A method for real-time measurement of the size of single particles or microbubbles based on optical interference

Abstract

The application discloses a kind of methods for measuring single particle or microbubble particle size in real time based on optical interference.The method comprises the following steps: pre-calibration known material different particle size of particle or microbubble particle size and the function relationship of weighted light intensity and defocus distance;Real-time optical interference of unknown particle or microbubble sample is recorded;The scattered light field of each particle or microbubble in three-dimensional space is reconstructed by numerical calculation;The defocus distance of target particle or microbubble is positioned;The centroid weighted light intensity of target particle or microbubble is determined;The function relationship of particle size and weighted light intensity and defocus distance is used to determine the real-time particle size of the individual particle or microbubble.The application makes up the shortcomings of existing particle or microbubble particle size measurement technology, which cannot measure the real-time particle size of specific particle or microbubble moving in space, and can measure the particle size of single particle or microbubble in real time, non-labeled, with the advantages of high accuracy, large measurement range and non-destructive.

Description

Technical Field

[0001] This invention relates to a method for measuring the size of a single particle or microbubble, specifically a method for real-time measurement of the size of a single particle or microbubble based on optical interferometry. Background Technology

[0002] Currently, various methods exist for measuring the size of microparticles or microbubbles. Non-image-based methods mainly include laser dynamic light scattering and multi-wavelength extinction methods; image-based methods, depending on the principle of the microscope used, can be mainly divided into optical microscopy, electron microscopy, and atomic force microscopy. However, all of these methods have limitations in measuring the size of microparticles or microbubbles. Non-image-based methods can characterize the average particle size and distribution of a large number of microparticles or microbubbles over a period of time, but cannot measure individual target microparticles or microbubbles, let alone obtain real-time particle size changes. For image-based characterization methods, atomic force microscopy is only suitable for observing the morphology of microparticles on the surface and interface, and cannot observe microparticles dispersed in the bulk phase. Furthermore, its slow scanning speed and destructive measurement method make it difficult to observe microparticles or microbubbles with rapidly changing particle sizes, and it cannot observe fragile particles such as microbubbles. Similarly, electron microscopy generally cannot observe moving microparticles or microbubbles, and its destructive nature in sample preparation makes it difficult to observe fragile particulate systems. Common optical microscopy methods can flexibly observe and track moving particles in solutions or surfaces by adjusting the design of the optical path, obtaining the particle size of single or multiple particles based on their motion patterns. However, its observation accuracy is relatively lower than other particle size measurement methods mentioned above. Furthermore, it is limited by the imaging depth of the objective lens and the two-dimensional imaging principle, making it unable to track the dynamic motion of flexible, fast-moving particles or microbubbles in three dimensions. Therefore, it often requires methods such as fluorescent labeling to enhance resolution, but fluorescent labeling is also limited by the sample. While microscopes based on the principle of total internal reflection improve resolution, they sacrifice the depth of field for three-dimensional observation.

[0003] It is evident that a major challenge in particle size characterization lies in the non-destructive tracking and measurement of individual particles that are difficult to label, easily disturbed and damaged, and whose size changes rapidly. Therefore, we have developed a method for real-time measurement of single particles or microbubbles based on optical interferometry. This method utilizes a label-free, non-destructive in-situ three-dimensional observation technique—digital holographic microscopy. By collecting the scattered light from particles or microbubbles under a designed optical interferometry path, and based on the principles of light propagation, the light field of the particles or microbubbles in space is reconstructed computationally, thereby obtaining the three-dimensional trajectory and morphology of the target individual in space in real time. This method can simultaneously obtain two parameters closely related to the particle or microbubble size in the optical interferogram: scattered light intensity and defocus distance. Combined with pre-calibrated curves of standard-sized particles or microbubbles, the particle size of each specified particle or microbubble in the optical interferogram can be obtained quickly and accurately. Summary of the Invention

[0004] This invention proposes a method for real-time measurement of the size of single particles or microbubbles based on optical interferometry. This method is an image analysis approach based on the principle of optical interferometry, enabling real-time particle size measurement of any specified particle or microbubble in an optical interferogram. It is suitable for systems that are easily disturbed, unsuitable for labeling, or where particle size can change rapidly, such as nanoparticles and colloids, solid particles, micro / nanobubbles, micro / nanovacuoles, droplets, and microorganisms.

[0005] The objective of this invention is achieved by at least one of the following technical solutions.

