Exosome particle size distribution detection processing method and system

Abstract

The invention relates to the technical field of biomedical detection, in particular to an exosome particle size distribution detection processing method and system which are used for accurately measuring and analyzing the size distribution characteristics of exosomes in a biological sample. The method comprises the following steps: firstly, extracting and marking an exosome from a cell culture supernatant by using an exosome extraction kit; arranging a capture probe in a light scattering detection system, wherein the system comprises a microscope lens and a three-color camera which are connected with a calculation module; an exosome image is transmitted to a three-color camera through a microscope lens, and detection is carried out by utilizing the fluorescence wavelength and a light scattering signal of a super-continuous laser; in the continuous change of the laser emission angle, calculating the particle size distribution of the exosome according to an optical signal; according to the method, the detection precision and accuracy are improved, the improvement from two-dimensional information to three-dimensional information is realized, the characterization problem of the non-spherical exosome is solved, and a more accurate and comprehensive tool is provided for exosome research.

Description

Technical Field

[0001] The present invention relates to the field of biomedical testing technology, and in particular to a method and system for detecting and processing exosome particle size distribution, which are used to accurately measure and analyze the size distribution characteristics of exosomes in biological samples. Background Art

[0002] Exosomes are nanoscale membrane vesicles with a diameter of approximately 30-150 nm secreted by cells. They carry specific proteins, nucleic acids, and lipids and play an important role in intercellular communication, disease development, and biomarker research. Accurately measuring the size distribution of exosomes is crucial for understanding their biological functions, quality control, and clinical application.

[0003] Currently, methods for measuring exosome size distribution primarily include dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy (EM), and atomic force microscopy (AFM). However, these methods have a number of limitations: DLS has low resolution and difficulty distinguishing polydisperse samples; NTA has limited sensitivity for detecting small exosomes (<50 nm); while EM provides high-resolution imaging, sample preparation is complex and may alter exosome morphology; and AFM is complex, requires high sample handling, and is time-consuming and labor-intensive.

[0004] In particular, traditional light scattering techniques typically capture scattered signals at only a single or fixed angle, resulting in limited measurement dimensionality and difficulty in capturing the true three-dimensional properties of exosomes. Furthermore, most existing technologies lack the ability to accurately characterize non-spherical exosomes, leading to biased measurement results. Furthermore, significant background interference in biological samples can compromise the accuracy and reliability of detection.

[0005] Therefore, developing a new method and system that can achieve high-precision, multi-dimensional detection of exosome particle size distribution is of great value for exosome-related research and applications. Summary of the Invention

[0006] This invention provides a method and system for detecting and processing exosome particle size distribution, aiming to address existing technical issues such as insufficient precision, limited dimensionality, and difficulty characterizing non-spherical exosomes. This invention innovatively upgrades the acquisition of exosome particle size distribution from a single-angle to a three-dimensional exosome optical signal, incorporating Rayleigh scattering theory to accurately calculate the actual three-dimensional size range and number of exosomes.

[0007] The present invention proposes a method for detecting and processing exosome particle size distribution, comprising:

[0008] an acquisition step, obtaining a cell culture supernatant sample using an exosome extraction kit and labeling the exosomes in the cell culture supernatant sample to obtain labeled exosomes;

[0009] a setting step of setting an exosome capture probe in a light scattering detection system, wherein the light scattering detection system includes a microscope lens and a three-color camera connected to a computing module;

[0010] a detection step, transmitting the exosome image to the three-color camera through the microscope lens, and the three-color camera detecting the labeled exosomes using the fluorescence wavelength and light scattering signal in the supercontinuum laser;

[0011] The calculation step is to calculate the particle size distribution of the exosomes in the cell culture supernatant sample according to the light signal emitted by the labeled exosomes during the process of continuously changing the laser emission angle.

[0012] Preferably, the calculation step specifically includes:

[0013] Obtain the data on the changes in exosome optical signals as the laser incident angle continuously changes;

[0014] Based on the change data, the exosome refractive index and the Rayleigh scattering coefficient, and in combination with the Rayleigh scattering formula, the exosome size distribution is improved from single-angle exosome light signal acquisition to three-dimensional exosome light signal acquisition;

[0015] According to the change data, combined with the refractive index, the Rayleigh scattering coefficient and the Rayleigh scattering formula, the actual three-dimensional size range of the exosomes and the number of exosomes are calculated.

