A parallel three-dimensional isotropic super-resolution imaging method and device based on a Bessel beam self-interference field

Abstract

The application discloses a kind of parallel three-dimensional isotropic super-resolution imaging methods based on Bessel beam self-interference field, comprising the following steps: S1: double-focus phase function is loaded to excitation light, and Bessel beam with axial symmetry bifocal point is formed;S2: double-focus Bessel beam is focused to sample region, and reflective interface is introduced in bifocal symmetric center position, so that incident light and reflected light occur self-interference, and multiple isotropic interference focal points of axial equidistant distribution are generated in sample space;S3: the superhigh-order nonlinear fluorescent probe contained in sample is excited using interference focal point, and the point spread function is compressed by superhigh-order nonlinear effect, and fluorescence signal is generated;S4: the fluorescence signal of different axial positions is separated to different regions of detector by multi-plane prism;S5: the fluorescence signal after separation is synchronously collected and three-dimensional image is reconstructed.The application has the advantages compared with prior art: axial parallel scanning and three-dimensional isotropic super-resolution imaging are realized under single objective architecture, and resolution improvement, imaging speed and system structure simplification are considered.

Description

Technical Field

[0001] This invention relates to the field of optical microscopy imaging technology, specifically to a parallel three-dimensional isotropic super-resolution imaging method and apparatus based on the self-interference field of Bessel beams. Background Technology

[0002] Optical microscopy is a core technique for revealing the microscopic structure and dynamic processes of living systems. Confocal laser scanning microscopy, due to its advantages of being non-contact, non-invasive, and easy to operate, is widely used in three-dimensional imaging research of cells and tissues.

[0003] However, due to the optical diffraction limit, the lateral resolution of traditional confocal microscopes is about 200 nm and the axial resolution is about 500 nm. The axial resolution is significantly lower than the lateral resolution, resulting in significant anisotropy of the three-dimensional point spread function, making it difficult to finely resolve three-dimensional structures at the subcellular scale.

[0004] The advent of super-resolution microscopy has broken through the optical diffraction limit. Methods such as stimulated emission depletion microscopy (STED) and single-molecule localization microscopy (PALM / STORM) can improve the resolution to tens of nanometers. However, these techniques mostly rely on high-power laser excitation or point-by-point scanning, resulting in slow imaging speed and high phototoxicity, which severely limits their application in the study of living cells and dynamic processes.

[0005] To improve axial resolution, some researchers have proposed a 4Pi microscopy imaging technique based on a dual-objective structure, which achieves improved axial resolution by generating coherent interference through two opposing objective lenses.

[0006] However, the system has a complex structure, is difficult to debug, has poor stability, and suffers from severe interference sidelobes, usually requiring complex deconvolution algorithms to suppress artifacts.

[0007] Subsequently, mirror self-interference technology based on single objective lens and mirror configuration was proposed to simplify the system structure, but it still has problems such as sidelobe interference, limited improvement in lateral resolution, and difficulty in achieving high-speed three-dimensional imaging.

[0008] Therefore, there is an urgent need to develop a novel microscopic imaging method and device that can simultaneously achieve three-dimensional isotropic super-resolution and high imaging speed in a single-objective architecture, so as to solve the technical bottleneck of the difficulty in balancing resolution, speed and system complexity in existing three-dimensional super-resolution imaging. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the above-mentioned technical defects and provide a parallel three-dimensional isotropic super-resolution imaging method and device based on the self-interference field of Bessel beam. By constructing the self-interference illumination field of Bessel beam, introducing the photon avalanche ultra-high-order nonlinear effect and combining it with multi-plane prism parallel detection, fast three-dimensional isotropic super-resolution imaging under single objective lens conditions is achieved, solving the problems of insufficient axial resolution, slow imaging speed and complex system structure in the prior art.

[0010] To solve the above-mentioned technical problems, the technical solution provided by the present invention is: a parallel three-dimensional isotropic super-resolution imaging method based on the self-interference field of Bessel beams, comprising the following steps: S1: Apply a bifocal phase function to the excitation light to form a Bessel beam with axially symmetrical bifocals; S2: Focus the bifocal Bessel beam onto the sample region and introduce a reflection interface at the bifocal symmetry center position to cause the incident light and the reflected light to interfere with each other, generating multiple isotropic interference focal points that are axially equidistantly distributed in the sample space. S3: Utilize the interference focus to excite the ultra-high-order nonlinear fluorescent probe contained in the sample, and compress the point diffusion function through the ultra-high-order nonlinear effect to generate a fluorescent signal; S4: Fluorescence signals at different axial positions are separated into different regions of the detector using a multi-plane prism; S5: Synchronously acquire the separated fluorescence signal and reconstruct a three-dimensional image.