[0006] A method for real-time measurement of single particle or microbubble size based on optical interferometry is characterized by the following steps: (1) using an optical interferometric microscopic optical path, and employing image processing steps of the image method and the interferometric image, pre-calibrating the functional relationship between the particle size, weighted light intensity, and defocus distance of particles or microbubbles of known material with different particle sizes; (2) recording the real-time optical interferogram of the measured sample; (3) reconstructing the three-dimensional light field of the particles or microbubbles in space through numerical reconstruction; (4) locating the defocus distance of the particles or microbubbles based on the result of the numerically reconstructed three-dimensional light field; (5) determining the centroid weighted light intensity of the particles or microbubbles based on the result of the numerically reconstructed three-dimensional light field combined with the noise reduction method in image processing; (6) determining the particle size of the particles or microbubbles using the three-dimensional weighted light intensity and defocus distance of the particles or microbubbles.

[0007] Furthermore, the quantitative relationship between centroid-weighted light intensity, defocus distance, and real-time microbubble particle size is derived from a pre-calibration experiment based on known particle size and known particles / microbubbles of the same material; for experiments conducted in devices with the same optical path design, this step only needs to be performed on the first experiment.

[0008] Furthermore, the optical interference pattern is an interference image generated by incident light irradiating a microparticle or microbubble sample.

[0009] Furthermore, the measured particles or microbubbles do not exhibit photoluminescence, or the excitation spectrum of the particles / microbubbles does not contain the wavelength of the incident light.

[0010] Furthermore, the dispersion medium of the measured particles or microbubbles is a relatively transparent and uniform liquid or solid, and the dispersion medium is colored, with the wavelength range corresponding to the color being different from the wavelength of the incident light.

[0011] A method for real-time measurement of single particle or microbubble size based on optical interferometry specifically includes the following steps:

[0012] S1. Pre-calibrate the functional relationship between the particle size or microbubble size of known material with different particle sizes and the centroid-weighted light intensity and defocus distance; see Example 1 for specific steps; for samples with the same optical path design, this step only needs to be performed during the first experiment.

[0013] S2. Record real-time optical interferograms of microparticles or microbubbles;

[0014] S3. Reconstruct the scattered light field of each particle or microbubble in three-dimensional space through calculation;

[0015] S4. Use the scattered light field to locate the defocus distance of the target particles or microbubbles;

[0016] S5. Use the scattered light field to determine the centroid-weighted light intensity of the target particle or microbubble;

[0017] S6. Determine the particle size of an individual particle or microbubble by using the centroid-weighted light intensity and defocus distance.

[0018] Furthermore, the optical interference pattern is an interference image generated by incident light irradiating a microparticle or microbubble sample.

[0019] Furthermore, the object-beam optical interferogram is obtained by subtracting the average light intensity from the aforementioned optical interferogram. The calculation is as follows:

[0020]

[0021] Among them, I b (x,y) represents the light intensity value of the pixel at position (x,y) in the background image, which is typically discretized into a 16-bit grayscale value. N is the total number of frames in the high-speed captured optical interferogram, and I is the specific frame number. i (x,y) represents the light intensity value of the pixel at position (x,y) in the i-th frame of the original optical interference image.

[0022] After obtaining the background light intensity, the object-light optical interference pattern can be calculated as follows:

[0023] I s (x,y)=I(x,y)-I b (x,y)

[0024] Furthermore, considering the reconstruction of the light field in space, according to the principles of diffractive optics, the light field I(r,-z) at any defocus distance z can be transformed into the focal plane light field I in polar coordinates. s The scattered light field of the target particle or microbubble in space is calculated and restored by (r,0) and a specific light field propagation operator h(r,-z).

[0025] I(r,-z)=FT -1 (FT(I s (r,0)·H(r,-z)))

[0026] Further, for the obtained scattered light field, noise reduction and peak searching processes need to be performed to obtain the defocus distance of the target particles or microbubbles and the weighted light intensity of the centroid of the target particles or microbubbles.

[0027] Further, the particle size range of the measured particles or microbubbles is: 10 nm < d < 500 μm. And the image resolution of the corresponding optical interference pattern should be adapted to the particle size of the particles or microbubbles.

[0028] Further, the concentration range of the measured particles or microbubbles is: c < 10 10 per mL.

[0029] Further, the measured particles or microbubbles have no photoluminescence effect, or the excitation spectrum of the particles or microbubbles does not contain the wavelength of the incident light, or the excitation spectrum and emission spectrum of the particles or microbubbles do not overlap.

[0030] Further, the dispersion medium of the measured particles or microbubbles is a transparent and homogeneous liquid or solid. If the dispersion medium has a color, the wavelength corresponding to the color should be different from the wavelength of the incident light.

