Calibration method and program for particle analysis system

Abstract

This invention provides a calibration method for a particle analysis system that prevents halation and insufficient light immediately after the start of measurement, even when dealing with unknown samples that have different optical properties from standard samples, and enables the rapid establishment of appropriate measurement conditions. [Solution] The calibration method of the present invention comprises: a reference setting step (S201) in which a reference laser output value and a reference brightness value are obtained using standard particles; an output adjustment step (S202) in which the ratio of scattered light intensity with that of a standard sample is estimated based on the refractive index information of the sample to be measured, and an initial laser output value for the sample to be measured is determined based on this ratio; a determination step (S203) in which the average brightness value of the particles measured with the initial laser output value is determined to be within a predetermined range of the reference brightness value; and an output control step (S202-S203) in which, if outside the range, the laser output value is changed, and measurement and determination are repeated until it falls within the range.

Description

Technical Field

[0001] The present invention relates to a calibration method for a particle analysis system that irradiates fine particles dispersed in a liquid with light and measures the particle size and the like using the scattered light. In particular, the present invention relates to a technique capable of improving the measurement accuracy when the characteristics of the particles and the solvent to be measured are different from those of the standard sample.

Background Art

[0002] In recent years, fine particles such as ultra-fine bubbles (UFB) have been used in a wide range of fields such as cleaning, food, and medicine. As an evaluation method for these particles, a PTA (Particle Tracking Analysis) method that tracks Brownian particles using laser light scattering is known (for example, Patent Document 1).

[0003] In the PTA method, in order to ensure the reproducibility of measurement, calibration of the laser output and the sensitivity (gain) of the imaging device is performed using standard particles with known particle size and concentration (for example, polystyrene latex (PSL) with a particle size of 100 nm).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, the actual sample to be measured does not necessarily have the same optical properties as the standard particles. For example, when the solvent to be measured has a higher refractive index than water, such as edible oil, the scattered light intensity increases extremely, and when measured under the same conditions as the standard sample, there is a problem that halation (white bleeding) occurs and particle recognition becomes difficult.

[0006] Furthermore, even with the same solvent, "water," the scattered light intensity fluctuates significantly if the refractive index of the particles being measured (e.g., bubbles or metal particles) differs from that of standard particles (PSL), or if the particle size is unknown. In the Rayleigh scattering region, the scattered light intensity is proportional to the sixth power of the particle size, so even a slightly larger particle size than expected can cause the brightness to saturate, while a smaller particle size can be too dimmer to be detected.

[0007] In other words, conventional calibration methods have made it difficult to consistently set optimal exposure conditions for "unknown samples" that have different characteristics from standard samples. The present invention has been made in view of the above problems, and aims to provide a calibration method that enables high-precision measurement by appropriately controlling the scattered light intensity regardless of the type of solvent or particle size. [Means for solving the problem]

[0008] To achieve the above objective, the calibration method for a particle analysis system according to the present invention is a calibration method for a particle analysis system that irradiates particles in a liquid with light from a laser light source and analyzes particles based on an image of scattered light acquired by an imaging device, and includes: a reference setting step of determining a reference laser output value that can obtain an appropriate particle size distribution using a standard sample containing standard particles, and acquiring the average value of the brightness of the standard particles obtained at the reference laser output value as a reference brightness value; an output adjustment step of estimating the ratio of scattered light intensity between the standard sample and the sample to be measured based on the solvent and refractive index information of the particles of the sample to be measured, and determining an initial laser output value for the sample to be measured based on the ratio; a determination step of measuring the sample to be measured using the initial laser output value and determining whether the average value of the brightness of the particles obtained by the measurement is within a predetermined range corresponding to the reference brightness value; and an output control step of changing the laser output value and repeating the measurement and determination until the average value of the brightness of the particles obtained by the measurement is within the predetermined range. [Effects of the Invention]

[0009] According to this invention, the laser output is first roughly adjusted using theoretical calculations based on the refractive index difference, thereby preventing extreme halation and insufficient light intensity immediately after the start of measurement. Furthermore, by performing feedback control targeting a reference brightness value, brightness deviations due to unknown particle sizes are absorbed, making it possible to detect particles with the optimal signal-to-noise ratio at all times. As a result, highly accurate measurements can be achieved for all samples, regardless of whether they are aqueous or non-aqueous. [Brief explanation of the drawing]

[0010] [Figure 1] This figure shows a particle analysis system according to an embodiment of the present invention. [Figure 2] This is a flowchart of a calibration method for a particle analysis system according to the first embodiment of the present invention. [Figure 3] This is a flowchart illustrating a calibration method for a particle analysis system according to a second embodiment of the present invention. [Modes for carrying out the invention]

[0011] Embodiments of the present invention will be described below with reference to the drawings. However, the components described are illustrative and are not intended to limit the technical scope of the present invention to them alone.