[0016] Preferably, the calculation formula for the number of the exorotators is:

[0017] N=∫γ(θ,θ0)dθ,

[0018] where N represents the number of exo-rotators within the exo-rotator scattering angle, and γ(θ,θ0) represents the average number density of exo-rotators scattered at angle θ0 when the detector angle is θ.

[0019] Preferably, the change data includes two-dimensional angle information of the external rotator and three-dimensional angle information of the external rotator, the two-dimensional angle information of the external rotator includes the relationship between the two-dimensional angle of the external rotator, light energy and the two-dimensional angle, and the three-dimensional angle information of the external rotator includes the relationship between the angle of the external rotator, light energy and the three angles.

[0020] Preferably, the Rayleigh scattering formula is:

[0021] I=I0·f(θ),

[0022] Wherein, I represents the light signal of the exo-rotator, I0 represents the incident light intensity, and θ is the central angle of the exo-rotator; the three-dimensional particle size formula of the exo-rotator after the formula I is transformed is:

[0023] I=∫V(θ)·r 3 ·N·K·dθ,

[0024] Where V(θ) represents the angle of the exo-rotator along the θ direction, r represents the actual three-dimensional size range of the exo-rotator, θ0 represents the scattering angle of the exo-rotator, N represents the average number density, and K is a constant and K=π / 6.

[0025] Preferably, the change data is an angle distribution intensity relationship, wherein the angle refers to the angle of the incident light, and the intensity refers to the relative intensity of the light signal.

[0026] Preferably, the calculation formula of the optical signal is:

[0027]

[0028] Where β is the incident angle, I(β) is the intensity of the light signal with a scattering angle of β, and I θ is the intensity of the light signal at the detection angle θ, and θ is the angle of the light signal at the scattering angle β.

[0029] Preferably, f(θ) in the Rayleigh scattering formula is related to the particle size, the refractive index of the material and the wavelength of the incident light, and satisfies the scattering law when the particle size is much smaller than the wavelength of the incident light under the Rayleigh scattering condition.

[0030] Preferably, the term cos(θ-β) in the optical signal calculation formula is used to compensate for the geometric relationship between the detection angle and the scattering angle, ensuring that the optical signal intensity data obtained at different incident angles are comparable.

[0031] A system for detecting and processing exosome particle size distribution, the system comprising:

[0032] An exosome collection module is used to obtain an exosome sample from a collection source using a collection tube of an exosome extraction kit to obtain a cell culture supernatant sample;

[0033] an exosome labeling module, used to label exosomes in the cell culture supernatant sample to obtain labeled exosomes;

[0034] A light scattering detection module is used to set an exosome capture probe in a light scattering detection system. The light scattering detection system includes a microscope lens and a three-color camera connected to the computing module. The microscope lens and the three-color camera are both located below the exosome capture probe. The microscope lens transmits the labeled exosome image to the three-color camera. The three-color camera detects the labeled exosomes using the fluorescence wavelength of the supercontinuum laser and the light scattering signal;

[0035] A calculation module is used to calculate the particle size distribution of the exosomes in the cell culture supernatant sample based on the light signals emitted by the labeled exosomes during the process of continuously changing the laser emission angle.

[0036] The beneficial effects of this invention include: 1) improving the precision and accuracy of exosome size distribution detection; 2) achieving a dimensional upgrade from two-dimensional information to three-dimensional characteristics; 3) resolving the difficulty of characterizing non-spherical exosomes; 4) establishing a method for calculating the number of exosomes based on the exosome model; and 5) reducing the impact of background interference on measurement results. These technical effects give this invention broad application prospects in fields such as biomedical research, disease diagnosis, and drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a structural block diagram of the exosome particle size distribution detection and processing system provided by an embodiment of the present invention.

[0038] Figure 2 Flowchart of a method for detecting and processing exosome particle size distribution provided in an embodiment of the present invention.

[0039] Figure 3 A schematic diagram of the principle of a light scattering detection system provided by an embodiment of the present invention.

[0040] Figure 4 A schematic diagram of data acquisition under continuously changing laser emission angles provided by an embodiment of the present invention.

[0041] Figure 5 This is an example diagram of the exosome particle size distribution detection results provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0042] Please refer to the attached Figure 1-5 The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0043] Example 1

[0044] Reference Figure 1 As shown, the exosome particle size distribution detection and processing system provided by the present invention includes an exosome collection module 1, an exosome labeling module 2, a light scattering detection module 3 and a calculation module 4.

