Isotropic three-dimensional super-resolution imaging method of single objective lens

Abstract

The invention relates to a single-objective isotropic three-dimensional super-resolution imaging method, which comprises the following steps of: outputting a linear polarization excitation light beam through a picosecond pulse laser, and outputting a linear polarization loss light beam through a nanosecond pulse laser, adjusting the polarization direction and power of the excitation light beam and the loss light beam by using a half-wave plate and a polarization beam splitter, enabling the loss light beam to enter a spatial light modulator, modulating a light field by loading a preset phase diagram, generating three focuses with different topological charges, axial positions and energy distribution, and outputting the three focuses. The high-order vortex phase retarder is used for carrying out polarization state modulation on the focus and separating loss light beams with different polarization components, the imaging resolution ratio smaller than 100 nm in the transverse direction and the axial direction is achieved, the transverse resolution ratio is similar to the axial resolution ratio, the three-dimensional real structure of an object can be accurately reflected when three-dimensional super-resolution imaging is carried out, and the imaging precision is improved. The imaging resolution is in direct proportion to the loss light intensity, and the higher the loss light intensity is, the higher the three-dimensional imaging resolution is.

Description

Technical Field

[0001] The present invention relates to the field of microscopic imaging, and in particular to an isotropic three-dimensional super-resolution imaging method using a single objective lens. Background Art

[0002] Limited by the optical diffraction limit, the maximum imaging resolution of conventional optical microscopes is only half a wavelength of light. This inadequate optical imaging resolution severely restricts researchers' understanding of relevant mechanisms in the biomedical field. In recent decades, with the development of fluorescent probes and advanced imaging methods, super-resolution microscopy, such as STED and STORM, has become a vital tool in basic biological research. Thanks to its ultra-high lateral resolution (approximately 20-40 nm), super-resolution imaging techniques enable two-dimensional visualization of cellular structures at the nanoscale, providing an indispensable tool for cellular and molecular research. However, while the lateral resolution of optical imaging has surpassed the diffraction limit, the axial resolution of optical imaging systems with a single objective lens configuration remains constrained by the optical diffraction limit. Dual-objective illumination-based iso-STED microscopy offers an effective approach to addressing this mismatch between lateral and axial resolution. This technique combines a 4Pi dual-objective illumination configuration with stimulated emission depletion (STED) technology to generate a hollow spherical PSF in the focal plane through interference enhancement between two opposing objective lenses. By precisely controlling the intensity of the erasing light, isotropic super-resolution imaging with a lateral and axial resolution of 50 nm can be achieved. However, the iso-STED microscope based on dual-objective illumination imaging requires that the two beams of illumination light from the dual objective lenses interfere in the focal area, which places strict requirements on the transparency and thickness of the sample (usually less than a few microns). Therefore, the iso-STED microscopy technology based on dual-objective illumination imaging is difficult to apply to the imaging of living samples, limiting its feasibility in conventional biological imaging. Therefore, in order to expand the compatibility of the technology in diverse applications, such as imaging of living samples and imaging at great depths, researchers have developed a three-dimensional STED microscope based on single-objective illumination. Specifically, by using a traditional lateral loss beam (STED xy ) based on the introduction of an additional axial loss beam (STED z ), effectively improving the lateral and axial resolution. This method minimizes the STED by introducing phase delay. xy and STED z interference between them, allowing them to operate independently. zThis significantly improves axial resolution. However, because a single objective can only collect a hemispherical wavefront, this design inevitably leads to an extension of the axial PSF. In other words, the lateral and axial resolutions of existing optical super-resolution imaging techniques based on single-objective illumination do not match. This resolution mismatch fundamentally limits the accuracy of these techniques in three-dimensional (3D) structural analysis, resulting in distortion of the sample's true 3D structure.

