Nondestructive positioning and imaging method based on double-helix point spread function

Abstract

A nondestructive positioning and imaging method based on a double-helix point spread function (DH-PSF) comprises: utilizing the DH-PSF to phase modulate a target beam transmitted through a scattering medium; performing scanning detection by varying the target's axial defocus, thereby acquiring a plurality of modulated images; evaluating the DH-PSF's positioning capability in a scattering environment using a signal-to-background ratio; calculating the target's three-dimensional position based on the series of modulated images; and performing a deconvolution optical sectioning algorithm on the series of modulated images using a system focal plane (DH-PSF) to obtain a clear structure of the target and the scattering medium, thereby reconstructing the target's three-dimensional distribution within the scattering medium. This method utilizes the DH-PSF's positioning capability in scattering environments and designs a corresponding algorithm based on its high recognition capability to reconstruct a three-dimensional image of the target within the scattering medium, achieving high resolution and a large penetration depth in situ and even within living tissue.

Description

Technical Field

[0001] The present invention belongs to the technical field of imaging through scattering media, and in particular relates to a three-dimensional positioning and imaging method through scattering media based on a double-helix point spread function. Background Art

[0002] In recent years, optical imaging has been widely used in biomedical imaging due to its high resolution and rich information content. However, the propagation of light in biological tissue is disturbed by scattering, resulting in very limited imaging penetration depth and resolution. To overcome the problems caused by scattering, many methods and techniques have been proposed, such as wavefront shaping and transfer matrix, deconvolution imaging technology, and speckle autocorrelation imaging. These methods can restore the target image through scattering media by utilizing ballistic light or speckle spread functions, but these methods and techniques are not suitable for super-resolution microscopy through or in biological tissue, especially non-invasive microscopy.

[0003] Fluorescence microscopy, using sparsely excited fluorescent particles and corresponding algorithms, can achieve nanometer-scale resolution, but this is typically limited to discrete cells rather than in situ environments. Current microscopes have limited penetration depth and resolution, making it impossible to observe subcellular structures and molecular arrangements beneath cells or within scattering biological tissues. Therefore, methods for high-precision three-dimensional localization and super-resolution imaging through or within scattering media are highly desirable. Summary of the Invention

[0004] Given that there are few technologies for three-dimensional imaging through scattering media and that axial positioning accuracy needs to be improved, the present invention proposes a method for three-dimensional positioning and imaging through scattering media based on a double-helix point spread function. By utilizing the super-resolution microscopy technology of the double-helix point spread function, which has excellent positioning capabilities in a scattering-free environment, a fluorescence microscopy system based on the double-helix point spread function is proposed to achieve three-dimensional positioning and imaging through biological tissue layers.

[0005] The technical solution of the present invention:

[0006] A three-dimensional positioning method through a scattering medium based on a double helix point spread function comprises the following steps:

[0007] The double helix point spread function is used to phase modulate the light beam emitted from the target through the scattering medium;

[0008] Changing the axial defocus of the target, performing scanning detection, and acquiring a number of modulated images;

[0009] The signal-to-background ratio is used to evaluate the localization capability of the double-helix point spread function in a scattering environment.

[0010] Calculating a three-dimensional position of a target according to the plurality of modulated images;

[0011] Selecting the focal plane DH-PSF of the system to perform deconvolution on the modulated images, obtaining a clear structure of the target and the scattering medium, and reconstructing the three-dimensional distribution of the target in the scattering medium;

[0012] The scattering medium may be frosted glass or biological tissue, such as egg shell membrane, onion epidermal tissue, etc.

[0013] The method of changing the target axial defocus amount, performing scanning detection, and acquiring a plurality of modulated images includes: changing the target axial defocus amount by moving a detector, a sample, or a microscope objective lens.

[0014] The signal-to-background ratio is used to evaluate the localization capability of the double helix point spread function in a scattering environment, including:

[0015] Distinguishing signal from background, i.e., ballistic component and scattered light component. Reading the plurality of modulated images, dividing the dual-spot matrix area into signal and the surrounding speckle into background;

[0016] The signal-to-background ratios (SBRs) of the plurality of modulated images are calculated.

