A multi-dimensional encoding long focal depth light field two-photon microscopic imaging method and system

Abstract

This invention relates to the field of microscopic optical imaging technology, specifically to a multi-dimensional encoded long focal depth optical field two-photon microscopic imaging method and system. The method and system include: designing a multi-dimensional encoded optical field, configured to flexibly adjust the intensity and position distribution of the optical field based on multifocal spatial multiplexing; inserting an optical field distribution shaping module into a traditional two-photon imaging system to change the phase position relative to the objective lens's back aperture; performing two two-dimensional scans of the sample based on the multi-dimensional encoded optical field scanning imaging to obtain two two-dimensional projection images, which are then determined by combining the lateral spacing distribution of the projection points and the fluorescence intensity ratio. This invention can simultaneously encode the sample distribution into the multi-dimensional information of intensity and position distribution in the projection images during the scanning imaging process, achieving efficient and rapid three-dimensional imaging by analyzing and locating the sample's three-dimensional distribution, improving the accuracy of sample depth analysis, and accelerating the three-dimensional imaging of the sample.

Description

Technical Field

[0001] This invention relates to the field of microscopic optical imaging technology, and more specifically, to a multidimensional encoded long focal depth optical field two-photon microscopic imaging method and system. Background Technology

[0002] Traditional two-photon microscopy, based on the two-photon excitation effect, excites fluorescence signals only at the focal plane of the objective lens. Achieving three-dimensional imaging of samples typically involves a three-dimensional traversal scan using a laser focal point. This method not only significantly reduces the speed of three-dimensional imaging but also leads to the accumulation of phototoxicity. Axially extended depth-of-field optical fields based on optical field shaping design can achieve longitudinal imaging while simultaneously scanning laterally, effectively improving imaging speed. However, existing long-depth-of-field optical fields only change a single parameter with transmission, thus primarily suitable for structural and functional imaging of sparsely labeled samples, and remain a significant challenge for imaging densely labeled samples.

[0003] Existing technology 1: Bessel beams are the most representative light fields with large depth of field. This light field achieves vertical imaging by expanding the depth of field along the axis while performing lateral grating scanning. This method can greatly improve the imaging speed by reducing one scanning dimension. Figure 1 (a)

[0004] Existing technology 2: Further designing the long focal depth light field as a V-shaped or mirror-symmetrically distributed Airy beam can transform the depth information of the sample into the lateral spacing information of paired projection points for characterization. Figure 1 (b)

[0005] Existing technology 3: The long focal depth light field can also be designed as an intensity-complementary variation light field to transform the depth information of the sample into the fluorescence intensity distribution of paired projection points for characterization. Figure 1 (c)

[0006] Existing technologies each have their limitations:

[0007] Existing technology 1 is based on Bessel beam scanning imaging, which improves the speed of 3D imaging, but only obtains the projection distribution map of the sample, and cannot characterize the depth information, thus lacking depth information.

[0008] The V-shaped light field design in the existing technology sacrifices the numerical aperture of the objective lens, and the two-photon fluorescence interference from the Airy beam side ring is significant, both of which limit the lateral resolution of the two-photon imaging system. Furthermore, this technology relies on the projection morphology when decomposing the fluorescent target projection, making it difficult to distinguish projections with similar morphologies, thus limiting the accuracy of depth acquisition and resulting in low spatial resolution.

[0009] In the existing third technology, which is based on axially elongated and intensity-complementary light field scanning imaging, when there are multiple imaging targets in the direction of the excitation light axis, their projections are superimposed and difficult to separate. This method cannot effectively determine the depth of each imaging target, and is therefore only suitable for three-dimensional imaging of sparsely labeled samples. It cannot distinguish multiple imaging targets along the axis. Summary of the Invention

[0010] This invention provides a multi-dimensional encoded long focal depth light field two-photon microscopy imaging method and system to at least solve the technical problem of low efficiency in existing three-dimensional rapid imaging.