[0003] In response to the technical bottleneck that the existing technology cannot achieve isotropic three-dimensional super-resolution imaging under a single objective lens architecture, the present invention proposes an isotropic three-dimensional super-resolution imaging method based on single objective lens illumination. Specifically, the single objective lens illumination STED microscope must meet two key conditions to achieve three-dimensional isotropic super-resolution. First, multiple focal points are generated in the axial direction, and their orbital angular momentum, light intensity distribution and three-dimensional position are precisely controlled to achieve three-dimensional erasing light. Second, interference between these focal points is prevented to maintain the isotropy of three-dimensional resolution. In order to meet the above conditions, a special vector beam with different phase responses is used as the erasing light in the STED erasing light path. By phase-controlling polarization, the intrinsic polarization mode is extracted from the special vector erasing beam within the focal area of ​​the illumination objective lens, and the left-handed circular polarization is assigned to the lateral loss beam and the right-handed circular polarization is assigned to the axial loss beam, thereby effectively avoiding the light field distortion caused by interference between the two. By precisely controlling the position, light intensity and energy distribution of the focus, this method can construct a uniform three-dimensional loss field that meets the requirements of isotropic three-dimensional super-resolution. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of existing technologies and meet current needs by providing a single-objective-lens isotropic 3D super-resolution imaging method. This method achieves sub-100nm 3D isotropic resolution using a single lossy laser. This technique, based on a conventional 2D STED system, eliminates the need for additional objective lenses or lossy light sources. By simply modulating the lossy beam once, the inherent anisotropic resolution limitations of a single-objective-lens configuration can be overcome. This method enables 3D imaging of the true structure of cellular subcellular organelles, exploring the relationship between their 3D morphology and relevant disease mechanisms, and addressing current technical challenges.

[0005] In order to achieve the purpose of the present invention, the technical solution adopted by the present invention is to design an isotropic three-dimensional super-resolution imaging method using a single objective lens, comprising the following steps:

[0006] S1. Output a linearly polarized excitation beam through a picosecond pulse laser, and output a linearly polarized loss beam through a nanosecond pulse laser. Use a half-wave plate and a polarization beam splitter to adjust the polarization direction and power of the excitation beam and the loss beam. The loss beam is incident on a spatial light modulator, and the light field is modulated by loading a preset phase diagram to generate three focal points with orthogonal polarization, different topological charges, axial positions and energy distribution. The intermediate focus along the axial direction mainly contributes to STED. xy , used to improve the lateral resolution, the two focal points at both ends of the axis constitute STED z For improving axial resolution, the focus is polarization-modulated by a high-order vortex retarder to separate loss beams of different polarization components;

[0007] S2. The Jones matrix of the high-order vortex delay plate is:

[0008]

[0009] in, represents a universal function of phase, m is the order of the high-order vortex delay plate; and β0 determine the polarization distribution of the light beam emerging from the polarization converter.

[0010] S3, the phase of the spatial light modulator is;

[0011]

[0012] Among them, s j is the amplitude weight factor of the jth focus; the value range of j is 1 to N, where N = 3 is the number of focuses; k = 2π / λ, λ is the wavelength of the incident light; θ, are the convergence angle and azimuth angle of the objective lens respectively; (r j , z j ,φ j ) The cylindrical coordinate position parameter of the jth focus within the focal area of ​​the illumination objective lens, r j is the radius of the jth focus in the horizontal position, φ j is the horizontal position angle of the jth focus; z j is the axial position of the jth focus; ψ j is the parameter that controls the polarization state of the jth focus. Phase is the phase function.

[0013] S4, the linearly polarized loss beam output by the nanosecond pulse laser is phase-controlled by the spatial light modulator and polarized by the high-order vortex delay plate, and three focal points with orthogonal polarization states, different axial positions, topological charges and energy distribution are generated in the focal area of ​​the illumination objective lens, among which the intermediate focus along the axial direction mainly contributes to the STED xy, used to improve the lateral resolution; the two focal points at both ends of the axis constitute STED z Used to improve axial resolution. The three focal points together form an isotropic lossy light field.

[0014] S5, the intermediate focus along the axial direction corresponding to the isotropic lossy light field (STED xy ) and two focal points along the axis (STED z There are two combinations of polarization states:

[0015] The first type is left-handed and right-handed circularly polarized light, in which the topological charge of the middle focus is ±1, and the topological charge of the two focuses at both ends along the axis is 0; the second type is angularly polarized light and radially polarized light, in which the middle focus is angularly polarized light, and the two focuses at both ends along the axis are radially polarized light.

[0016] S6. The isotropic lossy light field needs to pass through s j To adjust STED xy and STED z energy distribution to achieve a three-dimensional isotropic resolution, where 0≤s j ≤1, STED xy With STED z The distance between them is adjusted by z j Further optimize the isotropic erasing properties of lossy light.