[0011] Preferably, the ultra-high-order nonlinear fluorescent probe in S3 is a photon avalanche upconversion fluorescent probe; The ultra-high-order nonlinear fluorescence effect is used to suppress the inherent sidelobes of the Bessel beam and compress the main lobe width, thereby compressing the point spread function both laterally and axially.

[0012] Preferably, the bifocal Bessel beam is formed by loading a bifocal phase function onto the back focal plane of the objective lens using a spatial light modulator.

[0013] Preferably, the reflective interface in S2 is a reflector, which is set at the symmetrical center of the two focal points of the bifocal Bessel beam, so that the incident Bessel beam and the reflected Bessel beam undergo 4Pi-type self-interference in the sample space, forming a multifocal interference light field with axially equidistant distribution.

[0014] Preferably, the multi-plane prism in S4 includes multiple partial reflective interfaces, which separate fluorescence signals at different axial positions to different regions of the detector through different optical path differences.

[0015] Preferably, step S5 further includes: S5.1: Perform region separation on the multiplanar image signals obtained from different regions of the detector; S5.2: Spatial registration is performed on the separated multi-planar images to correct spatial deviations between different imaging channels; S5.3: Based on the interlayer signal intensity correction model, the fluorescence intensity at different axial positions is normalized and corrected; S5.4: Perform 3D registration and volume reconstruction on the corrected multi-plane image to obtain a 3D isotropic super-resolution image.

[0016] Another aspect of the present invention discloses a parallel three-dimensional isotropic super-resolution imaging device based on the self-interference field of a Bessel beam, which realizes axial parallel scanning and three-dimensional isotropic super-resolution imaging in a single objective lens architecture, including a laser excitation module, a bifocal Bessel beam phase modulation module, a self-interference field construction module, a scanning imaging module, and a multi-plane prism parallel detection module.

[0017] The advantages of this invention compared to the prior art are: Achieving true three-dimensional isotropic super-resolution imaging 1. By leveraging the synergistic effect of the Bessel beam self-interference field and the ultra-high-order nonlinear effect of photon avalanche, the three-dimensional point spread function is significantly compressed, achieving simultaneous improvement in both the lateral and axial directions, and obtaining sub-50 nm level three-dimensional isotropic resolution; 2. Imaging speed is significantly improved. By employing a multi-focal parallel scanning and multi-plane prism parallel detection method, axial parallel imaging is achieved, and the three-dimensional imaging speed is increased by about 5 to 8 times compared with the traditional layer-by-layer scanning method, which is suitable for rapid dynamic imaging of live cells. 3. The system has a simple structure and high stability. Based on a single objective lens and mirror self-interference configuration, it eliminates the need for a complex dual-objective 4Pi system. The optical path structure is simple, the debugging difficulty is low, and the system stability is high, making it easy to integrate and promote on existing commercial confocal microscopy systems. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the optical path structure of a parallel three-dimensional isotropic super-resolution imaging system based on a confocal microscope architecture.

[0019] Figure 2 This is a schematic diagram of the physical structure of a parallel three-dimensional isotropic super-resolution imaging system based on a confocal microscope architecture.

[0020] Figure 3 This is a single nanoparticle imaging result of the modulated bifocal Bessel beam in the embodiment. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the accompanying drawings.

[0022] Combined with appendix Figure 1-3As shown, a parallel three-dimensional isotropic super-resolution imaging method based on the self-interference field of Bessel beams includes the following steps: S1: A single laser beam is phase-modulated to form a bifocal Bessel beam, and a mirror is introduced at the symmetrical center of the two focal points to make the bifocal Bessel beam self-interfere under the single objective lens structure, thereby constructing a self-interfering illumination field of the Bessel beam and generating multiple isotropic focal points that are axially equidistantly distributed in the sample space. S2: The sample labeled with the fluorescent probe is excited by the self-interference illumination field of the Bessel beam. Combined with the ultra-high-order nonlinear effect generated by the photon avalanche upconversion fluorescent probe, the three-dimensional point spread function is compressed to achieve three-dimensional isotropic super-resolution imaging. S3: Introduce a multi-plane prism in the fluorescence detection optical path to separate fluorescence signals at different axial positions to different regions of the detector, thereby realizing multi-focal parallel scanning and synchronous detection; S4: Separate, register, intensity correct, and reconstruct the multi-plane image signals obtained from the detection to obtain a three-dimensional isotropic super-resolution image.