[0012] <First Embodiment> The particle analysis system 100 according to the first embodiment of the present invention comprises a laser light source 10, a sample cell 20, an imaging device 30, a processing device 40, and a display device 50 (see Figure 1).

[0013] The laser light source 10 can be a semiconductor laser. The laser light source 10 emits laser light 11 into the sample cell 20 containing the dispersion, and outputs light of a wavelength that can cause Rayleigh scattering light to be emitted from the particles contained in the dispersion. For example, a semiconductor laser with a wavelength of 405 [nm] can be used. As shown in Figure 1, the irradiation direction of the laser light 11 is the X direction when the XYZ directions are set as shown in Figure 1. Alternatively, lenses or the like can be added to the optical path to form a sheet of laser light 11. This expands the irradiation area, thereby increasing the imaging range. For example, a sheet can be formed by using a GRIN lens or a cylindrical lens.

[0014] The sample cell 20 is made of glass, transparent plastic, or the like, and can hold a dispersion. For example, the sample cell 20 may have a capacity of up to 500 mL of dispersion, but is not limited to this.

[0015] The imaging device 30 is an imaging device such as a camera equipped with a lens such as a telecentric lens, and it images a predetermined area of ​​the sample cell 20 that is irradiated with laser light 11. The imaging device 30 may also be equipped with an optical filter that selectively transmits the wavelength of the laser light 11 (scattered light) and blocks other wavelengths (fluorescence, ambient light, etc.). The imaging device 30 is connected to the processing device 40 and can transmit the captured image to the processing device 40. As shown in Figure 1, the imaging device 30 is positioned to capture scattered light from a direction perpendicular to the X direction, which is the irradiation direction of the laser light 11 (Y direction). The imaging range can be set to, for example, 5 [mm] × 1.4 [mm], but is not limited to this.

[0016] The processing unit 40 is, for example, a personal computer, and performs various image processing operations on the captured images sent from the imaging device 30, such as determining the number concentration of a dispersion. The processing unit 40 is also connected to the laser light source 10 and can control its output, etc. The processing unit 40 can also control the gain of the imaging device 30.

[0017] The display device 50 is, for example, a liquid crystal monitor. The display device 50 is connected to the processing device 40 and can display the captured image captured by the imaging device 30, the image processed by the processing device 40, and the like.

[0018] Next, according to the flowchart of FIG. 2, the calibration and measurement procedures will be described. In this embodiment, as an example, pure water containing standard particles (PSL: polystyrene) as a standard sample and rapeseed oil containing UFB (bubbles, unknown particle size) as a measurement target sample are used. However, the solvent of the measurement target sample is not limited to this and may be water, alcohol, or the like. Also, the particles may be different from the standard sample.

[0019] First, the reference setting step is executed in S201. The reference setting step is a step of determining the "reference laser output value" as the reference measurement condition for correctly measuring the particle size distribution using the standard sample, and acquiring the average luminance of the standard particles obtained under that condition as the "reference luminance value".

[0020] Specifically, first, a reference state is defined using the standard sample. Pure water (refractive index 1.33) in which standard particles (refractive index 1.59, particle size 100 nm) are dispersed and adjusted to 25°C is set in the sample cell 20, and laser light 11 is irradiated from the laser light source 10. At this time, the processing device 40 tracks the Brownian motion of each particle based on the captured consecutive captured images, and calculates the particle size distribution using the Stokes-Einstein equation.