[0045] The exosome collection module 1 is used to obtain an exosome sample from a collection source using the collection tube of an exosome extraction kit to obtain a cell culture supernatant sample. In practical applications, the exosome collection module 1 typically uses ultracentrifugation, density gradient centrifugation, ultrafiltration, or commercial exosome extraction kits to separate exosomes from biological samples such as cell culture medium, blood, and urine. Preferably, a commercial exosome extraction kit using a polymer precipitation method (such as ExoQuick, TotalExosomeIsolationKit, etc.) can achieve simple and efficient exosome extraction. In one embodiment of the present invention, the exosome collection module 1 separates exosomes from the culture supernatant of human lung cancer A549 cells by ultracentrifugation (centrifugation at 300×g, 2000×g, and 10,000×g to remove cells and cell debris, and then ultracentrifugation at 100,000×g for 2 hours to precipitate exosomes).

[0046] The exosome labeling module 2 is used to label the cell culture supernatant sample with exosomes to obtain labeled exosomes. Preferably, the exosome labeling module 2 uses fluorescent dyes, specific antibodies or other markers to label exosomes. In one embodiment of the present invention, the exosome labeling module 2 uses PKH26 lipid membrane dye (maximum excitation wavelength of 551nm, maximum emission wavelength of 567nm) to label exosomes. During the labeling process, the separated exosomes are suspended in Diluent C solution and a final concentration of 2×10 -6 The PKH26 dye was added to 50 μM and incubated at room temperature for 5 minutes. Then, an equal volume of 1% bovine serum albumin (BSA) was added to terminate the reaction. The unbound dye was removed by ultracentrifugation to obtain the labeled exosomes.

[0047] The light scattering detection module 3 is used to set the exosome capture probe in the light scattering detection system. The light scattering detection system includes a microscope lens 31 and a three-color camera 32 connected to the calculation module 4. The microscope lens 31 and the three-color camera 32 are both located below the exosome capture probe. The microscope lens 31 transmits the labeled exosome image to the three-color camera 32. The three-color camera 32 uses the fluorescence wavelength and light scattering signal in the supercontinuum laser 33 to detect the labeled exosomes.

[0048] like Figure 3As shown, the microscope lens 31 in the light scattering detection module 3 is a high-resolution microscope objective lens with a magnification of preferably 60x or 100x oil-immersed objective lens and a numerical aperture (NA) preferably greater than 1.3 to ensure the ability to detect nanoscale exosomes. The three-color camera 32 has three RGB channels and can simultaneously capture light signals of different wavelengths. The resolution is preferably at least 4K (3840×2160 pixels) and the frame rate is preferably at least 30fps to ensure accurate and real-time data acquisition. The light source generated by the supercontinuum laser 33 covers a wide spectrum (400-800nm) and can simultaneously provide excitation light for fluorescence and a continuous light source for scattering.

[0049] Preferably, the exosome capture probe uses a microfluidic chip or microwell array with a surface modified with specific antibodies (such as CD9, CD63, CD81, etc.), which can specifically capture and immobilize exosomes, facilitating subsequent light scattering measurements. In one embodiment of the present invention, the exosome capture probe uses a glass-based microfluidic chip with an anti-CD63 antibody on its surface to capture exosomes through specific antigen-antibody interactions.

[0050] The calculation module 4 is used to calculate the particle size distribution of the exosomes in the cell culture supernatant sample according to the light signal emitted by the labeled exosomes during the continuous change of the laser emission angle. Figure 4 As shown, the computing module 4 controls the laser to continuously change the incident angle within a preset angle range (typically 0° to 180°), scanning at a rate of 0.5° to 2° per second, while collecting scattered light signal intensity data at different angles through the three-color camera 32.

[0051] Example 2

[0052] Reference Figure 2 As shown, the exosome particle size distribution detection and processing method provided by the present invention includes the following steps:

[0053] Step S1: using an exosome extraction kit to obtain a cell culture supernatant sample and labeling the exosomes in the cell culture supernatant sample to obtain labeled exosomes.

