Method for measuring particle size of colloidal particle based on microscopy imaging particle tracer technology

Abstract

The invention belongs to the field of physical chemistry, and discloses a method for measuring the particle size of colloidal particles based on a microscopy imaging particle tracer technology, which comprises the following steps of: adding particles to be measured into Newtonian fluid at a proper concentration, and shooting a video of random thermal motion of the particles in the fluid by using a microscope and a high-speed camera; the method comprises the following steps of: obtaining a movement track of a particle through a series of image processing technologies such as de-noising, positioning and track connection, calculating a mean square displacement MSD of the particle, fitting a particle diffusion coefficient D based on a linear relation between the MSD and a time interval tau, and calculating the particle size of the colloidal particle according to a Stokes-Einstein relation in combination with known solvent viscosity eta, temperature T and a Boltzmann constant kB. The particle size of the added colloidal particles can be completely and accurately measured based on the particle tracer technology of microscope imaging, and a new method is provided for measuring the particle size of the particles.

Description

Technical Field

[0001] The invention belongs to the field of physical chemistry, and in particular relates to a method for measuring the particle size of colloidal particles by using a particle tracing technique based on microscope imaging. Background Art

[0002] Particle size and its distribution are key parameters characterizing the physicochemical properties of granular materials, directly influencing key performance indicators such as flowability, solubility, reactivity, and bioavailability. In fields such as materials science, pharmaceuticals, environmental monitoring, nanotechnology, and biomedicine, accurate particle size measurement is crucial for optimizing product performance, ensuring process stability, and assessing safety.

[0003] Dynamic light scattering (DLS), with its wide particle size range and high sensitivity, has become the preferred technique for characterizing nanoparticle liquid-phase systems. It is particularly suitable for standardized analysis of low-concentration homogeneous samples. However, it also has many limitations: 1. DLS testing assumes that only the particles to be tested generate scattered light, which places high demands on the preparation of the experimental suspension and the need for dust-free handling of the experimental materials, otherwise the accuracy of the results will be affected; 2. It is highly dependent on particle size and concentration, and multiple optimizations of the concentration range are required to obtain reliable experimental results (Dynamic light scattering: a practical guide and applications in biomedical sciences. Biophysical Reviews, 2016, 8, 409-427). In contrast, particle tracking technology ensures that only the particles to be tested are observed. At the same time, the particle distribution in the field of view can be directly observed and rationally controlled based on the distribution density. Moreover, combined with corresponding analysis algorithms, experiments have shown that the particle size values ​​are directly close to the actual values. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention provides a method for measuring the particle size of colloidal particles based on a particle tracing technique using microscope imaging.

[0005] In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions:

[0006] The particle tracing technology based on microscope imaging is a method for measuring the size of colloidal particles. It combines particle screening, positioning, identification, connection and statistics to more accurately and reliably obtain the mean square displacement of particles in the fluid and measure the particle size. It includes:

[0007] The particles to be tested are added to the Newtonian fluid at a suitable concentration, and a microscope and a high-speed camera are used to capture a video of the random thermal motion of the particles in the fluid;

[0008] After obtaining the particle's motion trajectory through a series of image processing techniques such as denoising, positioning, and trajectory connection, its mean square displacement (MSD) is calculated;

[0009] The particle diffusion coefficient D is fitted based on the linear relationship between MSD and time interval τ, and then the Stokes-Einstein relationship is combined with the known solvent viscosity η, temperature T and Boltzmann constant k. B Calculate the particle size of the colloidal particles.

[0010] Preferably, after obtaining the particle thermal motion video, image processing is mainly divided into three steps: first, denoising the image, then accurately locating the particle position, and finally connecting the particle trajectories.

[0011] Preferably, the main implementation method of denoising is to use the bpass function in the program to perform noise reduction processing on the video image, set the brightness threshold according to the brightness generated by the particles, and the brightness of the area below this threshold will automatically be set to 0.

[0012] Furthermore, positioning requires first processing the first frame. The particle diameter and error parameters are set based on the actual pixel size of the particle in the image. The program then automatically locates the particle in the image. The particle positioning information for several additional frames is then retrieved to ensure that all frames in the video can accurately locate the particles within the set parameters. The program then runs the processing program to locate the particles in all frames of the video one by one.