[0031] Based on the high-resolution optical interference pattern generated by irradiating a particle or microbubble sample with incident light, the present invention obtains two parameters, namely the centroid light intensity and the defocus distance, which have a high correlation with the particle size of the particles or microbubbles, and can batch measure the real-time particle sizes of all particles or microbubbles within the field of view.

[0032] Compared with the prior art, the advantages of the present invention are as follows:

[0033] The present invention compensates for the defect that the prior art for measuring the particle size of particles or microbubbles cannot measure the real-time particle size of specific particles or microbubbles moving in space, can measure the particle size of a single particle or microbubble in real time and non-invasively, and has the advantages of high precision, large measurement range, and non-destructiveness. Description of the Drawings

[0034] Figure 1 is a flowchart of a method for real-time measuring the particle size of a single particle or microbubble based on optical interference according to the present invention;

[0035] Figure 2 is a schematic diagram for determining the particle size of a target particle or microbubble using the three-dimensional weighted light intensity and defocus distance of the target particle or microbubble;

[0036] Figure 3 is a graph of the particle size measurement results of micro-nano bubbles in Example 2;

[0037] Figure 4 is a graph of the particle size measurement results of the growth of multiple liquid bubbles in a polyacrylic resin material in Example 3. Detailed Embodiments

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0039] Example 1

[0040] A method for real-time measurement of single particle or microbubble size based on optical interferometry, such as Figure 1 As shown, it includes the following steps.

[0041] first step:

[0042] Samples were prepared and observed under a digital holographic microscope capable of producing incident light to observe microparticles or microbubbles. Real-time digital optical interferograms of the samples were recorded using a camera. In this embodiment, polystyrene microspheres with an average particle size of 0.5 μm were dispersed in ultrapure water at a concentration of 10. 7 / mL.

[0043] Step Two:

[0044] The light source used was an LED light source with a center wavelength of 505nm and a 40x objective lens. The camera parameters were set as follows: frame rate of 40fps, exposure time of 0.025s, and image resolution of 1024×1024. 4800 grayscale images of optical interferograms with a bit depth of 16bit were continuously captured. The square field of view of each optical interferogram had a side length of 166μm, and the side length of the magnified unit pixel was 162.5nm. The position information of polystyrene microspheres with an average particle size of 0.5μm in space was calculated and restored by a program based on the principle of light propagation. The light field within a certain spatial range containing polystyrene microspheres with an average particle size of 0.5μm was numerically reconstructed.

[0045] Based on the original optical interferogram, a background subtraction algorithm is used to obtain a background image containing only the stationary image within the background by summing and averaging the optical interferograms over a longer time period. This background image is then further subtracted from each frame of the optical interferogram to obtain an object-optical interferogram containing only polystyrene microspheres with an average particle size of 0.5 μm. The formula used is as follows:

[0046]

[0047] Among them, I i (x,y) represents the light intensity value of the pixel at position (x,y) in the original optical interference image of polystyrene microspheres with an average particle size of 0.5μm in the i-th frame. This value is discretized into a 16-bit grayscale value. N is the total number of frames in the high-speed optical interference image, and I is the specific frame number. b(x, y) is the light intensity value of the pixel at position (x, y) in the background image after processing using the above formula. Finally, from:

[0048] I s (x,y)=I i (x,y)-I b (x,y)

[0049] Obtain an object-optical interferogram containing only polystyrene microspheres with an average particle size of 0.5 μm.

[0050] Step 3:

[0051] In the reconstructed spatial light field, the defocus distance of the target polystyrene microsphere is located through noise filtering and signal peak finding. All of the above information is contained in the calculated and restored spatial scattered light field I(r,-z), and the formula used is as follows:

[0052] I(r,-z)=FT -1 (FT(I s (r,0)·H(r,-z)))

[0053] Where r is the horizontal coordinate of a polystyrene microsphere with an average particle size of 0.5 μm, and z is its initial axial coordinate; FT -1 is the inverse Fourier transform; FT is the Fourier transform; H(r,-z) is the Fourier transform of the light propagation operator h(r,-z).

[0054] The noise filtering program uses the light intensity of the brightest point in the spatial light field as a reference. If the light intensity at any point is less than 20% of the brightest point's intensity, it is set as noise and is not considered during signal peak finding. The light intensity filtering threshold can be adjusted according to the sample's signal-to-noise ratio.

