CN117420099A - Method and device for detecting heterogeneous solution based on optical diffraction chromatography - Google Patents
Method and device for detecting heterogeneous solution based on optical diffraction chromatography Download PDFInfo
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Abstract
The invention relates to a method and a device for detecting heterogeneous solution based on optical diffraction chromatography. The method comprises the following steps: obtaining interference fringe information and a time domain autocorrelation function of a fluorescence signal; extracting interference fringe information to obtain refractive index distribution information of a solution to be detected; based on preset excitation light parameter information, preset objective lens parameter information and the refractive index distribution information, a three-dimensional point spread function for representing an observation volume is obtained; acquiring a transverse radius and an axial radius of an observation volume based on the three-dimensional point spread function; and fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume, and calculating the local concentration, the molecular fluid dynamic radius and the diffusion coefficient of the solution to be detected. The invention aims to solve the problems that the operation flow of the existing method is complex, and the detection result is easy to generate errors due to inaccurate characterization of the observed volume in the heterogeneous solution.
Description
Technical Field
The invention relates to the technical field of spectrum detection, in particular to a method and a device for detecting an inhomogeneous solution based on optical diffraction chromatography.
Background
Fluorescence correlation spectroscopy (fluorescence correlation spectroscopy, FCS) is a powerful method for detecting molecular dynamics in solutions, and has wide application in the fields of biology, chemistry, medicine, pharmacy and the like. By detecting and analyzing the temporal fluctuations of the fluorescence signal generated by the labeled biomolecules, the concentration of the biomolecules, the hydrodynamic radius and the diffusion coefficient of the molecules, the interactions of different biomolecules (especially proteins) and the like can be measured.
A currently popular FCS implementation is to use a confocal microscope. Confocal microscopy can provide a very fine observation volume (fly-by-stage, fL,10 -15 L), stray fluorescence outside the focal plane is effectively filtered out, so as to improve the signal-to-noise ratio of the measurement result. However, the FCS measurement method based on confocal microscopy relies on accurate calibration of the three-dimensional point spread function (point spread function, PSF) of the focused excitation light, since it will furtherThe precise calibration of the observed volume (approximately three-dimensional ellipsoidal) is affected, and thus the precise measurement of the concentration of molecules, the hydrodynamic radius and the diffusion coefficient is finally affected.
The physical quantity to be solved of many biomolecules is directly or indirectly influenced by the lateral and axial radii of the observed volume. Since the sensitivity of FCS can be accurate to a single molecular level, if the three-dimensional PSF in actual measurement is distorted, the obtained observed volume may deviate greatly from the true value, resulting in that the measured value of the above-mentioned physical quantity may differ from the true value by more than 4 times. In performing FCS experiments, it is first necessary to calibrate the three-dimensional PSF with a standard homogeneous solution (e.g., fluorescent nanoparticle solution, etc.) to reduce the effects of distortion to some extent. However, since the heterogeneous solution (e.g., living cells) belongs to the heterogeneous sample, the local refractive index of the interior of the heterogeneous solution cannot be consistent with that of the calibrated homogeneous solution, and the possibility of distortion of the three-dimensional PSF is greatly increased due to the change of the physiological activities of biomolecules in the heterogeneous solution. It is therefore necessary to correct the three-dimensional PSF distortion problem in FCS.
The prior art is modified by Adaptive Optics (AO) measurement methods based on wavefront sensing. The method measures the wave front characteristics of excitation light in a sample through a wave front detector, and feeds the wave front characteristics back to a deformable mirror (deformable mirror, DM) array to correct the wave front characteristics before entering an objective lens, thereby correcting the three-dimensional PSF in the sample. Although many studies have demonstrated the effectiveness of this method in standard solutions and FCS measurements inside cells, it still suffers from the following major drawbacks including: due to the limited number of guide satellites, the wavefront features of the focused excitation light at all locations in the cell cannot be restored; meanwhile, since the iterative algorithm involved requires time, measurement deviation caused by flow of intracellular substances cannot be excluded. In addition, the existing method requires high-precision calibration of the DM array in advance, so that the method still depends on pre-experiments made of standard liquid, and the operation flow for measuring the local concentration of biomolecules, the hydrodynamic radius of molecules and the diffusion coefficient is complicated, the detection time is long, and the reliability is low.