[0013] Furthermore, trajectory connection distinguishes different particle displacements and sets a maximum displacement for a particle between two frames. Any particle exceeding this displacement will be identified and located as two particles. The positions of the same particle in each frame are then connected to obtain the particle's trajectory.

[0014] Furthermore, the measurement sample is a particle suspension, the solute is a Newtonian fluid, the viscosity is known or can be obtained by other viscosity measurement methods, the shape of the colloidal particles to be measured is close to or can be approximated to a sphere, the upper limit of the particle size is 10 μm, and the lower limit is the observation limit of the imaging microscope.

[0015] Furthermore, the experimental temperature is controlled, and the sample is allowed to stand at the experimental temperature for a period of time before a video of the thermal motion of the particles is captured.

[0016] Furthermore, for Newtonian fluids, the mean square displacement of colloidal particles within a time interval τ satisfies:

[0017] <Δr 2 (τ)>=2dDτ;

[0018] At the same time, if the particle is spherical, according to the Stokes-Einstein equation:

[0019]

[0020] The particle diffusion coefficient D is obtained by fitting the linear relationship between MSD and time interval τ, and then combined with the known solvent viscosity η, temperature T and Boltzmann constant k B Calculate the particle size of the colloidal particles.

[0021]

[0022] Compared with the prior art, the present invention has at least the following advantages and effects:

[0023] 1. DLS requires a high purity of the experimental suspension. Measuring nanoparticle size based on multi-particle tracking microrheology ensures that only the particles to be measured are observed, eliminating interference from impurities in the experimental suspension.

[0024] 2. DLS test results only output the average particle size and polydispersity index (PDI), but multi-particle tracer microrheology technology can simultaneously obtain the particle size distribution, diffusion coefficient and rheological properties of the medium. It is suitable for fields such as soft materials and biomaterials that require multi-parameter collaborative analysis, and also supports real-time monitoring of dynamic processes and analysis of local inhomogeneities.

[0025] 3. DLS measurement is highly dependent on particle size and concentration; however, multi-particle tracer microrheology can rationally control the concentration of the particles to be measured by directly observing the particle concentration in the field of view. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a flow chart of a method for measuring the particle size of nanoparticles using a particle tracing technique based on microscope imaging in one embodiment of the present invention.

[0027] Figure 2 The particle motion MSD curve is obtained by measuring particles with a standard particle size of 2 μm at 25° C. using the present invention.

[0028] Figure 3 The particle motion MSD curve is obtained by measuring particles with a standard particle size of 1 μm at 25° C. using the present invention.

[0029] Figure 4 The particle motion MSD curve is obtained by measuring particles with a standard particle size of 2 μm at 30° C. using the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments, but the protection scope and implementation methods of the present invention are not limited thereto.

[0031] The present embodiment provides a method for measuring colloidal particle size using a particle tracing technique based on microscopic imaging. This method combines particle screening, location, identification, connection, and statistics techniques to more accurately and reliably obtain the mean square displacement of particles in a fluid and the particle size of the particles being measured. The method includes:

[0032] The particles to be tested are added to the Newtonian fluid at a suitable concentration, and a microscope and a high-speed camera are used to capture a video of the random thermal motion of the particles in the fluid;

[0033] After obtaining the particle's motion trajectory through a series of image processing techniques such as denoising, positioning, and trajectory connection, its mean square displacement (MSD) is calculated;

[0034] The particle diffusion coefficient D is fitted based on the linear relationship between MSD and time interval τ, and then the Stokes-Einstein relationship is combined with the known solvent viscosity η, temperature T and Boltzmann constant k. B Calculate the particle size of the colloidal particles.

[0035] As an embodiment, taking water and carboxyl-modified polystyrene particles as an example, the measurement method specifically includes the following steps:

[0036] (1) Preparation of particle suspension and particle tracing experiment

[0037] Probe particles were added to ultrapure water at a specific dilution factor and shaken to ensure uniform particle distribution. The sample was then injected into a homemade glass chamber and sealed with vacuum silicone grease. The sample was placed on the sample stage of a fluorescence confocal microscope. The experimental temperature was controlled, and a 100× oil immersion lens was used to focus the image. The high-speed camera was set to 2000 frames per second, a frame rate of 34 fps, an exposure time of 0.01 s, and a pixel size of 1412 px x 1412 px per frame. A video of the thermal motion of the particles under the microscope was captured using the high-speed camera.