[0017] S7, spatially combining the modulated loss beam and the excitation beam through a dichroic mirror, performing synchronous transverse and axial scanning on the combined beam using an X-axis galvanometer and a Y-axis galvanometer, and conjugating the light field to the objective lens entrance pupil through a scanning lens and a tube lens;

[0018] S8, focusing the combined beam onto the sample through the objective lens to stimulate the fluorescence signal, then adjusting the signal delay device to make the pulses of the excitation beam and the loss beam coincide in time domain, realizing the stimulated emission depletion effect, collecting the fluorescence signal emitted by the sample, filtering it through the filter and pinhole, and converting it into an electrical signal by the photodetector;

[0019] S9. The electrical signal is collected and reconstructed through a data acquisition card, and transmitted to a computer for three-dimensional super-resolution image processing to obtain a three-dimensional imaging result with an isotropic resolution of less than 100 nm.

[0020] Preferably, the output pulse width of the picosecond pulse laser is 10-200 picoseconds, and the output pulse width of the nanosecond pulse laser is on the order of 1 nanosecond.

[0021] Preferably, the high-order vortex phase retarder achieves spatial separation of different polarization components and suppresses inter-focal interference by regulating the spin angular momentum and orbital angular momentum of the light field.

[0022] Preferably, the high-order vortex delay plate is configured to control the phase and ψ j The parameters of the focal polarization state achieve orthogonality of the polarization states among multiple axial foci, minimizing the mutual interference terms during fluorescence excitation, thereby improving the consistency and stability of three-dimensional resolution.

[0023] Preferably, the linear polarization state of the excitation beam is modulated into a left-handed circular polarization state by a quarter-wave plate, and the linear polarization state of the loss beam is modulated into a horizontal linear polarization state by a half-wave plate, thereby meeting the polarization requirements of the incident light of the spatial light modulator.

[0024] Preferably, the scanning paths of the X-axis galvanometer and the Y-axis galvanometer are controlled by a computer to achieve layer-by-layer three-dimensional scanning of the sample.

[0025] Preferably, the stimulated emission depletion effect is achieved by adjusting the time delay of the signal delayer so that the excitation light beam and the loss light beam completely overlap in space and time.

[0026] Preferably, the fluorescence signal is collected by the objective lens and then returns to the original path, and is reflected to the photodetector through the tube lens, the scanning lens, the galvanometer mirror and the dichroic mirror in sequence.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. Solved the problem that a single objective lens can only collect half of the spherical wavefront, resulting in poor axial resolution.

[0029] 2. The present invention achieves an imaging resolution of less than 100 nm in both the lateral and axial directions, and the lateral resolution performs similarly to the axial resolution. When performing three-dimensional super-resolution imaging, it can accurately reflect the three-dimensional real structure of the object. The imaging resolution is proportional to the loss light intensity. The stronger the loss light intensity, the higher the three-dimensional imaging resolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the single objective lens isotropic three-dimensional super-resolution imaging system of the present invention;

[0031] Figure 2 Schematic diagram of the loss spot formed by orthogonal polarization of the present invention; Figure 3 This is a schematic diagram of the three-dimensional isotropic super-resolution effect achieved by the present invention. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0033] A single objective lens isotropic three-dimensional super-resolution imaging method, see Figures 1 to 2, including an improved stimulated emission depletion super-resolution imaging system, the system includes:

[0034] Picosecond pulse laser, which outputs high-frequency pulsed linearly polarized laser with a pulse width of picoseconds, is used as the excitation beam;

[0035] Nanosecond pulse laser, which outputs high-frequency pulsed linearly polarized laser with a pulse width of nanoseconds, used as loss beam;

[0036] Half-wave plate, used to adjust the polarization direction of the laser;

[0037] A quarter-wave plate, used to modulate the linear polarization state of the excitation light source into a left-handed circular polarization state;

[0038] Polarization beam splitter, used for laser beam splitting, can be used with half-wave plate to adjust the laser output power;

[0039] A spatial light modulator is used to modulate the lossy beam emitted by the nanosecond laser, adjust the number of focal spots to 3, and modulate the topological charge, energy distribution, and relative spatial position of each focal spot;

[0040] Lens, two lenses are combined into a 4f system to conjugate the light field to the back focal plane of the second lens;

[0041] High-order vortex retarders are used to impart focal points of different topological directions to different orthogonal polarization states;