Disclosure of Invention
The invention provides a detection method and a detection device for an uneven solution based on optical diffraction chromatography, which are used for solving the problems that the existing method is complex in operation flow of detecting the local concentration, hydrodynamic radius and diffusion coefficient of biomolecules in the uneven solution, and the detection result is easy to generate errors due to inaccurate observation volume characterization in the uneven solution.
The invention provides a detection method of an inhomogeneous solution based on optical diffraction chromatography, which comprises the following steps:
acquiring interference fringe information of a scattered light signal and reference light of a solution to be detected by an optical diffraction tomography device, and acquiring a time domain autocorrelation function of a fluorescent signal of the solution to be detected by a fluorescence correlation spectroscopy device;
extracting the interference fringe information to obtain refractive index distribution information of the solution to be detected;
based on preset excitation light parameter information, preset objective lens parameter information and the refractive index distribution information, a three-dimensional point spread function for representing an observation volume is obtained;
acquiring a transverse radius and an axial radius of an observation volume based on the three-dimensional point spread function;
and fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume, and calculating the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected.
According to the detection method of the inhomogeneous solution based on optical diffraction chromatography, the time domain autocorrelation function of the fluorescence signal is as follows:
wherein F (t) refers to the intensity of the fluorescent signal as a function of time t, and τ represents the time delay.
According to the method for detecting the inhomogeneous solution based on the optical diffraction chromatography, which is provided by the invention, the interference fringe information is extracted to obtain the refractive index distribution information of the solution to be detected, and the method comprises the following steps:
and extracting the interference fringe information by using a Fourier scattering model to obtain refractive index distribution information of the solution to be detected.
According to the method for detecting the inhomogeneous solution based on the optical diffraction chromatography provided by the invention, based on preset excitation light parameter information, preset objective lens parameter information and refractive index distribution information, a three-dimensional point spread function for representing an observation volume is obtained, and the method comprises the following steps:
and calculating the light ray track or light field distribution by using preset excitation light parameter information, preset objective lens parameter information and the refractive index distribution information to obtain a three-dimensional point spread function for representing the observation volume.
According to the method for detecting the heterogeneous solution based on the optical diffraction chromatography, which is provided by the invention, the transverse radius and the axial radius of the observed volume are obtained based on the three-dimensional point spread function, and the method comprises the following steps:
and extracting parameters of the observed volume based on the three-dimensional point spread function to obtain the transverse radius and the axial radius of the observed volume.
According to the method for detecting the inhomogeneous solution based on the optical diffraction chromatography, provided by the invention, the time domain autocorrelation function is fitted with a three-dimensional free diffusion model based on the transverse radius and the axial radius of an observation volume, and the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected are calculated, and the method comprises the following steps:
fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume to obtain the average fluorescence labeling molecule number in the observation volume and the average time length required by the molecules to enter and exit the observation volume through free diffusion;
and respectively calculating the diffusion coefficient, the molecular hydrodynamic radius and the local concentration of the solution to be detected by utilizing a molecular diffusion coefficient formula, a hydrodynamic radius formula and an average local concentration formula based on the average fluorescence labeling molecular number in the observation volume and the average time length required for the molecules to pass in and out of the observation volume through free diffusion.
According to the method for detecting the heterogeneous solution based on the optical diffraction chromatography, the three-dimensional free diffusion model is as follows:
wherein N is the average number of fluorescent labeling molecules in the observed volume, τ D Mean length of time required for molecules to pass into and out of the observed volume by free diffusion, r 0 Refers to the transverse radius, z, of the observation volume 0 Refers to the axial radius of the observed volume information.
The invention also provides a device for detecting the heterogeneous solution based on optical diffraction chromatography, which comprises:
the acquisition module is used for acquiring interference fringe information of a scattered light signal and reference light of the solution to be detected by the optical diffraction tomography device and acquiring a time domain autocorrelation function of a fluorescence signal of the solution to be detected by the fluorescence correlation spectroscopy device;
the extraction module is used for extracting the interference fringe information to obtain refractive index distribution information of the solution to be detected;
the PSF calculation module is used for obtaining a three-dimensional point spread function for representing the observation volume based on preset excitation light parameter information, preset objective lens parameter information and the refractive index distribution information;
the radius acquisition module is used for acquiring the transverse radius and the axial radius of the observed volume based on the three-dimensional point spread function;
and the solution parameter calculation module is used for fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume, and calculating the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected.
