An isotropic three-dimensional super-resolution microscopic imaging method and system

Abstract

The application belongs to the technical field of optical microscopic imaging, and specifically discloses an isotropic three-dimensional super-resolution microscopic imaging method and system. The application shapes an excitation light beam, so that a linear, point-like or multi-point array form excitation spot is generated at the target sample after focusing. Meanwhile, the phase of the loss beam is modulated, so that the phase information for generating the axial stimulated radiation loss spot is carried. Then, the two light beams are combined and focused on the target sample, forming superimposed excitation spots and axial stimulated radiation loss spots, and generating axial limited fluorescence signals. At least four original images with different excitation intensities are collected and digitally array modulated demodulated and reconstructed, so that the isotropic super-resolution image with improved lateral resolution and axial resolution is obtained. The imaging technology can realize 100nm level isotropic three-dimensional super-resolution.

Description

Technical Field

[0001] This application belongs to the field of optical microscopy imaging technology, and more specifically, relates to an isotropic three-dimensional super-resolution microscopy imaging method and system. Background Technology

[0002] Optical microscopy is an indispensable observational tool in life science research, but its resolution has long been limited by the Abbe diffraction limit. The lateral resolution limit of traditional wide-field microscopes is approximately 200-250 nm, and the axial resolution is approximately 600-750 nm. This anisotropic resolution has long constrained researchers' ability to accurately analyze subcellular structures and nanoscale biomolecules in three dimensions. Because conventional microscopy systems can only collect the approximately hemispherical wavefront emitted by fluorescent molecules, a spectral gap exists along the axis, resulting in an axial resolution that is typically only about one-third of the lateral resolution, severely distorting the true three-dimensional morphology of biological samples.

[0003] Currently, among existing super-resolution microscopy techniques, Structured Illumination Microscopy (SIM) and its variant, Digital Array Modulation Microscopy (DaMo), can achieve rapid, low-phototoxicity live-cell imaging with a lateral resolution of up to 100 nm, but their axial resolution is limited to 300 nm, and anisotropy remains. Stimulated Emission Depletion (STED) microscopy improves resolution by physically compressing the effective fluorescence area, but traditional STED is based on point scanning, resulting in slow imaging speed and high phototoxicity, making it unsuitable for long-term dynamic observation of live cells. Currently, no technology can achieve 100 nm three-dimensional isotropic super-resolution imaging while maintaining high imaging speed and low phototoxicity. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this application is to provide an isotropic three-dimensional super-resolution microscopy imaging method and system, which aims to solve the technical problem that the prior art cannot achieve 100nm three-dimensional isotropic super-resolution imaging at high speed.

[0005] The first aspect of this application relates to an isotropic three-dimensional super-resolution microscopy imaging method, comprising the following steps: An excitation beam is emitted and shaped so that, after being focused, it generates an excitation spot in the form of a line, a dot, or a multi-point array at the target sample; a loss beam is emitted and its phase is modulated so that the loss beam carries phase information for generating an axially stimulated emission loss spot at the target sample. The shaped excitation beam and the phase-modulated loss beam are combined and focused on the target sample to form a superimposed excitation spot and an axially stimulated emission loss spot to generate an axially confined fluorescence signal. The axially stimulated emission loss spot has a dark nucleus with zero intensity at the focal plane and forms loss regions at symmetrical positions above and below the focal plane, so that the axial effective fluorescence emission range is compressed only through the stimulated emission process. The axially confined fluorescence signal is acquired to obtain at least four original images with different excitation intensities; The at least four original images are digitally arrayed to modulate and demodulate them to reconstruct an isotropic super-resolution image with improved lateral and axial resolution.

[0006] Preferably, phase modulation of the lossy beam includes: introducing inner and outer zone phase modulation on the pupil surface, so that the central region and the outer ring region generate The phase difference, through the destructive interference between the central region and the outer ring region, forms the dark nucleus at the focal plane.

[0007] Preferably, the modulation function for the inner and outer partition phase modulation is specifically: :

[0008] in, It is an exponential function with the natural constant as its base. It is a step function. To normalize the pupil radius, As the boundary between the inner and outer partitions, It is pi. It is the imaginary unit.

[0009] Preferably, phase modulation of the loss beam includes: constructing a pair of out-of-focus phase distributions with opposite signs on the pupil surface and superimposing them antisymmetrically, so that the complex amplitudes at the focal center cancel each other out, and forming the dark nucleus at the focal plane.

[0010] Preferably, the antisymmetric superposition modulation function is specifically: :

[0011] in, It is based on the Zernike polynomial definition of the phase term corresponding to defocus, which satisfies... , The weighting coefficient for the defocus term. To normalize the pupil radius, Pi It is the imaginary unit.

