A fluorescent dipole three-dimensional orientation analytical imaging system and method thereof

Abstract

The application discloses a fluorescent dipole three-dimensional orientation analytical imaging system and a method thereof. The application adopts loading different direction stripes on a spatial light modulator to change the transmission direction of excitation light obliquely emitted from an objective lens, the transmission direction of excitation light is changed, the polarization state is changed accordingly, and thus polarization excitation modulation is carried out to enhance the imaging sparseness of adjacent fluorophores; the excitation of fluorescent dipoles distributed along the vertical direction of a sample plane is enhanced through the excitation light obliquely emitted from the objective lens, and the detection capability of the original technology for the fluorescent dipole emitter distributed along the vertical direction of the sample plane is improved; the application realizes the polarization excitation technology, realizes the intensity and three-dimensional orientation dimension imaging of the fluorescent dipole, and analyzes the polarization phase information of the fluorescent dipole, and has a great function improvement on the original technology.

Description

Technical Field

[0001] This invention relates to fluorescence molecular analysis technology, specifically to a three-dimensional orientation analysis imaging system and method for fluorescence dipoles. Background Technology

[0002] Fluorescent molecules are not isotropic sources and exhibit dipole behavior, acting as oscillating electric dipoles during absorption and / or emission. In these processes, the polarization state of the electromagnetic field is crucial because it relates to molecular orientation and provides insights into coupling properties. Conventional fluorescence polarization microscopy (FPM) can measure fluorescent dipole orientation through polarization excitation (linear dichroism) or polarization detection (fluorescence anisotropy). FPM has proven valuable in studying the biological mechanisms of molecular arrangement and lipid membranes. However, conventional fluorescence microscopy based on polarization studies faces limitations due to optical diffraction.

[0003] To address this issue, two super-resolution fluorescence microscopy (FPM) techniques have been developed: single-molecule localization microscopy (SMLM) and polarization demodulation techniques. While SMLM offers high spatial resolution, it sacrifices temporal resolution and requires specialized sample preparation, limiting its applicability to dynamic cellular processes. On the other hand, polarization demodulation techniques, such as super-resolution by polarization demodulation (SPoD) and super-resolution dipole orientation mapping (SDOM), offer rapid imaging speeds and compatibility with conventional fluorescent labeling. However, these techniques lack the ability to fully capture the three-dimensional orientation of fluorescent dipoles. SDOM was introduced to address the limitation of lost orientation information in SPoD, but it is limited to the determination of two-dimensional orientation.

[0004] In two-dimensional orientation solutions, the calculation of the sample's projection onto the sample plane introduces angular biases. Furthermore, the projection results used in two-dimensional orientation solutions cannot reflect the sample's perpendicularity to the sample plane. Three-dimensional orientation mapping can solve this problem, enabling a comprehensive spatial understanding of the subcellular structure of three-dimensional biological samples. However, achieving three-dimensional orientation based on polarization modulation techniques without complex sample processing is quite challenging. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention proposes a fluorescence dipole three-dimensional orientation analytical imaging system and method.

[0006] One object of the present invention is to provide a three-dimensional orientation analytical imaging system for dipoles.

[0007] The fluorescence dipole three-dimensional orientation analytical imaging system of the present invention includes: a laser source, first to third half-wave plates, a polarization beam splitter, a spatial light modulator, first to third lenses, collimating and beam expanding elements, a mask, a dichroic mirror, an objective lens, and a detector; wherein, the laser emitted by the laser source is converted into linearly polarized light through a single-mode polarization-maintaining fiber; the linearly polarized light is collimated and parallelized by the first lens; the parallel light is deflected at an angle by the first half-wave plate and incident on the spatial light modulator through the polarization beam splitter, generating diffracted light of different orders; alternating black and white stripes of a set frequency and direction are loaded on the spatial light modulator to adjust the direction of the diffracted light emitted from the spatial light modulator; the diffracted light is polarized by a second half-wave plate and then sent to the second lens through the polarization beam splitter; the second lens converges the diffracted light to the third half-wave plate. The first half-wave plate adjusts the polarization direction of diffracted light of different orders to p-polarization. After passing through the mask, only the +1 or -1 order diffracted light is transmitted. The +1 or -1 order diffracted light is collimated and converged by collimating and expanding elements, and then incident perpendicularly on the objective lens through a dichroic mirror. The incident position is not located at the center of the back focal plane of the objective lens, that is, the +1 or -1 order diffracted light is not located on the optical axis of the objective lens. This allows the +1 or -1 order diffracted light to exit the objective lens at an angle after passing through the objective lens, and then incident at an angle onto the sample. A fluorescent probe is placed on the sample for marking, and the fluorescent probe acts as a fluorescent dipole. The excitation light incident at an angle onto the sample excites the fluorescent probe to emit fluorescence. The emitted fluorescence is received by the detector after passing through the dichroic mirror and the third lens, generating a fluorescence image. The modulation frequency of the detector is synchronized with that of the spatial light modulator.

