contact manner.

[0008] Patent document 6 discloses a technology related to a detection device, which includes a control unit that illuminates a laser onto an object through a projection window, detects reflected light from the object, and generates measurement information. The detection device corrects the signal value based on either the detection time or the detection intensity of the reflected light from the projection window.

[0009] Patent Document 1: Japanese Patent No. 06559555

[0010] Patent Document 2: WO2020 / 144754

[0011] Patent Document 3: Japanese Patent Application Publication No. 2015-49204

[0012] Patent Document 4: Japanese Patent Application Publication No. 2008-267853

[0013] Patent Document 5: Japanese Patent Application Publication No. 2000-292127

[0014] Patent Document 6: Japanese Patent Application Publication No. 2019-045224 Summary of the Invention

[0015] As described above, the technology described in Patent Documents 1 and 2, by pre-obtaining the correspondence data between particle size and the maximum detection signal obtained at the focal position, can determine the particle size based on the measured maximum detection signal Amax. Furthermore, the particle concentration can be determined based on the number of detected particles. On the other hand, when managing performance by suppressing machine differences among multiple devices of the same model, due to the inherent characteristics of each device, such as the emission angle, coherence length, and objective lens mounting position of the semiconductor laser as the light source, variations in the energy of the light spot and the intensity of interference with the reference light occur during three-dimensional scanning of the light spot within the measurement object. Therefore, it is difficult to keep the magnitude of the detection signal constant, i.e., it is difficult to suppress variations in detection sensitivity.

[0016] In Patent Documents 4-6, for measurement objects with wavelengths sufficiently large than the light source, the position correction of the detection signal is achieved based on the position of the reflected light, not the intensity of the reflected light, using geometric optics methods such as triangulation. Therefore, it is difficult to suppress fluctuations in detection sensitivity based on the wave optical response from tiny particles with wavelengths equal to or smaller than the wavelength of the light source targeted by this invention.

[0017] The present invention was made in view of the above-mentioned problems, and its object is to suppress the variation in detection sensitivity caused by individual characteristics (mechanical differences) of the device in a particle measuring device that measures the size of particles by irradiating them with light in a liquid sample.

[0018] The particle measuring device of the present invention measures the size of particles by irradiating them with light in a liquid sample, and corrects the particle size using a correction function that uses the irradiation position of the light spot in at least one of the XYZ directions as the input value.

[0019] According to the present invention, in a particle measuring apparatus that measures the size of particles by irradiating them with light within a liquid sample, variations in detection sensitivity caused by individual characteristics (mechanical differences) of the apparatus can be suppressed. Other issues, structures, and effects not shown herein will become clear from the following description of embodiments. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the relationship between focus offset and detection signal, as shown in Patent Document 1 and Patent Document 2.

[0021] Figure 2 The simulation results of this invention use the interferometric detection optical system described in Patent Documents 1 and 2 to represent the relationship between particle size and maximum detection signal.

[0022] Figure 3 An example representing measurement data.

[0023] Figure 4A This is an example of an experimental result that quantifies changes in detection sensitivity.

[0024] Figure 4B This is an example of an experimental result that quantifies changes in detection sensitivity.

[0025] Figure 5 This is a schematic diagram showing the relationship between the intensity distribution of the semiconductor laser as a light source and the emitted light.

[0026] Figure 6A This is a schematic diagram showing a laser beam forming a spot in the center of the X or Y direction.

[0027] Figure 6BThis is a schematic diagram showing a laser beam forming a spot at the end in the X or Y direction.

[0028] Figure 7 This is a schematic diagram illustrating the change in detection sensitivity when scanning the light spot in the Z direction.

[0029] Figure 8 It is a flowchart illustrating the steps for calibrating the individual characteristics of each device.

[0030] Figure 9 It is a flowchart showing the actions that make the device operate in the operating environment.

[0031] Figure 10A The result is based on the correction model of the first-order polynomial shown in Equation 1.

[0032] Figure 10B The result is based on the correction model of the quadratic polynomial shown in Equation 2.

[0033] Figure 10C The result is based on the correction model of the cubic polynomial shown in Equation 3.

[0034] Figure 10D The result is based on the correction model of the fourth-order polynomial shown in Equation 4.

[0035] Figure 10E The result is based on the three-dimensional Gaussian distributed correction model shown in Equation 5.

[0036] Figure 10F The results are based on the Gaussian distributed correction model with three-dimensional compensation (offset) shown in Equation 6.

[0037] Figure 11 It is the size frequency distribution of the original data before applying the detection sensitivity correction function of the present invention.

[0038] Figure 12A The result is based on the correction model of the first-order polynomial shown in Equation 1.

[0039] Figure 12B The result is based on the correction model of the quadratic polynomial shown in Equation 2.

[0040] Figure 12C The result is based on the correction model of the cubic polynomial shown in Equation 3.

[0041] Figure 12D The result is based on the correction model of the fourth-order polynomial shown in Equation 4.

[0042] Figure 12E The result is based on the three-dimensional Gaussian distributed correction model shown in Equation 5.

[0043] Figure 12F The results are based on the Gaussian distributed correction model with three-dimensional compensation shown in Equation 6.

[0044] Figure 13 This is a graph summarizing the comparison results of RMS values ​​of standard particle sizes that represent the effectiveness of the detection sensitivity correction method of the present invention.

[0045] Figure 14A The result is based on the correction model of the quadratic polynomial shown in Equation 7.

[0046] Figure 14B It is the result of a correction model that combines the quadratic polynomial and Gaussian distribution shown in Equation 8.

[0047] Figure 15A The result is based on the correction model of the quadratic polynomial shown in Equation 7.

[0048] Figure 15B It is the result of a correction model that combines the quadratic polynomial and Gaussian distribution shown in Equation 8.

[0049] Figure 16 This is a graph summarizing the comparison results of RMS values ​​of standard particle sizes that represent the effectiveness of the detection sensitivity correction method of the present invention.

