An isotropic three-dimensional super-resolution imaging method with a single objective

Abstract

This invention relates to an isotropic three-dimensional super-resolution imaging method using a single objective lens, comprising the following steps: outputting a linearly polarized excitation beam through a picosecond pulsed laser and an outputting a linearly polarized loss beam through a nanosecond pulsed laser; adjusting the polarization direction and power of the excitation and loss beams using a half-wave plate and a polarizing beam splitter; incident the loss beam onto a spatial light modulator; modulating the light field by loading a preset phase diagram to generate three focal points with different topological charges, axial positions, and energy distributions; and modulating the polarization state of the focal points using a high-order vortex phase delay plate to separate the loss beams with different polarization components. This invention achieves an imaging resolution of less than 100 nm in both the lateral and axial directions, with similar lateral and axial resolution performance. In three-dimensional super-resolution imaging, it can accurately reflect the true three-dimensional structure of an object. The imaging resolution is directly proportional to the intensity of the loss beam; the stronger the loss beam intensity, the higher the three-dimensional imaging resolution.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging, specifically to an isotropic three-dimensional super-resolution imaging method using a single objective lens. Background Technology

[0002] Limited by the optical diffraction limit, the maximum imaging resolution of traditional optical microscopes is only half a wavelength of light. This insufficient optical imaging resolution severely restricts researchers' exploration of related mechanisms in the biomedical field. In recent decades, with the development of fluorescent probes and advanced imaging methods, super-resolution microscopy has become an important tool in basic biological research, such as STED and STORM. Thanks to its ultra-high lateral resolution (approximately 20-40 nm), super-resolution imaging technology can achieve two-dimensional visualization of cell structures at the nanoscale, providing an indispensable means for cell and molecular research. However, although the lateral resolution of optical imaging has broken through the diffraction limit, the axial resolution of single-objective optical imaging systems is still limited by the optical diffraction limit. The iso-STED microscopy technique based on dual-objective illumination imaging is an effective method to solve the mismatch between lateral and axial resolution. This technique combines a 4Pi dual-objective illumination structure with stimulated emission depletion (STED) technology, generating a hollow spherical PSF at the focal plane through interference enhancement between the two opposing objectives. By precisely controlling the erasure light intensity, isotropic super-resolution imaging with both lateral and axial resolutions of 50 nm can be achieved. However, iso-STED microscopy based on dual-objective illumination requires the interference of two illumination beams from the dual objectives within the focal region, which imposes strict requirements on sample transparency and thickness (typically less than a few micrometers). Therefore, iso-STED microscopy based on dual-objective illumination is difficult to apply to in vivo sample imaging, limiting its feasibility in routine biological imaging. Therefore, to expand the compatibility of the technology in diverse applications, such as in vivo sample imaging and deep-depth imaging, researchers have developed a three-dimensional STED microscope based on single-objective illumination. Specifically, by using a conventional transverse loss beam (STED)... xy Introducing additional axial loss beams (STED) on top of the existing structure z This method effectively improves both lateral and axial resolution. It minimizes STED by introducing a phase delay. xy and STED z The interference between them allows them to operate independently. Despite STED zWhile this approach can achieve a significant improvement in axial resolution, the design inevitably leads to an extension of the axial PSF (Positive Surface Range) because a single objective lens can only collect hemispherical wavefronts. In other words, existing optical super-resolution imaging techniques based on single-objective illumination suffer from a mismatch between lateral and axial resolution. This resolution mismatch fundamentally limits the accuracy of these techniques in three-dimensional (3D) structural analysis, resulting in distortion of the true 3D structure of the sample.