[0008] The direction of the fringes loaded in the spatial light modulator changes the angle between the projection of the excitation light emitted from the objective lens onto the sample plane and the x-axis, where the x-axis is the horizontal direction of the sample plane. The frequency of the loaded fringes changes the angle between the emitted excitation light and the objective lens optical axis, with the objective lens optical axis as the z-axis. N fringes with different directions (N≥2) are loaded onto the spatial light modulator, where the directions of the fringes differ by 2π / N. The angle between the nth tilted light emitted from the objective lens and the x-axis is α. n =α0 + 2π(n-1) / N, n = 1,…,N, where α0 is the angle between the first obliquely emitted excitation beam and the x-axis; the frequency of the nth fringe loaded on the spatial light modulator is f. n The angle between the nth obliquely emitted excitation beam and the objective lens optical axis is β. nThe polarization state of the excitation light incident on the sample is related to its transmission direction. A change in the transmission direction of the excitation light results in a corresponding change in its polarization state. A spatial light modulator loads stripes in different directions, causing N different angles of excitation light to exit from the objective lens. The change in the transmission direction of the excitation light alters the polarization state of the corresponding excitation light exiting from the objective lens. This process is called polarization excitation modulation, and N polarization excitation modulations constitute one modulation cycle. The change in the polarization state of the excitation light exiting from the objective lens alters the excitation efficiency of the fluorescent dipole, resulting in a change in the emission fluorescence intensity. By acquiring fluorescence images using a detector, constructing a response model of the fluorescent dipole to polarization excitation modulation, and extracting angle information, the three-dimensional orientation information of the fluorescent dipole is obtained.

[0009] The collimating and expanding element includes a fourth lens and a fifth lens; the beam is collimated by the fourth lens and converged by the fifth lens. β1~β N No angle restriction.

[0010] Spatial light modulators employ gratings, liquid crystal spatial light modulators (SLMs), digital micromirror devices (DMDs), or galvanometers.

[0011] Another objective of this invention is to propose a method for analyzing the three-dimensional orientation of fluorescent dipoles.

[0012] The method for analyzing the three-dimensional orientation of fluorescent dipoles according to the present invention includes the following steps:

[0013] 1) Construct a three-dimensional orientation resolution imaging system for fluorescent dipoles:

[0014] The laser light emitted from the laser source is converted into linearly polarized light through a single-mode polarization-maintaining fiber; the linearly polarized light is collimated and parallelized by a first lens; the parallel light is deflected by a first half-wave plate, and then incident on a spatial light modulator via a polarization beam splitter, generating diffracted light of different orders; alternating black and white fringes of a set frequency and direction are loaded onto the spatial light modulator to adjust the direction of the diffracted light emitted from the spatial light modulator; the diffracted light is polarized by a second half-wave plate, and then passes through a polarization beam splitter to a second lens; the second lens converges the diffracted light to a third half-wave plate; the third half-wave plate adjusts the polarization direction of the diffracted light of different orders to p-polarization, and after passing through a mask, only +1 or -1 polarization passes through. The first-order diffracted light; the +1st or -1st-order diffracted light is collimated and converged by collimating and expanding elements, and then incident perpendicularly on the objective lens through a dichroic mirror, with the incident position not located at the center of the back focal plane of the objective lens, that is, the +1st or -1st-order diffracted light is not located on the optical axis of the objective lens, so that the +1st or -1st-order diffracted light exits the objective lens at an angle after passing through the objective lens, and is incident at an angle on the sample; a fluorescent probe is placed on the sample for marking, and the fluorescent probe acts as a fluorescent dipole; the excitation light incident at an angle on the sample excites the fluorescent probe to produce emission fluorescence, and the emission fluorescence is received by the detector after passing through the dichroic mirror and the third lens to generate a fluorescence image, and the modulation frequency of the detector is synchronized with that of the spatial light modulator.