[0011] According to an embodiment of the present invention, a multi-dimensional encoded long focal depth light field two-photon microscopy imaging method is provided, comprising the following steps:

[0012] S101: Design a multi-dimensional encoded light field, which is configured to achieve flexible adjustment of light field intensity and position distribution based on multi-focal spatial multiplexing;

[0013] S102: Insert a light field distribution shaping module into a traditional two-photon imaging system to change the position of the phase at the back aperture of the objective lens;

[0014] S103: Based on multidimensional coded light field scanning imaging, two two-dimensional scans are performed on the sample to obtain two two-dimensional projection images, which are then determined by combining the lateral spacing distribution of the projection points and the fluorescence intensity ratio.

[0015] Furthermore, in step S101, the depth of focus of the light field is flexibly adjusted by changing the number of focal points; the side lobes of the light field are eliminated or reduced by superimposing tiny phase shifts on each focal point based on the mutual interference between focal points.

[0016] Furthermore, in step S101, the encoding of the light field intensity distribution with depth is achieved by superimposing mutually exclusive binarization matrices with different proportions of parameter 1 at each focal point, wherein the parameter of the binarization matrix is ​​0 or 1; the encoding of the light field lateral distribution with depth is achieved by adjusting the lateral misalignment between the center of the light field wavefront and the geometric center of the objective lens back aperture.

[0017] Further, in step S102, the light field distribution shaping module is configured as follows:

[0018] After passing through a series of relay lenses, the spatial light modulator is conjugate with the Galvo X and Galvo Y galvanometers and the back aperture of the objective lens. The sum of the single-focus phases is loaded onto the spatial light modulator to achieve the adjustment of the light field intensity distribution.

[0019] Furthermore, in step S102, the galvanometer Galvo 1 and the spatial light modulator are located on the front and rear focal planes of the same lens, respectively. Changing the angle of Galvo 1 causes the light spot to move on the galvanometer Galvo X, thereby changing the position of the phase at the back aperture of the objective lens.

[0020] Furthermore, in step S102, the tilting of the long focal depth light field is achieved by simply switching the voltage of the galvanometer Galvo 1, thereby generating the dual-encoded excitation light field.

[0021] Furthermore, step S103 specifically includes:

[0022] a) Set an intensity threshold to extract the effective projection area, number each projection, and extract the lateral position distribution and fluorescence intensity value of each projection;

[0023] b) Since the position of the same fluorescent sample in the two two-dimensional projection images differs only in the x-direction, and their y-coordinates are approximately the same, all possible projection pairs are matched based on this criterion.

[0024] c) For all possible combinations of projection pairs, based on the formula... The fluorescence intensity ratio is calculated based on the formula D = | x L -x R | Calculate the lateral spacing distribution;

[0025] The corresponding depth value is determined by combining the changes in the lateral spacing and intensity ratio of the multidimensional encoded light field with depth, and the two depth values ​​are matched and verified.

[0026] Furthermore, in step S103, when the two depth values ​​have a small difference, this projection pair corresponds to the same sample, and the sample depth is the average of the two values.

[0027] Furthermore, for projections that fail to pair, the depth value is first estimated based on the lateral spacing, and the fluorescence intensity of the projection is inferred from the estimated depth value. Then, the optimal projection pair combination is determined by comparing the intensity values ​​of one or more projection combinations, and the depth resolution of the sample is completed.

[0028] According to another embodiment of the present invention, a multi-dimensional encoded long focal depth light field two-photon microscopy imaging system is provided, comprising:

[0029] The light field design module is used to design multidimensional encoded light fields. The multidimensional encoded light fields are configured to achieve flexible adjustment of light field intensity and position distribution based on multi-focal spatial multiplexing.

[0030] An imaging modification module is used to insert a light field distribution shaping module into a traditional two-photon imaging system to change the position of the phase at the back aperture of the objective lens.

[0031] The sample depth analysis module is used to perform two two-dimensional scans on the sample based on multi-dimensional coded light field scanning imaging to obtain two two-dimensional projection images, which are then combined with the lateral spacing distribution of the projection points and the fluorescence intensity ratio to determine the depth.