[0050] Figure 17 This is an example of a structural diagram of the particle measuring device according to Embodiment 4. Detailed Implementation

[0051] <Background of the Invention>

[0052] The objects of measurement in this invention are submicron to micron-sized particles that float in solvents and undergo Brownian motion, similar to protein aggregates in biopharmaceuticals. The objective is to precisely measure the size and concentration of these objects using the wave optical response of a laser.

[0053] In the following description of the present invention, the optical axis direction is taken as the Z-axis, and an orthogonal coordinate system is formed with the X and Y axes in the plane orthogonal to it. The positions of the light spots in the X, Y, and Z axis directions are respectively set as x, y, and z.

[0054] Figure 1 This is a schematic diagram illustrating the relationship between focus shift and detection signal, as shown in Patent Documents 1 and 2. If the optical axis of the objective lens is set to z, the spot diameter at the z-position of the particle being measured is determined by the wavelength of the light source, the numerical aperture of the objective lens, and the focus shift. Based on the ratio of the spot diameter to the particle's area, the amount of reflected light from the particle approximately increases or decreases, thus affecting the detection signal. Figure 1The left side is a schematic diagram showing the particle of the measured object defocused from the focal point of the objective lens. In this case, the particle size is relatively small relative to the spot diameter, and the amount of reflected light received by the objective lens and the detection signal converted into an electrical signal are relatively small. On the other hand, Figure 1 The diagram on the right shows the particle of the measured object positioned at the focal point of the objective lens. In this case, the particle size is at its maximum relative to the spot diameter, resulting in the maximum amount of reflected light received by the objective lens and the maximum detection signal. (Patent Document 1) Figure 17 It is disclosed that, under the condition that the size (diameter) of the target particle is approximately three times or less than the minimum spot diameter at the focal point, the particle size can be determined by uniquely determining the maximum detection signal obtained from the particle size and focal position, and by obtaining the corresponding data in advance.

[0055] Figure 2 This is a simulation result of the present invention, which uses the interferometric detection optical system described in Patent Documents 1 and 2 to represent the relationship between particle size and maximum detection signal. The simulation is performed using Mie scattering and Fraunhofer diffraction methods. The wavelength of the light source is 785 nm, the numerical aperture of the objective lens is 0.45, and based on experimental results regarding protein condensates, the refractive index of the particles is 1.40, and the refractive index of the solvent corresponds to 1.333 for pure water. When the particle size is set as d, as shown in the figure, it can be seen that in the region d < 0.1 μm, corresponding to Rayleigh scattering, the maximum detection signal is proportional to the cube of d; in the region d > 0.3 μm, corresponding to Mie scattering, the maximum detection signal is proportional to the first power of d. The linearity of particle size and maximum detection signal in the region d > 0.3 μm is consistent with that in Patent Document 1. Figure 17 Consistent.

[0056] To avoid misunderstandings regarding the dependence on these particle sizes, further explanation is provided. It is generally known that the energy intensity of scattered light in Rayleigh scattering is proportional to the sixth power of the particle size *d*. Similarly, the magnitude of the electrical signal obtained by photodetector photoelectric conversion of the reflected light is also proportional to the sixth power of *d*. Patent Documents 1 and 2, as well as the optical system processed in this invention, utilize zero-difference interference, which amplifies the reflected light by interfering with a reference light. In this case, instead of converting the "energy" of the reflected light, the "electric field amplitude" is converted into an electrical signal, thus the maximum detection signal is proportional to the cube of *d*. By utilizing such an optical system, compared to methods that photoelectrically convert the "energy" of the reflected light, the change in the magnitude of the detection signal caused by particle size is slower, resulting in a wider dynamic range of measurable particle sizes. When using a semiconductor laser as the light source, superimposing a high frequency on the driving current using the technique described in Patent Document 3 can reduce laser noise, primarily caused by reflected light.

[0057] Rayleigh scattering, as is well known, detects light energy proportional to the sixth power of d. In contrast, this method converts the electric field amplitude of the reflected light into a detection signal through interference amplification, thus making it proportional to the cube of d. Compared to conventional methods, this method exhibits superior dynamic range, given that the variation in the magnitude of the detection signal is relatively small relative to the size range of the measured object.

[0058] Based on the above principles, the specific measurement method is explained. Setting the optical axis to Z, the detection signal obtained by three-dimensionally scanning the light spot focused by the objective lens inside the object being measured is called three-dimensional image data. Using an AD converter or similar device, the control signal or position monitoring signal of the light spot scanning in the X, Y, and Z directions, along with the detection signal, are saved as time-series data, and then processed to generate three-dimensional image data.

[0059] Figure 3 An example representing measurement data. Figure 3 The top left is a visualization of the 3D image data. Here, the scanning range of the light spot is set to 300μm, 300μm, and 50μm in the X, Y, and Z directions, respectively. Figure 3 The top right image shows a magnified view of the data of a single separated particle. The maximum value of the detection signal, Amax, obtained under the condition that the position of the light spot and the position of the particle are the same, is the size evaluation index of the particle. Figure 3 The lower part shows the result of superimposing the measured defocus of multiple particles with the magnitude of the detection signal. As shown in the figure, it can be seen that the detection signal Amax when the defocus is zero (focus) becomes the evaluation index of the particle size.

[0060] To facilitate understanding of the present invention, specific experimental results are shown here relating to the variation in detection sensitivity during spot scanning due to individual characteristics in two devices.

[0061] Figures 4A-4B This is an example of an experiment that quantifies changes in detection sensitivity. Here, the focus of the light spot is aligned with the bottom glass of the sample container to obtain the detection signal. Figure 4A This represents a two-dimensional image after converting the detection signal from one device into brightness information. As shown in the figure, the change in the detection signal when scanning the light spot along the X-direction is dominant, reaching its maximum in the center and its minimum at the right end. The reflectivity of the bottom glass of the sample container is constant; therefore, the variation in the detection signal, i.e., the variation in the detection sensitivity of the device, is approximately 15%. Figure 4B This represents a two-dimensional image after converting the detection signals from other devices into brightness information. As shown in the figure, the change in the detection signal when scanning the light spot along the X-direction is dominant, reaching its maximum at the right end and its minimum at the left end. The variation in the device's detection signal, i.e., the variation in detection sensitivity, is approximately 43%.