[0015] 2) Polarization-induced excitation light modulation:

[0016] The direction of the fringes loaded in the spatial light modulator changes the angle between the projection of the excitation light emitted from the objective lens onto the sample plane and the x-axis, where the x-axis is the horizontal direction of the sample plane. The frequency of the loaded fringes changes the angle between the emitted excitation light and the objective lens optical axis, with the objective lens optical axis as the z-axis. N fringes with different directions (N≥2) are loaded onto the spatial light modulator, where the directions of the fringes differ by 2π / N. The angle between the nth tilted light emitted from the objective lens and the x-axis is α. n =α0 + 2π(n-1) / N, n = 1,…,N, where α0 is the angle between the first obliquely emitted excitation beam and the x-axis; the frequency of the nth fringe loaded on the spatial light modulator is f. n The angle between the nth obliquely emitted excitation beam and the objective lens optical axis is β. n The polarization state of the excitation light incident on the sample is related to the transmission direction. When the transmission direction of the excitation light changes, the polarization state changes accordingly. The spatial light modulator loads stripes of different directions, which will cause N different angles of excitation light to be emitted from the objective lens. The change in the transmission direction of the excitation light causes the polarization state of the corresponding excitation light emitted from the objective lens to change. This process is called polarization excitation modulation. N polarization excitation modulations constitute one modulation cycle. The change in the polarization state of the excitation light emitted from the objective lens changes the excitation efficiency of the fluorescent dipole, and the corresponding emission fluorescence intensity also changes.

[0017] 3) Obtain fluorescence images using a detector;

[0018] 4) Obtain the emission fluorescence intensity of the fluorescent dipole:

[0019] Constructing a response model of a fluorescent dipole to modulation of polarized excitation light:

[0020] N stripes of different directions are applied to a spatial light modulator, and N excitation beams are emitted from the objective lens at different angles. The nth excitation beam with p-polarization is... The electric field of fluorescent dipoles

[0021] dipole moment The periodic response to polarization-excited modulation is correlated with the square of the cosine function:

[0022]

[0023] Among them, g n α represents the emission fluorescence intensity of the fluorescent dipole modulated by the nth polarization excitation, where n = 1, ..., N; n To determine the angle between the projection of the excitation light onto the horizontal plane and the x-axis, β n Let be the angle between the nth tilted excitation beam and the objective optical axis; ρ and η are the azimuth (in-plane angle) and polar angle (out-of-plane angle) of the fluorescence dipole, respectively, describing the angle between the projection of the fluorescence dipole onto the horizontal plane and the x-axis, and the angle between the fluorescence dipole and the z-axis; Q is the number of photons reaching the detector during the nth polarization excitation modulation. n =I0(∑g n *h+b), where h is the system point spread function used to describe diffraction phenomena during imaging; b represents the background, modeled as a polarization-invariant quantity over a short time; I0 represents the response of the optical system to non-uniformity, i.e., a polarization-dependent periodicity correction factor; the arrival of photons at the detector exhibits statistical uncertainty, typically described by a Poisson distribution statistical model. Therefore, the fluorescence image I of the fluorescence dipole acquired by the detector during the nth polarization excitation modulation is... n The number of photons Q reaching the detector n The Poisson distribution is represented by: I n ~Possion(Q n ); Fluorescence image I of the fluorescent dipole modulated by the nth polarization excitation acquired from the detector. n The number of photons reaching the detector during the nth polarization excitation modulation is obtained, and then the emission fluorescence intensity g of the fluorescent dipole during the nth polarization excitation modulation is obtained from the number of photons reaching the detector during the nth polarization excitation modulation. n ;

[0024] The fluorescence images of the fluorescent dipoles modulated by N polarization excitations are superimposed to obtain a wide-field fluorescence image I.

[0025] 5) Extracting the three-dimensional angle information of the fluorescent dipole: The emission fluorescence intensity g of the fluorescent dipole modulated by the nth polarization excitation n Expanding to the sum of N polynomials, its expression is as follows:

[0026]

[0027] Among them, M nm Let m be the polarization-modulation coefficient component under the nth polarization-modulation, where m = 1, ..., N and n = 1, ..., N, and related to α in the excitation light. n and β n Related; D m The m-th polarization excitation modulation coefficient component M is independent of polarization excitation modulation. nm The corresponding fluorescence dipole orientation component is related to ρ and η and does not change with the number of polarization excitation modulations. In angle extraction, the blurring caused by the system point spread function and background is not considered. The emission fluorescence intensity of the fluorescence dipole is expressed as the polarization response of each pixel unit of the detector. The set of polarization responses of all pixel units of the detector is the fluorescence image acquired by the detector. Therefore, the emission fluorescence intensity acquired under different spatial light modulators with different directional fringes is expressed as follows:

[0028]

[0029] Where A is a constant term related to the system, and M is the polarization excitation modulation coefficient; by utilizing the spatial domain inverse transformation of the polarization excitation modulation coefficient M, M... -1 The combination of orientation information is obtained in the reciprocal space:

[0030]

[0031] In D m (ρ,η) contains the orientation information of the fluorescent dipole to be solved. By using the acquired fluorescence image and the reciprocal space of the polarization excitation modulation coefficient, the information of the angular component of the fluorescent dipole is extracted, and an orientation result map of the same size as the wide-field fluorescence image is obtained.