[0032] A storage medium storing program files capable of implementing any of the above-mentioned multidimensional encoded long focal depth light field two-photon microscopy imaging methods.

[0033] A processor for running a program, wherein the program executes, during runtime, any of the above-mentioned multidimensional coded long focal depth light field two-photon microscopy imaging methods.

[0034] The multi-dimensional encoded long-focal-depth optical field two-photon microscopy imaging method and system in this invention can simultaneously encode the sample distribution into the intensity and positional distribution multi-dimensional information of the projection image during the scanning imaging process. By resolving and locating the three-dimensional distribution of the sample, efficient and rapid three-dimensional imaging is achieved. This invention overcomes the limitations of existing technologies for sparsely labeled samples, developing a rapid three-dimensional two-photon microscopy imaging technology suitable for densely distributed samples without sacrificing resolution or missing important biological processes. This improves the accuracy of sample depth resolution and accelerates the three-dimensional imaging of samples. Attached Figure Description

[0035] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0036] Figure 1 Figure showing the current status of two-photon microscopy research based on beam shaping.

[0037] Figure 2 This is a flowchart of the multi-dimensional encoded long focal depth light field two-photon microscopy imaging method of the present invention;

[0038] Figure 3 This is a schematic diagram illustrating the generation principle of the multidimensional encoded optical field in this invention;

[0039] Figure 4 This is a diagram of the light field distribution shaping module used in the experiment of this invention;

[0040] Figure 5 Flowchart of sample depth analysis in this invention;

[0041] Figure 6 A comparison of dual-coded optical field imaging (left) and traditional Gaussian beam imaging (right) in this invention, unit: micrometers;

[0042] Figure 7This is a preferred module diagram of the multi-dimensional encoded long focal length deep light field two-photon microscopy imaging system of the present invention. Detailed Implementation

[0043] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0044] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0045] Example 1

[0046] According to an embodiment of the present invention, a multi-dimensional encoded long focal depth light field two-photon microscopy imaging method is provided, see [link to relevant documentation]. Figure 2 This includes the following steps:

[0047] S101: Design a multi-dimensional encoded light field, which is configured to achieve flexible adjustment of light field intensity and position distribution based on multi-focal spatial multiplexing;

[0048] S102: Insert a light field distribution shaping module into a traditional two-photon imaging system to change the position of the phase at the back aperture of the objective lens;

[0049] S103: Based on multidimensional coded light field scanning imaging, two two-dimensional scans are performed on the sample to obtain two two-dimensional projection images, which are then determined by combining the lateral spacing distribution of the projection points and the fluorescence intensity ratio.

[0050] The multi-dimensional encoded long-focal-depth light field two-photon microscopy imaging method in this invention can simultaneously encode the sample distribution into the intensity and positional distribution information of the projection image during the scanning imaging process. By resolving and locating the three-dimensional distribution of the sample, efficient and rapid three-dimensional imaging is achieved. This invention overcomes the limitations of existing technologies for sparsely labeled samples, developing a rapid three-dimensional two-photon microscopy imaging technique suitable for densely distributed samples without sacrificing resolution or missing important biological processes. This improves the accuracy of sample depth resolution and accelerates the three-dimensional imaging of samples.

[0051] This invention proposes a multi-dimensional encoded long focal depth light field two-photon microscopy imaging method, which can simultaneously encode the sample distribution into the intensity and position distribution multi-dimensional information of the projection image during the scanning imaging process. By analyzing and locating the three-dimensional distribution of the sample, efficient three-dimensional rapid imaging can be achieved.

[0052] This invention overcomes the limitations of existing technologies for sparsely labeled samples, and develops a rapid three-dimensional two-photon microscopy technique suitable for densely distributed samples without sacrificing resolution or missing important biological processes. This improves the accuracy of sample depth resolution and accelerates three-dimensional imaging of samples.