[0062] To avoid misunderstanding regarding the sensitivity variation shown here, further explanation is provided. The measured object is tiny particles suspended within the sample container. If the standard measurement position for the three-dimensional image data is set to approximately 500 μm away from the bottom glass of the sample container, the optical system of the device performs aberration correction at that point with spherical aberration minimized. Therefore, when measuring the bottom glass of the sample container, spherical aberration occurs by approximately 2λ. Consequently, the sensitivity variation shown in Figure 4 is larger than the sensitivity variation that occurs when actually measuring tiny particles such as protein condensates, which are the measured object. The object of this invention is to suppress the sensitivity variation that occurs when measuring tiny particles such as protein condensates, which are measured objects undergoing Brownian motion in a solvent.

[0063] Next, the main reasons for the variation in detection sensitivity caused by the scanning of the light spot will be explained.

[0064] Figure 5 This is a schematic diagram illustrating the relationship between the intensity distribution of the emitted light and the semiconductor laser as a light source. The transverse (etching direction) dimension of the semiconductor laser chamber is larger than the longitudinal (stack direction), and the intensity distribution of the emitted laser becomes a half-value full-amplitude θ due to diffraction. ⊥ (≈16 degrees), θ / / The parallel beam, after being transformed by the collimating lens, has an intensity of approximately 8 degrees and a Gaussian distribution.

[0065] Figure 6A This is a schematic diagram illustrating a laser beam forming a spot at the center in the X or Y direction. As shown, the parallel beam is not deflected by the scanning mirror, and the light from the center of the parallel beam is focused by the objective lens.

[0066] Figure 6B This is a schematic diagram illustrating a laser beam forming a spot at one end in the X or Y direction. As shown, the parallel beam is deflected by the scanning mirror, and the light from the periphery of the parallel beam is focused by the objective lens. At this time, the light intensity focused on the spot is lower than that in the center.

[0067] For this main reason, as the light spot is scanned in the X or Y direction, the intensity of the light focused on the spot changes. In the two devices mentioned above, due to θ, which narrows the emission angle distribution of the semiconductor laser... / / The direction is the same as the X direction, so as... Figures 4A-4B As shown, the detection sensitivity changes more significantly in the X direction compared to the Y direction.

[0068] Figure 7This diagram illustrates the variation in detection sensitivity when scanning the laser spot in the Z-direction. It is well known that the magnitude of the detection signal, obtained by interfering the signal light reflected from the object with the reference light, follows a Gaussian distribution based on the difference in their optical path lengths. Here, the coherence length is defined as the optical path length difference (FWHM value) at which the detection signal reaches its maximum value. As shown in the diagram, the coherence length is approximately 200 μm. If the movement of the laser spot in the Z-direction when acquiring 3D image data is set to 50 μm, the sensitivity of the generated detection signal varies depending on the position in the Z-direction. This sensitivity variation varies for each device and for each output power, depending on the semiconductor laser element, the output power, and the conditions of high-frequency superposition.

[0069] In the following description of the present invention, the optical axis direction is taken as the Z-axis, and an orthogonal coordinate system is established with the X and Y axes taken in a plane orthogonal to it. The positions of the light spots in the X, Y, and Z axis directions are respectively defined as x, y, and z. Furthermore, to facilitate understanding of the detection sensitivity correction function, unless otherwise specified, the values ​​of x, y, and z are marked as positions standardized to ±1 within the measured scanning range. Additionally, for simplification, the maximum detection signal Amax obtained when the particle is located at the focal point of the objective lens is abbreviated as the detection signal. Furthermore, in each embodiment, unless otherwise specified, a method for... Figure 4B The calibration results of the present invention for a device with large variations in detection sensitivity are shown. In the present invention, individual characteristics of the device are stored as coefficients of the model formula in a storage device or the like during calibration, therefore... Figure 4A Needless to say, devices with small variations in detection sensitivity, as shown, can suppress errors in measurement results by correcting their detection sensitivity, as long as they meet certain performance standards.

[0070] <Implementation Method 1: Measurement and Correction Method for Sensitivity Variation During Particle Measurement>

[0071] The objects of measurement addressed in this invention are submicron to micron-sized particles, similar to protein condensates in biopharmaceuticals, that float in solvents and undergo Brownian motion. Since the location, size, and concentration of these microparticles are unknown, biopharmaceutical samples cannot be used to quantify variations in detection sensitivity for each device. Furthermore, as mentioned above, measurement data from the bottom glass of the sample container exhibit significant spherical aberration and can therefore only be used as a baseline for measuring variations in detection sensitivity.

[0072] This invention illustrates a method for measuring changes in detection sensitivity using a commercially available polystyrene standard particle suspension with uniform particle size and controllable concentration, and for determining the coefficients of the detection sensitivity correction function described later. Regarding the selection of polystyrene standard particles, any submicron region can be chosen, but here, polystyrene particles with an average size of 0.551 μm, accurately corrected beforehand by observation using a scanning electron microscope, were selected. These particles were diluted to a concentration of approximately 10 M particles / mL and sealed in a sample container, serving as a reference sample. Since the positions of the polystyrene particles within the reference sample vary randomly due to Brownian motion, obtaining a sufficient amount of three-dimensional image data allows for the acquisition of a detection signal with a roughly uniform standard deviation distribution in the X, Y, and Z directions. Here, approximately 12,600 calibration particle data points (x, y, z, Amax) were prepared.