[0032] 6) The obtained angular component information and fluorescence intensity information are mapped onto the fluorescent dipole to reveal the three-dimensional orientation information.

[0033] In step 5), compared to the spatial resolution capability of traditional methods, the present invention can provide three-dimensional angular information, thereby increasing the dimension for distinguishing the molecules being tested.

[0034] In step 6), the calculation of fluorescence image and polarization excitation modulation coefficient in formula (4) is pixel-by-pixel; the wide-field fluorescence image and the orientation result image are corresponding, and the orientation result image is superimposed on the wide-field fluorescence image. At the same pixel unit position, it reflects both the emission fluorescence intensity and the three-dimensional orientation information; finally, the resolution in the space-angle dimension is obtained, providing three-dimensional orientation information, thereby increasing the dimension for distinguishing the tested molecules.

[0035] Advantages of this invention:

[0036] This invention employs stripes of different directions loaded onto a spatial light modulator to alter the transmission direction of the excitation light emitted from the objective lens at an angle. This change in transmission direction alters the polarization state, thereby enhancing the imaging sparsity of adjacent fluorophores through polarization excitation modulation. By tilting the excitation light emitted from the objective lens, the excitation of fluorescent dipoles distributed perpendicular to the sample plane is enhanced, improving the detection capability of existing techniques for fluorescent dipole emitters distributed perpendicular to the sample plane. This invention achieves intensity and three-dimensional orientation imaging of fluorescent dipoles using polarization excitation technology, resolving the polarization phase information of the fluorescent dipoles, representing a significant functional improvement over existing techniques. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the fluorescence dipole three-dimensional orientation analytical imaging system of the present invention;

[0038] Figure 2 This is a schematic diagram showing the angle between the p-polarized, tilted-emission excitation light and the x-axis obtained according to an embodiment of the fluorescence dipole three-dimensional orientation analysis method of the present invention.

[0039] Figure 3 A fluorescence image acquired by a detector, obtained according to an embodiment of the fluorescence dipole three-dimensional orientation analysis method of the present invention;

[0040] Figure 4 This is an orientation result image superimposed on a wide-field fluorescence image obtained according to an embodiment of the fluorescence dipole three-dimensional orientation analysis method of the present invention. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0042] like Figure 1As shown, the fluorescence dipole three-dimensional orientation analytical imaging system of this embodiment includes: a laser source, a first lens 2, a first half-wave plate 3, a polarization beam splitter 6, a spatial light modulator 4, a second half-wave plate 5, a second lens 7, a third half-wave plate 8, a collimating and expanding element, a mask 9, a dichroic mirror 12, an objective lens 13, a third lens 15, and a detector 16; wherein, the laser 1 emitted by the laser source is converted into linearly polarized light through a single-mode polarization-maintaining fiber; the linearly polarized light is collimated and parallelized through the first lens 2; and the parallelized light is adjusted by the first half-wave plate 3. The deflection angle becomes vertical polarization, and the light is reflected by polarization beam splitter 6 and incident on spatial light modulator 4, generating diffracted light of different orders. Black and white alternating stripes of a set frequency and direction are loaded onto spatial light modulator 4 to adjust the direction of the diffracted light emitted from spatial light modulator 4. The diffracted light is then changed to horizontal polarization by second half-wave plate 5, with the horizontal polarization parallel to the plane where the polarization beam splitter is placed. The light is then transmitted through polarization beam splitter 6 to second lens 7. Second lens 7 converges the diffracted light to third half-wave plate 8. The diffracted light of different orders... The incident light is horizontally polarized to p-polarized by the third half-wave plate 8, where p-polarization is parallel to the plane formed by the wave vectors of the 0th and ±1st order diffracted light. After passing through the mask 9, only the +1st order diffracted light is transmitted. The +1st order diffracted light is then converged by collimating and expanding elements, including a fourth lens 10 and a fifth lens 11. The fourth lens 10 collimates and the fifth lens 11 converges the light, which then passes through the dichroic mirror 12 and is perpendicularly incident on the objective lens 13. The incident position is not located at the center of the rear focal plane of the objective lens 13, meaning the +1st order diffracted light is not located at the center of the objective lens. On the optical axis of 13, the +1 or -1 order diffracted light passes through objective lens 13 and is then obliquely emitted from objective lens 13 as excitation light, which is obliquely incident on sample 14. A fluorescent probe is set on the sample for marking, and the fluorescent probe acts as a fluorescent dipole. The excitation light obliquely incident on the sample excites the fluorescent probe to emit fluorescence. The emitted fluorescence is reflected by dichroic mirror 12 and then converged by third lens 15 before being received by detector 16 to generate a fluorescence image. Detector 16 is synchronized with the modulation frequency of spatial light modulator 4. Spatial light modulator 4 uses a grating.