[0054] Specifically, exosomes are first isolated from biological samples by ultracentrifugation or a commercial exosome extraction kit to obtain a cell culture supernatant sample. Then, fluorescent dyes (such as PKH26, Di O, DiI, etc.) or specific antibodies (such as fluorescently labeled anti-CD9, CD63, CD81 antibodies, etc.) are used to label the exosomes. Preferably, in one embodiment of the present invention, the culture medium is preliminarily treated (300×g centrifugation for 10 minutes to remove cells, 2000×g centrifugation for 20 minutes to remove cell debris, and 10000×g centrifugation for 30 minutes to remove large particles), and then the exosomes are collected by ultracentrifugation at 100000×g for 70 minutes. In the labeling step, PKH26 dye is used at 2×10 -6 A final concentration of M was incubated with exosomes in Diluent C solution at room temperature for 5 min, and unbound dye was subsequently removed by ultracentrifugation.

[0055] Step S2: setting an exosome capture probe in a light scattering detection system, wherein the light scattering detection system includes a microscope lens and a three-color camera connected to a computing module.

[0056] like Figure 3 As shown, the exosome capture probe is placed on the microscope stage, with the microscope lens located below the probe, transmitting the exosome image to the three-color camera. Preferably, a high numerical aperture (NA>1.3) objective lens is used to improve the detection sensitivity of nanoparticles. The three-color camera is connected to the microscope via a specific optical interface and can capture image signals of the three RGB channels. At the same time, the computing module is connected to the three-color camera via a data transmission interface to control image acquisition parameters (such as exposure time, gain, frame rate, etc.) and receive image data.

[0057] Step S3, transmitting the exosome image to the three-color camera through the microscope lens, and the three-color camera uses the fluorescence wavelength and light scattering signal in the supercontinuum laser to detect the labeled exosomes.

[0058] Specifically, excitation light generated by a supercontinuum laser is directed through a specific optical path system onto the exosome sample attached to the capture probe. Under the excitation light, the labeled exosomes simultaneously generate fluorescence and scattered light signals. A microscope lens captures these light signals and transmits the exosome image to a three-color camera. Preferably, the microscope system is equipped with an appropriate filter set to separate the fluorescence and scattered light signals. The three RGB channels of the three-color camera are used to capture light signals from different wavelength ranges, improving signal resolution.

[0059] Step S4, calculating the particle size distribution of the exosomes in the cell culture supernatant sample according to the light signals emitted by the labeled exosomes during the process of continuously changing the laser emission angle.

[0060] Example 3

[0061] Based on the above embodiment, the calculation steps of the present invention specifically include: obtaining change data of the exosome light signal during the continuous change of the laser incident angle; based on the change data, the exosome refractive index and the Rayleigh scattering coefficient, combined with the Rayleigh scattering formula, upgrading the exosome particle size distribution from single-angle exosome light signal acquisition to three-dimensional exosome light signal acquisition; based on the change data, combined with the refractive index, the Rayleigh scattering coefficient and the Rayleigh scattering formula, calculating the actual three-dimensional size range of the exosomes and the number of exosomes.

[0062] Preferably, the calculation module 4 includes a data collection submodule 41 , a particle distribution generation submodule 42 , and an exosome particle size calculation submodule 43 .

[0063] The data collection submodule 41 is used to acquire data on the exosome optical signal as the laser incident angle continuously changes, using the light scattering detection system. Specifically, as the laser incident angle continuously changes from 0° to 180°, the system collects optical signal data every 0.5° or 1°, recording the scattered light intensity at different angles. This data constitutes the angle-intensity relationship curve, also known as the variation data. Preferably, the data collection process is repeated 3-5 times, and the average is calculated to reduce the impact of random errors.

[0064] The particle distribution generation submodule 42 is used to upgrade the exosome particle size distribution from single-angle exosome light signal acquisition to three-dimensional exosome light signal acquisition based on the change data, the exosome refractive index and the Rayleigh scattering coefficient in combination with the Rayleigh scattering formula.

[0065] In the present invention, the refractive index of exosomes is determined according to their biological composition, with a typical value of 1.35-1.40, preferably 1.38. The Rayleigh scattering coefficient is related to the wavelength of incident light, the size of exosomes and the material properties, and is determined by calibration standard samples, with a typical value range of 0.8-1.2×10 -6 , preferably 1.0×10 -6 .

[0066] The basic form of the Rayleigh scattering formula is:

[0067] I=I0·f(θ),

[0068] Where I represents the light signal of the exo-rotator, I0 represents the incident light intensity, and θ is the central angle of the exo-rotator.

