Biological experiment automatic touch-free adding system based on optical control and operation method of biological experiment automatic touch-free adding system

Abstract

The invention discloses a biological experiment automatic touch-free adding system based on optical control and an operation method thereof, and relates to the technical field of biological experiment equipment, the system comprises the following components: an optical marking module, an optical imaging and detection module, an optical control module and an automatic control module; high automation of biological experiments is realized by integrating a plurality of modules including optical marking, optical imaging and detection, optical control and automatic control, efficient and stable marking of biological samples is ensured by utilizing specific marking of a quantum dot solution and combining accurate control of a microfluidic channel in the optical marking stage, and then, the accuracy of the biological samples is ensured. Through advanced equipment such as a high-resolution fluorescence microscope and an optical spectrum analyzer, the system can accurately obtain optical information of a marked biological sample, a solid foundation is provided for subsequent control and recognition, in the optical control stage, the optical tweezers system achieves accurate control over the biological sample in a touch-free mode, and the accuracy of the biological sample is improved. Pollution and damage possibly caused by traditional physical contact are avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of biological experiment equipment, and in particular to an automated non-touch addition system for biological experiments based on optical control and an operation method thereof. Background Art

[0002] With the rapid development of biotechnology, precise, efficient and pollution-free operation of biological samples has become a key requirement of scientific research. Traditional biological experimental methods often rely on manual operations, which have problems such as large operational errors, low experimental efficiency and cross-contamination risks.

[0003] There are some shortcomings in the traditional method. First, the labeling process of quantum dots and biological samples often lacks precise control, resulting in unstable labeling efficiency and susceptibility to interference from the external environment. Second, traditional light-controlled addition technology usually relies on physical contact to manipulate samples, which not only increases the risk of sample contamination and damage, but also limits the flexibility and accuracy of operations. In addition, the traditional system lacks efficient imaging and detection methods, making it difficult to accurately obtain and analyze optical information of labeled biological samples, thus affecting the accuracy and reliability of subsequent experiments.

[0004] In summary, the application of traditional quantum dot labeling light-controlled addition technology in biological experiments has many limitations and shortcomings. In order to overcome these shortcomings, it is particularly important to develop an automated touch-free addition system for biological experiments based on optical control and its operation method. Summary of the invention

[0005] The purpose of the present invention is to make up for the shortcomings of the prior art and to provide an automated touch-free addition system for biological experiments based on optical control and an operating method thereof. The system can realize high automation and touch-free operation of biological experiments by integrating multiple modules such as optical labeling, optical imaging and detection, optical manipulation, and automated control. The system significantly improves the accuracy and efficiency of biological experiments by precisely controlling the mixing ratio and mixing order of quantum dot solution and biological sample solution, and using an optical tweezers system to perform touch-free and precise manipulation of the labeled biological samples. At the same time, the introduction of high-resolution fluorescence microscopes and spectrometers provides strong support for the acquisition and analysis of optical information of labeled biological samples, further improving the accuracy and reliability of the experiments.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: an automated non-touch addition system for biological experiments based on optical control and an operation method thereof, the system comprising the following components: an optical marking module, an optical imaging and detection module, an optical manipulation module and an automated control module;

[0007] The optical labeling module is provided with a quantum dot solution reservoir, a microfluidic channel and a micropump. The quantum dot solution reservoir stores a quantum dot solution with a narrow emission spectrum, high fluorescence intensity and stability. The micropump drives the quantum dot solution and the biological sample solution to be labeled through the microfluidic channel to promote the quantum dot to specifically label the biological sample. The quantum dot labeling kinetic process follows the formula:

[0008]

[0009] Among them, M represents the mix The amount of biological samples successfully labeled within k a and k b is the kinetic constant related to the interaction between quantum dots and biological samples. A set of data that optimizes the labeling effect is selected as k a and k b The value of C QD represents the concentration of quantum dot solution, S sample represents the concentration of biological samples, t mix represents the mixing time of the quantum dot solution and the biological sample solution in the microfluidic channel;

[0010] The optical imaging and detection module includes a high-resolution fluorescence microscope, a spectrum analyzer and a light detector. The high-resolution fluorescence microscope collects fluorescence images of biological samples marked with quantum dots. The spectrum analyzer analyzes the fluorescence spectrum of the sample. The light detector detects the fluorescence intensity and obtains the optical information of the marked biological sample. The fluorescence spectrum analysis is based on a custom spectrum analysis algorithm, and the formula is:

[0011]

[0012] Where F(λ) is the fluorescence intensity at wavelength λ, A0, B0, C i is a parameter related to the optical properties of quantum dots and biological samples, λ0 is the central wavelength of the fluorescence spectrum, σ and σ i is the spectral width parameter, n is the number of spectral peaks considered, and the algorithm accurately models the fluorescence spectral characteristics of differently labeled biological samples to distinguish different biological samples and samples with different labeling states;

[0013] The optical manipulation module includes an optical pickup system and a microfluidic chip. The optical tweezers system consists of a laser source, a beam shaping element, and a focusing lens. The laser emitted by the laser source is shaped by the beam shaping element and focused by the focusing lens to form a laser beam with sufficient optical trapping force, so as to manipulate the marked biological sample in a touchless manner. The optical trapping force F trap The calculation formula is:

[0014]

[0015] Where Q is a parameter related to the optical properties of the laser beam and the biological sample, n m is the refractive index of the surrounding medium, P is the laser power, c is the speed of light, E is the electric field intensity, It is the electric field intensity gradient. By precisely controlling the laser power and beam focusing parameters, precise manipulation of biological samples of different sizes and characteristics can be achieved. The microfluidic chip is used to precisely control the flow and mixing of samples and reagents, and its fluid flow follows the improved form of the Navier-Stokes equation:

[0016]

[0017] Where ρ is the fluid density, v is the fluid velocity, t is the time, p is the pressure, μ is the fluid dynamic viscosity, and f ext is the external force, f op It is the light force;

[0018] The automation control module includes a computer system, an image recognition algorithm and a motion control algorithm. The computer system receives and processes data from the optical imaging and detection module. The image recognition algorithm identifies the target biological sample based on the fluorescent image and spectral information. The algorithm formula is:

[0019]

[0020] Among them, I(x,y) is the recognition result of the image at the (x,y) position, w mn It is a customized image convolution and weight, which is obtained by deep learning training of a large number of fluorescent images of labeled biological samples. The training samples cover images of various biological samples in different labeled states. The training process uses a training method based on adversarial networks to enable the convolution kernel weight to accurately extract sample feature information. f(xm,yn) is the pixel value of the input image. The motion control algorithm controls the optical camera system and the microfluidic chip to perform touchless addition operations according to the recognition results. The motion control follows the formula:

[0021]

[0022] Among them, x(t) represents the position at time t, x0 is the initial position, v0 is the initial velocity, a is the linear acceleration, and a i ,ω i and φ i It is a parameter related to the nonlinear motion characteristics of the sample.

[0023] Furthermore, the microfluidic channel includes a plurality of branch channels, the size and shape of each branch channel are customized according to the size and fluidity of different biological samples, each branch channel is provided with a microvalve, the opening and closing of the microvalve is controlled by a computer system, and by controlling the opening and closing sequence and time of the microvalve, a variety of mixing sequences and mixing ratios of different quantum dot solutions and biological sample solutions can be achieved to meet different experimental requirements. In the microfluidic channel design, the width w of each branch channel is i The diameter d of the biological sample i Satisfaction relationship:

[0024] w i =k c ·d i +k d

[0025] Among them, k c and k d is a coefficient obtained through experimental measurement and fluid dynamics simulation of different biological samples, ensuring smooth flow and effective mixing of samples in the channel to avoid sample blockage and damage. In addition, the opening and closing time interval t of the microvalve valve Follow the formula:

[0026] t valve =k e ·V sample / Q flow

[0027] Among them, k e is the time coefficient obtained by experimental optimization, V sample is the sample volume flowing through the microvalve, Q flow It is the fluid flow rate that can achieve precise control of different sample volumes. Through the precise design and control of microfluidic channels and microvalves, the system can be highly adaptable to a variety of biological samples and different experimental requirements.

[0028] Furthermore, the beam shaping element includes an adjustable diffractive optical element. By changing the phase modulation mode of the diffractive optical element, the shape, intensity distribution and light trap position of the laser beam can be adjusted. The phase modulation mode follows the formula:

[0029] φ(x,y)=k f ·sin(k g x+k h y+k i )

[0030] Among them, k f , k g , k h and k iis a parameter related to the desired beam shape and intensity distribution, and a beam expander is provided between the beam shaping element and the focusing lens to adjust the diameter of the laser beam, and its expansion factor m satisfies:

[0031] m=D1 / D0

[0032] Among them, D1 is the diameter of the expanded beam, and D0 is the original beam diameter. By precisely adjusting the beam expansion multiple, it is ensured that the appropriate light trapping force and control accuracy are maintained at different optical tweezers operating distances, thereby achieving effective control of biological samples at different distances. The beam shaping and expansion design improves the flexibility of the optical tweezers system and its adaptability to different samples and operating scenarios.

[0033] Furthermore, the high-resolution fluorescence microscope adopts multi-channel fluorescence imaging technology, which can simultaneously collect fluorescence signals of multiple different wavelength ranges. The fluorescence signal collection range of different channels is designed according to the emission spectrum characteristics of quantum dots and the fluorescence labeling characteristics of biological samples. The wavelength range (λ min ,λ max )Meet the conditions:

[0034] λ min =λ QD -k j ·Δλ,λ max =λ QD +k k ·Δλ

[0035] Among them, λ QD is the central emission wavelength of the quantum dot, Δλ is the half-height width of the quantum dot emission spectrum, k j and k k It is a coefficient obtained by analyzing the fluorescence signals of biological samples labeled with different quantum dots, ensuring the maximum capture of the fluorescence signals of quantum dots while avoiding interference from other background fluorescence. In addition, the fluorescence signals between different channels are separated and selected by a combination of a spectroscope and a filter. The splitting ratio R of the spectroscope satisfies:

[0036] R=I1 / I2=k l (λ1 / λ2) n

[0037] Among them, I1 and I2 are the fluorescence intensities of different channels, λ1 and λ2 are the central wavelengths of different channels, and k l and n are parameters obtained through experimental calibration to achieve efficient and accurate separation and collection of different fluorescence signals, providing more information sources for precise sample identification and analysis.