[0043] The method for analyzing the three-dimensional orientation of fluorescent dipoles in this embodiment includes the following steps:

[0044] 1) Construct a three-dimensional orientation analytical imaging system for fluorescent dipoles, such as... Figure 1 As shown:

[0045] 2) Polarization-induced excitation light modulation:

[0046] The direction of the fringes loaded in the spatial light modulator changes the angle between the projection of the excitation light emitted from the objective lens onto the sample plane and the x-axis, where the x-axis is the horizontal direction of the sample plane. The frequency of the loaded fringes changes the angle between the emitted excitation light and the optical axis of the objective lens. Sixth-order fringes with different directions are loaded onto the spatial light modulator, with the fringes differing by 2π / 6 in sequence. The angle between the nth tilted beam emitted from the objective lens and the x-axis is α. n =α0 + 2π(n-1) / 6, n = 1, ..., 6, where α0 is the angle between the first obliquely emitted excitation beam and the x-axis; the frequency of the nth fringe loaded on the spatial light modulator is f. n The angle between the nth obliquely emitted excitation beam and the objective lens optical axis is β. n β1 to β6 are not limited, with the objective optical axis as the z-axis; the polarization state of the excitation light incident on the sample is related to the transmission direction, and the polarization state changes accordingly when the transmission direction of the excitation light changes; the spatial light modulator loads stripes of different directions, which will cause six different angled excitation lights to be emitted from the objective lens, such as... Figure 2 As shown, the change in the direction of light transmission causes a change in the polarization state of the excitation light emitted from the objective lens. This process is called polarization excitation modulation, and six polarization excitation modulations constitute one modulation cycle. The change in the polarization state of the excitation light emitted from the objective lens causes a change in the excitation efficiency of the fluorescent dipole, and the corresponding emission fluorescence intensity also changes.

[0047] 3) Acquire fluorescence images:

[0048] Fluorescence images were acquired using a detector, and the fluorescence images obtained by six polarization excitation modulations are as follows: Figure 3 As shown;

[0049] 4) Obtain the emission fluorescence intensity of the fluorescent dipole:

[0050] A response model of a fluorescent dipole to polarized excitation light modulation is constructed: Six stripes of different directions are applied to a spatial light modulator, resulting in excitation light emitted from the objective lens at six different angles. The nth angled excitation light of p-polarized excitation light is... The electric dipole moment of a fluorescent dipole The periodic response to polarization-excited modulation is correlated with the square of the cosine function:

[0051]

[0052] Among them, g n α represents the emission fluorescence intensity of the fluorescent dipole modulated by the nth polarization excitation, where n = 1, ..., N; n To determine the angle between the projection of the excitation light onto the horizontal plane and the x-axis, β nρ is the angle between the nth inclined excitation beam and the objective lens optical axis; ρ and η are the azimuth angle (in-plane angle) and polar angle (out-plane angle) of the fluorescence dipole, respectively, describing the angle between the projection of the fluorescence dipole onto the horizontal plane and the x-axis and the angle between the fluorescence dipole and the z-axis;

[0053] The number of photons Q reaching the detector during the nth polarization excitation modulation. n =I0(∑g n *h+b), where h is the system point spread function used to describe diffraction phenomena during imaging; b represents the background, modeled as a polarization-invariant quantity over a short time; I0 represents the response of the optical system to non-uniformity, i.e., a polarization-dependent periodicity correction factor; the arrival of photons at the detector exhibits statistical uncertainty, typically described by a Poisson distribution statistical model. Therefore, the fluorescence image I of the fluorescence dipole acquired by the detector during the nth polarization excitation modulation is... n The number of photons Q reaching the detector n The Poisson distribution is represented by: I n ~Possion(Q n ); Fluorescence image I of the fluorescent dipole modulated by the nth polarization excitation acquired from the detector. n The number of photons reaching the detector during the nth polarization excitation modulation is obtained, and then the emission fluorescence intensity g of the fluorescent dipole during the nth polarization excitation modulation is obtained from the number of photons reaching the detector during the nth polarization excitation modulation. n ;

[0054] The fluorescence images of the fluorescent dipoles modulated by N polarization excitations are superimposed to obtain a wide-field fluorescence image I.