[0069] For nanoscale particles, the function f(θ) can be expressed as:

[0070]

[0071] Where R is the observation distance, λ is the wavelength of the incident light, and n p is the refractive index of the particle, n m is the refractive index of the medium, and d is the particle diameter.

[0072] The particle distribution generation submodule 42 generates a data set describing the three-dimensional distribution of exosomes by analyzing the variation patterns of scattering intensity at different angles and combining it with Rayleigh scattering theory. This process achieves a dimensional upgrade from single-angle planar information to three-dimensional spatial distribution.

[0073] The exosome particle size calculation submodule 43 is used to calculate the actual three-dimensional size range of the exosomes and the number of exosomes based on the change data, combined with the refractive index, the Rayleigh scattering coefficient and the Rayleigh scattering formula.

[0074] The three-dimensional particle size formula of the exo-rotator after formula transformation is:

[0075] I=∫V(θ)·r 3 ·N·K·dθ,

[0076] Where V(θ) represents the angular distribution function of the exo-rotator along the θ direction, r represents the actual three-dimensional size range of the exo-rotator, θ0 represents the scattering angle of the exo-rotator, N represents the average number density, and K is a constant and K=π / 6.

[0077] In practical applications, the actual three-dimensional size range r of exosomes is typically distributed between 30 and 150 nm. By inversely solving the above integral equation, the particle size distribution of exosomes can be obtained. Preferably, an iterative algorithm is used to solve the integral equation, with an initial value set to 30 nm and a step size of 1 nm. Iteration is performed until convergence (the difference between two adjacent iterations is less than 0.1 nm) or the maximum number of iterations (usually 100) is reached.

[0078] Example 4

[0079] Based on the above embodiment, the formula for calculating the number of external rotors of the present invention is:

[0080] N=∫γ(θ,θ0)dθ,

[0081] where N represents the number of exo-rotators within the exo-rotator scattering angle, and γ(θ,θ0) represents the average number density of exo-rotators scattered at angle θ0 when the detector angle is θ.

[0082] In practical applications, the average number density of exo-rotators γ(θ,θ0) can be estimated by the following formula:

[0083]

[0084] Where I(θ) is the scattered light intensity measured at angle θ, I0 is the incident light intensity, and Vscatt (θ,θ0) is the scattering volume function.

[0085] Scattering volume function V scatt (θ, θ0) is related to the optical parameters of the detection system and can be determined by calibration standards. Preferably, polystyrene microspheres with a diameter of 100 nm are used for system calibration to establish a calibration curve for the scattering volume function.

[0086] The total number of exosomes N can be obtained by numerical integration of the above formula. Preferably, the trapezoidal rule or Simpson's rule is used for numerical integration, with the integration interval from 0° to 180° and the step size of 1°. For typical exosome samples, the N value is usually around 10 8 to 10 12 The range is between 10 and 20 μg / mL, depending on the sample concentration and extraction method.

[0087] Example 5

[0088] Based on the above embodiments, the change data of the present invention includes two-dimensional angle information of the external rotator and three-dimensional angle information of the external rotator. The two-dimensional angle information of the external rotator includes the relationship between the two-dimensional angle of the external rotator, light energy and the two-dimensional angle. The three-dimensional angle information of the external rotator includes the relationship between the angle of the external rotator, light energy and the three angles.

[0089] The two-dimensional angular information of exosomes reflects the light intensity distribution within the scattering plane and is usually expressed as I(θ), where θ is the angle within the scattering plane, ranging from 0° to 180°. The relationship between the two-dimensional angle and the light energy can be described by the scattering phase function. For exosomes in the Rayleigh scattering region (particle size is much smaller than the wavelength), the phase function is approximately:

[0090]

[0091] The 3D angular exo-rotational information is extended to three dimensions, encompassing the azimuth angle φ (0° to 360°), the polar angle θ (0° to 180°), and the rotation angle ψ (0° to 360°). The 3D scattering intensity distribution can be expressed as I(θ, φ, ψ).

[0092] In the process of expanding two-dimensional information to three-dimensional space, the relationship between light energy and three angles can be expressed as:

[0093] I(θ,φ,ψ)=I0·S(θ,φ,ψ)·V(θ,φ,ψ),

[0094] Among them, S(θ,φ,ψ) is the scattering function and V(θ,φ,ψ) is the volume function.