[0038] (2) Particle screening, positioning, marking, and trajectory connection

[0039] First, call out the first frame of the image, and use the bpass function in the program to perform noise reduction optimization on the video image. Set the brightness threshold according to the brightness generated by the particles. The brightness of the area below this threshold will automatically be set to 0. Then set the particle size and error size according to the actual size of the particles in the image. At this time, the program will automatically locate the position of the particles in the image. Recall the particle positioning of several other frames to ensure that all frames contained in the video can accurately locate the particles under the set parameters. At the same time, different particles must be distinguished between each frame. Therefore, it is necessary to set the maximum displacement of the particles between two frames. If the displacement exceeds this value, they will be identified and located as two particles. Then run the processing program to locate all particles in the 2000 frames of the video one by one, and then connect the positions of the same particle in each frame to obtain the particle's motion trajectory.

[0040] (3) Calculation of particle size to be measured

[0041] The particle diffusion coefficient D is calculated based on the linear relationship between MSD and time interval τ, and then the Stokes-Einstein equation is combined with the known solvent viscosity η, temperature T and Boltzmann constant k. B Calculate the particle size of the colloidal particles.

[0042] The present invention will be further described below through several specific embodiments.

[0043] Example 1

[0044] 10 μL of polystyrene particle suspension (2 μm in diameter) was ultrasonicated for 3 min and then shaken for 2 min. 10 μL was taken with a pipette and added to 1 mL of ultrapure water. After shaking for about 1 min, the mixture was injected into the glass chamber and sealed with vacuum silicone grease.

[0045] The viscosity of ultrapure water was measured by macrorheology. The instrument selected was a rotational rheometer. The fixture was a coaxial cylinder with an inner diameter of 27 mm and made of stainless steel. The distance between the upper and lower fixtures was 3.6 mm during the experiment. 8 mL of liquid was added to the coaxial cylinder using a pipette. The temperature was set to 25 ° C. After 5 minutes of equilibrium, the test was started. -1 ~0.1s -1 The viscosity variation with shear rate was measured within a range of shear rates.

[0046] Place the sample on the microrheometer, set the chamber temperature to 25°C, use a 100× oil-immersion objective lens to focus, set the parameters of the high-speed camera to 2000 frames, a frame rate of 34fps, an exposure time of 0.01s, and the pixels of each frame are 1412px×1412px. After stabilization for 15 minutes, take a video of the thermal motion of the particles.

[0047] Read the video file, first call out the first frame image, use the bpass function to optimize the video image for noise reduction, set the brightness threshold to 10000, the particle size to 48px, the error size to 50px, locate all particles in each frame, set the maximum displacement to 10px to distinguish different particles, and then connect the position coordinates of the particles in each frame to obtain the particle motion trajectory.

[0048] For Newtonian fluids, the mean square displacement of colloidal particles within a time interval τ satisfies:

[0049] <Δr 2 (τ)>=2dDτ

[0050] At the same time, if the particle is spherical, according to the Stokes-Einstein equation:

[0051]

[0052] The particle diffusion coefficient D is obtained by fitting the linear relationship between MSD and time interval τ, and then combined with the known solvent viscosity η, temperature T and Boltzmann constant k B Calculate the particle size of the colloidal particles.

[0053]

[0054] The log-log plot of MSD versus time interval τ is used, and the mean square displacement obtained is almost a straight line, as shown in Figure 2 As shown, the slope of the fitted straight line is 81.13×10 -14 Since the MSD data is the average displacement of the particles in two-dimensional motion, the particle motion dimension d is taken as 2, and the calculated diffusion coefficient D is 20.28×10 -14 m 2 / s; the viscosity of the sample at 25°C was measured by a rotational rheometer to be 1.11 mPa s, and the particle size was calculated to be 1.94 μm.

[0055] Example 2

[0056] 10 μL of polystyrene particle suspension (1 μm in diameter) was ultrasonicated for 3 min and then shaken for 2 min. 10 μL was taken with a pipette and added to 10 mL of ultrapure water. After shaking for about 1 min, the mixture was injected into the glass chamber and sealed with vacuum silicone grease.