[0038] Furthermore, the spectrum analyzer includes a tunable filter, the center wavelength and bandwidth of which can be adjusted according to the needs of spectrum analysis, and the adjustment range and adjustment step are optimized according to the spectral characteristics of quantum dots and the fluorescence characteristics of biological samples. center And bandwidth BW satisfies the relationship:

[0039] λ center =k m ·λ QD +k n ,BW=k o ·Δλ+k p

[0040] Among them, k m to k p It is a parameter obtained through the spectrum analysis experiment of different quantum dot labeled biological samples, which realizes the fine analysis of the spectrum of different quantum dot labeled biological samples. In addition, the spectrum analyzer also includes a spectrum enhancement module, which adopts the photon multiplication technology, and its gain G satisfies:

[0041]

[0042] Among them, P detected is the detected optical power, k q and k r It is a parameter determined experimentally. Through precise adjustment of the spectrum analyzer and spectrum enhancement, the detection capability of weak fluorescence signals and the accuracy of spectral analysis are improved, which helps to more accurately identify and distinguish different biological samples.

[0043] Furthermore, the laser source of the optical tweezers system is a pulsed laser with modulatable frequency and pulse width, wherein the frequency f and pulse width t p It can be adjusted according to the characteristics and manipulation requirements of different biological samples. The adjustment relationship is as follows:

[0044] f = k s / t p +k t

[0045] Among them, k s and k t It is a coefficient obtained through manipulation experiments on different biological samples under different laser parameters to achieve the best light trapping force and manipulation effect for different biological samples. In addition, a polarization controller is provided between the laser source and the beam shaping element, and its polarization state adjustment follows the formula:

[0046] E x =E0·cos(θ),E y =E0·sin(θ)

[0047] Among them, E x and Ey is the electric field component of the laser beam in the x and y directions, E0 is the electric field intensity amplitude, and θ is the polarization angle. By adjusting the polarization angle, the direction and magnitude of the optical trapping force can be changed to adapt to the manipulation needs of biological samples of different shapes and characteristics and improve the manipulation performance and flexibility of the optical tweezers system.

[0048] Furthermore, the image recognition algorithm in the computer system includes a feature extraction sub-algorithm and a classification sub-algorithm, and the feature extraction sub-algorithm also combines local binary patterns and directional gradient histograms, and its feature vector V is represented by the formula:

[0049] V=[v conv ,v LBP ,v HOG ]

[0050] Among them, v conv is the feature vector extracted by the convolution kernel, v LBP is the local binary pattern eigenvector, v HOG is the eigenvector of the histogram of directional gradients, and the weights w of different eigenvectors conv、 w LBP and w HOG The use in the classification sub-algorithm follows the formula:

[0051] w conv =k u ·S conv / S total ,w LBP =k v ·S LBP / S total ,w HOG =k w ·S HOG / S total

[0052] Among them, S conv , S LBP and S HOG are the importance scores of convolution kernel, local binary pattern and oriented gradient histogram features, respectively, obtained through classification experiments and performance evaluation on a large number of labeled samples. total =S conv +S LBP +S HOG , through multi-feature fusion and weighted processing, the accuracy and robustness of image recognition are improved.

[0053] Furthermore, the microfluidic chip is provided with a plurality of micro chambers with different functions, including a mixing chamber, a reaction chamber and a storage chamber, each chamber is connected by a micro channel, a micro pump and a micro stirrer are provided in the micro channel, and the stirring speed v of the micro stirrer is s Follow the formula:

[0054] v s =k x Q mix / V cavity

[0055] Among them, k x is the stirring speed coefficient, Q mix is the mixed flow, V cavity is the volume of the chamber. By precisely controlling the stirring speed, different biological samples and reagents can be fully mixed and reacted in the chamber. Moreover, the flow control accuracy ΔQ of the micropump satisfies:

[0056] ΔQ=k y Q nominal / n pump

[0057] Among them, k y is the coefficient of precision, Q nominal is the nominal flow rate, n pump It is the operating frequency of the micropump. By precisely controlling the flow rate and stirring speed of the micropump, the precise flow, mixing and reaction process of samples and reagents in the microfluidic chip can be achieved, providing more precise experimental conditions for biological experiments.

[0058] Furthermore, the motion control algorithm in the automatic control module also takes into account the viscoelastic properties of the biological sample, and its motion equation is further modified as follows:

[0059]

[0060] Among them, F viscous is the viscous force on the sample, k z It is a coefficient related to the viscoelasticity of the sample, which is obtained through experimental measurement and fluid dynamics simulation of biological samples with different viscoelasticity. It can achieve more accurate motion control of biological samples with different viscoelasticity and ensure precise manipulation of biological samples during touch-free addition operations.