[0023] It also includes a parallel three-dimensional isotropic super-resolution imaging device based on the self-interference field of a Bessel beam, comprising: a laser excitation module, a bifocal Bessel beam phase modulation module, a self-interference field construction module, a scanning imaging module, and a multi-plane prism parallel detection module. These modules work collaboratively to achieve axial parallel scanning and three-dimensional isotropic super-resolution imaging within a single objective lens architecture. Specifically: (1) Laser excitation module, used to generate coherent laser beams; (2) Bessel beam shaping and bifocal phase modulation module, used to modulate the laser beam to form a bifocal Bessel beam; (3) Self-interference field construction module, used to introduce a reflector to make the bifocal Bessel beam undergo 4Pi type self-interference and construct the Bessel beam self-interference illumination field; (4) Scanning imaging module, which uses a scanning galvanometer to achieve high-speed lateral scanning of the sample; (5) Multi-plane prism parallel detection module, used to image fluorescence signals at different axial positions onto different regions of the detector to achieve multi-plane synchronous detection.

[0024] The bifocal Bessel beam is formed by loading a bifocal phase function onto the back focal plane of the objective lens using a spatial light modulator. Axial scanning is achieved by dynamically adjusting the bifocal phase parameters loaded by the spatial light modulator, without moving the stage or objective lens. The mirror is positioned at the symmetrical center of the two focal points of the bifocal Bessel beam, causing the incident Bessel beam and the reflected Bessel beam to undergo 4Pi-type self-interference in the sample space, forming a multifocal interference light field with axially equidistant distribution. This achieves axial compression of the three-dimensional isotropic point spread function under single objective lens conditions. The ultra-high-order nonlinear fluorescence effect is used to suppress the inherent side lobes of the Bessel beam and compress the main lobe width, thereby significantly compressing the point spread function in both the lateral and axial directions to achieve three-dimensional isotropic super-resolution imaging. The multi-plane prism is composed of multiple partial reflective interfaces. By constructing different optical path differences, isotropic focal scanning imaging signals at different axial levels are simultaneously recorded in different regions of the detector, achieving parallel detection of approximately 5–8 focal planes. The three-dimensional image reconstruction includes the following steps: performing region separation on the multi-plane image signals obtained by the multi-plane prism in different regions of the detector; performing spatial registration on the separated multi-plane images to correct spatial deviations between different imaging channels; normalizing the fluorescence intensity of different focal planes based on the interlayer signal intensity correction model; and performing three-dimensional registration and volume reconstruction on the corrected multi-plane images to obtain a three-dimensional isotropic super-resolution image.

[0025] This embodiment constructs a parallel three-dimensional isotropic super-resolution imaging system based on a confocal microscope architecture. The system optical path is shown in the appendix. Figure 1 (Illustrative diagram) and appendix Figure 2 (Actual photo) Its optical path structure includes two parts: excitation optical path and detection optical path.

[0026] Construction of Bessel self-interference field (excitation optical path) The excitation source is a continuous-wave near-infrared laser whose excitation energy matches that of the photon avalanche fluorescence probe. Its output beam is expanded and collimated by a beam-expanding lens group, ensuring that the lateral dimension of the laser beam covers the effective modulation region of the spatial light modulator. The expanded beam is then incident on the spatial light modulator, where a bifocal Bessel phase function is applied to its target surface, achieving the modulation conversion from a single-beam laser to a bifocal Bessel beam.

[0027] The bifocal Bessel phase function is used to form two Bessel focal points spaced 2z0 apart along the optical axis at the rear focal plane of the objective lens. Here, z0 is the bifocal modulation parameter, which can be dynamically adjusted to precisely control the bifocal distance. (See Appendix) Figure 3 .

[0028] The phase-modulated beam is conjugated to the scanning galvanometer surface through a 4f system consisting of a pair of relay lenses to reduce aberrations introduced during scanning. Subsequently, the beam passes sequentially through the scanning lens and the tube mirror, and is focused into the sample space by a high numerical aperture objective lens.

[0029] Reflectors are placed at the symmetrical centers of the two focal points of the bifocal Bessel beam, causing the incident and reflected Bessel beams to undergo equivalent 4Pi-type axial self-interference in the sample space, thus constructing a Bessel beam self-interference illumination field. The resulting self-interference field generates multiple equally spaced interference focal points along the axis, each focal point exhibiting a nearly isotropic point spread function shape in three-dimensional space. By adjusting the cone angle of the Bessel beam, the size of the annular aperture, and the bifocal phase parameters, precise control can be achieved over the axial focal point spacing, the number of focal points, and the intensity distribution.

