JP6726687B2 - Particle analyzer and particle analysis method - Google Patents

Description

The present invention relates to a particle analyzer that analyzes the state of particles in a dispersion medium based on the behavior of particles.

A particle analyzer is known that analyzes the state of particles in a dispersion medium (for example, particle size distribution, particle number concentration, agglomeration state, etc.) from the physical movement of each particle dispersed in the dispersion medium, that is, the behavior. ing.
For example, Patent Document 1 describes a particle analysis method using NTA (nanoparticle tracking analysis method) in paragraph 0004 and the like.

The NTA is, for example, a method of capturing scattered light from particles in a dispersion medium irradiated with laser light by a camera to acquire the behavior of each particle such as Brownian motion and calculating the particle state from the behavior. Is.

In this conventional method, a normal two-dimensional camera with a fixed focus is used, and the behavior of particles in the depth direction (z direction) cannot be detected. I can't.
Specifically, the following problems may occur.
(1) Since particles that move three-dimensionally are analyzed from a two-dimensional moving image, the movement in the depth direction (z direction) is ignored, and the accuracy of behavior analysis is poor.

(2) Particles outside the focused observation area cannot be measured. Therefore, one particle can be observed only within a very limited short time until it deviates from the observation region. On the other hand, if a cell with a depth as thin as the depth of focus is used, a problem that a sample with high viscosity cannot be used occurs.

(3) Since the particles can be measured only partially, a large error is included in the number measurement. Also, only particles illuminated by the depth of focus × camera angle of view can be observed, and the measurable concentration range is narrow.

Special table 2014-521967 gazette

The present invention has been made in view of such a problem, more accurately measures the behavior of the particles in the dispersion medium, based on which, while enabling the particle state in the dispersion medium can be accurately analyzed, The main objective is to enable observation of particle behavior for a long time.

That is, the particle analyzer according to the present invention analyzes the state of the particles in the dispersion medium based on the behavior of the particles in the dispersion medium, and three-dimensional information on the position of the particles in the dispersion medium. Three-dimensional information acquisition device for sequentially acquiring the three-dimensional information, and based on time-series data of the three-dimensional information obtained by the three-dimensional information acquisition device, three-dimensional behavior of particles is specified, and the dispersion of particles from the behavior A particle state analysis device for analyzing a state in a medium is provided.
The “three-dimensional information regarding the position of the particle” includes three-dimensional position information of the particle, as well as three-dimensional velocity vector and acceleration vector of the particle.

According to such a configuration, the particle state is analyzed from the three-dimensional behavior of the particle, so that the accuracy is greatly improved as compared with the conventional method that analyzes the particle state based on the two-dimensional behavior of the particle. Can be improved.

In addition, since it is possible to obtain all three-dimensional information for individual particles over a long period of time, it becomes possible to understand various particle behaviors as compared with the conventional two-dimensional method that can be observed only partially. As a result, it is possible to observe almost the entire number of observation areas. In addition, since a wide range is observed at the same time, it can be expected to be effective especially for low concentrations. Furthermore, three-dimensional observation is effective in the case of analysis of particles (spiral motion, etc.) that have anisotropic motion that can never be analyzed from a two-dimensional moving image. Further, since it is possible to observe three-dimensionally, simultaneous measurement such as Brownian motion observation in the vertical axis direction and electrophoretic observation in the horizontal axis direction and high-precision measurement are possible.
As a specific example, the behavior of particles is Brownian motion, and the state of particles is a particle size distribution.

It is also possible to analyze multiple particle states in parallel. As an example thereof, further comprising an electric field forming means for forming an electric field, wherein the dispersion medium and the particles are arranged in the electric field, the particle state analysis device, the behavior of particles generated by the electric field Based on barrel electrophoresis, the zeta potential of the particles is measured, and the particle diameter is measured based on Brownian motion, which is the behavior of the particles specified from the direction component different from the potential change direction due to the electric field. You can

In order to speed up the acquisition of the three-dimensional information and to observe the behavior of particles with high time resolution so that the particle state can be analyzed with high accuracy, a three-dimensional information acquisition device is required to have a plurality of different focal lengths. It is preferable that the three-dimensional information is obtained by integrating the two-dimensional images of the particles in the dispersion medium simultaneously captured by the two-dimensional cameras.