[0053] The basic contents of the technical solution of this invention include:

[0054] 1. Design of multi-dimensional encoded light field: Based on the spatial multiplexing of multiple focal points, the light field intensity and position distribution can be flexibly adjusted.

[0055] 2. Changes in Imaging Device: This invention achieves the generation of a pair of "tilted symmetrically distributed gradient focusing light fields" by inserting a light field distribution shaping module into a traditional two-photon imaging system (see...). Figure 3 The core of the light field distribution shaping module is a spatial light modulator and a one-dimensional galvanometer.

[0056] 3. Three-dimensional imaging process and sample depth analysis: Based on multi-dimensional coded light field scanning imaging, rapid movement of the sample or objective lens is no longer required; only two two-dimensional scans of the sample are needed. Based on the projection matching of the two two-dimensional projection images, combined with the changes in intensity ratio and lateral spacing with depth, the sample depth is analyzed.

[0057] The technical solution of the present invention is described in detail below:

[0058] 1. Design of multidimensional encoded light field

[0059] The principle of light field generation can be summarized as a series of spatially multiplexed multifocal points with certain intervals (see...). Figure 3The distance between adjacent focal points should be greater than half the Rayleigh length but less than one Rayleigh length. The depth of focus of the optical field can be flexibly adjusted by changing the number of focal points; the sidelobes of the optical field can be eliminated or reduced by superimposing tiny phase shifts at each focal point, based on the mutual interference between focal points. Encoding the optical field intensity distribution with depth is achieved by superimposing distinct binary matrices with different proportions of parameter 1 at each focal point; the parameters of the binary matrices are 0 or 1. Encoding the lateral distribution of the optical field with depth is achieved by adjusting the lateral misalignment between the center of the optical field wavefront and the geometric center of the objective lens back aperture.

[0060] 2. Changes in imaging devices

[0061] The light field distribution shaping module inserted into the system, such as Figure 4 As shown: Since the spatial light modulator is conjugate with galvanometers Galvo X and Galvo Y, and the objective lens back aperture, after passing through a series of relay lenses, the sum of the single-focal phases mentioned above can be applied to the spatial light modulator to adjust the light field intensity distribution. Galvo 1 and the spatial light modulator are located on the front and rear focal planes of the same lens, respectively. Therefore, changing the angle of Galvo 1 will cause the light spot to move on galvanometer Galvo X, thereby changing the phase position at the objective lens back aperture. Thus, in the experiment, the tilting of the long focal depth light field can be achieved by simply switching the voltage of galvanometer Galvo 1, realizing the generation of a dual-coded excitation light field.

[0062] 3. Three-dimensional imaging process and sample depth analysis

[0063] Multidimensional coded light field scanning imaging requires only two two-dimensional scans of the sample to obtain two two-dimensional projection images. The sample depth resolution process is as follows: Figure 5 As shown:

[0064] a) First, set an intensity threshold to extract the effective projection area, number each projection, and extract the lateral position distribution and fluorescence intensity value of the projection respectively.

[0065] b) Since the position of the same fluorescent sample differs only in the x-direction in the two two-dimensional projection images, and their y-coordinates are approximately the same, all possible projection pairs are matched based on this criterion.

[0066] c) For all possible combinations of projection pairs, based on the formula... The fluorescence intensity ratio is calculated based on the formula D = | x L -x R |Calculate the lateral spacing distribution. Combine the changes in lateral spacing and intensity ratio of the multidimensional encoded light field with depth to determine the corresponding depth values, and verify the matching of two depth values. When the two values ​​have a small difference, this projection pair corresponds to the same sample, and the sample depth is the average of the two values. For projections that fail to pair, there may be cases where projection overlap leads to incorrect intensity acquisition. In this case, first estimate the depth value based on the lateral spacing, and then infer the fluorescence intensity of the projection based on the estimated depth value. Further, by comparing the intensity values ​​of one or more projection combinations, determine the optimal projection pair combination and complete the sample depth resolution.