[0073] Figure 8 This is a flowchart illustrating the steps for calibrating the individual characteristics of each device. In step S801, a reference sample containing the aforementioned standard particles is prepared. In step S802, measurement standard conditions are set for the particle measuring device to be calibrated. In step S803, three-dimensional image data is acquired according to the method described above, and calibration particle data is obtained from the standard particles contained in the reference sample. In step S804, based on the model formula described later, the coefficients of the detection sensitivity correction function are determined in a manner that minimizes the RMS value of the detection signal. In step S805, the type of the model formula and the values ​​of each coefficient are stored in a storage device. Through the above, the individual characteristics of the device can be obtained and saved as the coefficients of the determined model formula.

[0074] Figure 9 This is a flowchart illustrating the actions of the device when operating in the environment. In step S901, the model type and coefficients are read from the storage device. In step S902, the sample is measured. In step S903, sensitivity correction is performed on the position of each particle and the detection signal (x, y, z, Amax) using the model and coefficients, thereby obtaining Amax' from Amax. In step S904, the size and concentration of the target particles contained in the sample are calculated and indicated based on Amax'.

[0075] Through the above, it is possible to perform calibration of the detection sensitivity of individual devices that are determined during calibration (mostly at the factory) in the operating environment, and obtain measurement data that suppresses the machine differences between multiple devices.

[0076] <Implementation Method 2: Correction Method for Detection Sensitivity of Three-Dimensional Position of Light Spot>

[0077] In Embodiment 2 of the present invention, a specific example of the correction model formula described in Embodiment 1 will be explained. The correction model formula (detection sensitivity correction function) is expressed as η(x, y, z).

[0078] The following formula is a correction model based on a first-order polynomial. C xyz0 C x1 C y1 C z1 It is a coefficient representing the correction amount for the detection sensitivity of individual characteristics of the device.

[0079] [Formula 1]

[0080] η(x, y, z) = G xyz0 +C x1 x+C y1 y+C z1 z (Equation 1)

[0081] The following formula is a correction model based on a quadratic polynomial. C xyz0 C x1 C x2 C y1 C y2 C z1 C z2 It is a coefficient representing the correction amount for the detection sensitivity of individual characteristics of the device.

[0082] [Formula 2]

[0083] η(x, y, z) = G xyz0 +C x1 x+C x2 x2+C y1 y+C y2 y2+C z1 z+C z2 z2 (Equation 2)

[0084] The following formula is a correction model based on a cubic polynomial. C xyz0 C x1 C x2 C x3 C y1 C y2 C y3 C z1 C z2 C z3 It is a coefficient representing the correction amount for the detection sensitivity of individual characteristics of the device.

[0085] [Formula 3]

[0086]

[0087] The following formula is a correction model based on a quartic polynomial. C xyz0 C x1 C x2 C x3 C x4 C y1 C y2 C y3 C y4 C z1 C z2 C z3 C z4 It is a coefficient representing the correction amount for the detection sensitivity of individual characteristics of the device.

[0088] [Formula 4]

[0089]

[0090] The above examples illustrate correction models based on polynomials of order one to four. However, it is noted in this invention that polynomials of order five and above can also be used as correction models for detection sensitivity.

[0091] The following formula is based on a three-dimensional Gaussian distributed correction model. C xyz0 μ x μ y μ z σ x σ y σ z It is a coefficient representing the correction amount for the detection sensitivity of individual characteristics of the device.

[0092] [Formula 5]

[0093]

[0094] The following formula is based on a Gaussian distributed correction model with three-dimensional compensation. x a y a z b x b y b z μ x μ y μ z σ x σ y σ z This is a coefficient representing the correction amount for the detection sensitivity specific to the characteristics of the device. The difference from Equation 5 is that a DC value 'a' for sensitivity correction is introduced. x a y a z And the sensitivity correction amount b based on Gaussian distribution x b yb z This improves the adaptability to individual characteristics of the device.

[0095] [Formula 6]

[0096]

[0097] In this invention, in addition to the Gaussian distribution shown here, various special functions corresponding to the characteristics of the device can also be used, such as the error function as its integral form, the Poisson distribution derived from the binomial distribution, and the chi-square distribution after squaring a random variable according to the Gaussian distribution.

[0098] The following is an explanation of how to follow the instructions. Figure 8 The results are shown after applying sensitivity correction to each model using the method described.

[0099] Figure 10A The results are based on the correction model of the first-order polynomial shown in Equation 1. The upper section of the figure shows the results of the original data before applying the detection sensitivity correction function of the present invention, and the lower section shows the results after applying the detection sensitivity correction function of the present invention. The horizontal axis of each figure represents the standardized spot position, and the vertical axis represents the size of the measured particle. Each plotted point in the figure corresponds to the experimental results for the particle data used for correction mentioned above. As shown in the figure, the results of the original data were confirmed to be dependent on the changes in the spot position x, y, z. This is the variation in detection sensitivity corresponding to each device and individual characteristics, as described above. On the other hand, it can be seen that the results of applying the detection sensitivity correction function of the present invention improve the deviation (deviation of measurement size) in the vertical direction of each plotted point. In particular, regarding the dependence in the X-axis direction, it can be seen that by applying the detection sensitivity correction function of the present invention, the characteristic of symmetry with the center (x=0) of the scanning range of the spot as the extreme value is improved.

[0100] Figure 10B This is the result of the correction model based on the quadratic polynomial shown in Equation 2. (And...) Figure 10A Similarly, the results of the original data and the results of applying the detection sensitivity correction function of the present invention are shown. As shown in the figure, it can be seen that by applying the detection sensitivity correction function of the present invention, the spot position dependence in the X, Y, and Z directions is suppressed to a level that is not visually discernible. The deviation in the vertical axis of the figure represents the SNR of the device and the size deviation of the standard particle itself. As explained above, the significant effect of the present invention demonstrated by the quadratic polynomial reflects the following empirical fact: the variation in detection sensitivity in the X, Y, and Z directions is caused by a Gaussian distribution, and the variation near the center of the Gaussian distribution can be approximated by the quadratic formula.

[0101] Figure 10CThis is the result based on the correction model using the cubic polynomial shown in Equation 3. As shown in the figure, it can be seen that by applying the detection sensitivity correction function of this invention, the spot position dependence in the X, Y, and Z directions is suppressed to a level that is visually unrecognizable. As defined, this correction model contains a quadratic polynomial, therefore it can be said that the suppression performance becomes... Figure 10B The above is self-evident.