[0055] 5) Extract the three-dimensional angle information of the fluorescent dipole:

[0056] The emission fluorescence intensity g of the fluorescent dipole modulated by the nth polarization excitation n Expanded into the sum of six polynomials, its expression is as follows:

[0057] g n =(sinηcosβ) n cos(ρ-α n )-cosηsinβ) 2

[0058] =M 1n +M 2n cos2η+M 3n cos2ρ n (1-cos2η)+M 4n sin2ρ n (1-cos2η)

[0059] +M 5n cosρsin2η+M 6n sinρsin2η

[0060] Among them, M mn The m-th polarization excitation modulation coefficient component of the n-th polarization excitation modulation, m = 1, ..., N, is related to the α component in the excitation light. n and β n Regarding angle extraction, without considering the blurring caused by the system point spread function and background, the emission fluorescence intensity of the fluorescent dipole is expressed as the polarization response of each pixel unit of the detector. The set of polarization responses of all pixel units of the detector constitutes the fluorescence image acquired by the detector. Therefore, the emission fluorescence intensity acquired under different stripes with different spatial light modulators is expressed as follows:

[0061]

[0062] By utilizing the spatial domain inverse transform M of the polarization excitation modulation coefficient M -1 The combination of orientation information is obtained in the reciprocal space:

[0063]

[0064] The left side of the above equation contains the orientation information of the fluorescent dipole to be solved. By solving the information of the angular component of the fluorescent dipole through the acquired fluorescence image and the reciprocal space of the polarization excitation modulation coefficient, an orientation result map of the same size as the wide-field fluorescence image is obtained.

[0065] 6) In step 4), the calculation of the fluorescence image and polarization excitation modulation coefficient is performed pixel-by-pixel; the wide-field fluorescence image corresponds to the orientation result image, and the orientation result image is superimposed on the wide-field fluorescence image, such as... Figure 4 As shown, at the same pixel unit position, both the emission fluorescence intensity and the three-dimensional orientation information are reflected; ultimately, the resolution in the spatial-angular dimension is obtained, providing three-dimensional orientation information, thereby increasing the dimension for distinguishing the tested molecules.

[0066] Figure 4 The reconstructed angle results are shown in the image. When the distance between two points is too small, the two points cannot be distinguished by the intensity of wide-field imaging alone. By adding information in the three-dimensional angle dimension, the two points can be distinguished by the angular difference in the azimuth and / or polar angle of the dipole, which can be applied to the orientation analysis of more refined biological structures.

[0067] Finally, it should be noted that the purpose of disclosing the embodiments is to help further understand the present invention. However, those skilled in the art will understand that various substitutions and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the present invention should not be limited to the content disclosed in the embodiments, and the scope of protection of the present invention is defined by the claims.

Claims

1. A fluorescence dipole three-dimensional orientation analytical imaging system, characterized in that, The fluorescence dipole three-dimensional orientation analytical imaging system includes: a laser source, first to third half-wave plates, a polarization beam splitter, a spatial light modulator, first to third lenses, collimating and beam expanding elements, a mask, a dichroic mirror, an objective lens, and a detector. The laser emitted from the laser source is converted into linearly polarized light via a single-mode polarization-maintaining fiber. The linearly polarized light is collimated and parallelized by the first lens. The parallel light has its deflection angle adjusted by the first half-wave plate and is incident on the spatial light modulator via the polarization beam splitter, generating diffracted light of different orders. Black and white alternating stripes of a set frequency and direction are loaded onto the spatial light modulator to adjust the direction of the diffracted light emitted from the spatial light modulator. The diffracted light has its polarization direction changed by the second half-wave plate and is then sent to the second lens via the polarization beam splitter. The second lens converges the diffracted light to the third half-wave plate. The third half-wave plate adjusts the polarization direction of diffracted light of different orders to p-polarization. After passing through the mask, only the +1 or -1 order diffracted light is transmitted. The +1 or -1 order diffracted light is collimated and converged by collimating and expanding elements, and then incident perpendicularly on the objective lens through a dichroic mirror. The incident position is not located at the center of the back focal plane of the objective lens, that is, the +1 or -1 order diffracted light is not located on the optical axis of the objective lens. This allows the +1 or -1 order diffracted light to exit the objective lens at an angle and then incident obliquely on the sample. A fluorescent probe is placed on the sample for marking, and the fluorescent probe acts as a fluorescent dipole. The excitation light incident obliquely on the sample excites the fluorescent probe to emit fluorescence. The emitted fluorescence is received by the detector after passing through the dichroic mirror and the third lens, generating a fluorescence image. The modulation frequency of the detector is synchronized with that of the spatial light modulator. The direction of the fringes loaded in the spatial light modulator changes the angle between the projection of the excitation light emitted from the objective lens onto the sample plane and the x-axis, where the x-axis is the horizontal direction of the sample plane. The frequency of the loaded fringes changes the angle between the emitted excitation light and the objective lens optical axis, with the objective lens optical axis as the z-axis. N fringes with different directions (N≥2) are loaded onto the spatial light modulator, where the directions of the fringes differ by 2π / N. The angle between the nth tilted light emitted from the objective lens and the x-axis is α. n =α0+2π(n-1) / N, n=1,…,N, where α0 is the angle between the first obliquely emitted excitation beam and the x-axis; the frequency of the nth fringe loaded on the spatial light modulator is f. n The angle between the nth obliquely emitted excitation beam and the objective lens optical axis is β. n The polarization state of the excitation light incident on the sample is related to the transmission direction. A change in the transmission direction of the excitation light results in a corresponding change in the polarization state. A spatial light modulator loads stripes in different directions, causing N different angles of excitation light to exit from the objective lens. The change in the transmission direction of the excitation light alters the polarization state of the corresponding excitation light exiting from the objective lens. This process is called polarization excitation modulation, and N polarization excitation modulations constitute one modulation cycle. The change in the polarization state of the excitation light exiting from the objective lens alters the excitation efficiency of the fluorescent dipole, resulting in a change in the corresponding fluorescence intensity. By acquiring fluorescence images using a detector, constructing a response model of the fluorescent dipole to polarization excitation modulation, and extracting angle information, the three-dimensional orientation information of the fluorescent dipole is obtained.