According to the present invention configured as described above, since the particle state is analyzed from the three-dimensional position information of the particle, it is possible to more accurately specify the behavior of the particle as compared with the conventional method, and based on that, It becomes possible to analyze the particle state in the dispersion medium more accurately and for a long time.

1 is an overall view schematically showing a particle analyzer according to a first embodiment of the present invention. The schematic diagram for demonstrating the particle imaging method of the particle analyzer in the same embodiment. The display image figure which shows an example which displayed the particle diameter distribution in the same embodiment by 3D graphic. The whole figure which shows typically the particle|grain analyzer in 2nd Embodiment of this invention. The schematic diagram which shows the electric field provision structure of the particle analyzer in other embodiment of this invention. The display image figure which shows an example of the display screen of the zeta potential distribution diagram in the same embodiment. The schematic diagram for demonstrating the particle|grain imaging method of the particle analysis apparatus in other embodiment of this invention.

100... Particle analysis device 3... Three-dimensional information acquisition device 4... Particle state analysis device

A particle analyzer according to an embodiment of the present invention will be described below with reference to the drawings.
<First Embodiment>
As shown in FIG. 1, the particle analyzer 100 according to this embodiment includes the following components.
(1) A cell 1 containing a sample in which particles S to be measured are dispersed in a dispersion medium.
(2) A light source device 2 for irradiating the sample with laser light R via a cell.
(3) The secondary light (for example, scattered light, reflected light, transmitted light, etc.) generated by irradiating the particles S with the laser light R is detected to determine the three-dimensional position of each particle S in the dispersion medium. A three-dimensional information acquisition device 3 that sequentially acquires the three-dimensional information shown in time series.
(4) The behavior of the particles S is specified based on the time-series data of the three-dimensional information obtained by the three-dimensional information acquisition device 3, and the state of the particles S in the dispersion medium is analyzed based on the behavior. Particle state analysis device 4.

Each component will be described.
As shown in FIG. 1, the cell 1 is a transparent cell formed of glass, quartz, resin or the like contained in a sample. The cell 1 in the illustrated example has a rectangular parallelepiped shape or a cubic shape, but may have another shape such as a cylindrical shape.

The light source device 2 includes a laser oscillator (not shown) that emits the laser light R as described above, and a lens optical system that outputs the laser light R emitted from the laser oscillator as parallel light having a predetermined diameter (shown in the figure). Not)). The laser light R output from the light source device R is applied to one surface of the cell 1. The light with which the sample is irradiated is not necessarily the laser light R. It may be LED light, light from a halogen lamp, or the like, or may not be completely parallel.

The three-dimensional information acquisition device 3 includes, for example, a two-dimensional camera (not shown) and an image processing device (not shown). The two-dimensional camera is a two-dimensional area sensor (not shown) composed of CCD or CMOS, a camera optical system (not shown) for forming an image on the two-dimensional area sensor, and a two-dimensional area sensor or camera. An image pickup control mechanism (not shown) for controlling an optical system to perform an image pickup operation is provided.

This two-dimensional camera is in a direction different from the optical axis direction of the laser light R (hereinafter, also referred to as x direction), specifically, for example, a direction perpendicular to the optical axis of the laser light R (hereinafter, also referred to as z direction). ) To image the sample in cell 1. This two-dimensional camera continuously captures a plurality of two-dimensional image data at a short time interval in a predetermined one image capturing cycle in response to a command from the image capturing control mechanism. Each two-dimensional image data is generated by capturing an image while shifting the focal position (focus) by a certain distance.

In this embodiment, as shown in FIG. 2, each of the focus positions is set in the cell 1, and each two-dimensional image data is taken in the xy plane P of the sample in the cell 1 which is different only in the z direction. It is configured to show an image. The three-dimensional image data in the cell 1 can be generated by integrating a plurality of two-dimensional image data obtained in this one photographing cycle.

The image processing apparatus is an information processing apparatus including a digital electric circuit such as a CPU, a memory, and a communication port, an analog electric circuit such as an analog amplifier and a buffer, and an ADC and a DAC that connect them, and is stored in the memory. The CPU and its peripherals cooperate with each other in accordance with the specified program to perform a predetermined process on each two-dimensional image data.