The invention also provides an electronic device comprising a memory, a processor and a computer program stored on the memory and capable of running on the processor, wherein the processor is used for realizing the detection method of the heterogeneous solution based on the optical diffraction chromatography when executing the program.
The present invention also provides a non-transitory computer readable storage medium having stored thereon a computer program which, when executed by a processor, implements a method of detecting a heterogeneous solution based on optical diffraction chromatography as described in any of the above.
The invention provides a detection method and a detection device for an inhomogeneous solution based on optical diffraction chromatography, wherein an optical diffraction tomography device is used for acquiring interference fringe information of a scattered light signal and reference light of a solution to be detected, and a fluorescence correlation spectroscopy device is used for acquiring a time domain autocorrelation function of a fluorescence signal of the solution to be detected; after the high-precision refractive index distribution information of the solution to be detected is obtained through the interference fringe information, the three-dimensional point spread function and the characterization of the observed volume are more accurate, the time domain autocorrelation function is further fitted with the three-dimensional free diffusion model through obtaining the transverse radius and the axial radius of the observed volume, and the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected are calculated, so that the operation steps of pre-experiment are reduced, the detection time is shortened, and the accuracy of the detection result is improved.
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In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a method for detecting heterogeneous solutions based on optical diffraction chromatography provided by the invention.
Fig. 2 is a schematic structural diagram of a device for detecting heterogeneous solution based on optical diffraction chromatography.
Fig. 3 is a schematic structural diagram of an electronic device provided by the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The following describes a method for detecting heterogeneous solution based on optical diffraction chromatography according to the present invention with reference to fig. 1, which comprises:
s1, acquiring interference fringe information of scattered light signals and reference light of a solution to be detected by an optical diffraction tomography device, and acquiring a time domain autocorrelation function of fluorescent signals of the solution to be detected by a fluorescence correlation spectroscopy device. Specifically, the time-domain autocorrelation function of the fluorescent signal is a characterization function in which the fluorescent signal maintains self-similarity over time.
In this embodiment, the optical diffraction tomography device adopts optical diffraction tomography (optical diffraction tomography, ODT), and does not need to perform fluorescent labeling on the cells to be detected, and the resolution can be as fine as a level breaking through the optical diffraction limit resolution, which belongs to a label-free and super-resolution microscopic imaging technology, and can perform damage-free and super-resolution observation on the solution to be detected for several hours. For cell samples, optical diffraction tomography does not adversely affect it by phototoxicity or metabolic interference. The imaging principle of the optical diffraction tomography is as follows: when parallel broad field light passes through a heterogeneous solution to be detected, such as biological cells, the scattered light naturally carries structural information of the cells due to the non-uniform distribution of refractive index inside the cells and weak scattering properties. By the process of back-scattering calculation based on the fourier scattering model, the three-dimensional refractive index distribution in the cell can be accurately restored. The optical diffraction tomography needs to use 2 beams of homologous narrow linewidth laser, and the coherence length is long and can reach the order of meters. Wherein, one beam is illumination light, and scattered light is generated after passing through the sample; the other beam is a reference beam that acts to interfere with the scattered light at a detector (e.g., camera) to produce fringe information.
The optical diffraction tomography device and the fluorescence correlation spectroscopy device use lasers of the same wavelength band to ensure the validity of the solved refractive index profile information to FCS (as a function of refractive index as a function of wavelength). The optical diffraction tomography device uses a narrow linewidth laser to split the light into illumination light and reference light, wherein the illumination light generates scattered light signals after passing through a sample, and the scattered light signals interfere with the reference light at a detector (such as a camera) to generate interference fringe information.
The fluorescence correlation spectroscopy device uses a laser with the same wave band as the narrow linewidth laser in the optical diffraction tomography device and a shorter coherence length, so that excitation light provided by the laser does not have obvious interference phenomenon with illumination light in the optical diffraction tomography device in an overlapped light path. The excitation light parameter information comprises the wavelength of excitation light (such as 488nm wavelength), the appearance and area of a light spot when entering the objective lens, and the objective lens parameter information comprises parameters such as the numerical aperture, working distance, focal length and the like of the objective lens.
S2, extracting the interference fringe information to obtain refractive index distribution information of the solution to be detected. Namely, the refractive index distribution in the nonuniform solution is accurately calculated through a weak scattering theory (the intensity of a scattered light field is far smaller than that of an original incident light field, namely, the weak scattering condition) and a Fourier diffraction theorem.