[0012] Preferably, the same location point on the target sample is made to correspond to different positions of the light spot diffusion function at different scanning times and / or in different spatial channels, thereby being excited by different excitation intensities and generating a fluorescence signal.

[0013] The second aspect of this application relates to an isotropic three-dimensional super-resolution microscopy system, comprising the following parts: Excitation light source, used to emit an excitation beam; An excitation beam shaping module is disposed in the downstream optical path of the excitation source and is used to shape the excitation beam so that the excitation beam generates a linear, dotted, or multi-point array excitation spot at the target sample after being focused by the focusing device. A lossy light source for emitting a lossy light beam with a wavelength longer than the excitation beam; A phase modulation module is disposed in the downstream optical path of the loss light source and is used to perform phase modulation on the loss beam so that the loss beam carries phase information for generating an axially stimulated emission loss spot at the target sample. A beam combining module is located in the downstream optical path of the excitation beam shaping module and the phase modulation module. It is used to receive the shaped excitation beam and the phase-modulated loss beam, and combine the two beams for output. A focusing device, disposed in the downstream optical path of the beam combining module, is used to focus the combined beam onto the target sample, forming a superimposed excitation spot and an axially stimulated emission loss spot at the target sample to generate an axially confined fluorescence signal; wherein, the axially stimulated emission loss spot has a dark nucleus with zero intensity at the focal plane and forms loss regions at symmetrical positions above and below the focal plane, so as to compress only the effective axial fluorescence emission range through the stimulated emission process; The signal separation module is used to separate the axially confined fluorescence signal generated by the target sample when stimulated from the return optical path; The detection module is located in the downstream optical path of the signal separation module and is used to collect the separated axially confined fluorescence signal to obtain at least four original images with different excitation intensities. The computational reconstruction module is communicatively connected to the detection module and is used to receive the at least four original images and perform digital array modulation and demodulation reconstruction to output an isotropic super-resolution image with improved lateral and axial resolution.

[0014] Preferably, the phase modulation module includes a spatial light modulator loaded with a phase hologram obtained by superposition calculation. The phase hologram simultaneously encodes the following information: amplitude information for generating a loss spot that matches the spatial morphology of the excitation spot; and phase modulation information for constructing the axially stimulated emission loss spot.

[0015] Preferably, the phase modulation module further includes a 4f relay system and a spatial filter located on the Fourier surface of the 4f relay system. The 4f relay system conjugates the encoding surface of the spatial light modulator with the back pupil surface of the focusing device, and the spatial filter is used to select a specified diffraction order.

[0016] Preferably, the excitation beam shaping module includes a microlens array disk and a pinhole array disk, used to divide the excitation beam into a parallel multi-point array; the axial stimulated emission loss spot generated by the phase modulation module is coupled to a position conjugate with the excitation multi-point in the form of a multi-point array through the beam combining module, and then focused by the focusing device and applied to the target sample.

[0017] Overall, the technical solutions conceived in this application have the following beneficial effects compared with the prior art: (1) This application combines axial stimulated radiation loss modulation with digital array modulation demodulation reconstruction to achieve both lateral and axial resolution at the 100nm level. While maintaining high imaging speed and low phototoxicity, it realizes isotropic three-dimensional super-resolution imaging and eliminates the anisotropic distortion problem in traditional super-resolution technology where the axial resolution is inherently lower than the lateral resolution.

[0018] (2) This application generates an axially stimulated emission loss spot that matches the spatial morphology of the excitation spot by phase modulation of the loss beam. This allows the axially stimulated emission loss spot to adapt to linear, dot-shaped, or multi-point array forms of excitation spots, significantly expanding the technical application scenarios of isotropic super-resolution imaging. It also has portability. For commercial confocal systems, by simply adding an axial STED modulation module, a multi-array detection module, and a digital array modulation reconstruction module, a leap from an ideal lateral resolution of 200nm and an axial resolution of 600nm to a stable isotropic resolution of 100nm can be achieved.

[0019] (3) This application uses an internal and external partition phase modulation method to generate axial stimulated emission loss spot. This modulation method can effectively compress the axial resolution of the system to about 100nm level while ensuring that the loss effect along the excitation line direction or array direction is uniform and consistent, providing the best axial compression performance for realizing isotropic super-resolution imaging. Attached Figure Description

[0020] Figure 1 This is a flowchart of an isotropic three-dimensional super-resolution microscopy imaging method provided in an embodiment of this application.