[0067] The key points and areas to be protected in this invention are:

[0068] 1. This invention designs a multi-dimensional encoded long focal depth light field whose focal depth length, intensity distribution, and tilt angle can be flexibly adjusted simultaneously, and is applicable to other types of imaging methods, such as photoacoustic imaging and OCT.

[0069] 2. This invention proposes a sample depth analysis algorithm that combines multidimensional information from projection images. The supplementary verification of multidimensional information can overcome the limitations of existing imaging methods on sparse labeled samples, providing a research method with a wider range of applications for the field of neuroscience.

[0070] Compared with the prior art, the advantages of the present invention are:

[0071] 1. In terms of imaging speed, the two-photon microscopy imaging technology of this invention has a fast imaging speed, which is generally 20-30 times faster than that of a conventional microscope;

[0072] 2. In terms of imaging resolution, the light field developed in this invention has a designable intensity distribution, is less affected by sidelobe interference, and has high spatial resolution, making it suitable for three-dimensional imaging of microbial samples.

[0073] 3. In terms of depth resolution accuracy, this technique combines the lateral spacing distribution of projection points and the fluorescence intensity ratio to jointly determine the axial position of the fluorescent target. Compared to relying on a single indicator, which is susceptible to interference from signal noise, optical distortion, or sample structural complexity, this technique, by combining the lateral spacing distribution and fluorescence intensity ratio to introduce a dual verification module of depth information, forms a multi-dimensional cross-validation system, which has significant advantages in handling depth resolution of complex biological samples.

[0074] This invention has been tested and used, such as Figure 6 As shown, compared with traditional Gaussian two-photon microscopy based on layer-by-layer scanning for 3D imaging, this technique can acquire depth information of samples with near-accuracy, and the 3D imaging speed is improved by nearly 20 times. Furthermore, this technique can effectively reconstruct submicron-level dendritic spine structures, making it suitable for recording the activity of tiny structures such as neural synapses.

[0075] The present invention achieves light fields with multiple indices that vary with depth through other means, and then relies on multiple indices to analyze the three-dimensional distribution of samples, all of which fall within the scope of the present invention.

[0076] Example 2

[0077] According to an embodiment of the present invention, a multi-dimensional encoded long focal depth light field two-photon microscopy imaging system is provided, see [link to relevant documentation]. Figure 7 ,include:

[0078] The light field design module 201 is used to design a multi-dimensional encoded light field, which is configured to achieve flexible adjustment of light field intensity and position distribution based on multi-focal spatial multiplexing.

[0079] The imaging modification module 202 is used to insert a light field distribution shaping module into a traditional two-photon imaging system to change the position of the phase at the back aperture of the objective lens.

[0080] The sample depth analysis module 203 is used to perform two two-dimensional scans on the sample based on multi-dimensional coded light field scanning imaging to obtain two two-dimensional projection images, which are then combined with the lateral spacing distribution of the projection points and the fluorescence intensity ratio to determine the depth.

[0081] The multi-dimensional encoded long-focal-depth light field two-photon microscopy imaging system in this embodiment of the invention can simultaneously encode the sample distribution into the intensity and positional distribution information of the projection image during the scanning imaging process. By resolving and locating the three-dimensional distribution of the sample, efficient and rapid three-dimensional imaging is achieved. This invention overcomes the limitations of existing technologies for sparsely labeled samples, developing a rapid three-dimensional two-photon microscopy imaging technology suitable for densely distributed samples without sacrificing resolution or missing important biological processes. This improves the accuracy of sample depth resolution and accelerates the three-dimensional imaging of samples.

[0082] This invention proposes a multi-dimensional encoded long focal depth light field two-photon microscopy imaging system, which can simultaneously encode the sample distribution into the intensity and position distribution multi-dimensional information of the projection image during the scanning imaging process. By analyzing and locating the three-dimensional distribution of the sample, efficient three-dimensional rapid imaging can be achieved.