[0003] To address the technical bottleneck of existing technologies that cannot achieve isotropic 3D super-resolution imaging with a single-objective architecture, this invention proposes an isotropic 3D super-resolution imaging method based on single-objective illumination. Specifically, achieving 3D isotropic super-resolution in a single-objective illumination STED microscope requires meeting two key conditions. First, multiple focal points must be generated along the axial direction, and their orbital angular momentum, intensity distribution, and 3D position must be precisely controlled to achieve 3D erasing light. Second, interference between these focal points must be prevented to maintain isotropic 3D resolution. To meet these conditions, a special vector beam with a different phase response is used as the erasing light in the STED erasing light path. By controlling the polarization through phase, the intrinsic polarization mode of this special vector erasing beam is extracted from the focal region of the illumination objective. Left-handed circular polarization is assigned to the transverse loss beam, and right-handed circular polarization is assigned to the axial loss beam, thereby effectively avoiding optical field distortion caused by interference between the two. By precisely controlling the position, intensity, and energy distribution of the focal points, this method can construct a uniform 3D loss field that meets the requirements of isotropic 3D super-resolution. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies, adapt to practical needs, and provide a single-objective, isotropic three-dimensional super-resolution imaging method. This method achieves sub-100nm three-dimensional isotropic resolution using a single-loss laser. Based on a traditional two-dimensional STED system, this technique requires no additional objectives or lossy light sources; it overcomes the inherent anisotropic resolution limitations of single-objective configurations simply by modulating the lossy beam once. Using this invention, three-dimensional imaging of the true structure of subcellular organelles can be achieved, allowing for the exploration of the relationship between their three-dimensional morphology and related disease mechanisms, thus solving current technical problems.

[0005] To achieve the objectives of this invention, the technical solution adopted is as follows: A single-objective isotropic three-dimensional super-resolution imaging method is designed, comprising the following steps:

[0006] S1. A linearly polarized excitation beam is output from a picosecond pulsed laser, and a linearly polarized loss beam is output from a nanosecond pulsed laser. The polarization direction and power of the excitation and loss beams are adjusted using a half-wave plate and a polarizing beam splitter. The loss beam is then incident on a spatial light modulator. By applying a preset phase diagram to modulate the light field, three focal points with orthogonal polarization, different topological charges, axial positions, and energy distributions are generated. The central focal point along the axial direction mainly contributes to the... It is used to improve lateral resolution, and consists of two focal points at both ends along the axial direction. To improve axial resolution, a high-order vortex delay plate is used to modulate the polarization state of the focal point, separating loss beams with different polarization components.

[0007] S2, the Jones matrix of the high-order vortex delay plate is

[0008]

[0009] in, This represents a general function of phase, where m is the order of the higher-order vortex delay plate; and It determines the polarization distribution of the beam emitted from the polarization converter.

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

[0011]

[0012] in, is the amplitude weighting factor for the j-th focus; the value of j ranges from 1 to N, where N=3 is the number of focuses; , The wavelength of the incident light; , These are the objective lens convergence angle and azimuth angle, respectively; , , The cylindrical coordinate position parameters of the j-th focal point within the focal region of the illumination objective. Let the radius of the j-th focus be at its horizontal position. Let the angle of the j-th focus be at its horizontal position. Let j be the position of the j-th focus along the axial direction; These are parameters that control the polarization state of the j-th focal point. Phase is the phase function.

[0013] S4. After phase modulation by a spatial light modulator and polarization modulation by a high-order vortex delay plate, the linearly polarized loss beam output from the nanosecond pulsed laser generates three focal points with orthogonal polarization states, different axial positions, topological charges, and energy distributions within the focal region of the illumination objective. The central focal point along the axial direction primarily contributes to... Used to improve lateral resolution; two focal points at both ends along the axial direction form... Used to improve axial resolution. The three focal points together form an isotropic lossy optical field.

[0014] S5, the axial center focal point corresponding to the isotropic loss optical field ( ) and the two focal points at both ends along the axial direction ( The polarization state of () can be combined in two ways:

[0015] The first type is left- and right-circularly polarized light, where the topological charge at the central focal point is... The topological charge at the two focal points at both ends along the axial direction is 0; the second type is angularly polarized light and radially polarized light, wherein the angularly polarized light at the middle focal point and the radially polarized light at the two focal points at both ends along the axial direction.