[0021] Figure 2 This is a phase distribution diagram obtained by using an internal and external partition phase modulation method, as provided in the embodiments of this application.

[0022] Figure 3 This is a schematic diagram of the intensity distribution of the stimulated emission loss spot obtained by using an internal and external partition phase modulation method, provided in an embodiment of this application.

[0023] Figure 4 This is a schematic diagram of the effective point spread function distribution before and after the stimulated radiation loss spot obtained by superimposing the inner and outer partition phase modulation method provided in the embodiments of this application.

[0024] Figure 5 This is a phase distribution diagram obtained by using a dual-defocus odd-symmetric modulation method, as provided in the embodiments of this application.

[0025] Figure 6 This is a schematic diagram of the intensity distribution of the stimulated emission loss spot obtained by using a dual-defocus odd-symmetric modulation method, provided in an embodiment of this application.

[0026] Figure 7 This is a schematic diagram of the effective point spread function distribution before and after the stimulated radiation loss spot obtained by superimposing a double-defocus odd-symmetric modulation method according to an embodiment of this application.

[0027] Figure 8 This is a diagram illustrating the composition of an isotropic three-dimensional super-resolution microscopy system provided in an embodiment of this application.

[0028] In all the accompanying drawings, the same reference numerals are used to denote the same elements or structures, wherein: 1 is the excitation source; 2 is the beam expander lens group; 3 is the cylindrical lens; 4 is the excitation light-fluorescence dichroic mirror; 5 is the tube lens; 6 is the loss source; 7 is the spatial light modulator; 8 is the 4f relay system; 9 is the spatial filter; 10 is the excitation light-loss light dichroic mirror; 11 is the objective lens; 12 is the target sample; 13 is the detector. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0030] This application provides an isotropic three-dimensional super-resolution microscopy imaging method and system. The core idea is to introduce stimulated emission loss modulation only in the axial dimension within a digital array modulation microscopy framework. Through a two-stage synergy of physical compression and computational reconstruction, isotropic three-dimensional super-resolution imaging with both lateral and axial resolutions reaching approximately 100 nm is achieved. This method is applicable not only to line illumination architectures but also to point illumination architectures and turntable confocal architectures.

[0031] The embodiments of this application are described below with reference to the accompanying drawings.

[0032] like Figure 1 The diagram shown is a flowchart of an isotropic three-dimensional super-resolution microscopy imaging method provided in an embodiment of this application, which specifically includes the following steps: Step S1: Excitation and shaping of the excitation beam.

[0033] An excitation beam is emitted and shaped so that after being focused, the excitation beam produces an excitation spot in the form of a line, dot, or multi-point array at the target sample.

[0034] Specifically, when a linear excitation spot is required, the excitation beam can be one-dimensionally compressed using a cylindrical lens and then focused by a focusing device. When a point-shaped excitation spot is required, the excitation beam can be two-dimensionally deflected using a scanning galvanometer and then focused by a focusing device. When a multi-point array excitation spot is required, the excitation beam can be divided into a parallel multi-point array using a microlens array disk and a pinhole array disk, and then focused by a focusing device.

[0035] Step S2: Emission and phase modulation of the lossy beam.

[0036] A loss beam with a wavelength longer than the excitation beam is emitted, and the loss beam is phase-modulated so that the loss beam carries phase information for generating an axially stimulated emission loss spot at the target sample.

[0037] The phase modulation can be achieved using a spatial light modulator (SLM). As a phase-type wavefront modulation device, the spatial light modulator can simultaneously perform functions such as axial STED light field generation, aberration compensation, blazed grating beam splitting, and SLM body phase calibration.

[0038] For a phase-type spatial light modulator, its total phase distribution can be written as:

[0039] in, To represent the spatial light modulator plane coordinates, this plane is conjugate with the objective lens's back focal plane, i.e., the pupil plane; The desired complex amplitude distribution to be applied to the pupil surface; To map the amplitude and phase information of the target complex amplitude together into a phase hologram that can be loaded by a phase-type spatial light modulator by using a computational holographic algorithm; For blazed grating terms, it is used to deflect the effective reconstructed light field to the positive first order diffraction order, thereby filtering out the zeroth order and other stray orders on the Fourier surface of the 4f relay system and improving the purity of the STED light field. This is a correction term for the spatial light modulator itself, used to compensate for the device's static wavefront error, spatial phase non-uniformity, and gray-scale-phase response deviation.

[0040] To obtain an axially STED loss spot that matches the spatial morphology of the excitation spot on the focusing focal plane, the target complex amplitude can be uniformly expressed as:

[0041] in, This is the target amplitude term used to generate a loss spot that matches the spatial morphology (linear, dotted, or multi-point array) of the excitation spot; This is the modulation term used to construct the axial STED optical field; For aberration compensation; The imaginary unit; It is an exponential function with the natural constant as its base.