And S3, obtaining a three-dimensional point spread function for representing the observation volume based on preset excitation light parameter information, preset objective lens parameter information and the refractive index distribution information. In step S1, the optical diffraction tomography device collects interference fringe information of a scattered light signal and a reference light of a solution to be detected, and the fluorescence correlation spectroscopy device collects a time domain autocorrelation function of a fluorescence signal of the solution to be detected; the method can also comprise the steps of firstly acquiring interference fringe information of scattered light signals and reference light of the solution to be detected by an optical diffraction tomography device, acquiring a three-dimensional point spread function used for representing an observation volume, and then acquiring a time domain autocorrelation function of fluorescent signals of the solution to be detected by a fluorescence correlation spectroscopy device.
And S4, acquiring the transverse radius and the axial radius of the observed volume based on the three-dimensional point spread function.
And S5, fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume, and calculating the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected. Specifically, the three-dimensional free diffusion model is an ideal physical model that describes the time dependence of the diffusion motion of molecules in a solution under a thermally balanced environment and within an observation volume determined by a confocal microscope and a specific excitation light wavelength.
The method comprises the steps of acquiring interference fringe information of scattered light signals and reference light of a solution to be detected by an optical diffraction tomography device, and acquiring a time domain autocorrelation function of fluorescent signals of the solution to be detected by a fluorescence correlation spectroscopy device; after the high-precision refractive index distribution information of the solution to be detected is obtained through the interference fringe information, the representation of the three-dimensional point spread function and the observed volume is more accurate, the time domain autocorrelation function is further fitted with the three-dimensional free diffusion model through obtaining the transverse radius and the axial radius of the observed volume, and the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected are calculated, so that the measurement precision of FCS on each physical quantity of fluorescent marker molecules in the heterogeneous solution is improved. Compared with the prior art, the method not only reduces the influence caused by calculation time delay, but also omits the pre-experiment step, thereby shortening the detection time and improving the accuracy of the detection result.
On the basis of the above embodiments, the intensity of the fluorescent signal may fluctuate over time when observing the volume due to the diffusion movement of the molecules themselves. The time-domain autocorrelation function of the fluorescent signal is shown in formula (1):
wherein F (t) refers to the intensity of the fluorescent signal as a function of time t, and τ represents the time delay.
On the basis of the above embodiment, the extracting process is performed on the interference fringe information to obtain refractive index distribution information of the solution to be detected, including:
and extracting the interference fringe information by using a Fourier scattering model to obtain refractive index distribution information of the solution to be detected. In this embodiment, the fourier scattering model is a combination of the weak scattering theory (the scattering light field intensity is far smaller than the original incident light field, i.e. weak scattering condition) and the fourier diffraction theorem, so as to accurately restore refractive index distribution information from interference fringe information.
Based on the above embodiments, based on preset excitation light parameter information, preset objective lens parameter information, and the refractive index distribution information, a three-dimensional point spread function for characterizing an observation volume is obtained, including:
and calculating the light ray track or light field distribution by using the obtained refractive index distribution information, excitation light parameter information and objective lens parameter information to obtain a three-dimensional point spread function for representing the observation volume. Wherein the observation volume approximates a three-dimensional ellipsoidal shape.
Based on the above embodiment, based on the three-dimensional point spread function, acquiring a lateral radius and an axial radius of an observation volume includes:
and extracting parameters of the observed volume based on the three-dimensional point spread function to obtain the transverse radius and the axial radius of the observed volume.
On the basis of the above embodiment, fitting the time-domain autocorrelation function with a three-dimensional free diffusion model based on the lateral radius and the axial radius of the observed volume, and calculating the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected, including:
fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume to obtain the average fluorescence labeling molecule number in the observation volume and the average time length required by the molecules to enter and exit the observation volume through free diffusion.
And respectively calculating the diffusion coefficient, the molecular hydrodynamic radius and the local concentration of the solution to be detected by utilizing a molecular diffusion coefficient formula, a hydrodynamic radius formula and an average local concentration formula based on the average fluorescence labeling molecular number in the observation volume and the average time length required for the molecules to pass in and out of the observation volume through free diffusion.
Specifically, a three-dimensional free diffusion model is shown in formula (2):
wherein N is the average number of fluorescent labeling molecules in the observed volume, τ D Mean length of time required for molecules to pass into and out of the observed volume by free diffusion, r 0 Refers to the transverse radius, z, of the observation volume 0 Refers to the axial radius of the observed volume information.