The function will be specifically described.
In each of the two-dimensional image data, the light scattered by the laser light R striking each particle S is photographed. Focusing on one image data, secondary light from each particle S existing on the xy plane P at that focal length is photographed in the image data, and the secondary light is represented as a bright spot having a high light intensity density. Has been done. On the other hand, in this image data, the scattered light from the particles S existing on each xy plane P other than the xy plane P is also captured as an out-of-focus state, that is, as dim background noise with low light intensity density. There is.

The image processing apparatus removes the background noise from each of the two-dimensional image data, extracts only the scattered light from the particles S existing on the corresponding xy plane P, and displays the scattered light as a bright spot. The two-dimensional image data whose position on the xy plane P can be specified is converted.

Note that the particles S existing on the xy plane P are not limited to the particles S existing strictly only on the xy plane, and as shown in FIG. 2, a minute constant width before or after the particle S, that is, a minute constant width in the z direction. The particles S in the region of. This is because the camera optical system has a certain depth of focus, and therefore the focused region also has a certain thickness in the z direction.

As described above, the plurality of two-dimensional image data obtained in the one photographing cycle are noise-removed by the image processing device, and the two noise-removed two-dimensional image data are arranged side by side in the z direction and integrated. Thus, the three-dimensional image data in one photographing cycle is obtained. The bright spot in the three-dimensional image data, that is, the position of the particle S can be grasped from the xy coordinates in the two-dimensional data and the z coordinate of the two-dimensional image data. 3D information indicating the position in 3D.

Then, the two-dimensional camera is set to repeat the photographing cycle a predetermined number of times at a constant time interval, and the three-dimensional image data sequentially obtained in each photographing cycle is associated with the photographing time, It is output from the image processing device either sequentially or all at once after the end of shooting. The shooting time may be attached to each three-dimensional image data, or more strictly, to each two-dimensional image data.

The particle state analysis device 4 is an information processing device including a CPU, a memory, a communication port, and the like, and the behavior of the particles S in the cell 1 is determined by the cooperation of the CPU and its peripheral devices according to the memory. It has a function of identifying and analyzing the state of the particles S in the dispersion medium from the behavior.
This will be specifically described.

The particle state analysis device 4 receives a series of three-dimensional image data transmitted from the image processing apparatus with a photographing time. The series of three-dimensional image data is time-series data of three-dimensional information indicating the three-dimensional position of the particle S in the cell 1, that is, three-dimensional moving image data.

Then, the behavior of each particle S, that is, the moving direction, moving speed, and moving direction of each particle S is specified from this series of three-dimensional image data. Specifically, the three-dimensional image data at a certain time is compared with the three-dimensional image data at the next time, and from the change in the position of the bright spot indicating each particle S, the behavior of each particle S described above is determined. Specify.

At this time, it is necessary to specify which particle S in the three-dimensional image data at the next time the particle S in the first three-dimensional image data corresponds to. Therefore, the particle state analysis device 4 identifies the same particle S between image data as described below, for example.

For explanation, one particle S is focused. Since the particle S appearing in the first three-dimensional image data has a limit in its moving speed, in the next three-dimensional image data, the particle S falls within a predetermined radius (sphere) defined by the limit moving speed from the first position. Exists.

Therefore, the particle state analysis device 4 first searches for particles S existing within the predetermined radius in the next three-dimensional image data. If there is only one, it is specified that the particle S is the same as the particle S that appeared in the initial three-dimensional image data.

On the other hand, in the next three-dimensional image data, when a plurality of particles S exist within the predetermined radius, the brightness of the particle S satisfying other conditions, for example, the brightness of the particle S appearing in the first three-dimensional image data is the highest. The particles S having similar brightness are specified as the particles S that are the same as the particles S that appeared in the initial three-dimensional image data.

In this way, the particle state analysis device 4 clarifies the correspondence relationship between the respective particles S appearing in the respective three-dimensional image data, and then specifies the behavior of each particle S from the difference in the position of the same particle S.
Next, the particle state analysis device 4 analyzes the state of the particles S in the dispersion medium from the behavior of each of the particles S.

The state of the particles S here is, for example, a particle size distribution here. Since the above-mentioned behavior shows, for example, Brownian motion, the particle size of each particle S is calculated from this Brownian motion to calculate the particle size distribution of the sample.