[0017] The three-dimensional positioning and imaging method based on the double-helix point spread function through a scattering medium is characterized in that the three-dimensional position of the target is calculated based on the plurality of modulated images, comprising the following steps:

[0018] Place the calibration target in the system and load a double-helix point spread function phase plate for modulation.

[0019] The double Gaussian fitting algorithm is used to calculate, determine the centers of the two light spots to obtain the target lateral position, determine the azimuth of the line connecting the two light spots, and record the azimuth and axial depth of the line connecting the two light spots.

[0020] By performing multiple measurements, a relationship curve between azimuth angle and axial depth is obtained;

[0021] According to the predetermined relationship curve between the rotation angle and the axial defocus, the double Gaussian positioning algorithm is used to process the modulated images to determine the three-dimensional position of the target at different depths.

[0022] Selecting the focal plane DH-PSF of the system to deconvolve the series of modulated images to obtain a clear structure of the target and the scattering medium includes the following steps:

[0023] From the calibration data, the double helix point spread function when the axial depth is equal to 0 is obtained, that is, the focal plane DH-PSF of the system;

[0024] Deconvolution is performed on the modulated images using the focal plane DH-PSF of the system as the deconvolution kernel. This approach blurs the out-of-focus cross-sectional information, enhances the in-focus cross-sectional information, and clearly restores the scattering medium image of each cross-sectional plane.

[0025] The double-spot area in the plurality of modulated images is selected as a convolution kernel, and the selected area is deconvolved to obtain a clear image of the target.

[0026] Compared to existing methods, this invention applies the double-helix point spread function (DH-PSF) to scattering environments and proposes a signal-to-background ratio to measure the reliability of positioning accuracy. After calibration, the system leverages the positioning capabilities of the DH-PSF to achieve nanometer-level lateral positioning accuracy and tens of nanometers axial positioning accuracy. In conjunction with a translation stage, slices of samples of a certain thickness are scanned. Based on the high recognition characteristics of the double-helix point spread function (DH-PSF), a corresponding algorithm is designed to clearly reconstruct the structure of the scattering medium and the three-dimensional distribution of the target within it. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of a three-dimensional positioning and imaging structure through a scattering medium based on a double-helix point spread function according to an embodiment of the present invention;

[0028] Figure 2 is a flow chart of three-dimensional positioning and imaging through a scattering medium in an embodiment of the present invention;

[0029] Figure 3 This is the experimental result diagram of Example 1, showing the distribution of 8 fluorescent microspheres in onion epidermal tissue and the three-dimensional reconstruction of the tissue.

[0030] In the figure: 1: laser, 2: filter, 3: lens, 4: dichroic mirror, 5: mirror, 6: microscope objective, 7: sample, 8: piezoelectric nano-stage, 9: filter, 10: lens, 11: polarizer, 12: mirror, 13: lens, 14: right-angle mirror, 15: spatial light modulator, 16: lens, 17: CCD. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and effect of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and detailed implementation methods and specific operating processes. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept and implementation system of the present invention, and these all fall within the scope of protection of the present invention. This embodiment is only used to explain the present invention, but is not intended to limit the present invention.

[0032] Example 1:

[0033] A three-dimensional positioning and imaging method through a scattering medium based on a double helix point spread function comprises the following steps:

[0034] S01 uses a double-helix point spread function to phase modulate the light beam emitted by a target through a scattering medium. The specific steps are as follows:

[0035] An optical module loaded with a double-helix point spread function is created. In this embodiment, lenses 10 and 13 with a focal length of 100 mm are selected to form a 4f system. A pure phase spatial light modulator 15 is selected and placed on the middle plane of the 4f system. An optimized double-helix point spread function phase plate is loaded on the spatial light modulator 15.