[0102] Figure 10D This is the result of a correction model based on the fourth-order polynomial shown in Equation 4. As shown in the figure, it can be seen that by applying the detection sensitivity correction function of this invention, the spot position dependence in the X, Y, and Z directions is suppressed to a level that is visually unrecognizable. As defined, this correction model contains a second-order polynomial, therefore it can be said that the suppression performance becomes... Figure 10B The above is self-evident.

[0103] Figure 10E The results are based on the three-dimensional Gaussian distributed correction model shown in Equation 5. As shown in the figure, it can be seen that by applying the detection sensitivity correction function of this invention, the spot position dependence in the X, Y, and Z directions is improved compared with the original data. However, compared with... Figure 10A Similarly, the sensitivity variation in the X direction remains to a degree that can be visually recognized, leaving room for improvement.

[0104] Figure 10F The results are based on the Gaussian distributed correction model with three-dimensional compensation shown in Equation 6. As mentioned above, this correction model improves the applicability of Equation 5. As a result, it can be seen that by applying the detection sensitivity correction function of the present invention, the spot position dependence in the X, Y, and Z directions is suppressed to a level that is not visually discernible.

[0105] To provide a clear and quantitative comparison of the results above, the frequency distribution of the measured standard particle sizes is shown below.

[0106] Figure 11 This is the size frequency distribution of the raw data before applying the detection sensitivity correction function of this invention. As shown in the figure, the distribution is asymmetrical about the left and right sides relative to the peak. The tail of the distribution on the side with larger size (right side) reflects the device characteristics of the X-direction + side (the side with higher detection sensitivity) shown above. In the case of precisely measuring the size of standard particles, the size frequency distribution must be symmetrical about the left and right sides relative to the peak. The RMS value representing the deviation of the measured size of the standard particles is 4.60%.

[0107] Figure 12A This is the result of the correction model based on the first-order polynomial shown in Equation 1. As shown in the figure, it can be seen that... Figure 11 Compared to the original data, the symmetry of the size frequency distribution relative to the peak value was improved. This is consistent with... Figure 10AThe same as described in the specification. The RMS value of the measured size of the standard particle is 3.66%.

[0108] Figure 12B The results are based on the correction model of the quadratic polynomial shown in Equation 2. As shown in the figure, it can be seen that by applying the detection sensitivity correction function of this invention, the symmetry of the size frequency distribution relative to the peak value is improved, and this improvement is achieved by narrowing the distribution width. The RMS value of the measured size of the standard particle is 2.74%.

[0109] Figure 12C The results are based on the correction model using the cubic polynomial shown in Equation 3. As shown in the figure, by applying the detection sensitivity correction function of this invention, the symmetry of the size frequency distribution relative to the peak value is improved, and this improvement is achieved by narrowing the distribution width. The RMS value of the measured size of the standard particle is 2.74%. As defined, this correction model contains a quadratic polynomial, therefore it can be said that the suppression performance becomes… Figure 12B The above is self-evident.

[0110] Figure 12D The results are based on the correction model using the fourth-order polynomial shown in Equation 4. As shown in the figure, by applying the detection sensitivity correction function of this invention, the symmetry of the size frequency distribution relative to the peak value is improved, and this improvement is achieved by narrowing the distribution width. The RMS value of the measured size of the standard particle is 2.73%. As defined, this correction model contains a second-order polynomial, therefore it can be said that the suppression performance becomes… Figure 12B The above is self-evident.

[0111] Figure 12E The results are based on the three-dimensional Gaussian distributed correction model shown in Equation 5. As shown in the figure, it can be seen that by applying the detection sensitivity correction function of this invention, the symmetry of the size frequency distribution relative to the peak value is improved, and this improvement is achieved by narrowing the distribution width. The RMS value of the measured size of the standard particle is 3.25%.

[0112] Figure 12F The results are based on the Gaussian distributed correction model with three-dimensional compensation shown in Equation 6. As shown in the figure, by applying the detection sensitivity correction function of this invention, the symmetry of the size frequency distribution relative to the peak value is improved, and this improvement is achieved by narrowing the distribution width. The RMS value of the measured size of the standard particle is 2.75%. As mentioned above, this correction model improves the application range of Equation 5, and it can be said that the RMS value becomes... Figure 12E Small values ​​are to be expected.

[0113] Figure 13This is a graph summarizing the comparison results of the RMS values ​​of the standard particle size, representing the effectiveness of the detection sensitivity correction method of the present invention. In the graph, the horizontal axis represents the correction model formula shown above, and the vertical axis represents the measured RMS value of the standard particle size. As shown, compared with the original data without the application of the present invention, by applying the present invention, the RMS value of the standard particle size decreases for all correction model formulas, quantitatively demonstrating the effectiveness of the detection sensitivity correction method of the present invention. Furthermore, the RMS values ​​of the standard particle size obtained by applying quadratic polynomials, cubic polynomials, quartic polynomials, and Gaussian distributions with three-dimensional compensation are approximately the same and the smallest, thus indicating that these model formulas are suitable for the characteristics of the device shown here. The fewer the number of coefficients contained in the model formula, the simpler and more stable the correction can be implemented, and the higher the robustness when operating in the operating environment. Therefore, for example, the quadratic polynomial of Equation 2 can be considered as a preferred sensitivity correction model formula.