In this embodiment, the formula of the diffusion coefficient of the molecules is shown in formula (3):
wherein D refers to the diffusion coefficient of the molecule.
In this embodiment, the hydrodynamic radius formula, see formula (4):
wherein R is hydrodynamic radius, k B Is the boltzmann constant, T is the ambient temperature, and η is the kinetic viscosity of the irregular solution.
In this embodiment, the average local concentration formula is shown in formula (5):
wherein C is the local concentration of the solution to be detected.
The apparatus for detecting an uneven solution based on optical diffraction chromatography provided by the invention is described below, and the apparatus for detecting an uneven solution based on optical diffraction chromatography described below and the method for detecting an uneven solution based on optical diffraction chromatography described above can be referred to correspondingly with each other.
Referring to fig. 2, an apparatus for detecting an inhomogeneous solution based on optical diffraction chromatography includes: the acquisition module 210, the extraction module 220, the PSF calculation module 230, the radius acquisition module 240, and the solution parameter calculation module 250.
The acquisition module 210 is configured to acquire interference fringe information of a scattered light signal and a reference light of a solution to be detected by using an optical diffraction tomography device, and acquire a time domain autocorrelation function of a fluorescence signal of the solution to be detected by using a fluorescence correlation spectroscopy device.
The extraction module 220 is configured to perform extraction processing on the interference fringe information to obtain refractive index distribution information of the solution to be detected.
The PSF calculation module 230 obtains a three-dimensional point spread function for characterizing the observed volume based on the preset excitation light parameter information, the preset objective lens parameter information, and the refractive index distribution information.
The radius acquisition module 240 is configured to acquire a lateral radius and an axial radius of the observation volume based on the three-dimensional point spread function.
The solution parameter calculation module 250 is configured to fit the time-domain autocorrelation function to a three-dimensional free diffusion model based on a lateral radius and an axial radius of an observation volume, and calculate a local concentration, a molecular hydrodynamic radius, and a diffusion coefficient of the solution to be detected.
The invention acquires interference fringe information of scattered light signals and reference light of a solution to be detected by an optical diffraction tomography device through an acquisition module 210, and acquires a time domain autocorrelation function of fluorescent signals of the solution to be detected by a fluorescence correlation spectroscopy device. The extraction module 220 obtains refractive index distribution information of the solution to be detected. The PSF calculation module 230 obtains a three-dimensional point spread function for characterizing the observed volume. The radius acquisition module 240 acquires a lateral radius and an axial radius of the observation volume. The solution parameter calculation module 250 detects the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected, so that the operation steps of pre-experiments are reduced, the detection time is shortened, and the accuracy of the detection result is improved.
The extraction module 220 is specifically configured to: and extracting the interference fringe information by using a Fourier scattering model to obtain refractive index distribution information of the solution to be detected.
The PSF calculation module 230 specifically is configured to: and calculating the light ray track or light field distribution by using preset excitation light parameter information, preset objective lens parameter information and the refractive index distribution information to obtain a three-dimensional point spread function for representing the observation volume.
The radius acquisition module 240 is specifically configured to: and extracting parameters of the observed volume based on the three-dimensional point spread function to obtain the transverse radius and the axial radius of the observed volume.
The solution parameter calculation module 250 is specifically configured to: fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume to obtain the average fluorescence labeling molecule number in the observation volume and the average time length required by the molecules to enter and exit the observation volume through free diffusion;
and respectively calculating the diffusion coefficient, the molecular hydrodynamic radius and the local concentration of the solution to be detected by utilizing a molecular diffusion coefficient formula, a hydrodynamic radius formula and an average local concentration formula based on the average fluorescence labeling molecular number in the observation volume and the average time length required for the molecules to pass in and out of the observation volume through free diffusion.
Fig. 3 illustrates a physical schematic diagram of an electronic device, as shown in fig. 3, where the electronic device may include: processor 310, communication interface (communication interface) 320, memory 330 and communication bus 340, wherein processor 310, communication interface 320, memory 330 accomplish communication with each other through communication bus 340. The processor 310 may invoke logic instructions in the memory 330 to perform a method for detecting heterogeneous solutions based on optical diffraction chromatography, the method comprising:
s1, acquiring interference fringe information of scattered light signals and reference light of a solution to be detected by an optical diffraction tomography device, and acquiring a time domain autocorrelation function of fluorescent signals of the solution to be detected by a fluorescence correlation spectroscopy device.