[0061] On the other hand, the method for automated touchless addition operation of biological experiments based on optical control is characterized in that the method comprises the following specific steps:

[0062] S1. Optical labeling step: a quantum dot solution reservoir, a microfluidic channel and a micropump are provided. The quantum dot solution reservoir stores a quantum dot solution with a narrow emission spectrum, high fluorescence intensity and stability. The micropump drives the quantum dot solution and the biological sample solution to be labeled through the microfluidic channel to promote quantum dot specific labeling of the biological sample. The quantum dot labeling kinetic process follows the formula:

[0063]

[0064] Where M represents the mix The number of biological samples successfully labeled within k a and k b is the kinetic constant related to the interaction between quantum dots and biological samples. A set of data that optimizes the labeling effect is selected as k a and k b The value of C QD represents the concentration of quantum dot solution, S sample represents the concentration of biological samples, t mix represents the mixing time of the quantum dot solution and the biological sample solution in the microfluidic channel;

[0065] S2, optical imaging and detection step: including a high-resolution fluorescence microscope, a spectrum analyzer and a light detector, wherein the high-resolution fluorescence microscope collects the fluorescence image of the biological sample marked with quantum dots, the spectrum analyzer analyzes the fluorescence spectrum of the sample, and the light detector detects the fluorescence intensity to obtain the optical information of the marked biological sample. The fluorescence spectrum analysis is based on a custom spectrum analysis algorithm, and the formula is:

[0066]

[0067] Where F(λ) is the fluorescence intensity at wavelength λ, A0, B0, C i is a parameter related to the optical properties of quantum dots and biological samples, λ0 is the central wavelength of the fluorescence spectrum, σ and σ i is the spectral width parameter, n is the number of spectral peaks considered, and the algorithm accurately models the fluorescence spectral characteristics of differently labeled biological samples to distinguish different biological samples and samples with different labeling states;

[0068] S3, optical manipulation step: including an optical camera system and a microfluidic chip, the optical tweezers system is composed of a laser source, a beam shaping element and a focusing lens, the laser emitted by the laser source is shaped by the beam shaping element and focused by the focusing lens to form a laser beam with sufficient optical trapping force, so as to manipulate the marked biological sample in a touchless manner, and the optical trapping force F trap The calculation basis formula is:

[0069]

[0070] Where Q is a parameter related to the optical properties of the laser beam and the biological sample, n m is the refractive index of the surrounding medium, P is the laser power, c is the speed of light, E is the electric field intensity, It is the electric field intensity gradient. By precisely controlling the laser power and beam focusing parameters, precise manipulation of biological samples of different sizes and characteristics can be achieved. The microfluidic chip is used to precisely control the flow and mixing of samples and reagents, and its fluid flow follows the improved form of the Navier-Stokes equation:

[0071]

[0072] Where ρ is the fluid density, v is the fluid velocity, t is the time, p is the pressure, μ is the fluid dynamic viscosity, and f ext is the external force, f op It is the light force;

[0073] S4, automated control step: including a computer system, an image recognition algorithm and a motion control algorithm. The computer system receives and processes data from the optical imaging and detection module. The image recognition algorithm identifies the target biological sample based on the fluorescent image and spectral information. The algorithm formula is:

[0074]

[0075] Among them, I(x,y) is the recognition result of the image at the (x,y) position, w mn It is a customized image convolution and weight, which is obtained by deep learning training of a large number of fluorescent images of labeled biological samples. The training samples cover images of various biological samples in different labeled states. The training process uses a training method based on adversarial networks to enable the convolution kernel weight to accurately extract sample feature information. f(xm,yn) is the pixel value of the input image. The motion control algorithm controls the optical camera system and the microfluidic chip to perform touchless addition operations according to the recognition results. The motion control follows the formula:

[0076]

[0077] Among them, x(t) represents the position at time t, x0 is the initial position, v0 is the initial velocity, a is the linear acceleration, and a i ,ω i and φ i It is a parameter related to the nonlinear motion characteristics of the sample.

[0078] Compared with the prior art, the optically controlled biological experiment automated touch-free addition system and its operation method have the following beneficial effects:

[0079] 1. This system achieves high automation of biological experiments by integrating multiple modules such as optical labeling, optical imaging and detection, optical manipulation, and automatic control. In the optical labeling stage, the specific labeling of quantum dot solutions is combined with the precise control of microfluidic channels to ensure efficient and stable labeling of biological samples. Subsequently, through high-resolution fluorescence microscopes and advanced equipment such as spectrometers, the system can accurately obtain the optical information of labeled biological samples, providing a solid foundation for subsequent manipulation and identification. In the optical manipulation stage, the optical tweezers system can achieve precise manipulation of biological samples in a touchless manner, avoiding the contamination and damage that may be caused by traditional physical contact. At the same time, the precise control of the microfluidic chip further improves the mixing and reaction efficiency of samples and reagents. The automatic control module of the entire system makes intelligent decisions based on the data obtained in real time, achieving comprehensive control of the experimental process, thereby significantly improving the experimental efficiency and accuracy.

[0080] 2. This system achieves precise control of multiple mixing orders and mixing ratios through customized branch channels and microvalve control. In addition, the tunable filter and spectrum enhancement module of the spectrometer can be finely adjusted according to the spectral characteristics of quantum dots and the fluorescence characteristics of biological samples, improving the detection capability of weak fluorescence signals and the accuracy of spectral analysis. The laser source and beam shaping elements of the optical tweezers system can also be flexibly adjusted according to the characteristics and manipulation requirements of different biological samples, ensuring the optimal light trapping force and manipulation effect. This high adaptability and flexibility enables this system to be widely used in different types of biological experiments, providing strong technical support for scientific researchers.