[0042] This embodiment integrates the illumination pattern-matched STED light field generation and axial STED modulation into the same target complex field, and then achieves this in one step through computational holographic encoding using a phase-type spatial light modulator. For purely phase-based targets, It can be degenerated into direct phase loading; for targets with complex amplitude constraints, it can be encoded using the Gerchberg–Saxton (GS) iterative algorithm, the weighted GS iterative algorithm, or the dual-phase holographic coding method.

[0043] Step S21: Internal and external partition phase modulation scheme.

[0044] As a preferred implementation of axial STED modulation, an approximate "bottle-shaped beam" in the form of a one-dimensional extension, dot, or multi-point array can be used as the axial STED spot. The core idea is to introduce inner and outer partition phase modulation on the pupil plane, so that the focusing fields from the central disk and the outer ring region cancel each other out near the focal center, thereby forming a central dark nucleus and axial loss double lobes above and below the focal plane.

[0045] Its axial modulation term can be written as:

[0046] in, It is an exponential function with the natural constant as its base. It is a step function. To normalize the pupil radius, satisfying , As the boundary between the inner and outer partitions, It is pi. It is the imaginary unit.

[0047] like ,but Modulation function Values ; like ,but Modulation function Values ; Therefore, on the pupil surface As a boundary, apply [measures] to the central area and the outer ring area. The phase difference causes the two beams to completely destructively interfere at the focal point, forming a dark nucleus with zero central intensity. On the defocused surfaces above and below the focal plane, the optical path difference between the two beams changes, no longer satisfying the phase inversion condition, resulting in increased light intensity and the formation of axially lossy double lobes.

[0048] The simulation used an excitation wavelength of 488 nm, an STED loss wavelength of 595 nm, an objective lens NA of 1.5, and an objective lens magnification of 100. Through parameter scanning, when... , At this time, the system's axial resolution can be improved to 100.8 nm. When SLM adopts such Figure 2 After phase encoding as shown, an axial STED optical field is generated at the focal plane, and its xz and yz profile distributions are as follows: Figure 3 As shown in sections (a) and (b), the xz profile distributions of the system PSF with and without superimposed STED loss light are respectively shown in Figures (a) and (b). Figure 4 As shown in parts (a) and (b), the intensity distribution of PSF along the x and z directions before and after superposition are as follows. Figure 4 As shown in sections (c) and (d), the axial half-height full width is significantly compressed, while the lateral strength distribution is almost unaffected.

[0049] Step S22: Dual-defocus odd-symmetric modulation scheme.

[0050] As another implementation of axial STED modulation, dual defocus odd-symmetric modulation can be used to construct the axial STED optical field. The basic idea is to construct a pair of defocused phase distributions with opposite signs on the pupil surface and superimpose them antisymmetrically, so that the complex amplitudes at the focal center cancel each other out, thereby forming an axial dark nucleus.

[0051] First, define the phase term corresponding to defocusing according to the Zernike polynomial. :

[0052] in, The weighting coefficient for the defocus term. This is the defocus term of the Zernike polynomial, describing the phase change distributed along a quadratic surface along the radial direction. The corresponding axial modulation term is written as:

[0053] Essentially, this modulation term simultaneously generates two out-of-focus modes of opposite sign and equal magnitude on the pupil surface. The complex amplitudes of these two modes at the focal center are equal in magnitude but opposite in sign, canceling each other out through subtraction to form a dark nucleus of absolute zero. In the out-of-focus region, the wavefronts of the two modes differ, no longer satisfying the complete cancellation condition, and the light intensity is actually enhanced, forming a lossy double lobe. Replacing the step function with a continuous form of a sine function reduces the spatial frequency requirement for phase encoding on the pupil surface.

[0054] In the simulation, the excitation wavelength was set to 488 nm, the STED loss wavelength to 592 nm, the objective lens NA=1.5, and the magnification to 100. Through parameter scanning, when... , At this point, the axial resolution reaches its minimum value of 148.2 nm. When SLM employs... Figure 5 After phase encoding as shown, an axial STED optical field is generated at the focal plane, and its xz and yz profile distributions are as follows: Figure 6 As shown in sections (a) and (b), the xz profile distributions of the system PSF with and without superimposed STED loss light are respectively shown in Figures (a) and (b). Figure 7 Parts (a) and (b) are shown in the figure. The intensity distribution of PSF along the x and z directions before and after superposition are shown in the figures. Figure 7 Parts (c) and (d) are shown in the diagram.