[0016] S6, the isotropic lossy optical field needs to be passed through To adjust and The energy distribution is such that it achieves three-dimensional isotropic resolution. , and The distance between them is adjusted Further optimize the isotropic erasure properties of lossy light.

[0017] S7. The modulated loss beam and the excitation beam are spatially combined using a dichroic mirror. The combined beam is then scanned laterally and axially using an X-axis galvanometer and a Y-axis galvanometer. The light field is conjugated to the entrance pupil of the objective lens through a scanning lens and a tube mirror.

[0018] S8. The combined beam is focused onto the sample through the objective lens to excite the fluorescence signal. Then, the signal delay device is adjusted to make the pulse time domain of the excitation beam and the loss beam coincide, so as to realize the stimulated emission loss effect. The fluorescence signal emitted by the sample is collected and converted into an electrical signal by a photodetector after being filtered by a filter and a pinhole.

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

[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 delay plate achieves spatial separation of different polarization components and suppresses interference between focal points by controlling the spin angular momentum and orbital angular momentum of the optical field.

[0022] Preferably, the higher-order vortex delay plate controls the phase. as well as By adjusting the parameters of the focal polarization state, orthogonality of the polarization states between multiple axial focal points is achieved, minimizing their mutual interference terms during fluorescence excitation, thereby improving the consistency and stability of the 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, thus satisfying the polarization requirements of the incident light of the spatial light modulator.

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

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

[0026] Preferably, the fluorescence signal is collected by the objective lens and returned along the same path, and is reflected sequentially through the tube mirror, scanning lens, galvanometer and dichroic mirror to the photodetector.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

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

[0029] 2. This invention achieves an imaging resolution of less than 100nm in both the lateral and axial directions, and the lateral resolution is similar to the axial resolution. When performing three-dimensional super-resolution imaging, it can accurately reflect the true three-dimensional structure of the object. The imaging resolution is proportional to the light intensity at loss; the stronger the light intensity at loss, the higher the three-dimensional imaging resolution. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the single-objective isotropic three-dimensional super-resolution imaging system of the present invention;

[0031] Figure 2 This is a schematic diagram of the loss spot formed by polarization orthogonality according to the present invention;

[0032] Figure 3 This is a schematic diagram illustrating the three-dimensional isotropic super-resolution implementation effect of the present invention. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0034] A single-objective isotropic three-dimensional super-resolution imaging method, see [link to relevant documentation]. Figures 1 to 3This includes an improved stimulated emission depletion super-resolution imaging system, the system comprising:

[0035] Picosecond pulsed lasers output high-frequency linearly polarized pulsed lasers with pulse widths on the order of picoseconds, which are used as excitation beams;

[0036] Nanosecond pulsed lasers output high-frequency linearly polarized pulsed lasers with pulse widths on the order of nanoseconds, and are used as loss beams;

[0037] A half-wave plate is used to adjust the polarization direction of a laser beam.

[0038] A quarter-wave plate is used to modulate the linear polarization state of the excitation source into a left-handed circular polarization state.

[0039] Polarizing beam splitter, used for laser beam splitting, can be used in conjunction with a half-wave plate to adjust the laser output power;

[0040] Spatial light modulators are used to modulate the lossy beam emitted by nanosecond lasers, modulating the number of focal points to 3, and modulating the topological charge, energy distribution, and spatial relative position of each focal point.

[0041] A lens, two lenses combined into a 4f system, is used to conjugate the light field to the back focal plane of the second lens;

[0042] Higher-order vortex delay plates are used to impart different orthogonal polarization states to the focus of different topological orientations;

[0043] A mirror reflects laser light, changing the direction of light propagation;

[0044] Dichroic mirrors are used to combine different laser beams in space, specifically to combine loss beams and excitation beams, or to reflect fluorescence.