[0114] Further explanation is provided regarding the selection of preferred model formulas corresponding to the design of the device and the characteristics of its constituent components. If necessary and sufficient correction of the detection sensitivity can be achieved, the robustness of a simple model formula with fewer coefficients is naturally higher. In devices designed to reduce variations in the individual detection sensitivity of the device through the selection of constituent components, the first-order polynomial of Equation 1 and the Gaussian distribution of Equation 5 are preferred as simple model formulas. When the particles being measured are not only protein aggregates, but also inorganic nanoparticles, silicone oil spheres, and other particles with different physical properties (refractive indices), devices capable of simultaneously measuring these particles irradiate them with lasers of multiple wavelengths simultaneously to utilize the wavelength dependence of these physical properties. In such cases, it is preferable to select a higher-order polynomial, including the third-order polynomial of Equation 3 and the fourth-order polynomial of Equation 4, as the sensitivity correction model formula. In devices designed to shorten measurement time by expanding the spot scanning range in the Z-direction, the Gaussian distribution of Equation 6 with three-dimensional compensation, which can perform more stringent detection sensitivity correction than polynomials, is preferred to address variations in the optical path length difference in the Z-direction.

[0115] In devices designed with sufficiently small sensitivity variations in a portion of the X, Y, and Z directions, selective correction of detection sensitivity in at least one axis direction can be easily and is a preferred option by transforming the formula that eliminates one or two coefficients of particle position x, y, and z from the model formula disclosed in this invention.

[0116] As described above, according to the present invention, in accordance with Figure 8 and Figure 9The method shown selects an appropriate model from a variety of models that include the above model formulas, saves the different characteristics of each device as coefficients, and corrects the detection signal according to them in the customer environment, thereby improving the deviation of the measurement results and suppressing the machine difference between multiple devices.

[0117] <Example 2: Summary>

[0118] The particle measuring device of Embodiment 2 uses a detection sensitivity correction function with a polynomial and Gaussian distribution containing the position of the light spot in each of the X, Y, and Z directions as variables to correct the magnitude of the detection signal, thereby suppressing the variation in detection sensitivity caused by the individual characteristics of the particle measuring device.

[0119] <Implementation Method 3: Correction Method for Sensitivity Variation When the Refractive Index of the Measured Particles is Different>

[0120] The objects of measurement addressed in this invention are submicron to micron-sized particles, such as protein condensates in biopharmaceuticals, that float in solvents and undergo Brownian motion. As described above, in addition to protein condensates, commercially available polystyrene beads can also be measured in this invention. Furthermore, Au particles, Ag particles, TiOx particles, silicone oil beads, etc., can also be measured. Compared to protein condensates, these generally have a higher refractive index, so even for particles of the same size, the amount of reflected light increases depending on the object being measured, and the magnitude of the detection signal also increases. It is sufficient to have a detection signal and particle size correction data corresponding to the type of particle being measured pre-installed in the device. In this case, to avoid saturation of the detection signal, the output light intensity of the semiconductor laser can be reduced. As described above, in the variation of detection sensitivity corresponding to the scanning position of the light spot, the main reason for the variation in the X and Y directions is determined by the luminous intensity distribution based on the diffraction phenomenon dependent on the shape of the semiconductor laser chamber; therefore, even if the output light intensity of the semiconductor laser is reduced, the change is negligible. On the other hand, in the variation of detection sensitivity corresponding to the scanning position of the light spot, the main reason for the variation in the Z direction is the change in coherence length. Therefore, it is necessary to select the correction amount appropriately according to the emission power of the semiconductor laser.

[0121] Here, based on the factors affecting the variation of detection sensitivity, a preferred model formula corresponding to the emission power conditions of multiple semiconductor lasers is shown. Hereinafter, the emission power of the semiconductor laser is expressed as p, and the detection sensitivity correction function is expressed as η(x, y, z, p).

[0122] The following formula is a correction model based on a quadratic polynomial. C xyz0 C x1 C x2 C y1 C y2 Cz0 (p), C z1 (p), C z2 (p) is a coefficient representing the correction amount for the detection sensitivity based on the individual characteristics of the device. By assigning dependence on p as coefficients in the Z direction, it is possible to appropriately suppress factors affecting the detection sensitivity that are related to changes in the emission power of the semiconductor laser.

[0123] [Formula 7]

[0124]

[0125] The following equation is a correction model that combines corrections based on quadratic polynomials in the X and Y directions with a Gaussian distribution in the Z direction. C xyz0 C x1 C x2 C y1 C y2 μ z (p), σ z (p) is a coefficient representing the correction amount for the detection sensitivity based on the individual characteristics of the device. Similarly, by assigning dependence on p as coefficients in the Z direction, it is possible to appropriately suppress factors affecting the detection sensitivity that are related to changes in the emission power of the semiconductor laser.

[0126] [Formula 8]

[0127]

[0128] according to Figure 8 The method shown performs sensitivity calibration on each model. The difference from the method described above is that calibration is performed under two different emission power conditions: a preferred emission power p-p0 for measuring protein condensates and a preferred emission power p-p1 (p1 = 0.42p0) for measuring polystyrene beads.

[0129] Figure 14A The results are based on the correction model of the quadratic polynomial shown in Equation 7. The original data is omitted. The upper part of the figure shows the application results of the detection sensitivity correction function of the present invention at a luminous power p = p0. The lower part of the figure shows the application results of the detection sensitivity correction function of the present invention at a luminous power p = p1. It can be seen that by applying the detection sensitivity correction function of the present invention, under any luminous power condition, the spot position dependence in the X, Y, and Z directions is suppressed to a level that is visually unrecognizable.

[0130] Figure 14BThis is the result of a correction model combining the quadratic polynomial and Gaussian distribution shown in Equation 8. The upper part of the figure shows the application results of the present invention at a luminous power p = p0, and the lower part shows the application results of the present invention at a luminous power p = p1. According to the present invention, it can be seen that under any luminous power condition, the spot position dependence in the X, Y, and Z directions is suppressed to a level that is not visually discernible.

[0131] Figure 15A The results are based on the correction model using the quadratic polynomial shown in Equation 7. The upper part of the figure shows the results of the original data before applying the detection sensitivity correction function of this invention, and the lower part shows the results after applying the detection sensitivity correction function of this invention. It can be seen that when the luminous power p = p0, the symmetry of the size frequency distribution of the standard particles relative to the peak value is improved before and after the application of this invention, and the RMS value of the measured size improves from 4.60% to 2.73%. It can also be seen that when the luminous power p = p1, the symmetry of the size frequency distribution of the standard particles relative to the peak value is also improved before and after the application of this invention, and the RMS value of the measured size improves from 4.98% to 2.82%.