[0055] Step S3: Beam combining, focusing, and superimposing.

[0056] The shaped excitation beam and the phase-modulated loss beam are combined and focused onto the target sample to form a superimposed excitation spot and an axially stimulated emission loss spot, thereby generating an axially confined fluorescence signal.

[0057] The axially stimulated emission loss spot has a dark nucleus with zero intensity at the focal plane and loss regions are formed at symmetrical positions above and below the focal plane, so as to compress only the effective axial fluorescence emission range through the stimulated emission process.

[0058] When the excitation spot and the axial STED loss spot are superimposed to illuminate the sample, the STED light loses the excited state particles in the axial peripheral region through the stimulated emission process, so that the fluorescence emission is compressed to the unlossed region near the zero intensity in the center, thereby obtaining an axially confined fluorescence signal.

[0059] After superimposing the STED loss beam, the effective fluorescence distribution of the system is mainly determined by two factors: first, the spatial shape of the loss spot near the focal point, especially the intensity gradient around the central zero point; and second, the magnitude of the loss intensity relative to the saturation loss intensity of the fluorescent molecules. Under the saturation loss approximation used in this scheme, after superimposing the STED beam, in shaft and The effective fluorescence distribution in the plane containing the axis can be written as:

[0060] in, The normalized STED loss light intensity, This represents the ratio of the peak intensity of the loss spot to the saturation intensity. Theoretically, increasing the loss intensity can further compress the effective point spread function (PSF) of the system, thereby improving spatial resolution. However, in practical experiments, the loss intensity cannot be increased indefinitely. On the one hand, the loss intensity is limited by the laser output power, the damage threshold of optical components, and system stability; on the other hand, excessively high STED intensity can increase fluorescence bleaching, phototoxicity, and non-ideal background signals. Therefore, the loss intensity should be selected as a trade-off between improving resolution and ensuring the sample's tolerance to fluorescence labeling.

[0061] Step S4: Fluorescence signal acquisition.

[0062] The axially confined fluorescence signal was acquired to obtain at least four original images with different excitation intensities.

[0063] The same point on the target sample is made to correspond to different positions of the light spot diffusion function at different scanning times and / or in different spatial channels, thereby being excited by different excitation intensities and generating a fluorescence signal.

[0064] Different scan times and / or different spatial channels specifically include the following two cases: The same point on the target sample is made to correspond to different positions of the light spot diffusion function at different scanning times or in different spatial channels, thereby being excited by different excitation intensities and generating a fluorescence signal.

[0065] The same point on the target sample is made to correspond to different positions of the light spot diffusion function at different scanning times and in different spatial channels, thereby being excited by different excitation intensities and generating a fluorescence signal.

[0066] Step S5: Digital array modulation, demodulation, and reconstruction.

[0067] The at least four original images are digitally arrayed to modulate and demodulate them to reconstruct an isotropic super-resolution image with improved lateral and axial resolution.

[0068] Without axial STED modulation, the DaMo system achieves lateral and axial resolutions of 100 nm and 300 nm, respectively. After superimposing the axial STED modulated spot, the axial full width at half maximum (FWHM) of the system's point spread function can be compressed to approximately 100 nm, while the lateral intensity distribution maintains an approximately Gaussian shape. DaMo's single-spectrum reconstruction algorithm performs frequency domain high-frequency information demodulation and deconvolution background suppression on the original image after axial STED physical compression, further refining the axial resolution to a level consistent with the lateral resolution.

[0069] The isotropic three-dimensional super-resolution microscopy method of this application is applicable to various illumination architectures, as detailed below: Linear lighting implementation In online illumination, the excitation beam is compressed one-dimensionally by a cylindrical lens and then focused by a focusing device, forming a one-dimensional extended and two-dimensional focused line spot at the sample. The loss beam is modulated by an SLM to form a one-dimensional extended axial STED line loss spot, producing a uniformly distributed dark nucleus and upper and lower loss lobes at the focal plane. When the excitation line spot and the loss spot are superimposed to illuminate the sample, a central bright line with a smaller axial dimension than the original line spot appears at the focal plane. The intensity distribution of this bright line in the lateral focusing direction approximately follows a Gaussian distribution.

[0070] The fluorescence signal is received by a multi-column linear array detector. Taking a detector consisting of a 4-column, 2048-row pixel array as an example, in a single exposure, the 4-column detector array can simultaneously record four images composed of one 2048-row pixel array. The imaging range is extended through line scanning. As the relative position of the illumination line spot and the fluorescence sample changes, each point on the sample is excited by excitation light of different Gaussian intensities over time. Images captured by the same column of the detector array are stitched together according to their imaging positions to obtain four complete original images with different excitation intensities.