[0045] X-axis galvanometer, used for transverse scanning of two laser beams;

[0046] The Y-axis galvanometer is used for longitudinal scanning of two laser beams and, when used in conjunction with the X-axis galvanometer, enables surface imaging of the sample.

[0047] The scanning lens is positioned at a location conjugate to the y-axis galvanometer on its front focal plane.

[0048] The tube lens, together with the scanning lens, forms a 4f system, which conjugates the modulated light field to the entrance pupil of the objective lens.

[0049] Objective lens, used to focus the light beam and collect fluorescence;

[0050] Filters are used to reflect fluorescence signals and remove stray light other than fluorescence.

[0051] The pinhole is used to filter fluorescence signals outside the focal plane, thereby improving image contrast.

[0052] A photodetector converts light signals into electrical signals and amplifies the collected signals. It typically uses a photomultiplier tube or an avalanche photodiode.

[0053] The data acquisition card is used to output level signals for controlling the scanning of the galvanometer and other motor components, and to acquire and reconstruct the electrical signals of the photodetector.

[0054] A signal delay unit 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.

[0055] A computer is used for user interaction with hardware and software, controlling image acquisition software, storing data, and processing data.

[0056] Example 1

[0057] Specific workflow of single-objective isotropic 3D super-resolution imaging

[0058] S1, Loss Beam Modulation and Generation

[0059] Nanosecond lasers output linearly polarized light with a pulse width of 1 ns. Figure 1 (Black dashed line) The power is adjusted and the linear polarization ratio is optimized by combining a half-wave plate and a polarizing beam splitter. Then, the half-wave plate is rotated to adjust the polarization state of the incident light to horizontal linear polarization in order to maximize the modulation efficiency of the spatial light modulator (SLM). After the phase map is calculated by loading the SLM, the light field is modulated into three high-order orbital angular momentum foci with topological charges of m, -(m+1), and m, respectively. The topological charge, direction, axial position and energy distribution are dynamically adjusted to lay the foundation for subsequent polarization modulation.

[0060] S2, Higher-order focal polarization state conversion and beam combining

[0061] The three modulated higher-order focal points are conjugated to an m-order vortex delay plate through a 4f system consisting of two lenses, and are respectively assigned right-hand circular polarization (RCP) and left-hand circular polarization (LCP) states (e.g., ...). Figure 2 a) or radial polarization (RP) or azimuthal polarization (AP) states (such as...) Figure 2 b) The loss beam that has completed polarization modulation is reflected by a dichroic mirror and spatially merged with the excitation beam path, entering the synchronous scanning stage;

[0062] S3. Excitation Beam Path Optimization and Beam Combining

[0063] Picosecond lasers output linearly polarized light with pulse widths of 10-200 ps. Figure 1 Similarly, the power and polarization ratio are adjusted by a half-wave plate and a polarizing beam splitter. Then, the half-wave plate (HWP) and the quarter-wave plate (QWP) are combined to convert the linearly polarized light into circularly polarized light. After the beam passes through the dichroic mirror and combines with the loss light, it is conjugated to the X-axis mirror and the Y-axis mirror in sequence through the 4f system, completing the synchronous preparation for transverse (XY) and axial (Z) scanning.

[0064] S4, 3D scanning and fluorescence excitation

[0065] The combined beam passes through a scanning lens and a conjugate tube to the entrance pupil of the objective lens, and is then focused by the objective lens onto the stage to excite fluorescent molecules. During this process, a signal delay device precisely controls the time delay between the excitation beam and the loss beam (erase beam), ensuring that the two pulses completely overlap spatially, triggering the stimulated emission loss (STED) effect, thereby compressing the three-dimensional point spread function (PSF) of the fluorescence signal to a super-resolution scale (e.g., ...). Figure 3 );

[0066] S5. Fluorescence signal collection and filtering

[0067] 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 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 filter and pinhole to filter out stray light, and is finally captured by the photodetector.