[0095] By analyzing the scattering intensity change pattern under different angle combinations, the three-dimensional morphology and size distribution of exosomes can be reconstructed. Especially for non-spherical exosomes, this three-dimensional angle analysis can provide more accurate morphological characterization.

[0096] Example 6

[0097] Based on the above embodiment, the Rayleigh scattering formula of the present invention is:

[0098] I=I0·f(θ),

[0099] Wherein, I represents the light signal of the exo-rotator, I0 represents the incident light intensity, and θ is the central angle of the exo-rotator; the three-dimensional particle size formula of the exo-rotator after the formula I is transformed is:

[0100] I=∫V(θ)·r 3 ·N·K·dθ,

[0101] Where V(θ) represents the angle of the exo-rotator along the θ direction, r represents the actual three-dimensional size range of the exo-rotator, θ0 represents the scattering angle of the exo-rotator, N represents the average number density, and K is a constant K=π / 6.

[0102] Preferably, the derivation process of the three-dimensional particle size formula of the exo-rotator is as follows:

[0103] Rayleigh scattering theory is applicable to particles whose diameter is much smaller than the wavelength of the incident light. Rayleigh scattering theory can be applied to the scattering of exosomes (diameter is about 30-150nm) in the visible light range (400-700nm). The basic Rayleigh scattering formula is:

[0104]

[0105] Where α is the polarizability of the particle, which is related to the particle volume and material properties, R is the observation distance, and λ is the wavelength of the incident light.

[0106] For spherical particles, the polarizability α is related to the particle volume V and the relative refractive index m:

[0107]

[0108] in, r is the particle radius, n p is the refractive index of the particle, n m is the refractive index of the medium.

[0109] Substituting these relations into the scattering formula, we obtain:

[0110]

[0111] Here, d = 2r is the particle diameter.

[0112] Considering the possible non-spherical characteristics of exosomes, an exosome model is introduced, and its volume element can be expressed as:

[0113] dV=V(θ)·dθ,

[0114] Where V(θ) is the volume distribution function in the direction of angle θ.

[0115] For an exosome sample with a number density N, the total scattering intensity is the integral of the scattering from all exosomes:

[0116]

[0117] Where K = π / 6 is the shape factor constant and θ0 is the scattering angle range.

[0118] By inversely solving this integral equation, the actual three-dimensional size range r and number N of exosomes can be calculated.

[0119] Example 7

[0120] On the basis of the above embodiments, the change data of the present invention is the angle distribution intensity relationship, wherein the angle refers to the angle of the incident light, and the intensity refers to the relative intensity of the light signal.

[0121] In actual measurements, the system records the scattered light intensity I(β) at different incident angles β, forming an angle-intensity curve. Typically, the angle β ranges from 0° to 180°, scanned in steps of 1° or less. For typical exosome samples, the angle-intensity curve exhibits a characteristic distribution pattern, with higher intensities in the forward scatter (near 0°) and backscatter (near 180°) directions and lower intensity in the side scatter (near 90°).

[0122] Accurate measurement of the angular distribution intensity relationship is crucial for subsequent particle size calculation. To ensure measurement accuracy, the following steps are used in one embodiment of the present invention:

[0123] 1. System calibration: Use standard polystyrene microspheres (diameter 100 nm, refractive index 1.59) to calibrate the system and establish a standard curve of the angle-intensity relationship.

[0124] 2. Background correction: Collect buffer without exosomes as background and subtract the background signal from the sample measurement results.

[0125] 3. Repeat multiple times: Repeat the measurement of each angle point 3-5 times and take the average value to reduce random errors.

[0126] 4. Data Smoothing: Use a Savitzky-Golay filter to smooth the raw data to reduce the impact of noise. The filter window size is preferably 5-9 data points, and the polynomial order is 2-3.

[0127] The processed angle-intensity relationship data serves as the basic input for subsequent calculations.

[0128] Example 8

[0129] Based on the above embodiment, the calculation formula of the optical signal of the present invention is: I(β)=I θ / cos(θ-β), where β is the incident angle, I(β) is the intensity of the light signal with a scattering angle of β, and I θ is the intensity of the light signal at the detection angle θ, and θ is the angle of the light signal at the scattering angle β.

[0130] This formula accounts for the geometric relationship between the scattering angle and the detection angle, ensuring that the light signal intensity data obtained at different incident angles are comparable. Specifically, when the light source is incident at an angle β and the detector is positioned at an angle θ, the actual scattering angle is θ-β. Therefore, a geometric correction is required using the term cos(θ-β).