[0071] Point lighting implementation In point illumination, the excitation beam is deflected in two dimensions by a scanning galvanometer and then focused to form a point-like excitation focal spot on the sample. The loss beam is modulated by an SLM to form an axial loss double lobe around the point-like focal spot. The imaging range is expanded by point-by-point scanning. At the detector end, a multi-pinhole detector array can be used, with signals from different pinhole positions constructing different excitation intensity channels to obtain at least four original images with different excitation intensities.

[0072] Turntable confocalization implementation method In the rotary confocal method, the excitation beam is split into a parallel multi-point array by a microlens array disk and a pinhole array disk, forming a multi-point array excitation spot at the sample through a focusing device. The loss beam, after SLM modulation, is coupled to a position conjugate to the excitation multi-points through a beam combining module in the form of a multi-point array, forming a multi-point array axial STED loss distribution. The imaging range is expanded by using the multi-point parallel scanning formed by the rotation of the rotary disk, and original images with different excitation intensities are constructed by switching between time-sequential frames or illumination coding patterns during rotary disk rotation.

[0073] Corresponding to the imaging methods described above, this application also provides an isotropic three-dimensional super-resolution microscopy imaging system. For example... Figure 8 As shown, the system includes: Excitation source 1 is used to emit an excitation beam. In one embodiment, the excitation source is a continuous laser.

[0074] The beam expanding lens group 2 includes a beam expanding lens disposed in the downstream optical path of the excitation source 1, which is used to expand the excitation beam into a parallel beam with a certain diameter.

[0075] The cylindrical lens 3, serving as the excitation beam shaping module, is positioned downstream of the excitation source 1 and the beam expander lens group 2 in the optical path. It is used to compress the parallel beam in one dimension, causing it to produce a linear excitation spot after being focused by the focusing device. For point illumination, the cylindrical lens 3 can be replaced by a scanning galvanometer pair. For rotary confocal focusing, the cylindrical lens 3 can be replaced by a rotary confocal module composed of a microlens array disk and a pinhole array disk.

[0076] The loss light source 6 is used to emit a loss beam with a wavelength longer than the excitation beam. In one embodiment, the loss light source is a continuous laser with a wavelength that allows the loss light to effectively deplete excited-state fluorescent molecules through stimulated emission.

[0077] The beam expander lens group 2, wherein another beam expander lens is disposed in the downstream optical path of the loss light source 6, is used to expand the loss beam into a parallel beam with a certain diameter.

[0078] The spatial light modulator 7, as the core device of the phase modulation module, is located in the downstream optical path of the lossy light source 6 and the beam expander lens group 2, and is used to apply phase modulation to the lossy beam. The SLM is a phase-type wavefront modulator device, loaded with a phase hologram calculated by superposition. This phase hologram simultaneously encodes the amplitude information of illumination mode matching and the phase information of axial STED modulation.

[0079] The 4f relay system 8, located downstream of the SLM, is used to ensure strict conjugation between the SLM encoding plane and the rear pupil plane of the objective lens. A spatial filter 9 is located at the Fourier surface of the 4f system to filter out stray light of non-positive first-order diffraction orders.

[0080] The excitation-loss dichroic mirror 10, as a beam combining module, is located in the downstream optical path of the excitation beam shaping module and the phase modulation module. It is used to receive the shaped excitation beam and the phase-modulated loss beam, and then combine the two beams for output.

[0081] Objective lens 11, as a focusing device, is located in the downstream optical path of the beam combiner module to focus the combined beam onto the target sample 12, forming a superimposed excitation spot and an axially stimulated emission loss spot at the sample. Objective lens 11 also serves as a fluorescence collection device.

[0082] The excitation-fluorescence dichroic mirror 4, as part of the signal separation module, is used to separate the excitation light path from the fluorescence return light path.

[0083] Tube lens 5 is used to focus the fluorescence signal onto the detection module.

[0084] Detector 13, as a detection module, is located in the downstream optical path of the signal separation module and is used to acquire axially confined fluorescence signals. In online illumination mode, the detector is a multi-column linear array detector; in point illumination mode, the detector can be a point detector array; in turntable confocal mode, the detector can be an area array camera.

[0085] In online illumination methods, sample scanning or beam scanning is equivalent to each other in two-dimensional imaging. As the corresponding position of the illumination line spot and the fluorescent sample changes, each point on the sample is excited by excitation light of different Gaussian distribution intensities over time and acquired by the corresponding detection channel. The original frames captured under the same excitation intensity are stitched together according to the imaging position to form four complete original images with different excitation intensities.