[0042] Reflector, which reflects the laser and changes the direction of light propagation;

[0043] A dichroic mirror is used to spatially combine different laser beams, specifically, to combine the loss beam and the excitation beam, or to reflect fluorescence;

[0044] X-axis galvanometer, used for horizontal scanning of the two laser beams;

[0045] The Y-axis galvanometer is used to scan the two laser beams longitudinally and works with the X-axis galvanometer to achieve surface imaging of the sample;

[0046] The scanning lens is placed at a position where its front focal plane is conjugate with the y-axis galvanometer;

[0047] The tube lens forms a 4f system with the scanning lens, conjugating the modulated light field to the entrance pupil of the objective lens;

[0048] Objective lens, used to focus the light beam and collect fluorescence;

[0049] Filter, used to reflect fluorescence signals and remove stray light other than fluorescence;

[0050] Pinhole, used to filter out-of-focus fluorescence signals and improve imaging contrast;

[0051] Photodetectors convert light signals into electrical signals and amplify the collected signals, typically using photomultiplier tubes or avalanche photodiodes;

[0052] Data acquisition card, used to output level signals for controlling galvanometer scanning and other motor components, and to collect and reconstruct electrical signals from photodetectors;

[0053] The signal delayer is used to increase or decrease the delay of the pulse synchronization signal output by the nanosecond laser and output the synchronization input signal to the picosecond pulse laser to ensure that the two beams coincide in the time domain;

[0054] Computer, used for user interaction with software and hardware, controlling image acquisition software, storing data and data processing, etc.

[0055] Example 1

[0056] Specific workflow of single objective lens isotropic 3D super-resolution imaging

[0057] S1. Lossy beam modulation and generation

[0058] Nanosecond laser outputs linearly polarized light with a pulse width of 1ns ( Figure 1 The black dashed line) is used to adjust the power and optimize the linear polarization ratio through a half-wave plate and polarization beam splitter combination. The half-wave plate is then rotated to adjust the polarization state of the incident light to horizontal linear polarization to maximize the modulation efficiency of the spatial light modulator (SLM). After the calculated phase map is loaded into the SLM, the light field is modulated into three high-order orbital angular momentum foci with topological charges of order m, -(m+1), and m, respectively. The topological charge, direction, axial position, and energy distribution are dynamically controlled, laying the foundation for subsequent polarization modulation.

[0059] S2, High-order focal polarization state conversion and beam combining

[0060] The three high-order focal points after modulation are conjugated to the m-order vortex retarder through a 4f system composed of two lenses, and are respectively given right circular polarization (RCP) and left circular polarization (LCP) states (such as Figure 2 a) or radial polarization (RP), azimuthal polarization (AP) state (such as Figure 2 b) The polarization-modulated loss beam is reflected by a dichroic mirror and spatially combined with the excitation beam path, entering the synchronous scanning phase;

[0061] S3. Excitation beam path optimization and beam combining

[0062] Picosecond laser outputs linearly polarized light with a pulse width of 10-200ps ( Figure 1 The power and polarization ratio are similarly adjusted by a half-wave plate and a polarization beam splitter. Subsequently, a half-wave plate (HWP) and a quarter-wave plate (QWP) are combined to convert the linearly polarized light into circularly polarized light. This beam passes through a dichroic mirror and is combined with the lossy beam before being conjugated to the X-axis galvanometer and Y-axis galvanometer via a 4f system, completing the synchronous preparation for transverse (XY) and axial (Z) scanning.

[0063] S4, 3D scanning and fluorescence excitation

[0064] The combined beam is conjugated to the objective lens entrance pupil through the scanning lens and the tube lens, and is focused by the objective lens to the stage to excite the fluorescent molecules. In this process, the signal delay device accurately controls the time delay between the excitation beam and the loss beam (erasing beam) to ensure that the two pulses completely overlap in space, triggering the stimulated emission depletion (STED) effect, thereby compressing the three-dimensional point spread function (PSF) of the fluorescence signal to the super-resolution scale (such as Figure 3 );

[0065] S5. Fluorescence signal collection and filtering

[0066] The fluorescence signal generated by excitation ( Figure 1 The black dotted line returns along the original path, passing through the objective lens, tube lens, scanning lens, and galvanometer mirror in sequence, and is reflected by the dichroic mirror to the detection path. After the signal light is focused by the lens, it passes through a combination of a filter and a pinhole to filter out stray light, and is finally captured by the photodetector;

[0067] S6. Signal conversion and image reconstruction

[0068] The photodetector converts the fluorescence signal into an electrical signal, which is amplified by the amplifier and then digitally collected by the acquisition card and transmitted to the computer to reconstruct an isotropic three-dimensional super-resolution image. The imaging results are finally stored and output.