[0068] S6. Signal Conversion and Image Reconstruction

[0069] The photodetector converts the fluorescence signal into an electrical signal, which is then amplified by an amplifier and digitally acquired by a data acquisition card. The signal is then transmitted to a computer to reconstruct an isotropic three-dimensional super-resolution image, which is finally stored and output as an imaging result.

[0070] Example 2

[0071] The principle of generating uniform loss photocage by light field modulation

[0072] For a vortex phase retarder, incident light with different polarization states will obtain different polarization results after passing through the vortex phase retarder. Linearly polarized light can be composed of left-handed and right-handed circularly polarized light, and the polarization relationship can be expressed as:

[0073] (1)

[0074] Where m is the topological load. It is the right-hand circularly polarized light component. It is a left-handed circularly polarized light component, which is then passed through a vortex phase retarder:

[0075] (2)

[0076] If we take the sign of the above equation as positive, multiplying equation 1 and equation 2 will give us:

[0077] (3)

[0078] Equation 3 shows that the topological charge of right-handed circularly polarized light is 2m, while that of left-handed circularly polarized light is 0. Because the two beams have vastly different topological charges, they are successfully separated in space, breaking the original polarization entanglement. This means that by simply changing the sign of 'm' in Equation 1, we can achieve the control of a specific circular polarization state under the regulation of a vortex phase retardation plate.

[0079] 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 the focusing of each ray at the non-focal position is...

[0080] (4)

[0081] in, It is the radius in the spherical coordinate system near the focus. Here, θ represents the convergence angle and azimuth angle, and z represents the position along the optical axis. To focus at a position containing this optical path difference, the input phase must be compensated. The optical path difference is such that the required optical path length is [missing information].

[0082] (5)

[0083] in wave number, If the refractive index of the medium is , then the pupil function it produces is: If multiple focal points converge in the region near a single focal point, then the pupil function can be expressed as:

[0084] (6)

[0085] in The number of focal points, Indicates the nth focus. The amplitude adjustment coefficient is used. Since amplitude modulation results in low modulation efficiency in practice, this invention achieves the above focus adjustment by extracting the phase of the pupil. Therefore, the phase loaded into the SLM can be expressed by equation 7.

[0086] (7)

[0087] The 7-style includes Parameters such as the number of beam focal points, intensity distribution, three-dimensional spatial position adjustment, and topological charge phase can be used to generate two beams at different positions. and The topological charge is m at the two foci, in The position of the topological load is One focal point is used, and then the polarization extraction function of the vortex phase delay plate is utilized (Equation 1-3) to achieve two solid focal points that improve axial resolution. Right-handed circularly polarized light, transversely depleted light It is left-handed circularly polarized light, which can be adjusted by... and The size of the light cage ultimately achieves uniform intensity distribution, enabling isotropic three-dimensional loss. When the light cage coincides with the excitation beam in space and time, stimulated emission loss effect can be achieved, ultimately yielding a three-dimensional isotropic point spread function. Figure 3 a).

[0088] Example 3

[0089] Loss-based optical field modulation method based on radial and angular polarization state combination

[0090] The generation of the three-dimensional loss point spread function can also be achieved by combining radial and angular polarization states, that is, the polarization states of these two are also orthogonal, avoiding interference between focal points that affects the integrity of the final loss spot.

[0091] The linearly polarized light expressed by equation (1) can also be:

[0092] (8)

[0093] in , yes The mutually orthogonal polarization states of order allow for the adjustment of radial and angular polarization. .when hour, and These can be simplified to linear polarization modes in the x and y directions, respectively. When this occurs, it becomes a vector beam (including angular and radial polarization modes). Therefore, by adjusting... and This allows the extraction of angular and radial polarization states.

[0094] Specifically, equation (8) can be simplified as follows:

[0095] (9)

[0096] According to equation (9), the present invention is achieved through... To adjust the polarization state, where It is a carrier of polarization direction of A beam of light of order. When using When adjusted, two optical fields with orthogonal polarization directions can ultimately be obtained, achieving spatial separation:

[0097] (10)

[0098] At this time, if the polarization angle Angularly polarized light, when tightly focused, has a hollow distribution. This is the radial polarization mode. Similarly, these two orthogonal polarization modes separated from a single beam have different orders, which can achieve spatial separation. By adjusting m, the desired polarization mode can be obtained.