[0036] Sample preparation: A layer of fresh onion epidermal tissue was used as the scattering medium. The surface of the onion epidermal tissue is uneven, providing axial depth variation. Fluorescent microspheres (1 μm in diameter) diluted 5000 times were then brushed onto the back of the onion epidermal tissue. The tissue was then placed on a glass slide and covered with a coverslip. The coverslip was gently pressed to expel air.

[0037] See also Figure 1 Sample 7 is placed on piezoelectric nanostage 8, and the object distance can be changed by moving piezoelectric nanostage 8. The excited fluorescence is magnified by microscope objective 6 and lens 10 and imaged onto the front focal plane of lens 13. It is then modulated by the double-helix phase plate loaded on spatial light modulator 15 and imaged onto CCD 17.

[0038] S02 changes the target axial defocus, performs slice scanning, and obtains several modulated images. The specific steps are as follows:

[0039] The test sample 7 is fixed on the piezoelectric nano-displacement stage 8. The piezoelectric nano-displacement stage 8 is fine-tuned to focus the target light beam onto the upper surface of the onion tissue. The spatial light modulator 15 is then turned on and loaded with a double-helix point spread function phase plate.

[0040] The distance between the sample and the detection objective lens is changed by moving the translation stage or sample stage along the z-axis, and the slice scanning is performed at intervals of 0.5 m. The camera synchronously collects the fluorescent signal in the double helix form, and a total of 240 image data are obtained;

[0041] S03 uses the signal-to-background ratio to evaluate the positioning capability of the double helix point spread function in a scattering environment, including:

[0042] The double-Gaussian positioning algorithm primarily relies on dual-spot intensity for positioning. The modulated signal, after passing through a scattering medium, produces a chaotic speckle pattern. Only when the intensity of the double-Gaussian spot is significantly greater than the surrounding speckle and background can positioning errors be minimized. This paper proposes using the signal-to-background ratio (SBR), the ratio of the intensity of the ballistic light to the scattered light, to measure the confidence of positioning accuracy. Calculation of the SBR of the modulated image shows that when the SBR is greater than 1, the xyz positioning error is within 1%, ensuring the confidence of positioning accuracy. Calculations show that the SBR of the modulated image of fluorescent microspheres transmitted through onion epidermal tissue is greater than 1, ensuring data confidence.

[0043] S04 calculates the three-dimensional position of the target according to the plurality of modulated images, including:

[0044] The calibration sample (i.e., fluorescent microspheres) is placed in the system and loaded with a double-helix point spread function phase plate for modulation. A double Gaussian fitting algorithm is used for calculation to determine the center of the two light spots to obtain the target lateral position, determine the azimuth of the line connecting the two light spots, and record the azimuth and axial depth of the line connecting the two light spots. By performing multiple measurements, a relationship curve between the azimuth and axial depth is obtained. Based on the predetermined relationship curve between the rotation angle and the axial depth, a double Gaussian positioning algorithm is used to process the modulated images of the fluorescent microspheres that have passed through the onion epidermal tissue to determine the three-dimensional position of the fluorescent microspheres at different depths. In positioning, the position of the microsphere is extracted by fitting two points.

[0045] S05 selects the focal plane DH-PSF of the system to perform deconvolution on the plurality of modulated images, obtains a clear structure of the target and the scattering medium, and reconstructs the three-dimensional distribution of the target in the scattering medium, including:

[0046] From the calibration data, the double helix point spread function when the axial depth is equal to 0 is obtained, that is, the focal plane DH-PSF of the system;

[0047] The system's focal plane DH-PSF is used as the deconvolution kernel to deconvolve the modulated images of the fluorescent microspheres that penetrated the onion epidermal tissue. Because when the piezoelectric nanostage scans, only the focused section at each position has a double-helix point spread function with an azimuth angle of 0 degrees. In the defocused section, the double spot has rotated. The collected pattern is a superposition of all sections. Using the system's focal plane DH-PSF for deconvolution can blur the defocused section information and enhance the reconstruction of the focused section information, thereby clearly restoring the image of the onion epidermal tissue structure of each section, such as Figure 3 Please provide detailed explanation. Figure 3 d is the reconstructed three-dimensional distribution result of fluorescent microspheres in onion epidermal tissue, and the white dots are fluorescent microspheres. Figure 3a, 3b, 3c are the projections of the three-dimensional image in the xy, xz, and yz directions respectively, in which some fibrous structures of the onion epidermal tissue can be clearly seen.