[0086] In point illumination mode, point-by-point scanning is achieved by scanning mirrors, resonant scanners, acousto-optic or electro-optic deflectors or sample stages, and different excitation intensity channels are constructed by detecting differences in channel position or scanning timing.

[0087] In the rotary confocal method, multi-point parallel scanning is formed by rotating pinholes or microlens arrays on the rotary disk, and original images with different excitation intensities are constructed by rotating the rotary disk in time-sequential framing or switching illumination coding patterns.

[0088] The computational reconstruction module (not shown in the figure) is communicatively connected to detector 13. It is used to receive at least four original images and execute a digital array modulation and demodulation reconstruction algorithm to output an isotropic super-resolution image.

[0089] After obtaining at least four original images, a digital array modulation and demodulation reconstruction algorithm is used for processing. This algorithm is based on the single-spectrum reconstruction method and performs frequency domain high-frequency information demodulation and deconvolution background suppression processing on the original images after axial STED physical compression, so that the axial resolution is further refined to the same level as the lateral resolution.

[0090] Without axial modulation, the DaMo system itself has the ability to improve the axial resolution of ordinary confocal lasers from about 600 nm to about 300 nm. After superimposing the axial STED modulated spot, the axial full width at half maximum (FWHM) of the system's PSF is physically compressed to about 100-150 nm, and then enhanced by the DaMo algorithm, ultimately achieving an isotropic 3D super-resolution effect of about 100 nm.

[0091] In practical systems, the introduction of noise and background signals, the perturbation of axial STED spot morphology by system aberrations, and the center alignment error between the STED spot and the fluorescence excitation spot can all lead to a degradation in actual axial resolution compared to simulation results. However, DaMo's single-spectrum reconstruction algorithm can effectively suppress coaxial defocus background in confocal microscopy, improving the system's tomographic capabilities. Simultaneously, the spatial light modulator itself can also perform aberration compensation, providing system optimization space for subsequent introduction of adaptive optics to correct aberrations and the addition of precision alignment and modulation devices.

[0092] It is understood that the detailed implementation of each part of the system can be found in the description of the aforementioned method embodiments, and will not be repeated here.

[0093] The technical solution provided in this application, in terms of implementation, generates an axially stimulated emission loss spot that matches the spatial morphology of the excitation spot by phase modulation of the loss beam. This enables the STED loss spot to adapt to linear, dot-shaped, or multi-point array forms of excitation spots, and extends the axial resolution compression mechanism from the traditional point-scan STED architecture to line-scan confocal and turntable confocal architectures, significantly expanding the technical application scenarios of isotropic super-resolution imaging.

[0094] In terms of imaging performance, stimulated radiation loss modulation is applied only in the axial direction, and the lateral dimension relies entirely on digital array modulation, demodulation and reconstruction to achieve super-resolution. This avoids the high light intensity irradiation and rapid photobleaching problems caused by lateral loss light in traditional STED imaging, significantly reduces phototoxicity in the live cell imaging process, and makes long-term dynamic observation possible.

[0095] In terms of engineering applications, through modular optical architecture design, this solution can be built as an independent imaging system or upgraded in a modular way to upgrade existing point scanning confocal microscopes or rotating confocal microscopes, reducing the deployment cost and hardware threshold of isotropic super-resolution imaging technology.

[0096] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent three cases: A existing alone, A and B existing simultaneously, and B existing alone. In this application, the symbol " / " indicates that the related objects are in an "or" relationship, for example, A / B means A or B.

[0097] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. An isotropic three-dimensional super-resolution microscopic imaging method, characterized in that, Includes the following steps: An excitation beam is emitted and shaped so that, after being focused, it generates an excitation spot in the form of a line, a dot, or a multi-point array at the target sample; a loss beam is emitted and its phase is modulated so that the loss beam carries phase information for generating an axially stimulated emission loss spot at the target sample. The shaped excitation beam and the phase-modulated loss beam are combined and focused on the target sample to form a superimposed excitation spot and an axially stimulated emission loss spot to generate an axially confined fluorescence signal. The axially stimulated emission loss spot has a dark nucleus with zero intensity at the focal plane and forms loss regions at symmetrical positions above and below the focal plane, so that the axial effective fluorescence emission range is compressed only through the stimulated emission process. The axially confined fluorescence signal is acquired to obtain at least four original images with different excitation intensities; The at least four original images are digitally arrayed to modulate and demodulate them to reconstruct an isotropic super-resolution image with improved lateral and axial resolution.