[0021] Here, if the laser intensity is inappropriate, a correct particle size distribution cannot be obtained. For example, when the laser intensity is too low, small particles with weak scattered light are buried in the noise and not detected, resulting in problems such as an apparent increase in the average particle size or a decrease in the detected particle concentration compared to the true value. Conversely, when the laser intensity is too high, the particle image may be enlarged due to halation, the accuracy of specifying the centroid position may decrease, and the width (dispersion) of the particle size distribution may be unduly widened.

[0022] Therefore, in this step, the brightness and S / N ratio of the particles in the captured image are within a predetermined range, and as a result, the mode diameter of the calculated particle size distribution matches the nominal value of the standard particles (e.g., within the range of 100 ± 10 nm). According to the instructions of the processing device 40, the laser output value and the gain are adjusted. Specifically, while the processing device 4 is instructing the laser light source 10 to change the laser output value, continuous captured images are acquired from the imaging device 30 to obtain the particle size distribution. If the particle size distribution matches the nominal value of the standard particles and the average value of the brightness of the particles in the captured image is within a predetermined range, then the number concentration is obtained based on the number of bright spots in the captured image. Then, the gain of the imaging device 30 is adjusted so that the obtained number concentration is within a predetermined appropriate range.

[0023] Assuming that the appropriate laser output value determined in this way is, for example, 80 mW, this value is stored in the memory of the processing device 40 as the reference laser output value. Also, assuming that the average brightness of the particles as bright spots in the captured image at that time is 1315, this value is stored in the memory of the processing device 40 as the reference brightness value.

[0024] Next, in S202, an output adjustment step is executed. The output adjustment step is a step of determining, by calculation, the initial value or the changed value of the laser output value to be irradiated to the measurement target sample.

[0025] First, let's explain how to determine the initial values. The processing device 40 acquires refractive index information of the solvent and particles of the sample to be measured. The refractive index information is specified, for example, by user input. In this embodiment, it is assumed that the solvent of the sample to be measured is rapeseed oil (refractive index approximately 1.47) and the particles are UFB (bubbles: refractive index approximately 1.00). The solvent of the sample to be measured may be pure water, and the same applies if the particles are metal nanoparticles or other combinations of refractive indices different from the standard sample. Next, the processing device 40 compares the refractive index conditions of the standard sample (water / PSL) used in the reference setting step with the refractive index conditions of the sample to be measured (oil / UFB), and estimates the ratio of the scattered light intensity of the sample to the standard sample (luminance magnification) based on predetermined scattering theory or correlation information. As for this estimation method, an approximate formula of Rayleigh scattering (scattering intensity is proportional to the square of the relative refractive index term) may be used to reduce the computational load, or an exact calculation based on the theory of Mie scattering may be performed to perform a more accurate estimation. Alternatively, the system may refer to a table (lookup table) that has been pre-calculated based on these theories, showing the relationship between refractive index difference and scattering intensity change. In this embodiment, as a measure to determine the initial value, it is assumed that the particle size of the sample to be measured is the same as that of the standard particle, and the calculation is performed by applying the approximate formula for Rayleigh scattering. As a result of this calculation, it is calculated that under the conditions of this embodiment, the sample to be measured will be approximately three times brighter than the standard sample. Therefore, the processing unit 40 performs a correction calculation by multiplying the reference laser output value (80mW) stored in S201 by its reciprocal (approximately 1 / 3), and determines the resulting value of 26mW as the initial laser output value for the sample to be measured.

[0026] On the other hand, if the process is re-executed via the loop from S203 described later (to determine the change value), the refractive index calculation described above is not performed, and a value (command value) obtained by adding or subtracting a predetermined amount from the previous output value is determined as the new laser output value. This makes it possible to suppress extreme halation immediately after the start of measurement when measuring the sample to be measured, or conversely, the failure to detect particles due to insufficient light intensity.

[0027] Next, in S203, the determination step is executed. In the determination step, the sample to be measured is actually measured using the laser output value (initial value or changed value) determined in S202, and it is determined whether the measurement conditions are appropriate based on the particle detection status and average brightness. The processing device 40 instructs the laser light source 10 to output at the laser output value determined in S202. Then, the image of the sample to be measured captured by the imaging device 30 is acquired, particles (bright spots) in the image are extracted, and their average brightness is calculated. In this case, if no particles are extracted due to insufficient output value or other reasons, the average brightness is treated as zero or an undeterminable value.