[0055] The signal peak finding process further filters suspected signal points after noise removal. First, an expansion algorithm is used to confirm whether nearby points originate from the same polystyrene microsphere based on spatial continuity. Then, by maintaining the spatiotemporal consistency of the polystyrene microsphere signal across multiple frames of optical interferograms, signals with continuous and stable trajectories for more than 30 frames are preserved, while randomly discontinuous noise signals are filtered out. As an auxiliary noise reduction step, increasing the minimum frame rate limit can further filter noise, but this may also inadvertently delete genuine signals; adjustments can be made based on the sample's signal-to-noise ratio.

[0056] Step 4:

[0057] In the reconstructed spatial light field, the weighted light intensity of the tiny spatial region where the target particle or microbubble is located is determined. In this embodiment, the particle or microbubble has a similar particle size, so the sum of the light intensities within a 9×9×9 pixel range with the strongest light intensity of each particle or microbubble as the centroid is used as the weighted light intensity.

[0058] Step 5:

[0059] The defocus distance of the target particles or microbubbles and the weighted light intensity are used as input parameters, and then... Figure 2 The corresponding mapping algorithm of the standard particle size microparticle or microbubble calibration determines the particle size of the target microparticle or microbubble and obtains the particle size measurement results.

[0060] Example 2

[0061] The sample in Example 1 was replaced with a 3:7 volume mixture of ethanol and ultrapure water containing dispersed bulk microbubbles. After microbubble generation, the near-interfacial bubble concentration was 10. 8 / mL.

[0062] The light source is still an LED light source with a center wavelength of 505nm, a 40x objective lens, and the camera parameters are set as follows: frame rate is set to 40fps, exposure time is 0.025s, image resolution is 1024×1024, 1000 original grayscale images of optical interferograms with a bit depth of 16bit are continuously captured, the side length of the square field of view of each original optical interferogram is 166μm, and the side length of the magnified unit pixel is 162.5nm. The position information of particles or microbubbles in space is calculated and restored by a program based on the principle of light propagation, and the light field within a certain spatial range containing microbubbles is numerically reconstructed.

[0063] The threshold for filtering noise points based on light intensity after reconstructing the signal points remains 20%, and the threshold for filtering noise points based on spatiotemporal consistency remains 30 frames. All other experimental and data processing steps and formulas used remain unchanged.

[0064] After the above image capture and data processing, the continuous particle size measurement results of a microbubble within 25 seconds are obtained as follows: Figure 3 .

[0065] Example 3

[0066] The sample in Example 1 was replaced with a transparent polyacrylic acid sheet at room temperature. This sheet was heated in solution to 50°C, then cooled to 35°C, inducing the formation of vacuoles within it. The vacuole density was approximately 10. 9 / mL.

[0067] The light source was still an LED light source with a center wavelength of 505nm, a 40x objective lens, and the camera parameters were set as follows: frame rate of 40fps, exposure time of 0.025s, image resolution of 2048×2048, and 12,000 grayscale original images of optical interferograms with a bit depth of 16bit were continuously captured. The side length of the square field of view of each original optical interferogram was 333μm, and the side length of the magnified unit pixel was 162.5nm. The position information of the particles or microbubbles in the space was calculated and restored by a program based on the principle of light propagation, and the light field within a certain spatial range containing the internal vacuole of polyacrylic acid was numerically reconstructed.

[0068] The threshold for filtering noise points based on light intensity after reconstructing the signal points remains at 20%, and the threshold for filtering noise points based on spatiotemporal consistency remains at 30 frames. The obtained defocus distance of each bubble and the centroid-weighted light intensity are then input. Figure 2 The diagram above illustrates the determination of the particle size of the target microparticle or microbubble using three-dimensional weighted light intensity and defocus distance. All other experimental and data processing steps and formulas remain unchanged.

[0069] After the above image capture and data processing, the continuous particle size measurement results of several vacuoles within a few minutes are obtained, as shown below. Figure 4 .

[0070] It should be understood that the above detailed description of the technical solutions of the present invention with reference to optimized embodiments is illustrative and not restrictive. It should not be considered that the specific implementation of the present invention is limited to this. For those skilled in the art, any modifications to the technical solutions described in the embodiments or equivalent substitutions of some technical features without departing from the concept of the present invention should be considered as falling within the scope of patent protection defined by the claims submitted by the present invention.