[0099] 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 equation (7) in Example 2), the following spatial distribution is constructed: located at the axial center position ( The focal point is tuned to angularly polarized light, exhibiting a hollow intensity center under tight focusing conditions, suitable for improving lateral resolution; located at both ends of the axial direction ( The two focal points are modulated to radially polarized light, forming a sharply centered spot in the focusing region to enhance axial resolution. To further optimize the spatial separation between focal points, the system modulates the amplitude weighting factor of each focal point in the phase diagram of the spatial light modulator. Axial position This process achieves optimal configuration of the three focal points in terms of spatial location, energy distribution, and polarization direction, ultimately constructing a three-dimensional lossy optical field with good axial symmetry and no inter-focal interference. The constructed three-dimensional lossy optical field then achieves complete temporal and spatial overlap with the excitation beam, triggering stimulated emission loss effect, compressing the three-dimensional point spread function of the fluorescence signal, and finally obtaining isotropic three-dimensional super-resolution imaging results (such as...). Figure 3 (b)).

[0100] Based on the above principles, this invention, through modulation of the pupil function, achieves the generation of isotropic three-dimensional lossy beams under the conditions of a single objective lens and a single lossy beam. This is a novel method that extends traditional 2D-STED microscopy into three-dimensional super-resolution imaging without adding objectives or light sources. Its innovation lies in solving the problem that the axial resolution of the fluorescence dot spread function excited by a single objective lens is always inferior to the lateral resolution, and achieving super-resolution imaging in three dimensions. This not only saves on setup costs and reduces implementation difficulty, but also accurately reflects the true three-dimensional morphology of fluorescent materials, providing a new method for fields such as biomedical research and nanolithography.

[0101] In addition, all components designed in this invention are general standard parts or components known to those skilled in the art. Their structures and principles can be learned by those skilled in the art through technical manuals or conventional experimental methods. They can be fully implemented by those skilled in the art, so there is no need to elaborate. The content protected by this invention does not involve improvements to the internal structure and methods.

[0102] The embodiments disclosed in this invention are preferred embodiments, but are not limited thereto. Those skilled in the art can easily understand the spirit of this invention based on the above embodiments and make different extensions and variations, but as long as they do not depart from the spirit of this invention, they are all within the protection scope of this invention.