[0069] Example 2

[0070] Principle of uniform loss optical cage generation by light field manipulation

[0071] For vortex phase retarder, incident light of different polarization states will obtain different polarization results after passing through the vortex phase retarder. Linearly polarized light can be composed of left-handed circularly polarized light and right-handed circularly polarized light. The polarization relationship can be expressed as:

[0072]

[0073] Where m is the topological charge, |R> is the right-handed circularly polarized light component, |L> is the left-handed circularly polarized light component, and then passes through the vortex phase retarder:

[0074]

[0075] If the signs in the above formula are positive, multiplying formula 1 and formula 2 will give:

[0076]

[0077] Equation 3 shows that the topological charge of right-handed circularly polarized light is 2m, while the topological charge of left-handed circularly polarized light is 0. Because the two beams have vastly different topological charges, they are successfully spatially separated, destroying the polarization entanglement of the original beams. This means that by simply changing the sign of m in Equation 1, a specific circular polarization state can be manipulated using the vortex phase retarder.

[0078] To achieve this goal, the present invention further generates three focal points with different axial positions, topological charges, topological directions, and light intensities by deriving the characteristics of the light field distribution after focusing by the objective lens. After focusing by the objective lens, in the space near the focal point, the optical path difference formed by each light beam focusing at the non-focal position is

[0079]

[0080] Among them, ρ is the radius of the spherical coordinate system in the space near the focus, θ, is the convergence angle and azimuth angle, and z is the position along the optical axis. If the focus is to be formed at a position containing this optical path difference, the input phase must compensate for the optical path difference of Δs. Therefore, the optical path that needs to be introduced is

[0081] ψ=kΔs (5)

[0082] Where k = 2πn / λ wave number, n is the refractive index of the medium, then the pupil function generated is T = exp(iψ). If there are multiple focal points in the area near the focal point, the pupil function can be expressed as

[0083]

[0084] Where N=3 is the number of foci, j represents the number of foci, s j is the amplitude adjustment coefficient. In practice, amplitude modulation leads to low modulation efficiency. Therefore, the present invention realizes the above focus control by extracting the phase of the pupil. Then the phase we load in the SLM can be expressed by formula 7:

[0085]

[0086] Formula 7 includes parameters such as x, y, z, and δ. Therefore, the number of foci, light intensity distribution, three-dimensional spatial position control, and phase of topological charge can be used to generate two foci with a topological charge of m at z = Δz and z = -Δz, and a focus with a topological charge of -(m+1) at z = 0. The polarization extraction function of the vortex phase retarder (Formulas 1-3) is then used to achieve two solid focus STEDs with improved axial resolution. z Right-handed circularly polarized light, transverse loss light STED xy For left-handed circularly polarized light, by adjusting the size of Δz and s, the intensity distribution is finally uniform, and an isotropic three-dimensional loss light cage can be realized. After coinciding with the excitation beam in space and time, the stimulated emission loss effect can be realized, and finally a three-dimensional isotropic point spread function ( Figure 3 a).

[0087] Example 3

[0088] Lossy light field control method based on radial and angular polarization state combination

[0089] The generation of a three-dimensional loss point spread function can also be achieved by using a combination of radial and angular polarization states, that is, the polarization states of the two are also orthogonal, avoiding interference between the focal points that affects the integrity of the final loss spot.

[0090] The linear polarized light can also be expressed as follows:

[0091]

[0092] in It is a mutually orthogonal polarization state of order σ. For the adjustment of radial polarization and angular polarization, When σ=0, |RVB σ > and | LVB σ > can be simplified to linear polarization modes in the x and y directions respectively. When σ≠0, it is a vector beam (including angular and radial polarization modes). and The angular and radial polarization states can be extracted.