2. The fluorescence dipole three-dimensional orientation analytical imaging system as described in claim 1, characterized in that, The collimating and beam expanding element includes a fourth lens and a fifth lens, which collimates the beam through the fourth lens and converges it through the fifth lens.

3. The fluorescence dipole three-dimensional orientation analytical imaging system as described in claim 1, characterized in that, The spatial light modulator is a grating, a liquid crystal spatial light modulator, a digital micromirror device, or a galvanometer.

4. A method for resolving the three-dimensional orientation of a fluorescence dipole in a fluorescence dipole three-dimensional orientation resolution imaging system as described in claim 1, characterized in that, The method for analyzing the three-dimensional orientation of fluorescent dipoles includes the following steps: 1) Construct a three-dimensional orientation resolution imaging system for fluorescent dipoles: The laser light emitted from the laser source is converted into linearly polarized light through a single-mode polarization-maintaining fiber; the linearly polarized light is collimated and parallelized by a first lens; the parallel light is deflected by a first half-wave plate, and then incident on a spatial light modulator via a polarization beam splitter, generating diffracted light of different orders; alternating black and white fringes of a set frequency and direction are loaded onto the spatial light modulator to adjust the direction of the diffracted light emitted from the spatial light modulator; the diffracted light is polarized by a second half-wave plate, and then passes through a polarization beam splitter to a second lens; the second lens converges the diffracted light to a third half-wave plate; the third half-wave plate adjusts the polarization direction of the diffracted light of different orders to p-polarization, and after passing through a mask, only +1 or -1 polarization passes through. The first-order diffracted light; the +1st or -1st-order diffracted light is collimated and converged by collimating and expanding elements, and then incident perpendicularly on the objective lens through a dichroic mirror, with the incident position not located at the center of the back focal plane of the objective lens, that is, the +1st or -1st-order diffracted light is not located on the optical axis of the objective lens, so that the +1st or -1st-order diffracted light exits the objective lens at an angle after passing through the objective lens, and is incident at an angle on the sample; a fluorescent probe is placed on the sample for marking, and the fluorescent probe acts as a fluorescent dipole; the excitation light incident at an angle on the sample excites the fluorescent probe to produce emission fluorescence, and the emission fluorescence is received by the detector after passing through the dichroic mirror and the third lens to generate a fluorescence image, and the modulation frequency of the detector is synchronized with that of the spatial light modulator. 2) Polarization-induced excitation light modulation: The direction of the fringes loaded in the spatial light modulator changes the angle between the projection of the excitation light emitted from the objective lens onto the sample plane and the x-axis, where the x-axis is the horizontal direction of the sample plane. The frequency of the loaded fringes changes the angle between the emitted excitation light and the objective lens optical axis, with the objective lens optical axis as the z-axis. N fringes with different directions (N≥2) are loaded onto the spatial light modulator, where the directions of the fringes differ by 2π / N. The angle between the nth tilted light emitted from the objective lens and the x-axis is α. n =α0+2π(n-1) / N, n=1,…,N, where α0 is the angle between the first obliquely emitted excitation beam and the x-axis; the frequency of the nth fringe loaded on the spatial light modulator is f. n The angle between the nth obliquely emitted excitation beam and the objective lens optical axis is β. n The polarization state of the excitation light incident on the sample is related to the transmission direction. When the transmission direction of the excitation light changes, the polarization state changes accordingly. The spatial light modulator loads stripes of different directions, which will cause N different angles of excitation light to be emitted from the objective lens. The change in the transmission direction of the excitation light causes the polarization state of the corresponding excitation light emitted from the objective lens to change. This process is called polarization excitation modulation. N polarization excitation modulations constitute one modulation cycle. The change in the polarization state of the excitation light emitted from the objective lens changes the excitation efficiency of the fluorescent dipole, and the corresponding emission fluorescence intensity also changes. 