Claims

1. A single-objective, isotropic three-dimensional super-resolution imaging method, characterized in that, Includes the following steps: S1. A linearly polarized excitation beam is output from a picosecond pulsed laser, and a linearly polarized loss beam is output from a nanosecond pulsed laser. The polarization direction and power of the excitation and loss beams are adjusted using a half-wave plate and a polarizing beam splitter. The loss beam is then incident on a spatial light modulator. By applying a preset phase diagram to modulate the optical field, three focal points with different topological charges, axial positions, and energy distributions are generated. The central focal point along the axial direction mainly contributes to the... It is used to improve lateral resolution, and consists of two focal points at both ends along the axial direction. To improve axial resolution, a high-order vortex delay plate is used to modulate the polarization state of the focal point, separating loss beams with different polarization components. S2, the Jones matrix of the high-order vortex delay plate is ； in, This represents a general function of phase, where m is the order of the higher-order vortex delay plate; and It determines the polarization distribution of the beam emitted from the higher-order vortex delay plate; S3, the phase of the spatial light modulator is; ； in, is the amplitude weighting factor for the j-th focus; the value of j ranges from 1 to N, where N=3 is the number of focuses; , The wavelength of the incident light; , These are the objective lens convergence angle and azimuth angle, respectively; , , The cylindrical coordinate position parameters of the j-th focal point within the focal region of the illumination objective. Let the radius of the j-th focus be at its horizontal position. Let the angle of the j-th focus be at its horizontal position. Let j be the position of the j-th focus along the axial direction; The parameter controls the polarization state of the j-th focal point; Phase is the phase function. S4. After phase modulation by a spatial light modulator and polarization modulation by a high-order vortex delay plate, the linearly polarized loss beam output from the nanosecond pulsed laser generates three focal points with orthogonal polarization states, different axial positions, topological charges, and energy distributions within the focal region of the illumination objective. The central focal point along the axial direction primarily contributes to... Used to improve lateral resolution; two focal points at both ends along the axial direction form... Used to improve axial resolution; the three focal points together form an isotropic loss optical field; S5, the axial center focal point corresponding to the isotropic loss optical field. and the two focal points at both ends along the axial direction There are two ways to combine the polarization states: The first type is left- and right-circularly polarized light, where the topological charge at the central focal point is... The topological charge at the two foci along the axial direction is 0; the second type is angularly polarized light and radially polarized light, wherein the angularly polarized light at the middle focal point and the radially polarized light at the two foci along the axial direction are respectively. S6, the isotropic lossy optical field needs to be passed through To adjust and The energy distribution is such that it achieves three-dimensional isotropic resolution. , and The distance between them is adjusted Further optimize the isotropic erasure properties of lossy light; S7. The modulated loss beam and the excitation beam are spatially combined using a dichroic mirror. The combined beam is then scanned laterally and axially using an X-axis galvanometer and a Y-axis galvanometer. The light field is conjugated to the entrance pupil of the objective lens through a scanning lens and a tube mirror. S8. The combined beam is focused onto the sample through the objective lens to excite the fluorescence signal. Then, the signal delay device is adjusted to make the pulse time domain of the excitation beam and the loss beam coincide, so as to realize the stimulated emission loss effect. The fluorescence signal emitted by the sample is collected and converted into an electrical signal by a photodetector after being filtered by a filter and a pinhole. S9. The electrical signal is acquired and reconstructed through the data acquisition card and transmitted to the computer for three-dimensional super-resolution image processing to obtain a three-dimensional imaging result with an isotropic resolution of less than 100nm.

2. The isotropic three-dimensional super-resolution imaging method with a single objective lens as described in claim 1, characterized in that, The picosecond pulse laser has an output pulse width of 10-200 picoseconds, and the nanosecond pulse laser has an output pulse width on the order of 1 nanosecond.

3. The isotropic three-dimensional super-resolution imaging method with a single objective lens as described in claim 1, characterized in that, The high-order vortex phase delay plate achieves spatial separation of different polarization components and suppresses interference between focal points by controlling the spin angular momentum and orbital angular momentum of the optical field.

4. The isotropic three-dimensional super-resolution imaging method with a single objective lens as described in claim 1, characterized in that, The high-order vortex delay plate controls the phase. In conjunction with a spatial light modulator, the focal polarization state parameters are adjusted. By regulating the polarization states of multiple axial focal points, orthogonality can be achieved, minimizing their 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 with a single objective lens as described in claim 1, characterized in that, 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, thus satisfying the polarization requirements of the incident light of the spatial light modulator.

6. The isotropic three-dimensional super-resolution imaging method with a single objective lens as described in claim 1, characterized in that, The scanning paths of the X-axis and Y-axis galvanometers are controlled by a computer, enabling layer-by-layer three-dimensional scanning of the sample.

7. The isotropic three-dimensional super-resolution imaging method with a single objective lens as described in claim 1, characterized in that, The stimulated emission loss effect is achieved by adjusting the time delay of the signal delay device so that the excitation beam and the loss beam completely overlap in space and time.

8. The isotropic three-dimensional super-resolution imaging method with a single objective lens as described in claim 1, characterized in that, The fluorescence signal is collected by the objective lens and returned along the same path, then reflected sequentially through the tube mirror, scanning lens, galvanometer, and dichroic mirror to the photodetector.

Images