[0093] The details are as follows: Equation (8) can be simplified as:

[0094] E=|RVB 2n+σ,-β >+|RVB σ,β > (9)

[0095] According to equation (9), the present invention is achieved by To adjust the polarization state, is a beam of order η with polarization direction ω. When adjusting, we can finally get two light fields with orthogonal polarization directions, but achieve separation in space:

[0096] E=|RVB 2m,-β >+|RVB 0,β > (10)

[0097] At this time, if the polarization angle β is angular polarized light, which is hollow in the case of tight focus, then -β is the radial polarization mode. Similarly, the two orthogonal polarization modes separated from a single light beam have different orders, which can achieve spatial separation. By adjusting m, the desired polarization mode can be obtained.

[0098] On the spatial light modulator, by loading a specific form of polarization structure function and combining it with the spatial position of the light field (refer to formula (7) in Example 2), the following spatial distribution is constructed: the focus at the middle axial position (z = 0) is controlled to be angularly polarized light, which appears as a hollow intensity center under tight focusing conditions and is suitable for improving lateral resolution; the two focuses at the two ends of the axial direction (z = ± Δz) are controlled to be radially polarized light, which forms a light spot with a sharp center distribution in the focusing area, which is used to enhance axial resolution. To further optimize the spatial separation effect between the focuses, the system changes the amplitude weight factor s of each focus in the phase diagram of the spatial light modulator. j , axial position z j , achieving the optimal configuration of the three focal points in spatial position, energy distribution and polarization direction, and finally constructing a three-dimensional loss light field with good axial symmetry and no interference between focal points. The constructed three-dimensional loss light field is then completely overlapped with the excitation beam in time and space, triggering the stimulated emission loss effect, compressing the three-dimensional point spread function of the fluorescence signal, and finally obtaining a three-dimensional super-resolution imaging result with isotropic resolution (such as Figure 3 (b)).

[0099] Based on the above principles, the present invention achieves the generation of an isotropic three-dimensional loss beam by modulating the pupil function using a single objective lens and a single loss beam. This method expands traditional 2D-STED microscopy to 3D super-resolution imaging without adding an objective lens or light source. Its innovation addresses the problem that the axial resolution of the fluorescence point spread function (PSF) excited by a single objective lens is always inferior to its lateral resolution, achieving super-resolution imaging in three dimensions. This approach not only reduces construction costs and implementation complexity, but also accurately reflects the true 3D morphology of fluorescent objects, offering a new approach for fields such as biomedical research and nanolithography.

[0100] In addition, the components designed in the present invention are all universal standard parts or components known to those skilled in the art. Their structures and principles can be known to those skilled in the art through technical manuals or conventional experimental methods. They can be fully implemented by those skilled in the art. Needless to say, the content protected by the present invention does not involve improvements to internal structures and methods.

[0101] The embodiments disclosed in the present invention are preferred embodiments, but are not limited to them. Ordinary technicians in this field can easily understand the spirit of the present invention based on the above embodiments and make different extensions and changes. As long as they do not deviate from the spirit of the present invention, they are all within the scope of protection of the present invention.