S2, extracting the interference fringe information to obtain refractive index distribution information of the solution to be detected.
And S3, obtaining a three-dimensional point spread function for representing the observation volume based on preset excitation light parameter information, preset objective lens parameter information and the refractive index distribution information.
And S4, acquiring the transverse radius and the axial radius of the observed volume based on the three-dimensional point spread function.
And S5, fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume, and calculating the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected.
Further, the logic instructions in the memory 330 described above may be implemented in the form of software functional units and may be stored in a computer-readable storage medium when sold or used as a stand-alone product. Based on this understanding, the technical solution of the present invention may be embodied essentially or in a part contributing to the prior art or in a part of the technical solution in the form of a software product stored in a storage medium, comprising several instructions for causing a computer device (which may be a personal computer, a server, a network device, etc.) to perform all or part of the steps of the method according to the embodiments of the present invention. And the aforementioned storage medium includes: a U-disk, a removable hard disk, a read-only memory (ROM), a random access memory (RAM, random access memory), a magnetic disk, or an optical disk, or other various media capable of storing program codes.
In another aspect, the present invention also provides a computer program product, the computer program product comprising a computer program, the computer program being storable on a non-transitory computer readable storage medium, the computer program, when executed by a processor, being capable of performing the method for detecting an inhomogeneous solution based on optical diffraction chromatography provided by the above methods, the method comprising:
s1, acquiring interference fringe information of scattered light signals and reference light of a solution to be detected by an optical diffraction tomography device, and acquiring a time domain autocorrelation function of fluorescent signals of the solution to be detected by a fluorescence correlation spectroscopy device.
S2, extracting the interference fringe information to obtain refractive index distribution information of the solution to be detected.
And S3, obtaining a three-dimensional point spread function for representing the observation volume based on preset excitation light parameter information, preset objective lens parameter information and the refractive index distribution information.
And S4, acquiring the transverse radius and the axial radius of the observed volume based on the three-dimensional point spread function.
And S5, fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume, and calculating the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected.
In yet another aspect, the present invention also provides a non-transitory computer readable storage medium having stored thereon a computer program which, when executed by a processor, is implemented to perform the method for detecting a heterogeneous solution based on optical diffraction chromatography provided by the above methods, the method comprising:
s1, acquiring interference fringe information of scattered light signals and reference light of a solution to be detected by an optical diffraction tomography device, and acquiring a time domain autocorrelation function of fluorescent signals of the solution to be detected by a fluorescence correlation spectroscopy device.
S2, extracting the interference fringe information to obtain refractive index distribution information of the solution to be detected.
And S3, obtaining a three-dimensional point spread function for representing the observation volume based on preset excitation light parameter information, preset objective lens parameter information and the refractive index distribution information.
And S4, acquiring the transverse radius and the axial radius of the observed volume based on the three-dimensional point spread function.
And S5, fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume, and calculating the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected.
The apparatus embodiments described above are merely illustrative, wherein the elements illustrated as separate elements may or may not be physically separate, and the elements shown as elements may or may not be physical elements, may be located in one place, or may be distributed over a plurality of network elements. Some or all of the modules may be selected according to actual needs to achieve the purpose of the solution of this embodiment. Those of ordinary skill in the art will understand and implement the present invention without undue burden.
From the above description of the embodiments, it will be apparent to those skilled in the art that the embodiments may be implemented by means of software plus necessary general hardware platforms, or of course may be implemented by means of hardware. Based on this understanding, the foregoing technical solution may be embodied essentially or in a part contributing to the prior art in the form of a software product, which may be stored in a computer readable storage medium, such as ROM/RAM, a magnetic disk, an optical disk, etc., including several instructions for causing a computer device (which may be a personal computer, a server, or a network device, etc.) to execute the method described in the respective embodiments or some parts of the embodiments.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for detecting an inhomogeneous solution based on optical diffraction chromatography, comprising:
acquiring interference fringe information of a scattered light signal and reference light of a solution to be detected by an optical diffraction tomography device, and acquiring a time domain autocorrelation function of a fluorescent signal of the solution to be detected by a fluorescence correlation spectroscopy device;
extracting the interference fringe information to obtain refractive index distribution information of the solution to be detected;
based on preset excitation light parameter information, preset objective lens parameter information and the refractive index distribution information, a three-dimensional point spread function for representing an observation volume is obtained;
acquiring a transverse radius and an axial radius of an observation volume based on the three-dimensional point spread function;
and fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume, and calculating the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected.