Furthermore, the particle state analysis device 4 in this embodiment displays the particle size distribution calculated in this way in a distribution graph in which the horizontal axis represents the particle diameter and the vertical axis represents the number of particles, as shown in FIG. A three-dimensional map as shown is displayed on the display 41 (shown in FIG. 1). This three-dimensional map is constructed so that the view direction can be changed.

In the display example of FIG. 3, after setting a plurality of particle diameter ranges and determining to which particle diameter range each particle S belongs, each particle S is displayed at the corresponding coordinates of the three-dimensional position graph. The display mode is changed for each particle size range (for example, as dot symbols having different colors and shapes).

With such a configuration, since the particle state is analyzed from the three-dimensional position information of the particle S, it is possible to more accurately specify the behavior of the particle S as compared with the conventional 2D method. For example, the particle size distribution in the dispersion medium can be measured more accurately.

Further, since the display as shown in FIG. 3 is made possible, for example, large particles S tend to be sunk, or particles S having a particle diameter within a certain range tend to aggregate. Analysis became intuitive and various tendencies that could not be found in the past were clarified, which could open up new possibilities for analysis of particle size distribution.

<Second Embodiment>
In the particle behavior measurement of the three-way component according to the present invention, when focusing on one particle state (for example, particle diameter) as in the first embodiment, the accuracy is significantly higher than that of the conventional particle behavior measurement using the two-way component. It is possible to achieve such effects as the improvement in performance and the continuous measurement for a long time.

On the other hand, from a different point of view, it can be said that the present invention can obtain a large amount of information for the one-way component as compared with the conventional particle behavior measurement using the two-way component. Thus, the increased one-way component allows the different states of the particle to be measured simultaneously. An example of this is the second embodiment.

In the second embodiment, the zeta potential can also be measured. As is well known, the zeta potential is calculated from the moving speed (electrophoretic speed) of the particles S when the sample is placed in an electric field.

Here, a pair of electrode plates 5 are provided spaced apart in the y-axis direction so as to be sandwiched on the outside of the cell 1, and an AC or DC voltage is applied to these electrode plates 5 so that the y-axis direction is applied to the sample. An electric field that creates a potential difference is applied to.

The camera of the three-dimensional information acquisition device 3 is arranged so that the cell 1 is photographed between the electrode plates 5 so that the electrode plates 5 do not interfere with the photographing. Specifically, the optical axis direction (shooting optical axis) C of the camera is set to be perpendicular to the surface direction of the electrode plate 5.
Other physical configurations are the same as those in the first embodiment.

However, in this embodiment, as a function of the particle state analysis device 4, a function of simultaneously measuring the zeta potential of the particle S and measuring the particle diameter based on the analysis of Brownian motion is added.

That is, in order to measure the zeta potential, the behavior of the y-axis direction component of the particle S is extracted and the zeta potential is calculated from this, while the Brownian motion is calculated from the behavior of the x-axis direction component and the z-axis direction component of the particle S. Analysis is performed to calculate the particle size.

It is also possible to simultaneously measure two types of zeta potentials. For example, as in the above-described embodiment, in addition to the pair of first electrode plates 5 that are spaced apart in the y-axis direction, a pair of second electrode plates 6 that are spaced apart in the z-axis direction are provided, and in the y-axis direction. In addition, an electric field that causes a potential difference is also applied in the z-axis direction.

At this time, a low-frequency or direct-current voltage is applied in the y-axis direction, and a high-frequency AC voltage is applied in the z-axis direction. Then, the behavior of the y-direction component and the behavior of the z-axis direction component of the particle S are separated and analyzed. In this way, the zeta potential distribution can be measured from the behavior of the y-direction component of the particle S, and the average value (or absolute value) of the zeta potential can be measured from the behavior of the z-direction component of the particle S. Things can be done in parallel. FIG. 6 shows a display example of the two measurement results on the display. It should be noted that the photographing may be performed through the through hole, for example, by forming a through hole in the second electrode plate 6.

<Other embodiments>
The present invention is not limited to the above embodiment.
Since the 3D observation of the behavior of the particle S by three-dimensional animation can be performed, the behavior of the particle S can be tracked for a long time. Therefore, for example, it is possible to add a function of observing not only the Brownian motion of the particle S described above but also the behavior of the particle S such as settling or floating in the cell 1 or convection movement due to temperature.