3) Obtain fluorescence images using a detector; 4) Obtain the emission fluorescence intensity of the fluorescent dipole: Constructing a response model of a fluorescent dipole to modulation of polarized excitation light: N stripes of different directions are applied to a spatial light modulator, and N excitation beams are emitted from the objective lens at different angles. The nth excitation beam with p-polarization is... The electric dipole moment of a fluorescent dipole The periodic response to polarization-excited modulation is correlated with the square of the cosine function: （1） in, Let be the emission fluorescence intensity of the fluorescent dipole modulated by the nth polarization excitation, n=1,…,N; To excite the angle between the projection of light onto the horizontal plane and the x-axis, The angle between the nth inclined excitation beam and the objective lens optical axis; and These are the azimuth and polar angles of the fluorescent dipole, respectively, describing the angle between the projection of the fluorescent dipole onto the horizontal plane and the x-axis, and the angle between the fluorescent dipole and the z-axis; The number of photons reaching the detector during the nth polarization excitation modulation h is the system point spread function, used to describe diffraction phenomena during imaging; b represents the background, which is modeled as a quantity that remains polarization-invariant over a short time. This represents the periodicity correction factor in response to non-uniformity in an optical system, which depends on polarization. The arrival of photons at the detector exhibits statistical uncertainty, which is described by a Poisson distribution statistical model. Therefore, the fluorescence image of the fluorescent dipole modulated by the nth polarization excitation modulated by the detector is obtained during the nth polarization excitation modulation. The number of photons Q reaching the detector n The Poisson distribution is represented as follows: ; Fluorescence image of the fluorescent dipole modulated by the nth polarization excitation acquired by the detector. The number of photons reaching the detector during the nth polarization excitation modulation is obtained, and then the emission fluorescence intensity of the fluorescent dipole during the nth polarization excitation modulation is obtained from the number of photons reaching the detector during the nth polarization excitation modulation. ; The fluorescence images of the fluorescent dipoles modulated by N polarization excitations are superimposed to obtain a wide-field fluorescence image I. 5) Extract the three-dimensional angle information of the fluorescent dipole: The emission fluorescence intensity of the fluorescent dipole modulated by the nth polarization excitation Expanding to the sum of N polynomials, its expression is as follows: （2） Among them, M nm Let m be the polarization-modulation coefficient component under the nth polarization-modulation, where m=1,…,N and n=1,…,N, and it is related to the excitation light. and related; The m-th polarization excitation modulation coefficient component M is independent of polarization excitation modulation. nm The corresponding fluorescent dipole orientation components, and and The fluorescence intensity is correlated and does not change with the number of polarization excitation modulations. In angle extraction, the blurring caused by the system point spread function and background is not considered. The emission fluorescence intensity of the fluorescent dipole is expressed as the polarization response of each pixel unit of the detector, and the set of polarization responses of all pixel units of the detector is the fluorescence image acquired by the detector. Therefore, the emission fluorescence intensity acquired under different directional fringes with different spatial light modulators is expressed as follows: （3） Where A is a constant term related to the system. These are the polarization excitation modulation coefficients; by utilizing the polarization excitation modulation coefficients... Inverse spatial domain transformation The combination of orientation information is obtained in the reciprocal space: （4） exist It contains the orientation information of the fluorescent dipole to be solved. By using the acquired fluorescence image and the reciprocal space of the polarization excitation modulation coefficient, the information of the angular component of the fluorescent dipole is solved to obtain the orientation result map of the same size as the wide-field fluorescence image. 6) The obtained angular component information and fluorescence intensity information are mapped onto the fluorescent dipole to reveal the three-dimensional orientation information.

5. The method for analyzing the three-dimensional orientation of fluorescent dipoles as described in claim 4, characterized in that, In step 6), the calculation of fluorescence image and polarization excitation modulation coefficient in formula (4) is pixel-by-pixel; the wide-field fluorescence image and the orientation result image are corresponding, and the orientation result image is superimposed on the wide-field fluorescence image. At the same pixel unit position, it reflects both the emission fluorescence intensity and the three-dimensional orientation information; finally, the resolution in the space-angle dimension is obtained, providing three-dimensional orientation information, thereby increasing the dimension for distinguishing the tested molecules.

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