2. The method for detecting heterogeneous solution based on optical diffraction chromatography according to claim 1, wherein the time-domain autocorrelation function of the fluorescence signal is:
wherein F (t) refers to the intensity of the fluorescent signal as a function of time t, and τ represents the time delay.
3. The method for detecting an inhomogeneous solution based on optical diffraction chromatography according to claim 1, wherein the extracting process of the interference fringe information to obtain refractive index distribution information of the solution to be detected includes:
and extracting the interference fringe information by using a Fourier scattering model to obtain refractive index distribution information of the solution to be detected.
4. The method for detecting an inhomogeneous solution based on optical diffraction chromatography according to claim 1, wherein obtaining a three-dimensional point spread function for characterizing an observation volume based on preset excitation light parameter information, preset objective lens parameter information, and the refractive index distribution information, comprises:
and calculating the light ray track or light field distribution by using preset excitation light parameter information, preset objective lens parameter information and the refractive index distribution information to obtain a three-dimensional point spread function for representing the observation volume.
5. The method for detecting an inhomogeneous solution based on optical diffraction chromatography according to claim 1, wherein acquiring a lateral radius and an axial radius of an observed volume based on the three-dimensional point spread function includes:
and extracting parameters of the observed volume based on the three-dimensional point spread function to obtain the transverse radius and the axial radius of the observed volume.
6. The method for detecting an inhomogeneous solution based on optical diffraction chromatography according to claim 1, wherein fitting the time-domain autocorrelation function to a three-dimensional free diffusion model based on a lateral radius and an axial radius of an observation volume, calculates a local concentration, a molecular hydrodynamic radius, and a diffusion coefficient of the solution to be detected, comprising:
fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume to obtain the average fluorescence labeling molecule number in the observation volume and the average time length required by the molecules to enter and exit the observation volume through free diffusion;
and respectively calculating the diffusion coefficient, the molecular hydrodynamic radius and the local concentration of the solution to be detected by utilizing a molecular diffusion coefficient formula, a hydrodynamic radius formula and an average local concentration formula based on the average fluorescence labeling molecular number in the observation volume and the average time length required for the molecules to pass in and out of the observation volume through free diffusion.
7. The method for detecting an inhomogeneous solution based on optical diffraction chromatography according to any one of claims 1-6, wherein the three-dimensional free diffusion model is:
wherein N is the average number of fluorescent labeling molecules in the observed volume, τ D Mean length of time required for molecules to pass into and out of the observed volume by free diffusion, r 0 Refers to the transverse radius, z, of the observation volume 0 Refers to the axial radius of the observed volume information.
8. A device for detecting a heterogeneous solution based on optical diffraction chromatography, comprising:
the acquisition module is used for acquiring interference fringe information of a scattered light signal and reference light of the solution to be detected by the optical diffraction tomography device and acquiring a time domain autocorrelation function of a fluorescence signal of the solution to be detected by the fluorescence correlation spectroscopy device;
the extraction module is used for extracting the interference fringe information to obtain refractive index distribution information of the solution to be detected;
the PSF calculation module is used for obtaining a three-dimensional point spread function for representing the observation volume based on preset excitation light parameter information, preset objective lens parameter information and the refractive index distribution information;
the radius acquisition module is used for acquiring the transverse radius and the axial radius of the observed volume based on the three-dimensional point spread function;
and the solution parameter calculation module is used for fitting the time domain autocorrelation function with a three-dimensional free diffusion model based on the transverse radius and the axial radius of the observation volume, and calculating the local concentration, the molecular hydrodynamic radius and the diffusion coefficient of the solution to be detected.
9. An electronic device comprising a memory, a processor and a computer program stored on the memory and executable on the processor, characterized in that the processor implements the method for detecting a heterogeneous solution based on optical diffraction chromatography as claimed in any one of claims 1 to 7 when executing the program.
10. A non-transitory computer readable storage medium, on which a computer program is stored, characterized in that the computer program, when executed by a processor, implements the method for detecting a heterogeneous solution based on optical diffraction chromatography according to any one of claims 1 to 7.
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