[0131] In practical applications, the incident angle β usually varies from 0° to 180°, while the detection angle θ is usually fixed at a specific position (such as 90°). θ , the light signal intensity I(β) under different scattering angles can be calculated.

[0132] Preferably, to improve measurement accuracy, the system uses multiple detectors at fixed positions (for example, at 0°, 45°, 90°, 135°, and 180°) to simultaneously collect scattered signals at different angles, and then fuse the data using the above formula to obtain a more complete scattering intensity distribution.

[0133] Example 9

[0134] In one embodiment of the present invention, f(θ) in the Rayleigh scattering formula is related to the particle size, the material refractive index and the wavelength of the incident light, and satisfies the scattering law when the particle size is much smaller than the wavelength of the incident light under the Rayleigh scattering condition.

[0135] For particles that satisfy the Rayleigh scattering condition (particle size d < λ / 10, where λ is the wavelength of the incident light), the scattering function f(θ) can be expressed as:

[0136]

[0137] in: is the wave number, d is the particle diameter, n p is the refractive index of the particle, nm is the refractive index of the medium, θ is the scattering angle,

[0138] For exosomes, the refractive index n p Usually between 1.35 and 1.40, while the refractive index of aqueous solution n m When using a green laser with a wavelength of 532 nm, exosomes (diameter 30-150 nm) meet the Rayleigh scattering condition d < λ / 10.

[0139] Under this condition, the scattering intensity is proportional to the sixth power of the particle size (I∝d 6 ), which results in a significantly stronger scattering signal from large exosomes than from small exosomes. To prevent large exosomes from masking the signal from small exosomes, the present invention uses multi-angle measurement and special data processing algorithms (such as logarithmic transformation and weight adjustment) to enhance the detection sensitivity of small exosomes.

[0140] Example 10

[0141] In one embodiment of the present invention, the term cos(θ-β) in the optical signal calculation formula is used to compensate for the geometric relationship between the detection angle and the scattering angle, ensuring that the optical signal intensity data obtained at different incident angles are comparable.

[0142] In scattering experiments, the geometrical arrangement of the light source, sample, and detector has a significant impact on the measurement results. Figure 4 As shown in Figure 2, when the light source angle changes, the spatial distribution of the scattered signal will also change accordingly. In order to obtain comparable scattering data at different incident angles, the influence of geometric factors needs to be considered.

[0143] Formula I(β)=I θ The term cos(θ-β) in / cos(θ-β) is a geometric correction factor, and its principle is as follows:

[0144] 1. When the incident light angle is β, the projection intensity of the scattered light at angle θ is inversely proportional to cos(θ-β).

[0145] 2. For a detector at a fixed position (angle θ), the measured scattering intensity I θ It needs to be divided by cos(θ-β) to obtain the true scattered intensity I(β).

[0146] This geometric correction is particularly important in the following situations:

[0147] When θ-β approaches 90°, cos(θ-β) approaches 0, and the correction factor becomes very large. Special attention should be paid to the amplification effect of the measurement error.

[0148] When using multiple fixed-position detectors, the geometric correction factors of different detectors are different and need to be calculated separately.

[0149] Preferably, in an actual system, the accuracy of the geometric correction is verified by pre-calibrating with standard particles of known size (e.g., polystyrene microspheres with a diameter of 100 nm). For the standard particles, the corrected scattering intensity at different angles should conform to the theoretically predicted angular distribution.

[0150] Experimental verification shows that the use of geometrically corrected scattering data can significantly improve the accuracy of exosome size distribution measurements, especially for non-spherical exosome samples. This correction is crucial for the correct interpretation of scattering data.

[0151] Thus, the exosome particle size distribution detection and processing method and system of the present invention can achieve high-precision, multi-dimensional exosome characterization, providing advanced technical support for exosome-related research and applications.

[0152] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A method for detecting and processing exosome particle size distribution, characterized in that: include: an acquisition step, obtaining a cell culture supernatant sample using an exosome extraction kit and labeling the exosomes in the cell culture supernatant sample to obtain labeled exosomes; a setting step of setting an exosome capture probe in a light scattering detection system, wherein the light scattering detection system includes a microscope lens and a three-color camera connected to a computing module; a detection step, transmitting the exosome image to the three-color camera through the microscope lens, and the three-color camera detecting the labeled exosomes using the fluorescence wavelength and light scattering signal in the supercontinuum laser; The calculation step is to calculate the particle size distribution of the exosomes in the cell culture supernatant sample according to the light signal emitted by the labeled exosomes during the process of continuously changing the laser emission angle.