[0083] This invention overcomes the limitations of existing technologies for sparsely labeled samples, and develops a rapid three-dimensional two-photon microscopy technique suitable for densely distributed samples without sacrificing resolution or missing important biological processes. This improves the accuracy of sample depth resolution and accelerates three-dimensional imaging of samples.

[0084] The basic contents of the technical solution of this invention include:

[0085] 1. Design of multi-dimensional encoded light field: Based on the spatial multiplexing of multiple focal points, the light field intensity and position distribution can be flexibly adjusted.

[0086] 2. Changes in Imaging Device: This invention achieves the generation of a pair of "tilted symmetrically distributed gradient focusing light fields" by inserting a light field distribution shaping module into a traditional two-photon imaging system (see...). Figure 3 The core of the light field distribution shaping module is a spatial light modulator and a one-dimensional galvanometer.

[0087] 3. Three-dimensional imaging process and sample depth analysis: Based on multi-dimensional coded light field scanning imaging, rapid movement of the sample or objective lens is no longer required; only two two-dimensional scans of the sample are needed. Based on the projection matching of the two two-dimensional projection images, combined with the changes in intensity ratio and lateral spacing with depth, the sample depth is analyzed.

[0088] The technical solution of the present invention is described in detail below:

[0089] 1. Design of multidimensional encoded light field

[0090] The principle of light field generation can be summarized as a series of spatially multiplexed multifocal points with certain intervals (see...). Figure 3 The distance between adjacent focal points should be greater than half the Rayleigh length but less than one Rayleigh length. The depth of focus of the optical field can be flexibly adjusted by changing the number of focal points; the sidelobes of the optical field can be eliminated or reduced by superimposing tiny phase shifts at each focal point, based on the mutual interference between focal points. Encoding the optical field intensity distribution with depth is achieved by superimposing distinct binary matrices with different proportions of parameter 1 at each focal point; the parameters of the binary matrices are 0 or 1. Encoding the lateral distribution of the optical field with depth is achieved by adjusting the lateral misalignment between the center of the optical field wavefront and the geometric center of the objective lens back aperture.

[0091] 2. Changes in imaging devices

[0092] The light field distribution shaping module inserted into the system, such as Figure 4 As shown: Since the spatial light modulator is conjugate with galvanometers Galvo X and Galvo Y, and the objective lens back aperture, after passing through a series of relay lenses, the sum of the single-focal phases mentioned above can be applied to the spatial light modulator to adjust the light field intensity distribution. Galvo 1 and the spatial light modulator are located on the front and rear focal planes of the same lens, respectively. Therefore, changing the angle of Galvo 1 will cause the light spot to move on galvanometer Galvo X, thereby changing the phase position at the objective lens back aperture. Thus, in the experiment, the tilting of the long focal depth light field can be achieved by simply switching the voltage of galvanometer Galvo 1, realizing the generation of a dual-coded excitation light field.

[0093] 3. Three-dimensional imaging process and sample depth analysis

[0094] Multidimensional coded light field scanning imaging requires only two two-dimensional scans of the sample to obtain two two-dimensional projection images. The sample depth resolution process is as follows: Figure 5 As shown:

[0095] a) First, set an intensity threshold to extract the effective projection area, number each projection, and extract the lateral position distribution and fluorescence intensity value of the projection respectively.

[0096] b) Since the position of the same fluorescent sample differs only in the x-direction in the two two-dimensional projection images, and their y-coordinates are approximately the same, all possible projection pairs are matched based on this criterion.

[0097] c) For all possible combinations of projection pairs, based on the formula... The fluorescence intensity ratio is calculated based on the formula D = | x L -x R | Calculate the lateral spacing distribution. Combine the changes in lateral spacing and intensity ratio of the multidimensional encoded light field with depth to determine the corresponding depth values, and verify the matching of two depth values. When the two values ​​have a small difference, this projection pair corresponds to the same sample, and the sample depth is the average of the two values. For projections that fail to pair, there may be cases where projection overlap leads to incorrect intensity acquisition. In this case, first estimate the depth value based on the lateral spacing, and then infer the fluorescence intensity of the projection based on the estimated depth value. Further, by comparing the intensity values ​​of one or more projection combinations, determine the optimal projection pair combination and complete the sample depth resolution.