[0132] Figure 15B This is the result of a correction model combining the quadratic polynomial and Gaussian distribution shown in Equation 8. The upper part of the figure shows the results of the original data before applying the detection sensitivity correction function of this invention, and the lower part shows the results after applying the detection sensitivity correction function of this invention. At any emission power, the symmetry of the standard particle size frequency distribution relative to the peak value is also improved before and after the application of this invention. When the emission power p = p0, the RMS value of the measured size of the standard particle improves from 4.60% to 2.74% before and after the application of this invention. When the emission power p = p1, the RMS value of the measured size of the standard particle also improves from 4.98% to 2.83% before and after the application of this invention.

[0133] Figure 16 This is a graph summarizing the comparison results of the RMS values ​​of the standard particle size, representing the effectiveness of the detection sensitivity correction method of the present invention. In the graph, the horizontal axis represents the correction model shown above, and the vertical axis represents the measured RMS value of the standard particle size, showing the cases where the luminous power p = p0 and p = p1. As shown in the figure, compared with the original data without the application of the present invention, by applying the present invention, the RMS value of the standard particle size is reduced to the same extent, quantitatively demonstrating the effectiveness of the detection sensitivity correction method of the present invention.

[0134] As described above, according to the present invention, in accordance with... Figure 8 as well as Figure 9The method shown appropriately selects a model from a variety of models including the above-mentioned model formulas, saves the different characteristics of each device as coefficients, and corrects the detection signal based on them in the operating environment, thereby improving the deviation of the measurement results and suppressing the machine difference between multiple devices.

[0135] The above describes the preferred emission powers for protein aggregates and polystyrene beads, showing the cases where the emission power p = p0 and p = p1. However, other preferred emission powers can also be easily applied to metal nanoparticles, silicone beads, etc. Furthermore, σ in Equation 8 can easily be substituted. z (p) The FWHM value w, which is measured using a different coherence length, is used. z (p). In this case, it is well known that the conversion can be performed in the following way. Therefore, σ z (p) and w z (p) can be treated as physically equivalent expressions, so the expressions resulting from their interconversion can also be treated as equivalent expressions.

[0136] [Formula 9]

[0137]

[0138] Furthermore, in devices designed with sufficiently small sensitivity variations in either the X or Y direction, the present invention is readily implemented by transforming the formula that eliminates the coefficients in either the x or y direction of the particle position from the model formula disclosed in the present invention, making it a preferred option.

[0139] As described above, according to the present invention, in accordance with Figure 8 as well as Figure 9 The method shown appropriately selects from a variety of models including the above-mentioned model formulas, and saves the different characteristics of each device as coefficients of the model formula for multiple luminous power conditions preferred for the measurement object. Based on these coefficients, the detection signal is corrected in the customer environment, thereby improving the deviation of the measurement results and suppressing the machine difference between multiple devices.

[0140] <Implementation Method 4: Particle Measurement Device>

[0141] Figure 17 This is an example of the structural diagram of the particle measuring device according to Embodiment 4 of the present invention. The laser emitted from the light source 100, whose emission state is controlled by the laser driver 101 which controls the high-frequency superposition and emission power, is transformed into parallel light by the collimating lens 102. After the polarization orientation is adjusted by the λ / 2 plate 103 whose optical axis is set at approximately 22.5 degrees relative to the horizontal direction, it is separated into signal light and reference light by the polarization beam splitter 104.

[0142] After the reference light is transformed into circularly polarized light by the λ / 4 plate 105, it is reflected by the reference light mirror 106, becomes polarized light with a 90-degree rotation relative to the path after passing through the λ / 4 plate 105, and is reflected by the polarization beam splitter 104. The signal light, after being deflected in its direction of travel by the XY-direction composite deflection element 107 (scanning mechanism), is transformed into circularly polarized light by the built-in λ / 4 plate, and is focused and imaged within the sample 204 held in the sample container 200 by the objective lens 108. The drive mechanism 109 (scanning mechanism) that moves the sample in the Z-axis direction has the function of scanning the focal position of the signal light along the Z-axis direction (optical axis direction). The component of the signal light reflected from individual particles contained in the sample 204 is deflected in the same direction as the path by the XY-direction composite deflection element 107, and becomes polarized light with a 90-degree rotation relative to the path after passing through the built-in λ / 4 plate, and then passes through the polarization beam splitter 104. Here, the sample container 200 holds the sample 204 within the well, and signal light is introduced into the sample via the transparent window 202. 203 is a resin component forming the well of the sample container. The base plate 201 contacts the transparent window 202, mechanically holding the sample container 200 and stabilizing the sample temperature.

[0143] The signal light and the reference light are combined by the polarization beam splitter 104 and guided to the detection optical system 112. The signal light is then split into transmitted light and reflected light by the half-beam splitter 113 through the pinhole 111.

[0144] After the reflected light passes through the λ / 4 plate 114, whose optical axis is set at approximately 45 degrees relative to the horizontal direction, it is focused by the condenser lens 115 and split into two parts by the polarization beam splitter 116. The two parts are photoelectrically converted by the photodetectors 150 and 151 respectively, and differentially amplified by the current differential amplifier 152 to become the detection signal 123.

[0145] After the transmitted light is transmitted through the λ / 2 plate 118, whose optical axis is set at approximately 22.5 degrees relative to the horizontal direction, it is focused by the condenser lens 119 and split into two parts by the polarization beam splitter 120. The two parts are photoelectrically converted by the photodetectors 153 and 154 respectively, and differentially amplified by the current differential amplifier 155 to become the detection signal 122.

[0146] The detection optical system 112 shown here constitutes a homodyne phase diversity method. The real component 122 and the imaginary component 123 of the detection signal are electrical signals corresponding to the real and imaginary parts of the electric field amplitude of the signal light reflected from the individual particles contained in the sample 204. The detection signal is calculated by the signal processing unit 124 as a value obtained by calculating the square root after squaring these components.