[0057] The viscosity of ultrapure water was measured by macrorheology. The instrument was a rotational rheometer. The fixture was a coaxial cylinder with an inner diameter of 27 mm and made of stainless steel. The distance between the upper and lower fixtures was 3.6 mm. 8 mL of liquid was added to the coaxial cylinder using a pipette. The temperature was set to 25 ° C. After 5 minutes of equilibrium, the test was started. -1~0.1s -1 The viscosity variation with shear rate was measured within a range of shear rates.

[0058] Place the sample on the microrheometer, set the chamber temperature to 25°C, use a 100× oil-immersion objective lens to focus, set the parameters of the high-speed camera to 2000 frames, a frame rate of 34fps, an exposure time of 0.01s, and the pixels of each frame are 1412px×1412px. After stabilization for 15 minutes, take a video of the thermal motion of the particles.

[0059] Read the video file, first call out the first frame image, use the bpass function to optimize the video image for noise reduction, set the brightness threshold to 3000, the particle size to 24px, the error size to 26px, locate all particles in each frame, set the maximum displacement to 20px to distinguish different particles, and then connect the position coordinates of the particles in each frame to obtain the particle motion trajectory.

[0060] For Newtonian fluids, the mean square displacement of colloidal particles within a time interval τ satisfies:

[0061] <Δr 2 (τ)>=2dDτ

[0062] At the same time, if the particle is spherical, according to the Stokes-Einstein equation:

[0063]

[0064] The particle diffusion coefficient D is obtained by fitting the linear relationship between MSD and time interval τ, and then combined with the known solvent viscosity η, temperature T and Boltzmann constant k B Calculate the particle size of the colloidal particles.

[0065]

[0066] The log-log plot of MSD versus time interval τ is used, and the mean square displacement obtained is almost a straight line, as shown in Figure 3 As shown, the slope of the fitted straight line is 157.08×10 -14 Since the MSD data is the average displacement of particles in two-dimensional space, the particle motion dimension d is taken as 2, and the calculated diffusion coefficient D is 39.27×10 -14 m 2 / s; the viscosity of the sample at 25°C was measured by a rotational rheometer to be 1.11 mPa s, and the particle size was calculated to be 1.00 μm.

[0067] Example 3

[0068] 10 μL of polystyrene particle suspension (2 μm in diameter) was ultrasonicated for 3 min and then shaken for 2 min. 10 μL was taken with a pipette and added to 1 mL of ultrapure water. After shaking for about 1 min, the mixture was injected into the glass chamber and sealed with vacuum silicone grease.

[0069] The viscosity of the sample was measured by macrorheology. The instrument was a rotational rheometer. The fixture was a coaxial cylinder with an inner diameter of 27 mm and made of stainless steel. The distance between the upper and lower fixtures was 3.6 mm. 8 mL of liquid was added to the coaxial cylinder using a pipette. The temperature was set to 30 ° C. After 5 minutes of equilibrium, the test was started. -1 ~0.1s -1 The viscosity variation with shear rate was measured within a range of shear rates.

[0070] Place the sample on the microrheometer, set the chamber temperature to 30°C, use a 100× oil-immersion objective lens to focus, set the parameters of the high-speed camera to 2000 frames, a frame rate of 34fps, an exposure time of 0.01s, and the pixels of each frame are 1412px×1412px. After stabilization for 15 minutes, take a video of the thermal motion of the particles.

[0071] Read the video file, first call out the first frame image, use the bpass function to optimize the video image for noise reduction, set the brightness threshold to 10000, the particle size to 48px, the error size to 50px, locate all particles in each frame, set the maximum displacement to 10px to distinguish different particles, and then connect the position coordinates of the particles in each frame to obtain the particle motion trajectory.

[0072] For Newtonian fluids, the mean square displacement of colloidal particles within a time interval τ satisfies:

[0073] <Δr 2 (τ)>=2dDτ

[0074] At the same time, if the particle is spherical, according to the Stokes-Einstein equation:

[0075]

[0076] The particle diffusion coefficient D is obtained by fitting the linear relationship between MSD and time interval τ, and then combined with the known solvent viscosity η, temperature T and Boltzmann constant k B Calculate the particle size of the colloidal particles.