[0071] The above embodiments of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the implementation of the present invention. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for real-time measurement of single particle or microbubble size based on optical interferometry, characterized in that, Includes the following steps: (1) Using an optical interference microscope optical path, and by using the image method and the image processing steps of the interference image, pre-calibrate the functional relationship between the particle size, weighted light intensity and defocus distance of the known material particles or microbubbles of different sizes; (2) Record the real-time optical interference pattern of the measured sample; (3) Reconstruct the three-dimensional light field of the particles or microbubbles in space through numerical reconstruction; (4) Locate the defocus distance of the particles or microbubbles based on the result of the numerically reconstructed three-dimensional light field; (5) Determine the centroid weighted light intensity of the particles or microbubbles based on the result of the numerically reconstructed three-dimensional light field combined with the noise reduction method in image processing; (6) Determine the particle size of the particles or microbubbles using the three-dimensional weighted light intensity and defocus distance of the particles or microbubbles.

2. The method for real-time measurement of single particle or microbubble size based on optical interferometry according to claim 1, characterized in that, The quantitative relationship between centroid-weighted light intensity, defocus distance, and real-time particle size of microbubbles is derived from pre-calibration experiments based on known particle size and known particles / microbubbles of the same material; for experiments conducted in devices with the same optical path design, this step only needs to be performed on the first experiment.

3. The method for real-time measurement of single particle or microbubble size based on optical interferometry according to claim 1, characterized in that, The optical interference pattern is an interference image generated by incident light irradiating a microparticle or microbubble sample.

4. The method for real-time measurement of single particle or microbubble size based on optical interferometry according to claim 1, characterized in that, The range of particle size d measured for microparticles or microbubbles: 10 nm <d<500μm。 5. The method for real-time measurement of single particle or microbubble size based on optical interferometry according to claim 1, characterized in that, The measured particle or microbubble density range is no greater than 10. 10 per mL.

6. The method for real-time measurement of single particle or microbubble size based on optical interferometry according to claim 1, characterized in that, The measured particles or microbubbles do not exhibit photoluminescence, or the excitation spectrum of the particles / microbubbles does not contain the wavelength of the incident light.

7. The method for real-time measurement of single particle or microbubble size based on optical interferometry according to claim 1, characterized in that, The dispersion medium of the particles or microbubbles being measured is a relatively transparent and uniform liquid or solid. The dispersion medium is colored, and the wavelength range corresponding to its color should be different from the wavelength of the incident light.

8. A method for real-time measurement of single particle or microbubble size based on optical interferometry according to any one of claims 1 to 7, characterized in that, The specific operating steps are as follows: S1. Pre-calibrate the functional relationship between the particle size or microbubble size of known material with different particle sizes and the weighted light intensity and defocus distance; S2. Record real-time optical interferograms of microparticles or microbubbles; S3. Reconstruct the scattered light field of each particle or microbubble in three-dimensional space through calculation; S4. Based on the three-dimensional scattered light field, locate the defocus distance of the target particles or microbubbles; S5. Based on the three-dimensional scattered light field, obtain the centroid-weighted light intensity of the target particles or microbubbles; S6. Determine the real-time particle size of the target particle or microbubble based on the centroid-weighted light intensity and the defocus distance; the quantitative relationship between the centroid-weighted light intensity, the defocus distance, and the real-time particle size of the microbubble is derived from the pre-calibration experiment based on the standard sample in S1.

9. The method for real-time measurement of single particle or microbubble size based on optical interferometry according to claim 8, characterized in that, The optical interferogram is obtained by subtracting the average light intensity to obtain an object-optical optical interferogram that can be calculated and reconstructed. The calculation is as follows: Among them, I b (x,y) represents the light intensity value of the pixel at position (x,y) in the background image, which will be discretized into a 16-bit grayscale value. N is the total number of frames in the high-speed captured optical interferogram, and I is the specific frame number. i (x,y) represents the light intensity value of the pixel at position (x,y) in the i-th frame of the original optical interference image.

10. The method for real-time measurement of single particle or microbubble size based on optical interferometry according to claim 8, characterized in that, The calculation of the scattered light field U(r,-z) of the target particle or microbubble in space is as follows: U(r,-z)=FT -1 (FT(I s (r,0)·H(r,-z))) Where r is the initial horizontal coordinate of the particle or microbubble, and z is the initial axial coordinate of the particle or microbubble; FT -1 is the inverse Fourier transform; FT is the Fourier transform; H(r,-z) is the Fourier transform of the light propagation operator h(r,-z), the negative sign indicates that the calculation direction is opposite to the light propagation direction; I s (r, 0) = I i (r, 0) - I b (r, 0) represents the intensity of the particle-scattered light after subtracting the background light.

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