[0098] The key points and areas to be protected in this invention are:

[0099] 1. This invention designs a multi-dimensional encoded long focal depth light field whose focal depth length, intensity distribution, and tilt angle can be flexibly adjusted simultaneously, and is applicable to other types of imaging methods, such as photoacoustic imaging and OCT.

[0100] 2. This invention proposes a sample depth analysis algorithm that combines multidimensional information from projection images. The supplementary verification of multidimensional information can overcome the limitations of existing imaging methods on sparse labeled samples, providing a research method with a wider range of applications for the field of neuroscience.

[0101] Compared with the prior art, the advantages of the present invention are:

[0102] 1. In terms of imaging speed, the two-photon microscopy imaging technology of this invention has a fast imaging speed, which is generally 20-30 times faster than that of a conventional microscope;

[0103] 2. In terms of imaging resolution, the light field developed in this invention has a designable intensity distribution, is less affected by sidelobe interference, and has high spatial resolution, making it suitable for three-dimensional imaging of microbial samples.

[0104] 3. In terms of depth resolution accuracy, this technique combines the lateral spacing distribution of projection points and the fluorescence intensity ratio to jointly determine the axial position of the fluorescent target. Compared to relying on a single indicator, which is susceptible to interference from signal noise, optical distortion, or sample structural complexity, this technique, by combining the lateral spacing distribution and fluorescence intensity ratio to introduce a dual verification module of depth information, forms a multi-dimensional cross-validation system, which has significant advantages in handling depth resolution of complex biological samples.

[0105] This invention has been tested and used, such as Figure 6 As shown, compared with traditional Gaussian two-photon microscopy based on layer-by-layer scanning for 3D imaging, this technique can acquire depth information of samples with near-accuracy, and the 3D imaging speed is improved by nearly 20 times. Furthermore, this technique can effectively reconstruct submicron-level dendritic spine structures, making it suitable for recording the activity of tiny structures such as neural synapses.

[0106] The present invention achieves light fields with multiple indices that vary with depth through other means, and then relies on multiple indices to analyze the three-dimensional distribution of samples, all of which fall within the scope of the present invention.

[0107] Example 3

[0108] A storage medium storing program files capable of implementing any of the above-mentioned multidimensional encoded long focal depth light field two-photon microscopy imaging methods.

[0109] Example 4

[0110] A processor for running a program, wherein the program executes, during runtime, any of the above-mentioned multidimensional coded long focal depth light field two-photon microscopy imaging methods.

[0111] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0112] In the above embodiments of the present invention, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0113] In the several embodiments provided in this application, it should be understood that the disclosed technical content can be implemented in other ways. The system embodiments described above are merely illustrative; for example, the division of units can be a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be through some interfaces; the indirect coupling or communication connection of units or modules may be electrical or other forms.

[0114] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0115] Furthermore, the functional units in the various embodiments of the present invention can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit.

[0116] If the integrated unit is implemented as a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, or all or part of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the steps of the methods of the various embodiments of the present invention. The aforementioned storage medium includes various media capable of storing program code, such as USB flash drives, read-only memory (ROM), random access memory (RAM), portable hard drives, magnetic disks, or optical disks.

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multi-dimensional encoded long focal depth light field two-photon microscopy imaging method, characterized in that, Includes the following steps: S101: Design a multi-dimensional encoded light field, which is configured to achieve flexible adjustment of light field intensity and position distribution based on multi-focal spatial multiplexing; S102: Insert a light field distribution shaping module into a traditional two-photon imaging system to change the position of the phase at the back aperture of the objective lens; S103: Based on multidimensional coded light field scanning imaging, two two-dimensional scans are performed on the sample to obtain two two-dimensional projection images, which are then determined by combining the lateral spacing distribution of the projection points and the fluorescence intensity ratio.