[0081] Other advantages, objectives and features of the present invention will be set forth in part in the following description and, in part, will be apparent to those skilled in the art based on an examination of the following or may be taught from the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0083] Figure 1 Add system flow chart for touch-free automation of biological experiments based on optical control;

[0084] Figure 2 Add a flow chart of the operation method for touch-free addition of operation methods for optically controlled biological experiment automation. DETAILED DESCRIPTION

[0085] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0086] Embodiment 1

[0087] This embodiment describes that in the cutting-edge field of cell therapy research, in the exploration of treatment options for leukemia patients, hematopoietic stem cell transplantation is a highly promising treatment method. However, accurately separating hematopoietic stem cells from bone marrow samples of leukemia patients becomes a key link. Since the cellular composition in the bone marrow of leukemia patients is complex, in addition to hematopoietic stem cells, it also contains a large number of other types of cells, such as leukemia cells and immune cells. Traditional cell sorting methods are difficult to achieve ideal purity and activity requirements. This embodiment uses an automated touch-free addition system for biological experiments based on optical control to achieve efficient and accurate sorting of hematopoietic stem cells, providing a high-quality cell source for subsequent cell therapy.

[0088] Before the experiment began, the technicians carefully prepared the experimental materials and carefully injected the quantum dot solution containing hematopoietic stem cell-specific antibody markers into the quantum dot solution reservoir. The quantum dot solution has the characteristics of narrow emission spectrum, high fluorescence intensity and stability, and can effectively mark hematopoietic stem cells. At the same time, the pre-treated leukemia patient bone marrow sample solution was prepared, and the micropump was turned on to allow the two solutions to pass through the microfluidic channel at a set flow rate. Based on a large amount of previous experimental data and theoretical analysis, the k value that optimizes the labeling effect was selected. a , k b value, assuming that it is known from preliminary experiments that for this batch of quantum dots and bone marrow samples, k a =0.5, k b =0.3, determine the concentration of quantum dot solution C QD The concentration of bone marrow sample solution is 10 μmol / L. sample 1×10 6 / mL, mixing time t m ix is ​​5 minutes, according to the formula The amount M of hematopoietic stem cells successfully labeled during this period of time is calculated, thereby ensuring that as many hematopoietic stem cells as possible are specifically labeled while reducing the occurrence of non-specific labeling.

[0089] After labeling, a high-resolution fluorescence microscope is used to collect the fluorescence images of the labeled cells. The multi-channel fluorescence imaging technology of the microscope sets the fluorescence signal collection range of different channels according to the emission spectrum characteristics of quantum dots and the fluorescence labeling characteristics of hematopoietic stem cells. Assuming that the central emission wavelength of the quantum dots is λ QD=520nm, the half-width of the emission spectrum Δλ=20nm, and k is obtained by analyzing the fluorescence signals of biological samples labeled with different quantum dots. j =0.5,k k =0.5, then the wavelength range of a channel is (λ min ,λ max )=(520-0.5×20,520+0.5×20)=(510,530)nm, ensuring the maximum capture of the fluorescence signal of quantum dots while avoiding other background fluorescence interference. The spectrum analyzer conducts in-depth analysis of the fluorescence spectrum of the sample, and the light detector accurately detects the fluorescence intensity. The fluorescence spectrum analysis formula is used Combining the known parameters A0, B0, C related to the optical properties of quantum dots and hematopoietic stem cells i , fluorescence spectrum center wavelength λ0, spectrum width parameters σ and σ i , as well as the number of spectral peaks considered n, the fluorescence spectral characteristics of different cells are accurately analyzed, thereby clearly distinguishing hematopoietic stem cells from other cells.

[0090] The optical tweezers system starts working. The laser light emitted by the laser source is shaped by the beam shaping element and focused by the focusing lens to form a laser beam with sufficient light trapping force. The adjustable diffraction optical element in the beam shaping element is adjusted according to the formula By changing the phase modulation mode, the shape, intensity distribution and light trap position of the laser beam can be precisely adjusted. Assuming k f =2, k g =1, k h =1, k i =0, and the required beam characteristics are obtained. At the same time, the beam expander adjusts the diameter of the laser beam, and the expansion factor m = D1 / D0. Assuming that the original beam diameter D0 = 1mm and the expanded beam diameter D1 = 2mm, the expansion factor m = 2. According to the formula Precisely control the laser power P parameter, assuming that the parameter Q related to the optical properties of the laser beam and hematopoietic stem cells is 0.3, and the refractive index of the surrounding medium is n m =1.33, laser power P = 100mW, calculate the optical trap force F trap , to achieve precise capture and manipulation of labeled hematopoietic stem cells, the fluid flow in the microfluidic chip follows the improved Navier-Stokes equations By precisely controlling the pressure p and fluid dynamic viscosity μ parameters within the chip, precise control of the flow of samples and reagents can be achieved, ensuring that cells move stably and orderly within the chip.