2. The method for detecting and processing the exosome particle size distribution according to claim 1, wherein: The calculation steps specifically include: Obtain the data on the changes in exosome optical signals as the laser incident angle continuously changes; Based on the change data, the exosome refractive index and the Rayleigh scattering coefficient, and in combination with the Rayleigh scattering formula, the exosome size distribution is improved from single-angle exosome light signal acquisition to three-dimensional exosome light signal acquisition; According to the change data, combined with the refractive index, the Rayleigh scattering coefficient and the Rayleigh scattering formula, the actual three-dimensional size range of the exosomes and the number of exosomes are calculated.

3. The method for detecting and processing the exosome particle size distribution according to claim 2, wherein: The calculation formula for the number of the external rotor is: N=∫γ(θ,θ0)dθ, where N represents the number of exo-rotators within the exo-rotator scattering angle, and γ(θ,θ0) represents the average number density of exo-rotators scattered at angle θ0 when the detector angle is θ.

4. The method for detecting and processing the exosome particle size distribution according to claim 2, wherein: The change data includes two-dimensional angle information of the external rotator and three-dimensional angle information of the external rotator. The two-dimensional angle information of the external rotator includes the relationship between the two-dimensional angle of the external rotator, light energy and the two-dimensional angle. The three-dimensional angle information of the external rotator includes the relationship between the angle of the external rotator, light energy and the three angles.

5. The method for detecting and processing the exosome particle size distribution according to claim 2, wherein: The Rayleigh scattering formula is: I=I0·f(θ), Wherein, I represents the light signal of the exo-rotator, I0 represents the incident light intensity, and θ is the central angle of the exo-rotator; the three-dimensional particle size formula of the exo-rotator after the formula I is transformed is: I=∫V(θ)·r 3 ·N·K·dθ, Where V(θ) represents the angle of the exo-rotator along the θ direction, r represents the actual three-dimensional size range of the exo-rotator, θ0 represents the scattering angle of the exo-rotator, N represents the average number density, and K is a constant and K=π / 6.

6. The method for detecting and processing the exosome particle size distribution according to claim 2, wherein: The variation data is the angle distribution intensity relationship, wherein the angle refers to the angle of the incident light, and the intensity refers to the relative intensity of the light signal.

7. The method for detecting and processing the exosome particle size distribution according to claim 6, wherein: The calculation formula of the optical signal is: Where β is the incident angle, I(β) is the intensity of the light signal with a scattering angle of β, and I θ is the intensity of the light signal at the detection angle θ, and θ is the angle of the light signal at the scattering angle β.

8. The method for detecting and processing the exosome particle size distribution according to claim 5, wherein: f(θ) in the Rayleigh scattering formula is related to the particle size, the refractive index of the material and the wavelength of the incident light, and satisfies the scattering law when the particle size is much smaller than the wavelength of the incident light under Rayleigh scattering conditions.

9. The method for detecting and processing the exosome particle size distribution according to claim 7, wherein: The term cos(θ-β) in the optical signal calculation formula is used to compensate for the geometric relationship between the detection angle and the scattering angle, ensuring that the optical signal intensity data obtained at different incident angles are comparable.

10. An exosome particle size distribution detection and processing system for performing the method according to any one of claims 1 to 9, the system comprising: An exosome collection module is used to obtain an exosome sample from a collection source using a collection tube of an exosome extraction kit to obtain a cell culture supernatant sample; an exosome labeling module, used to label exosomes in the cell culture supernatant sample to obtain labeled exosomes; A light scattering detection module is used to set an exosome capture probe in a light scattering detection system. The light scattering detection system includes a microscope lens and a three-color camera connected to the computing module. The microscope lens and the three-color camera are both located below the exosome capture probe. The microscope lens transmits the labeled exosome image to the three-color camera. The three-color camera detects the labeled exosomes using the fluorescence wavelength of the supercontinuum laser and the light scattering signal; A calculation module is used to calculate the particle size distribution of the exosomes in the cell culture supernatant sample based on the light signals emitted by the labeled exosomes during the process of continuously changing the laser emission angle.

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