[0048] In summary, this invention applies the double-helix point spread function (DH-PSF) to scattering environments and proposes a signal-to-background ratio to measure the reliability of positioning accuracy. After calibration, the system leverages the positioning capabilities of the DH-PSF to achieve nanometer-level lateral positioning accuracy and tens of nanometers axial positioning accuracy. Combined with a translation stage, this technology allows for slice scanning of samples of a certain thickness. Based on the high recognition characteristics of the double-helix point spread function (DH-PSF), a corresponding algorithm is designed to clearly reconstruct the structure of the scattering medium and the three-dimensional distribution of the target within it.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A non-destructive positioning and imaging method based on a double helix point spread function, characterized in that: include: The double helix point spread function is used to phase modulate the light beam emitted from the target through the scattering medium; Changing the axial defocus of the target, performing scanning detection, and acquiring a number of modulated images; The signal-to-background ratio is used to evaluate the localization capability of the double-helix point spread function in a scattering environment. performing image processing on the plurality of modulated images to obtain a three-dimensional position of a target; Selecting the focal plane DH-PSF of the system to perform optical sectioning of the modulated images using a deconvolution algorithm, obtaining a clear structure of the target and the scattering medium, and reconstructing the three-dimensional distribution of the target in the scattering medium; The scattering medium is biological tissue; The use of the signal-to-background ratio to evaluate the positioning capability of the double helix point spread function in a scattering environment includes: Reading the plurality of modulated images, dividing the dual-spot matrix area into signals and the surrounding speckles into background, i.e., ballistic light and scattered light; Calculating the signal-to-background ratio (SBR) of the plurality of modulated images, i.e., the ratio of the intensity values ​​of the ballistic light to the scattered light, and ensuring that the SBR is greater than 1; Calculating a three-dimensional position of a target according to the plurality of modulated images includes: The calibration sample is placed in the system and loaded with a double-helix point spread function phase plate for modulation; Use the double Gaussian fitting algorithm to calculate, determine the center of the two spots to obtain the target lateral position, determine the azimuth angle of the line connecting the two spots, and record the azimuth angle and axial defocus of the line connecting the two spots; By performing multiple measurements, a relationship curve between the azimuth angle and the axial defocus amount is obtained; According to a predetermined relationship curve between the rotation angle and the axial defocus amount, the plurality of modulated images are processed using a double Gaussian positioning method to determine the three-dimensional positions of the targets at different depths.

2. The non-destructive positioning and imaging method based on double helix point spread function according to claim 1, characterized in that: The biological tissue is egg shell membrane or onion epidermal tissue.

3. The non-destructive positioning and imaging method based on double helix point spread function according to claim 1, characterized in that: The method of changing the target axial defocus amount, performing scanning detection, and acquiring a plurality of modulated images includes: changing the target axial defocus amount by moving a detector, a sample, or a microscope objective lens.

4. The non-destructive positioning and imaging method based on double helix point spread function according to claim 1, characterized in that: Select the focal plane DH-PSF of the system to deconvolve the series of modulated images to obtain the clear structure of the target and the scattering medium, including: From the calibration data, the double helix point spread function when the axial defocus is equal to 0 is obtained, that is, the focal plane DH-PSF of the system; Using the focal plane DH-PSF of the system as a deconvolution kernel, deconvolving the plurality of modulated images to restore the scattering medium image of each section; The double-spot area in the plurality of modulated images is selected as a convolution kernel, and the selected area is deconvolved to obtain a clear image of the target.

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