[0030] The system's lateral scanning is performed by a scanning galvanometer, enabling point-by-point scanning of the sample in the xy plane. Axial scanning is achieved by dynamically adjusting the bifocal phase parameter z0 loaded by the spatial light modulator, eliminating the need to move the stage or objective lens, thus significantly improving system stability and scanning speed.

[0031] Construction of a multi-plane prism detection system (detection optical path) In the detection optical path, a multi-plane prism is integrated after the tube mirror. The multi-plane prism is composed of multiple partially reflective interfaces, with different interfaces forming different optical path lengths, so that fluorescence signals from different axial positions are imaged in different areas of the detector, achieving synchronous detection of multiple focal planes.

[0032] The axial interlayer spacing Δz between adjacent focal planes satisfies the following relationship: ; Where d is the optical path difference constructed by the multi-plane prism, and n r M is the refractive index of the prism material. a This refers to the system amplification factor of the fluorescence detection optical path.

[0033] By rationally designing the structural parameters of the multi-plane prism, the interlayer spacing is precisely matched with the axial multifocal spacing generated by the self-interference field of the Bessel beam, thereby achieving synchronous imaging of approximately 5-8 focal planes.

[0034] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0035] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0036] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A parallel three-dimensional isotropic super-resolution imaging method based on Bessel beam self-interference field, characterized in that: Includes the following steps: S1: Apply a bifocal phase function to the excitation light to form a Bessel beam with axially symmetrical bifocals; S2: Focus the bifocal Bessel beam onto the sample region and introduce a reflection interface at the bifocal symmetry center position to cause the incident light and the reflected light to interfere with each other, generating multiple isotropic interference focal points that are axially equidistantly distributed in the sample space. S3: Utilize the interference focus to excite the ultra-high-order nonlinear fluorescent probe contained in the sample, and compress the point diffusion function through the ultra-high-order nonlinear effect to generate a fluorescent signal; S4: Fluorescence signals at different axial positions are separated into different regions of the detector using a multi-plane prism; S5: Synchronously acquire the separated fluorescence signal and reconstruct a three-dimensional image.

2. The parallel three-dimensional isotropic super-resolution imaging method based on Bessel beam self-interference field according to claim 1, characterized in that: The ultra-high-order nonlinear fluorescent probe in S3 is a photon avalanche upconversion fluorescent probe. The ultra-high-order nonlinear fluorescence effect is used to suppress the inherent sidelobes of the Bessel beam and compress the main lobe width, thereby compressing the point spread function both laterally and axially.

3. The parallel three-dimensional isotropic super-resolution imaging method based on Bessel beam self-interference field according to claim 1, characterized in that: The bifocal Bessel beam is formed by loading a bifocal phase function onto the back focal plane of the objective lens using a spatial light modulator.

4. The parallel three-dimensional isotropic super-resolution imaging method based on Bessel beam self-interference field according to claim 1, characterized in that: The reflective interface in S2 is a reflector, which is set at the symmetrical center of the two focal points of the bifocal Bessel beam, so that the incident Bessel beam and the reflected Bessel beam undergo equivalent 4Pi-type axial coherent self-interference in the sample space, forming a multifocal interference light field with equal axial spacing.

5. The parallel three-dimensional isotropic super-resolution imaging method based on Bessel beam self-interference field according to claim 1, characterized in that: The multi-plane prism in S4 includes multiple partial reflective interfaces, which separate fluorescence signals at different axial positions to different regions of the detector through different optical path differences.

6. The parallel three-dimensional isotropic super-resolution imaging method based on Bessel beam self-interference field according to claim 1, characterized in that: S5 also includes: S5.1: Perform region separation on the multiplanar image signals obtained from different regions of the detector; S5.2: Spatial registration is performed on the separated multi-planar images to correct spatial deviations between different imaging channels; S5.3: Based on the interlayer signal intensity correction model, the fluorescence intensity at different axial positions is normalized and corrected; S5.4: Perform 3D registration and volume reconstruction on the corrected multi-plane image to obtain a 3D isotropic super-resolution image.

7. A parallel three-dimensional isotropic super-resolution imaging device based on a Bessel beam self-interference field, implementing the method of any one of claims 1-6, achieving axial parallel scanning and three-dimensional isotropic super-resolution imaging in a single-objective architecture, characterized in that: It includes a laser excitation module, a bifocal Bessel beam phase modulation module, a self-interference field construction module, a scanning imaging module, and a multi-plane prism parallel detection module.

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