Moreover, the behavior when the two particles S approach or collide can also be accurately observed by three-dimensional measurement. This may be analyzed by a particle analysis device to calculate repulsive force or attractive force between particles (this is a particle state). Based on this, it becomes possible to analyze the interaction of particles. This also applies to particles that do not produce a zeta potential.

Also, a gel may be used as the sample. In such a case, the three-dimensional behavior of the particles S dispersed in the gel is observed, and the length of the cross-linking arm of each particle S (this is the particle state) is calculated accurately from the vibration state. You can Further, this makes it possible to specify the shearing force (so-called “sliding”) applied to the gel, its position, and the like.

Further, not only the electric field but also the behavior of the particle S due to other external fields (magnetic field, gravitational field, field due to centrifugal force, etc.) may be measured, and this may be mapped in various formats and visually. It is more preferable if it is easy to understand.

The three-dimensional image data was generated by sequentially photographing a plurality of two-dimensional image data with a single two-dimensional camera while shifting the focal length and integrating them, but as shown in FIG. It is also possible to capture two-dimensional image data by capturing images in parallel at once by a plurality of two-dimensional cameras 3 having different positions, and to integrate and generate these. The two-dimensional cameras 3 do not need to be physically completely independent, and may be housed in, for example, one housing and share a part of the configuration or function such as a control mechanism and a shutter. .. Further, in this case, a plurality of two-dimensional cameras having different focal positions in the z direction may be used to shoot moving images, and these may be synchronously integrated to generate a three-dimensional moving image.
Besides, it goes without saying that the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the spirit of the present invention.

With the particle analysis device according to the present invention, since the particle state is analyzed from the three-dimensional position information of the particle, it is possible to more accurately specify the behavior of the particle as compared with the related art, and based on that, It becomes possible to analyze the particle state in the dispersion medium more accurately and for a long time.

Claims (5)

By shifting the focus position in the dispersion medium along the imaging optical axis, a plurality of two-dimensional image data is generated by imaging different surfaces, and the particles included in the respective two-dimensional image data are present on the surfaces having different focus positions. The scattered light of is removed as background noise, and the two-dimensional image data from which the background noise is removed are integrated to sequentially obtain three-dimensional information regarding the positions of a plurality of particles in the dispersion medium in time series. A dimensional information acquisition device,
Based on the time-series data of the three-dimensional information obtained by the three-dimensional information acquisition device, the same particle is identified among a plurality of particles among the three-dimensional information at each time, and A particle state analysis device, comprising: a particle state analysis device that identifies each three-dimensional behavior and analyzes the state of particles in a dispersion medium from the behavior. The particle analyzer according to claim 1, wherein the behavior of the particles is Brownian motion, and the state of the particles is a particle size distribution. An electric field forming means for forming an electric field is further provided, and the dispersion medium and the particles are arranged in the electric field.
The particle state analysis device measures the zeta potential of the particle based on electrophoresis, which is the behavior of the particle generated by the electric field, and is a brown that is the behavior of the particle specified from a direction component different from the potential change direction by the electric field. The particle analyzer according to claim 1, wherein the particle size is measured based on movement. The three-dimensional information acquisition device includes a plurality of two-dimensional cameras having different focal lengths, and the three-dimensional images are obtained by integrating the two-dimensional images obtained by simultaneously capturing the particles in the dispersion medium with each two-dimensional camera. The particle analyzer according to claim 1, which acquires information. A particle analysis method using a three-dimensional information acquisition device that sequentially acquires three-dimensional information regarding the positions of a plurality of particles in a dispersion medium in a time series,
The three-dimensional information acquisition device shifts the focal position in the dispersion medium along the photographing optical axis to generate a plurality of two-dimensional image data obtained by imaging different surfaces. Based on time series data of three-dimensional information obtained by removing scattered light from particles existing on different surfaces as background noise and integrating the two-dimensional image data from which the background noise has been removed , A plurality of particles that are the same among the three-dimensional information at time are specified, the three-dimensional behavior of each specified particle is specified, and the state of the particles in the dispersion medium is analyzed from the behavior. A particle analysis method characterized by the above.

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