Claims

1. A method for isotropic three-dimensional super-resolution imaging using a single objective lens, characterized in that: The following steps are involved: S1. Output a linearly polarized excitation beam through a picosecond pulse laser, and output a linearly polarized loss beam through a nanosecond pulse laser. Use a half-wave plate and a polarization beam splitter to adjust the polarization direction and power of the excitation beam and the loss beam. The loss beam is incident on a spatial light modulator, and the light field is modulated by loading a preset phase diagram to generate three focal points with orthogonal polarization, different topological charges, axial positions and energy distribution. The intermediate focus along the axial direction mainly contributes to STED. xy , used to improve the lateral resolution, the two focal points at both ends of the axis constitute STED z For improving axial resolution, the focus is polarization-modulated by a high-order vortex retarder to separate loss beams of different polarization components; S2. The Jones matrix of the high-order vortex delay plate is: in, represents a universal function of phase, m is the order of the high-order vortex delay plate; and β0 determine the polarization distribution of the light beam emerging from the polarization converter. S3, the phase of the spatial light modulator is; Among them, s j is the amplitude weight factor of the jth focus; the value range of j is 1 to N, where N = 3 is the number of focuses; k = 2π / λ, λ is the wavelength of the incident light; θ, are the convergence angle and azimuth angle of the objective lens respectively; (r j , z j ,φ j ) The cylindrical coordinate position parameter of the jth focus within the focal area of ​​the illumination objective lens, r j is the radius of the jth focus in the horizontal position, φ j is the horizontal position angle of the jth focus; z j is the axial position of the jth focus; ψ j is the parameter that controls the polarization state of the jth focus. Phase is the phase function. S4, the linearly polarized loss beam output by the nanosecond pulse laser is phase-controlled by the spatial light modulator and polarized by the high-order vortex delay plate, and three focal points with orthogonal polarization states, different axial positions, topological charges and energy distribution are generated in the focal area of ​​the illumination objective lens, among which the intermediate focus along the axial direction mainly contributes to the STED xy , used to improve the lateral resolution; the two focal points at both ends of the axis constitute STED z Used to improve axial resolution. The three focal points together form an isotropic lossy light field. S5, the intermediate focus along the axial direction corresponding to the isotropic lossy light field (STED xy ) and two focal points along the axis (STED z There are two combinations of polarization states: The first type is left-handed and right-handed circularly polarized light, in which the topological charge of the middle focus is ±1, and the topological charge of the two focuses at both ends along the axis is 0; the second type is angularly polarized light and radially polarized light, in which the middle focus is angularly polarized light, and the two focuses at both ends along the axis are radially polarized light. S6. The isotropic lossy light field needs to pass through s j To adjust STED xy and STED z energy distribution to achieve a three-dimensional isotropic resolution, where 0≤s j ≤1, STED xy With STED z The distance between them is adjusted by z j Further optimize the isotropic erasing properties of lossy light. S7, spatially combining the modulated loss beam and the excitation beam through a dichroic mirror, performing synchronous transverse and axial scanning on the combined beam using an X-axis galvanometer and a Y-axis galvanometer, and conjugating the light field to the objective lens entrance pupil through a scanning lens and a tube lens; S8, focusing the combined beam onto the sample through the objective lens to stimulate the fluorescence signal, then adjusting the signal delay device to make the pulses of the excitation beam and the loss beam coincide in time domain, realizing the stimulated emission depletion effect, collecting the fluorescence signal emitted by the sample, filtering it through the filter and pinhole, and converting it into an electrical signal by the photodetector; S9. The electrical signal is collected and reconstructed through a data acquisition card, and transmitted to a computer for three-dimensional super-resolution image processing to obtain a three-dimensional imaging result with an isotropic resolution of less than 100 nm.

2. The isotropic three-dimensional super-resolution imaging method using a single objective lens according to claim 1, wherein: The output pulse width of the picosecond pulse laser is 10-200 picoseconds, and the output pulse width of the nanosecond pulse laser is on the order of 1 nanosecond.

3. The isotropic three-dimensional super-resolution imaging method using a single objective lens according to claim 1, wherein: The high-order vortex phase retarder achieves spatial separation of different polarization components and suppresses inter-focal interference by regulating the spin angular momentum and orbital angular momentum of the light field.

4. The isotropic three-dimensional super-resolution imaging method using a single objective lens according to claim 1, wherein: The high-order vortex delay plate controls the phase and ψ j The parameters of the focal polarization state achieve orthogonality of the polarization states among multiple axial foci, minimizing the mutual interference terms during fluorescence excitation, thereby improving the consistency and stability of three-dimensional resolution.

5. The isotropic three-dimensional super-resolution imaging method using a single objective lens according to claim 1, wherein: The linear polarization state of the excitation beam is modulated into a left-handed circular polarization state by a quarter-wave plate, and the linear polarization state of the loss beam is modulated into a horizontal linear polarization state by a half-wave plate, thereby meeting the polarization requirements of the incident light of the spatial light modulator.

6. The isotropic three-dimensional super-resolution imaging method using a single objective lens according to claim 1, wherein: The scanning paths of the X-axis galvanometer and the Y-axis galvanometer are controlled by a computer to achieve layer-by-layer three-dimensional scanning of the sample.

7. The isotropic three-dimensional super-resolution imaging method using a single objective lens according to claim 1, wherein: The stimulated radiation loss effect adjusts the time delay of the signal delay device so that the exciting light beam and the loss light beam completely overlap in space and time.

8. The isotropic three-dimensional super-resolution imaging method using a single objective lens according to claim 1, wherein: The fluorescence signal is collected by the objective lens and then returns to the original path, and is reflected to the photodetector through the tube lens, scanning lens, galvanometer and dichroic mirror in sequence.

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