[0147] The signal processing unit 124 synthesizes the time-series data of the acquired detection signal to form three-dimensional image data in the XYZ directions within the sample, and separates / tracks the data of individual particles contained therein, calculating the maximum value of the detection signal obtained under focusing conditions for each particle. The signal processing unit 124 has the following function: by implementing... Figure 8 and Figure 9 The corrections shown for variations in detection sensitivity and for the customer environment improve the deviation of measurement results and suppress machine errors between multiple devices. Finally, after the signal processing unit 124 organizes the measurement results corrected by the methods shown in Embodiments 1 to 3 of the present invention into charts or numerical tables, it displays the results to the user on the display unit 125.

[0148] The signal processing unit 124 shown in the figure can be installed using a microcomputer for assembly purposes built into the device, or a computer configured externally to the device that can perform necessary signal exchange via an interface board or the like. When the processing items of the signal processing unit 124 are subject to time or power constraints, a dedicated processing circuit mounted on an FPGA or similar device can be used as an auxiliary arithmetic unit.

[0149] The storage unit 126 can, for example, store the calibration model formula for each individual particle measuring device. The signal processing unit 124 uses the calibration model formula stored in the storage unit 126 to correct the error in the detection sensitivity variation using the method described in the above embodiments.

[0150] <Regarding variations of the present invention>

[0151] This invention is not limited to the embodiments described above, but includes various modifications. For example, the embodiments described above are detailed embodiments for the purpose of easily understanding and illustrating the invention, and are not limited to having all the structures described. Furthermore, a portion of the structure of one embodiment can be replaced with the structure of another embodiment, and it is also possible to add structures of other embodiments to the structure of one embodiment. Additionally, regarding a portion of the structure of each embodiment, other structures can be added, deleted, or replaced.

[0152] In the above implementation, the coefficient (C) provided as a function of optical power p z0 (p), C z1 (p), C z2 (p), μ z (p), σ z (p) can be a mathematical function expressed by a mathematical formula, a data table describing the numerical values ​​corresponding to p, or any other arbitrary form. That is, it is sufficient to define the functional relationship between p and these coefficients.

[0153] In the above embodiments, the signal processing unit 124 may be built into the particle measuring device, or the particle measuring device may be configured such that it only acquires data describing the results of the measured sample, and the signal processing unit 124 is disposed outside the particle measuring device and receives the data. In either case, the structure of the particle measuring device of the present invention is the same as that described in the above embodiments.

[0154] Symbol Explanation

[0155] 100: Light source

[0156] 101: Laser driver with high-frequency superposition function

[0157] 108: Objective lens

[0158] 109: Drive mechanism for sample container

[0159] 112: Detection optical system

[0160] 124: Signal Processing Department

[0161] 200: Sample container

[0162] 201: Base Plate

[0163] 202: Transparent Window

[0164] 203: Resin components that form cavities.

Claims

1. A particle measuring device for measuring particles in a liquid sample, characterized in that, The particle measuring device includes: A light source, the light it emits; A branch section that splits the light from the light source into a signal light and a reference light; A scanning mechanism that scans the position of the spot illuminated by the signal light; The irradiation unit focuses the signal light through an objective lens and irradiates the sample. The detection unit detects the interference signal obtained by interfering the reflected light from the particle with the reference light; and The processing unit uses the interference signal to measure the size of the particle. The scanning mechanism is configured to scan the irradiation position in a first direction and a second direction orthogonal to each other in a plane orthogonal to the optical axis of the light, and to scan the irradiation position upwards in a third direction parallel to the optical axis. The processing unit uses a correction function with at least one of the irradiation positions in the first direction, the second direction, and the third direction as input values ​​to correct the size of the particles.

2. The particle measuring device according to claim 1, characterized in that, The correction function is composed of a polynomial, which has at least one of the illumination positions in the first direction, the illumination positions in the second direction, and the illumination positions in the third direction as terms.

3. The particle measuring device according to claim 2, characterized in that, The correction function is constructed by performing linear addition on terms of the irradiation position at least once in the first direction, terms of the irradiation position at least once in the second direction, and terms of the irradiation position at least once in the third direction.

4. The particle measuring device according to claim 1, characterized in that, The correction function is configured to correct the particle size by taking the reciprocal of the normal distribution of at least one of the irradiation positions in the first direction, the second direction, and the third direction.

5. The particle measuring device according to claim 4, characterized in that, The correction function is composed of the average and standard deviation of the irradiation position in the first direction on the normal distribution, the average and standard deviation of the irradiation position in the second direction on the normal distribution, and the average and standard deviation of the irradiation position in the third direction on the normal distribution.

6. The particle measuring device according to claim 4, characterized in that, The correction function also includes a DC value to compensate for the normal distribution and a sensitivity correction amount that is a multiple of the normal distribution constant.

7. The particle measuring device according to claim 1, characterized in that, The correction function includes either the power of the light or the coherence length of the light as input values.

8. The particle measuring device according to claim 7, characterized in that, The correction function is composed of terms of degree one or more at the irradiation position upwards from the third point. The term of the third-party upward irradiation position, which is at least once, has a coefficient as a function with the power or the coherence length as input.

9. The particle measuring device according to claim 7, characterized in that, The correction function is composed of terms of degree one or more at the irradiation position in the first direction, terms of degree one or more at the irradiation position in the second direction, and a Gaussian distribution that corrects the particle size in the third direction by the reciprocal of the normal distribution of the irradiation position in the third direction. The Gaussian distribution includes the mean and standard deviation of the normal distribution. The average is constituted by a function with the power or the coherence length as input values. The standard deviation is constituted by a function with the power or the coherence length as input values.

10. The particle measuring device according to claim 1, characterized in that, The particle measuring device also includes a storage unit for storing data containing the correction function. The processing unit uses the correction function read from the data to correct the size of the particles.

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