2. The isotropic three-dimensional super-resolution microscopy imaging method according to claim 1, characterized in that, Phase modulation of the lost beam includes: introducing inner and outer zone phase modulation on the pupil surface to generate phase modulation between the central region and the outer ring region. The phase difference, through the destructive interference between the central region and the outer ring region, forms the dark nucleus at the focal plane.

3. The isotropic three-dimensional super-resolution microscopy imaging method according to claim 2, characterized in that, The modulation function for the inner and outer partition phase modulation is specifically as follows: : in, It is an exponential function with the natural constant as its base. It is a step function. To normalize the pupil radius, As the boundary between the inner and outer partitions, It is pi. It is the imaginary unit.

4. The isotropic three-dimensional super-resolution microscopy imaging method according to claim 1, characterized in that, Phase modulation of the lost beam includes: constructing a pair of out-of-focus phase distributions with opposite signs on the pupil surface and superimposing them antisymmetrically, so that the complex amplitudes at the focal center cancel each other out, and forming the dark nucleus at the focal plane.

5. The isotropic three-dimensional super-resolution microscopy imaging method according to claim 4, characterized in that, The modulation function of the antisymmetric superposition is specifically as follows: : in, It is based on the Zernike polynomial definition of the phase term corresponding to defocus, which satisfies... , The weighting coefficient for the defocus term. To normalize the pupil radius, Pi It is the imaginary unit.

6. The isotropic three-dimensional super-resolution microscopy imaging method according to claim 1, characterized in that, The same point on the target sample is made to correspond to different positions of the light spot diffusion function at different scanning times and / or in different spatial channels, thereby being excited by different excitation intensities and generating a fluorescence signal.

7. An isotropic three-dimensional super-resolution microscopic imaging system, characterized in that, Includes the following parts: Excitation light source, used to emit an excitation beam; An excitation beam shaping module is disposed in the downstream optical path of the excitation source and is used to shape the excitation beam so that the excitation beam generates a linear, dotted, or multi-point array excitation spot at the target sample after being focused by the focusing device. A lossy light source for emitting a lossy light beam with a wavelength longer than the excitation beam; A phase modulation module is disposed in the downstream optical path of the loss light source and is used to perform phase modulation on the loss beam so that the loss beam carries phase information for generating an axially stimulated emission loss spot at the target sample. A beam combining module is located in the downstream optical path of the excitation beam shaping module and the phase modulation module. It is used to receive the shaped excitation beam and the phase-modulated loss beam, and combine the two beams for output. A focusing device, disposed in the downstream optical path of the beam combining module, is used to focus the combined beam onto the target sample, forming a superimposed excitation spot and an axially stimulated emission loss spot at the target sample to generate an axially confined fluorescence signal; wherein, the axially stimulated emission loss spot has a dark nucleus with zero intensity at the focal plane and forms loss regions at symmetrical positions above and below the focal plane, so as to compress only the effective axial fluorescence emission range through the stimulated emission process; The signal separation module is used to separate the axially confined fluorescence signal generated by the target sample when stimulated from the return optical path; The detection module is located in the downstream optical path of the signal separation module and is used to collect the separated axially confined fluorescence signal to obtain at least four original images with different excitation intensities. The computational reconstruction module is communicatively connected to the detection module and is used to receive the at least four original images and perform digital array modulation and demodulation reconstruction to output an isotropic super-resolution image with improved lateral and axial resolution.

8. The isotropic three-dimensional super-resolution microscopic imaging system according to claim 7, characterized in that, The phase modulation module includes a spatial light modulator loaded with a phase hologram obtained by superposition calculation. The phase hologram simultaneously encodes the following information: amplitude information for generating a loss spot that matches the spatial morphology of the excitation spot; and phase modulation information for constructing the axially stimulated emission loss spot.

9. The isotropic three-dimensional super-resolution microscopic imaging system according to claim 8, characterized in that, The phase modulation module further includes a 4f relay system and a spatial filter located on the Fourier surface of the 4f relay system. The 4f relay system conjugates the encoding surface of the spatial light modulator with the back pupil surface of the focusing device. The spatial filter is used to select a specified diffraction order.

10. The isotropic three-dimensional super-resolution microscopic imaging system according to claim 7, characterized in that, The excitation beam shaping module includes a microlens array disk and a pinhole array disk, used to divide the excitation beam into a parallel multi-point array; the axial stimulated emission loss spot generated by the phase modulation module is coupled to a position conjugate with the excitation multi-point in the form of a multi-point array through the beam combining module, and then focused by the focusing device and applied to the target sample.

Images