[0077]

[0078] The log-log plot of MSD versus time interval τ is used, and the mean square displacement obtained is almost a straight line, as shown in Figure 4As shown, the slope of the fitted straight line is 90.24×10 -14 Since the MSD data is the average displacement of particles in two-dimensional space, the particle motion dimension d is taken as 2, and the diffusion coefficient D is calculated to be 22.56×10 -14 m 2 / s; the viscosity of the sample at 30°C was measured by a rotational rheometer to be 1.03 mPa s, and the particle size was calculated to be 1.91 μm.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for measuring colloidal particle size using a particle tracing technique based on microscope imaging, characterized in that: The following steps are involved: The particles to be tested are added to the Newtonian fluid at a suitable concentration, and a microscope and a high-speed camera are used to capture a video of the random thermal motion of the particles in the fluid; The particle thermal motion video is processed using image processing technology to obtain the particle trajectory, and its mean square displacement (MSD) is calculated. The particle diffusion coefficient D is fitted based on the linear relationship between MSD and time interval τ, and then the Stokes-Einstein relationship is combined with the known solvent viscosity η, temperature T and Boltzmann constant k. B Calculate the particle size of the colloidal particles.

2. The method for measuring colloidal particle size using particle tracing technology based on microscope imaging according to claim 1, characterized in that: The image processing includes: firstly, removing noise from the image, then accurately locating the particle positions, and finally, connecting the particle trajectories.

3. The method for measuring colloidal particle size using particle tracing technology based on microscope imaging according to claim 2, characterized in that: The denoising process uses the bpass function to perform noise reduction on the video image, and sets a brightness threshold according to the brightness generated by the particles. The brightness of the area below the threshold is automatically set to 0.

4. The method for measuring colloidal particle size using particle tracing technology based on microscope imaging according to claim 2, characterized in that: The positioning process requires first retrieving the first frame of image for processing, setting the particle diameter and error parameters based on the actual pixel size of the particles in the image, and automatically locating the particle position in the image. Then, the particle positioning conditions of several other frames of images are retrieved to ensure that all frames contained in the video can accurately locate the particles within the set parameters. Finally, the particles in all frames of the video are positioned one by one.

5. The method for measuring colloidal particle size using a particle tracing technique based on microscope imaging according to claim 2, wherein: To connect trajectories, we need to distinguish different particle displacements and set the maximum displacement value for a particle to move between two frames. If the displacement exceeds this value, the particle will be identified and located as two particles. Then, we connect the positions of the same particle in each frame to obtain the particle's motion trajectory.

6. The method for measuring colloidal particle size using particle tracing technology based on microscope imaging according to claim 1, characterized in that: The sample to be tested is a particle suspension, the solute is a Newtonian fluid, and the viscosity is known or can be obtained by other viscosity measurement methods.

7. The method for measuring colloidal particle size using particle tracing technology based on microscope imaging according to claim 6, characterized in that: The shape of the colloidal particles in the test sample is close to or considered to be spherical, with an upper limit of 10 μm and a lower limit of the particle size being the observation limit of the imaging microscope.

8. The method for measuring colloidal particle size using particle tracing technology based on microscope imaging according to claim 1, characterized in that: The test sample is left to equilibrate at a set temperature for a set time before a video of the particle thermal motion is taken.

9. The method for measuring colloidal particle size using particle tracing technology based on microscope imaging according to claim 1, characterized in that: For Newtonian fluids, the mean position of the colloidal particles in the time interval τ is <Δr 2 (τ)> shift satisfies: <Δr 2 (τ)>=2dDτ At the same time, if the particle is spherical, according to the Stokes-Einstein equation: The particle diffusion coefficient D is obtained by fitting the linear relationship between MSD and time interval τ, and then combined with the known solvent viscosity η, temperature T and Boltzmann constant k B Calculate the particle size of the colloidal particles.

10. An electronic device, characterized in that: The method comprises a memory, a processor and a computer program stored in the memory and executable on the processor, wherein the processor executes the program to implement the image processing and calculation steps of the method according to any one of claims 1 to 9.

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