[0091] The computer system receives the data from the optical imaging and detection module in real time, and the image recognition algorithm starts working. The algorithm formula is I(x,y)=∑ m,n w mnThe convolution kernel weights w in f(xm,yn) mn The training was obtained by deep learning training of a large number of fluorescent images of labeled biological samples. The training samples cover images of various biological samples in different labeled states. The training process adopts a training method based on adversarial networks, so that the convolution kernel weights can accurately extract sample feature information. The computer analyzes the fluorescent images and spectral information to accurately identify the target hematopoietic stem cells. The motion control algorithm is based on the formula Combined with the recognition results, the optical tweezers system and microfluidic chip are precisely controlled. Assume that the initial position x0 = 0, the initial velocity v0 = 0.1 mm / s, and the linear acceleration a = 0.01 mm / s 2 According to the nonlinear motion characteristics of the sample, k = 2, a1 = 0.05, ω1 = 1, a2=0.03,ω2=2, By achieving coordinated control of optical tweezers and microfluidic chips, the identified hematopoietic stem cells can be accurately sorted into specific culture containers to complete the cell sorting operation and provide high-quality hematopoietic stem cell samples for subsequent cell therapy research.

[0092] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. The biological experiment automation non-touch addition system based on optical control is characterized by: The system includes an optical marking module, an optical imaging and detection module, an optical manipulation module and an automated control module: The optical labeling module is provided with a quantum dot solution reservoir, a microfluidic channel and a micropump. The quantum dot solution reservoir stores a quantum dot solution with a narrow emission spectrum, high fluorescence intensity and stability. The micropump drives the quantum dot solution and the biological sample solution to be labeled through the microfluidic channel to promote the quantum dot to specifically label the biological sample. The quantum dot labeling kinetic process follows the formula: Where M represents the mix The number of biological samples successfully labeled within k a and k b is the kinetic constant related to the interaction between quantum dots and biological samples. A set of data that optimizes the labeling effect is selected as k a and k b The value of C QD represents the concentration of quantum dot solution, S sample represents the concentration of biological samples, t mix represents the mixing time of the quantum dot solution and the biological sample solution in the microfluidic channel; The optical imaging and detection module includes a high-resolution fluorescence microscope, a spectrum analyzer and a light detector. The high-resolution fluorescence microscope collects fluorescence images of biological samples marked with quantum dots. The spectrum analyzer analyzes the fluorescence spectrum of the sample. The light detector detects the fluorescence intensity and obtains the optical information of the marked biological sample. The fluorescence spectrum analysis is based on a custom spectrum analysis algorithm, and the formula is: Where F(λ) is the fluorescence intensity at wavelength λ, A0, B0, C i is a parameter related to the optical properties of quantum dots and biological samples, λ0 is the central wavelength of the fluorescence spectrum, σ and σ i is the spectral width parameter, n is the number of spectral peaks considered, and the algorithm accurately models the fluorescence spectral characteristics of differently labeled biological samples to distinguish different biological samples and samples with different labeling states; The optical manipulation module includes an optical pickup system and a microfluidic chip. The optical tweezers system consists of a laser source, a beam shaping element, and a focusing lens. The laser emitted by the laser source is shaped by the beam shaping element and focused by the focusing lens to form a laser beam with sufficient optical trapping force, so as to manipulate the marked biological sample in a touchless manner. The optical trapping force F trap The calculation formula is: Where Q is a parameter related to the optical properties of the laser beam and the biological sample, n m is the refractive index of the surrounding medium, P is the laser power, c is the speed of light, E is the electric field intensity, It is the electric field intensity gradient. By precisely controlling the laser power and beam focusing parameters, precise manipulation of biological samples of different sizes and characteristics can be achieved. The microfluidic chip is used to precisely control the flow and mixing of samples and reagents, and its fluid flow follows the improved form of the Navier-Stokes equation: Where ρ is the fluid density, v is the fluid velocity, t is the time, p is the pressure, μ is the fluid dynamic viscosity, and f ext is the external force, f op It is the light force; The automation control module includes a computer system, an image recognition algorithm and a motion control algorithm. The computer system receives and processes data from the optical imaging and detection module. The image recognition algorithm identifies the target biological sample based on the fluorescent image and spectral information. The algorithm formula is: Among them, I(x,y) is the recognition result of the image at the (x,y) position, w mn It is a customized image convolution and weight, which is obtained by deep learning training of a large number of fluorescent images of labeled biological samples. The training samples cover images of various biological samples in different labeled states. The training process uses a training method based on adversarial networks to enable the convolution kernel weight to accurately extract sample feature information. f(xm,yn) is the pixel value of the input image. The motion control algorithm controls the optical camera system and the microfluidic chip to perform touchless addition operations according to the recognition results. The motion control follows the formula: Among them, x(t) represents the position at time t, x0 is the initial position, v0 is the initial velocity, a is the linear acceleration, and a i ,ω i and φ i It is a parameter related to the nonlinear motion characteristics of the sample.