2. The multi-dimensional encoded long focal depth light field two-photon microscopy imaging method according to claim 1, characterized in that, In step S101, the depth of focus of the light field is flexibly adjusted by changing the number of focal points; the side lobes of the light field are eliminated or reduced by superimposing tiny phase shifts on each focal point based on the mutual interference between focal points.

3. The multi-dimensional encoded long focal depth light field two-photon microscopy imaging method according to claim 2, characterized in that, In step S101, the encoding of the light field intensity distribution with depth is achieved by superimposing mutually exclusive binarization matrices with different proportions of parameter 1 at each focal point, and the parameters of the binarization matrices are 0 or 1; the encoding of the light field lateral distribution with depth is achieved by adjusting the lateral misalignment between the center of the light field wavefront and the geometric center of the objective lens back aperture.

4. The multi-dimensional encoded long focal depth light field two-photon microscopy imaging method according to claim 1, characterized in that, In step S102, the light field distribution shaping module is configured as follows: After passing through a series of relay lenses, the spatial light modulator is conjugate with the Galvo X and Galvo Y galvanometers and the back aperture of the objective lens. The sum of the single-focus phases is loaded onto the spatial light modulator to achieve the adjustment of the light field intensity distribution.

5. The multi-dimensional encoded long focal depth light field two-photon microscopy imaging method according to claim 4, characterized in that, In step S102, the galvanometer Galvo 1 and the spatial light modulator are located on the front and rear focal planes of the same lens, respectively. Changing the angle of Galvo 1 causes the light spot to move on the galvanometer Galvo X, thereby changing the position of the phase at the back aperture of the objective lens.

6. The multi-dimensional encoded long focal depth light field two-photon microscopy imaging method according to claim 5, characterized in that, In step S102, the tilting of the long focal depth light field is achieved by simply switching the voltage of the Galvo 1 galvanometer, thereby generating the dual-encoded excitation light field.

7. The multi-dimensional encoded long focal depth light field two-photon microscopy imaging method according to claim 1, characterized in that, Step S103 specifically includes: a) Set an intensity threshold to extract the effective projection area, number each projection, and extract the lateral position distribution and fluorescence intensity value of each projection; b) Since the position of the same fluorescent sample in the two two-dimensional projection images differs only in the x-direction, and their y-coordinates are approximately the same, all possible projection pairs are matched based on this criterion. c) For all possible combinations of projection pairs, based on the formula... The fluorescence intensity ratio is calculated based on the formula D = | x L -x R | Calculate the lateral spacing distribution; The corresponding depth value is determined by combining the changes in the lateral spacing and intensity ratio of the multidimensional encoded light field with depth, and the two depth values ​​are matched and verified.

8. The multi-dimensional encoded long focal depth light field two-photon microscopy imaging method according to claim 7, characterized in that, In step S103, when the two depth values ​​have a small difference, this projection pair corresponds to the same sample, and the sample depth is the average of the two values.

9. The multi-dimensional encoded long focal depth optical field two-photon microscopy imaging method according to claim 7, characterized in that, For projections that fail to pair, the depth value is first estimated based on the lateral spacing, and the fluorescence intensity of the projection is inferred from the estimated depth value. Then, the optimal projection pair is determined by comparing the intensity values ​​of one or more projection combinations, and the depth resolution of the sample is completed.

10. A multi-dimensional encoded long focal depth light field two-photon microscopy imaging system, characterized in that, include: The light field design module is used to design multidimensional encoded light fields. The multidimensional encoded light fields are configured to achieve flexible adjustment of light field intensity and position distribution based on multi-focal spatial multiplexing. An imaging modification module is used to insert a light field distribution shaping module into a traditional two-photon imaging system to change the position of the phase at the back aperture of the objective lens. The sample depth analysis module is used to perform two two-dimensional scans on the sample based on multi-dimensional coded light field scanning imaging to obtain two two-dimensional projection images, which are then combined with the lateral spacing distribution of the projection points and the fluorescence intensity ratio to determine the depth.

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