[0028] Here, the particle size of the particles contained in the sample to be measured is unknown. Since the scattered light intensity is proportional to the sixth power of the particle size, even if the refractive index correction in S202 is correct, if the actual particle size is smaller than expected, the particles may be too faint to be detected, or the average brightness may be lower than the reference value. Therefore, the processing device 40 determines whether the particles have been detected correctly and whether their average brightness is within a predetermined tolerance range (e.g., ±5%) corresponding to the reference brightness value (1315). If particles are detected and the average brightness is within the predetermined range (S203; YES), it is determined that the measurement can be performed correctly with the current laser output value, and the flow shown in Figure 2 is terminated to proceed to the actual measurement. On the other hand, if particles are not detected, or if the average brightness is not within the predetermined range (S203; NO), it is determined that the current laser output value is inappropriate, and an output control step is executed as feedback control to increase or decrease the laser output, and the laser output value is obtained as the changed value.

[0029] Specifically, if the average brightness determined in the previous judgment is greater than the reference brightness value, the processing unit 40 determines that the output is "excessive" and generates a command value to reduce the laser output by a predetermined step (e.g., 2mW). Conversely, if the average brightness is less than the reference brightness value, or if no particles are detected, it determines that the output is "insufficient" and generates a command value to increase the laser output by a predetermined step. Then, the processing unit 40 returns to S202 to set the next output value using the newly generated command value. This loop processing allows the system to gradually increase the output to capture particles even if the unknown particle size is extremely small and cannot be detected at the initial value, and to converge the laser output until it reaches the optimal brightness (reference brightness value) for the imaging device 30.

[0030] Next, the main measurement of the sample to be measured is performed. Here, the processing device 40 uses the viscosity of the solvent of the sample to be measured as the particle size calculation parameter in this measurement. This is because, in the Stokes-Einstein equation used in the PTA method, particle size is inversely proportional to the viscosity of the solvent. For example, in this embodiment, since the solvent is rapeseed oil, the viscosity of rapeseed oil (for example, about 50 mPa·s) is used from the viscosity of water for the standard sample (about 0.89 mPa·s). If the solvent of the sample to be measured is water, it is not necessary to change the viscosity setting. In this way, in this measurement, the processing device 40 can analyze the video data (sequentially captured images) of the sample to be measured based on the calibrated laser output value and calculate the particle size distribution and number concentration.

[0031] <Second Embodiment> In the first embodiment, an example was described in which rapeseed oil is used as the solvent for the sample to be measured. When using an oil-based solvent such as rapeseed oil, strong scattered light from particles and impurities can cause multiple scattering, resulting in the entire image captured by imaging the scattered light from the particles appearing whitish and making it difficult to clearly identify the particles. Alternatively, fluorescent molecules dissolved in the rapeseed oil may be excited at the wavelength of the laser light source, 405 nm, and emit fluorescence, causing the entire image to appear whitish and making it difficult to clearly identify the particles. In response to this, it is effective to remove fluorescence by providing an optical filter (for example, a bandpass filter that transmits the scattered light wavelength of 405 nm and blocks the fluorescence wavelength band) in the imaging device 30. The present invention may also be configured to combine such optical filters. However, if background noise remains even when using an optical filter, or if an optical filter is not used to simplify the device configuration, this can be solved by reducing the intensity of the laser light. In the second embodiment, a background determination step (S303) for determining brightness abnormalities of the entire captured image is added to the calibration method of the particle analysis system according to the first embodiment shown in Figure 2 to address this issue. This allows for the appropriate setting of the laser light intensity necessary for particle size measurement, even if scattered light spreads across the entire captured image when an oil-based solvent is used for the sample being measured.

[0032] The particle analysis system according to the second embodiment is the same as the particle analysis system 100 shown in Figure 1. The calibration method flow for the particle analysis system according to the second embodiment follows Figure 3. The calibration method flow according to the second embodiment differs from that of the first embodiment in that a background determination step S303 is added.

[0033] Next, the flow of the calibration method for the particle analysis system according to the second embodiment will be described. As shown in Figure 3, after the start of the calibration method flow, a reference setting step is performed in S301. This is the same as the reference setting step (S201) shown in Figure 2. The output adjustment step (S302) performed next is also the same as the output adjustment step (S202).