2. The optically controlled biological experiment automated touchless addition system according to claim 1, characterized in that: The microfluidic channel comprises a plurality of branch channels, the size and shape of each branch channel are customized according to the size and fluidity of different biological samples, each branch channel is provided with a microvalve, the opening and closing of the microvalve is controlled by a computer system, and by controlling the opening and closing sequence and time of the microvalve, a variety of mixing sequences and mixing ratios of different quantum dot solutions and biological sample solutions can be achieved. In the microfluidic channel design, the width w of each branch channel is i The diameter d of the biological sample i Satisfaction relationship: w i =k c ·d i +k d Among them, k c and k d is a coefficient obtained through experimental measurement and fluid dynamics simulation of different biological samples, ensuring smooth flow and effective mixing of samples in the channel to avoid sample blockage and damage. In addition, the opening and closing time interval t of the microvalve valve Follow the formula: t valve =k e ·V sample / Q flow Among them, k e is the time coefficient obtained by experimental optimization, V sample is the sample volume flowing through the microvalve, Q flow is the fluid flow rate.

3. The optically controlled biological experiment automated touchless addition system according to claim 1, characterized in that: The beam shaping element includes an adjustable diffractive optical element. The shape, intensity distribution and light trap position of the laser beam can be adjusted by changing the phase modulation mode of the diffractive optical element. The phase modulation mode follows the formula: φ(x,y)=k f ·sin(k g x+k h y+k i Among them, k f , k g , k h and k i is a parameter related to the desired beam shape and intensity distribution, and a beam expander is provided between the beam shaping element and the focusing lens to adjust the diameter of the laser beam, and its expansion factor m satisfies: m=D1 / D0 Where D1 is the expanded beam diameter and D0 is the original beam diameter.

4. The optically controlled biological experiment automated touchless addition system according to claim 1, characterized in that: The high-resolution fluorescence microscope adopts multi-channel fluorescence imaging technology, which can simultaneously collect fluorescence signals of multiple different wavelength ranges. The fluorescence signal collection range of different channels is designed according to the emission spectrum characteristics of quantum dots and the fluorescence labeling characteristics of biological samples. The wavelength range (λ min ,λ max )Meet the conditions: l min =λ QD -k j ·Dl,l max =λ QD +k k ·Dl Among them, λ QD is the central emission wavelength of the quantum dot, Δλ is the half-height width of the quantum dot emission spectrum, k j and k k It is a coefficient obtained by analyzing the fluorescence signals of biological samples labeled with different quantum dots, ensuring the maximum capture of the fluorescence signals of quantum dots while avoiding interference from other background fluorescence. In addition, the fluorescence signals between different channels are separated and selected by a combination of a spectroscope and a filter. The splitting ratio R of the spectroscope satisfies: R=I1 / I2=k l ·(λ1 / λ2) n Among them, I1 and I2 are the fluorescence intensities of different channels, λ1 and λ2 are the central wavelengths of different channels, and k l and n are parameters obtained through experimental calibration.

5. The optically controlled biological experiment automated touchless addition system according to claim 1, characterized in that: The spectrum analyzer includes a tunable filter, whose central wavelength and bandwidth can be adjusted according to the needs of spectrum analysis, and the adjustment range and adjustment step length are optimized according to the spectral characteristics of quantum dots and the fluorescence characteristics of biological samples. center And bandwidth BW satisfies the relationship: l center =k m ·l QD +k n ,BW=k o ·Dl+k p Among them, k m to k p It is a parameter obtained through the spectrum analysis experiment of different quantum dot labeled biological samples, which realizes the fine analysis of the spectrum of different quantum dot labeled biological samples. In addition, the spectrum analyzer also includes a spectrum enhancement module, which adopts the photon multiplication technology, and its gain G satisfies: Among them, P detected is the detected optical power, k q and k r It is a parameter determined by experiment.

6. The optically controlled biological experiment automated touchless addition system according to claim 1, characterized in that: The laser source of the optical tweezers system is a pulsed laser with modulatable frequency and pulse width, and its frequency f and pulse width t p It can be adjusted according to the characteristics and manipulation requirements of different biological samples. The adjustment relationship is as follows: f=k s / t p +k t Among them, k s and k t It is a coefficient obtained through manipulation experiments on different biological samples under different laser parameters. In addition, a polarization controller is provided between the laser source and the beam shaping element, and its polarization state adjustment follows the formula: E x =E0·cos(θ),E y =E0 sin(θ) Among them, E x and E y are the electric field components of the laser beam in the x and y directions, E0 is the electric field intensity amplitude, and θ is the polarization angle.

7. The optically controlled biological experiment automated touchless addition system according to claim 1, characterized in that: The image recognition algorithm in the computer system includes a feature extraction sub-algorithm and a classification sub-algorithm. The feature extraction sub-algorithm also combines local binary patterns and directional gradient histograms, and its feature vector V is represented by the formula: V=[v conv ,v LBP ,v HOG ] Among them, v conv is the feature vector extracted by the convolution kernel, v LBP is the local binary pattern eigenvector, v HOG is the eigenvector of the histogram of directional gradients, and the weights w of different eigenvectors conv、 w LBP and w HOG The use in the classification sub-algorithm follows the formula: w conv =k u ·S conv / S total ,w LBP =k v ·S LBP / S total ,w HOG =k w ·S HOG / S total Among them, S conv , S LBP and S HOG are the importance scores of convolution kernel, local binary pattern and oriented gradient histogram features, respectively, obtained through classification experiments and performance evaluation on a large number of labeled samples. total =S conv +S LBP +S HOG .