[0034] After performing steps S301 to S302, the background determination step is then executed in S303. In the background determination step (S303), first, the laser light is irradiated onto the sample to be measured using the laser output value (initial value or modified value) determined in S302, and the resulting image is acquired to identify the region of particles (bright spots) in the image as the particle region. Next, a region of a predetermined size adjacent to the outside of the identified particle region is identified as the adjacent region. For example, the adjacent region can be defined as the area from the outer periphery of the particle region to a radius of several pixels starting from the center or centroid of the particle region.

[0035] Next, the difference between the average brightness of the particle region and the average brightness of the adjacent region is calculated. The larger this difference, the more scattered light directly from the particles is captured in the image. Here, it is determined whether this difference is greater than or equal to a predetermined threshold (background determination step). If it is greater than or equal to the threshold (S303; YES), the process proceeds to S304. On the other hand, if it is less than the threshold (S303; NO), the processing unit 40 generates a command value to reduce the laser output by a predetermined step (e.g., 2mW). Then, the process returns to the output adjustment step (S302), where this command value is determined as the new laser output value (changed value), and measurement and determination are performed again. In this way, the background control step is executed, repeating the reduction of the laser output and the measurement and determination until the difference is greater than or equal to the predetermined threshold. After this difference is greater than or equal to the threshold and the background control step is passed (S303; YES), the process proceeds to S304, where the same processing as the determination step (S203) of the first embodiment is performed.

[0036] Thus, in the second embodiment, a background determination step (S303) is added to handle solvents that cause multiple scattering, and the laser output value is adjusted while also considering the brightness state other than that of particles.

[0037] <Example 1> Furthermore, when the particles of the sample being measured are UFB, the number concentration may decrease over time. This tendency has been confirmed to be particularly pronounced when a viscous substance such as rapeseed oil is used as the solvent for the sample being measured. For this reason, for example, the change in number concentration over time may be observed when the particles of the sample being measured are UFB and the solvent is rapeseed oil. In such cases, the laser output value of the sample being measured is first set according to the first or second embodiment, and this laser output value may be used on subsequent days, but it is necessary to fine-tune and calibrate the laser output value depending on environmental conditions, etc.

[0038] First, the standard sample (water / PSL) is measured using a laser output value set for the sample to be measured according to the first or second embodiment (for example, a lower output value than that used for the standard). At this time, because the laser output is weaker than that of the standard sample, the number concentration of standard particles actually detected will be measured lower than the nominal number concentration. A reference number concentration acquisition step is then performed to record this measured value as the reference number concentration. The number concentration can be determined based on the number of bright spots of particles in the captured image.

[0039] Next, when analyzing a sample to be measured on a subsequent day, for example, first, the number concentration of a standard sample is measured using the same laser output setting. If the number concentration at this time is lower than the reference number concentration, it is determined that a change in the device (light source, etc.) has occurred over time, and the laser output value is slightly increased to calibrate it so that it falls within a predetermined range relative to the reference number concentration. On the other hand, if the number concentration at this time is higher than the reference number concentration, the laser output value is slightly decreased to calibrate it so that it falls within a predetermined range relative to the reference number concentration. In other words, the number concentration of the image captured with the changed laser output value is evaluated, and the output change step is repeated until it falls within the appropriate range. By performing this calibration for daily measurements in this way, the number concentration can be accurately measured.

[0040] <Modification 2> In the embodiment of the present invention, all particles in the captured image are included when calculating the average brightness. However, before calculating the average brightness, the user may be asked to specify a predetermined range within the captured image, and after the specification, the average brightness of only the particles within that range may be calculated. This makes it possible to exclude obviously foreign objects from the calculation and to shorten the calculation process when there are many particles. [Explanation of Symbols]

[0041] 10 Laser light source 20 sample cells 30 Imaging device 40 Processing Unit 50 Display device 100-particle analysis system

Claims

1. A calibration method for a particle analysis system that irradiates particles in a liquid with light from a laser light source and analyzes the particles based on an image of scattered light acquired by an imaging device, A reference setting step involves determining a reference laser output value that yields an appropriate particle size distribution using a standard sample containing standard particles, and obtaining the average value of the brightness of the standard particles obtained at the reference laser output value as the reference brightness value. An output adjustment step is performed to estimate the ratio of scattered light intensity between the standard sample and the sample to be measured based on the refractive index information of the solvent and particles of the sample to be measured, and to determine the initial laser output value for the sample to be measured based on the ratio. A determination step in which the sample to be measured is measured using the initial laser output value, and it is determined whether the average value of the brightness of the particles obtained by the measurement is within a predetermined range corresponding to the reference brightness value, If the average value of the particle brightness obtained by the measurement is not within the predetermined range, the output control step involves repeating the laser output value change, the measurement, and the determination until the value falls within the predetermined range. A calibration method for a particle analysis system, including the above.

2. In the output adjustment step, A calibration method for a particle analysis system according to claim 1, comprising the assumption that the particle size of the standard particles contained in the standard sample is the same as the particle size of the particles contained in the sample to be measured, and estimating the ratio of the scattered light intensity using a theoretical formula for Rayleigh scattering or Mie scattering, or a lookup table based on said theoretical formula.

3. In the output control step, A calibration method for a particle analysis system according to claim 1 or 2, wherein if no particles are detected when the sample to be measured is measured, control is performed to increase the output of the laser light source until particles are detected.

4. A calibration method for a particle analysis system according to claim 1 or 2, wherein the average value of the brightness of the standard particles or the average value of the brightness of the particles obtained by the measurement is based on particles present in a predetermined range in the captured image.

5. A calibration method for a particle analysis system that irradiates particles in a liquid with light from a laser light source and analyzes the particles based on an image of scattered light acquired by an imaging device, A reference setting step involves determining a reference laser output value that yields an appropriate particle size distribution using a standard sample containing standard particles, and obtaining the average value of the brightness of the standard particles obtained at the reference laser output value as the reference brightness value. An output adjustment step is performed to estimate the ratio of scattered light intensity between the standard sample and the sample to be measured based on the refractive index information of the solvent and particles of the sample to be measured, and to determine the initial laser output value for the sample to be measured based on the ratio. A background determination step in which the sample to be measured is measured using the initial laser output value, and it is determined whether the difference between the average brightness of the particle region in the captured image and the average brightness of the adjacent region adjacent to the particle region is greater than or equal to a predetermined threshold, If the difference is less than the predetermined threshold, a background control step is performed in which the laser output value is reduced and the measurement and determination are repeated until the difference is equal to or greater than the predetermined threshold. A determination step of measuring the sample to be measured and determining whether the average value of the brightness of the particles obtained by the measurement falls within a predetermined range corresponding to the reference brightness value, If the average value of the particle brightness obtained by the measurement is not within the predetermined range, the output control step involves repeating the laser output value change, the measurement, and the determination until the value falls within the predetermined range. A calibration method for a particle analysis system, including the above.

6. In the output adjustment step, A calibration method for a particle analysis system according to claim 5, comprising assuming that the particle size of the standard particles contained in the standard sample is the same as the particle size of the particles contained in the sample to be measured, and estimating the ratio of the scattered light intensity using a theoretical formula for Rayleigh scattering or Mie scattering, or a lookup table based on said theoretical formula.

7. In the output control step, Calibration method for a particle analysis system according to claim 5 or 6, wherein if no particles are detected when the sample to be measured is measured, control is performed to increase the output of the laser light source until particles are detected.

8. The calibration method for a particle analysis system according to claim 5 or 6, wherein the average value of the brightness of the standard particles or the average value of the brightness of the particles obtained by the measurement is based on particles present in a predetermined range in the captured image.

9. The calibration method for a particle analysis system according to claim 1 or 5, wherein the imaging device includes an optical filter that transmits the wavelength of light from the laser light source and blocks the wavelength band of fluorescence.

10. A reference number concentration acquisition step is performed, in which the standard sample is measured using the laser output value determined by the output control step, and the number concentration of the standard particles obtained by the measurement is acquired as the reference number concentration. After a predetermined period has elapsed, the standard sample is measured using the determined laser output value, and the laser output value is changed so that the number concentration obtained by the measurement falls within a predetermined range corresponding to the reference number concentration. A calibration method for a particle analysis system according to claim 1 or 5, further comprising:

11. A program for causing a computer to perform the calibration method for a particle analysis system according to claim 1 or 5.

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