8. The optically controlled biological experiment automated touchless addition system according to claim 1, characterized in that: The microfluidic chip is provided with a plurality of micro chambers with different functions, including a mixing chamber, a reaction chamber and a storage chamber. Each chamber is connected by a micro channel. A micro pump and a micro stirrer are provided in the micro channel. The stirring speed v of the micro stirrer is s Follow the formula: v s =k x ·Q mix / V cavity Among them, k x is the stirring speed coefficient, Q mix is the mixed flow, V cavity is the volume of the chamber. By precisely controlling the stirring speed, different biological samples and reagents can be fully mixed and reacted in the chamber. Moreover, the flow control accuracy ΔQ of the micropump satisfies: ΔQ=k y ·Q nominal / n pump Among them, k y is the coefficient of precision, Q nominal is the nominal flow rate, n pump is the operating frequency of the micropump.

9. The optically controlled biological experiment automated touchless addition system according to claim 1, characterized in that: The motion control algorithm in the automated control module also takes into account the viscoelastic properties of the biological sample, and its motion equation is further modified as follows: Among them, F viscous is the viscous force on the sample, k z is a coefficient related to the viscoelasticity of the sample.

10. A method for automated touchless addition of biological experiments based on optical control, characterized in that: The specific steps of this method are: S1. Optical labeling step: a quantum dot solution reservoir, a microfluidic channel and a micropump are provided. The quantum dot solution reservoir stores a quantum dot solution with a narrow emission spectrum, high fluorescence intensity and stability. The micropump drives the quantum dot solution and the biological sample solution to be labeled through the microfluidic channel to promote quantum dot specific labeling of the biological sample. The quantum dot labeling kinetic process follows the formula: Where M represents the mix The number of biological samples successfully labeled within k a and k b is the kinetic constant related to the interaction between quantum dots and biological samples. A set of data that optimizes the labeling effect is selected as k a and k b The value of C QD represents the concentration of quantum dot solution, S sample represents the concentration of biological samples, t mix represents the mixing time of the quantum dot solution and the biological sample solution in the microfluidic channel; S2, optical imaging and detection step: including a high-resolution fluorescence microscope, a spectrum analyzer and a light detector, wherein the high-resolution fluorescence microscope collects the fluorescence image of the biological sample marked with quantum dots, the spectrum analyzer analyzes the fluorescence spectrum of the sample, and the light detector detects the fluorescence intensity to obtain the optical information of the marked biological sample. The fluorescence spectrum analysis is based on a custom spectrum analysis algorithm, and the formula is: Where F(λ) is the fluorescence intensity at wavelength λ, A0, B0, C i is a parameter related to the optical properties of quantum dots and biological samples, λ0 is the central wavelength of the fluorescence spectrum, σ and σ i is the spectral width parameter, n is the number of spectral peaks considered, and the algorithm accurately models the fluorescence spectral characteristics of differently labeled biological samples to distinguish different biological samples and samples with different labeling states; S3, optical manipulation step: including an optical camera system and a microfluidic chip, the optical tweezers system is composed of a laser source, a beam shaping element and a focusing lens, the laser emitted by the laser source is shaped by the beam shaping element and focused by the focusing lens to form a laser beam with sufficient optical trapping force, so as to manipulate the marked biological sample in a touchless manner, and the optical trapping force F trap The calculation formula is: Where Q is a parameter related to the optical properties of the laser beam and the biological sample, n m is the refractive index of the surrounding medium, P is the laser power, c is the speed of light, E is the electric field intensity, It is the electric field intensity gradient. By precisely controlling the laser power and beam focusing parameters, precise manipulation of biological samples of different sizes and characteristics can be achieved. The microfluidic chip is used to precisely control the flow and mixing of samples and reagents, and its fluid flow follows the improved form of the Navier-Stokes equation: Where ρ is the fluid density, v is the fluid velocity, t is the time, p is the pressure, μ is the fluid dynamic viscosity, and f ext is the external force, f op It is the light force; S4, automated control step: including a computer system, an image recognition algorithm and a motion control algorithm. The computer system receives and processes data from the optical imaging and detection module. The image recognition algorithm identifies the target biological sample based on the fluorescent image and spectral information. The algorithm formula is: Among them, I(x,y) is the recognition result of the image at the (x,y) position, w mn It is a customized image convolution and weight, which is obtained by deep learning training of a large number of fluorescent images of labeled biological samples. The training samples cover images of various biological samples in different labeled states. The training process uses a training method based on adversarial networks to enable the convolution kernel weight to accurately extract sample feature information. f(xm,yn is the pixel value of the input image. The motion control algorithm controls the optical camera system and the microfluidic chip to perform touchless addition operations according to the recognition results. The motion control follows the formula: Among them, x(t) represents the position at time t, x0 is the initial position, v0 is the initial velocity, a is the linear acceleration, and a i ,ω i and φ i It is a parameter related to the nonlinear motion characteristics of the sample.

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