CN-121994764-A - Three-dimensional super-resolution microscopic imaging system and method

CN121994764ACN 121994764 ACN121994764 ACN 121994764ACN-121994764-A

Abstract

The application provides a three-dimensional super-resolution microscopic imaging system and method. The illumination unit is used for outputting uniform and parallel excitation light for the phase modulation unit, the phase modulation unit is used for generating a plurality of beams of structure light with different axial phases, the light conduction unit is used for accurately projecting the structure light and efficiently converging fluorescence, the multi-axial depth information is integrated by combining a super-resolution reconstruction algorithm, and finally the whole-volume optical super-resolution image acquisition of a sample to be detected is realized. The application can greatly improve the acquisition efficiency of a single volume image, has three-dimensional super-resolution imaging with high transverse resolution, high axial resolution and high fidelity, and expands the application scene and practical value of the structured light illumination microscopic imaging technology in the fields of life science and the like.

Inventors

PENG JUNXIN
MENG QUAN

Assignees

中国科学院生物物理研究所

Dates

Publication Date: 20260508
Application Date: 20260213

Claims (10)

1. A three-dimensional super-resolution microscopy imaging system, comprising: an illumination unit for emitting excitation light of at least one wavelength in parallel; A phase modulation unit, disposed on a downstream optical path of the illumination unit, for performing phase modulation on the incident excitation light and forming a plurality of structured lights having different axial phases; The light conduction unit is arranged on a downstream light path of the phase modulation unit and used for projecting and converging incident multiple beams of the structural light to a sample to be detected; the imaging unit is arranged in a common path with the light conduction unit and is used for collecting and super-resolution reconstructing a fluorescent signal formed by exciting the sample to be detected so as to obtain a three-dimensional super-resolution image of the sample to be detected.
2. The three-dimensional super-resolution microscopy imaging system of claim 1, wherein the plurality of structured light beams comprises at least 3 structured light beams having different axial phases.
3. The three-dimensional super-resolution microscopic imaging system according to claim 2, wherein the phase modulation unit at least comprises a spatial light modulator, and a preset phase modulation pattern is loaded on the spatial light modulator, so as to perform phase modulation on the incident excitation light and form a plurality of beams of the structured light.
4. The three-dimensional super-resolution microscopic imaging system according to claim 3, wherein the phase modulation pattern loaded on the spatial light modulator is composed of a plurality of fresnel zone plate patterns distributed in an array, and the coordinates of the center points of different fresnel zone plate patterns on the spatial light modulator are different.
5. The three-dimensional super-resolution microscopic imaging system according to claim 4, wherein the phase modulation pattern loaded on the spatial light modulator includes at least 3 fresnel zone plate patterns distributed in an array.
6. The three-dimensional super-resolution microscopic imaging system according to claim 3, wherein the phase modulation unit further comprises a first half-wave plate, a polarizing beam splitter, and a second half-wave plate, wherein, The first half-wave plate is arranged on an upstream optical path of the polarization beam splitter, and the second half-wave plate is arranged on an optical path between the polarization beam splitter and the spatial light modulator.
7. The three-dimensional super-resolution microscopic imaging system according to claim 1, wherein the illumination unit includes a light source, a first lens, a pinhole, and a second lens, wherein, The first lens is arranged on a downstream light path of the light source, the pinhole is arranged on the downstream light path of the first lens, the second lens is arranged on the downstream light path of the pinhole, and focuses of one sides of the first lens and the second lens, which are close to the pinhole, are overlapped and are jointly located in the aperture of the pinhole.
8. The three-dimensional super-resolution microscopic imaging system according to claim 1, wherein the light conducting unit comprises a third lens, a field stop, a fourth lens, a dichroic mirror, a fifth lens, a barrel lens and an objective lens which are sequentially arranged at intervals on a downstream light path of the phase modulating unit, The focuses of the third lens and the fourth lens close to one side of the field diaphragm are overlapped to form a 4F optical system, and the field diaphragm is positioned on the middle focal plane of the 4F system formed by the third lens and the fourth lens; the fifth lens, the barrel lens and the objective lens are sequentially arranged on a reflection or transmission light path of the dichroic mirror.
9. The three-dimensional super-resolution microscopic imaging system according to claim 8, wherein the imaging unit includes a multi-focal-plane depth-of-field expanding prism and at least one camera, and the multi-focal-plane depth-of-field expanding prism and the camera are sequentially disposed on a transmission or reflection light path of the dichroic mirror.
10. A three-dimensional super-resolution microscopic imaging method, comprising: acquiring a plurality of fluorescence image data sets of a plurality of phases in a plurality of directions of the sample to be detected; registering and correcting the acquired multiple fluorescent image data sets to obtain multiple calibrated fluorescent image data sets; performing full spectrum separation, extraction and frequency shift processing on the plurality of calibrated fluorescent image data sets by adopting a super-resolution reconstruction algorithm to obtain a plurality of super-resolution spectrum components; carrying out noise reduction and signal enhancement processing on the plurality of spectrum components by adopting a wiener filtering algorithm to obtain a plurality of optimized super-resolution spectrum components; Carrying out fusion reconstruction on a plurality of optimized super-resolution frequency spectrum components by utilizing a three-dimensional reconstruction algorithm to obtain a whole-volume optical super-resolution image of a sample to be detected; The three-dimensional super-resolution microscopic imaging system according to any one of claims 1-9 is used for acquiring fluorescence signals formed by excitation of the sample to be detected.

Description

Three-dimensional super-resolution microscopic imaging system and method Technical Field The application relates to the technical field of imaging, in particular to a three-dimensional super-resolution microscopic imaging system and method. Background The rapid development of super-resolution microscopy breaks the diffraction limit of the traditional optical microscope, and high-resolution observation of cell components becomes a reality. In various super-resolution technologies, the structured light illumination microscopic imaging technology (Structured Illumination Microscopy, SIM) is lower in phototoxicity to living cells due to the fact that low-power laser irradiation is adopted, and is particularly suitable for dynamically observing living cell samples for a long time. The three-dimensional structured light obvious micro-imaging technology (3D-SIM) developed on the basis realizes super-resolution imaging of a three-dimensional space by introducing structured light in an axial direction (Z direction), so that isotropic resolution is effectively improved, full-volume information of a sample can be completely captured, and the method plays an important role in the fields of biomedical research and the like. However, the conventional three-dimensional structure light illumination microscopic imaging technology (3D-SIM) mostly adopts a grating diffraction mode to generate structural light fringes, and the mode has inherent limitations, mainly is that the phase of the fringes formed in the axial direction cannot be flexibly adjusted, so that the complete super-resolution information acquisition can be completed only by moving a sample layer by layer through an axial displacement table, further, the imaging speed is directly slow, and the acquisition time of a single volume image can be as long as a few seconds. The slower imaging efficiency is not only difficult to meet the real-time observation requirement of the three-dimensional rapid biological process, but also can generate motion artifacts due to the motion of the sample, and the accuracy and the reliability of the final imaging are affected. Disclosure of Invention The present application aims to solve at least one of the technical problems in the related art to some extent. Therefore, a first object of the present application is to provide a three-dimensional super-resolution microscopic imaging system and method, which can equally separate fluorescent signals excited by structural light illumination of a sample to be measured by a prism, acquire three-dimensional information through one-time camera acquisition, and greatly improve the efficiency of volume imaging. To achieve the above object, an embodiment of a first aspect of the present application provides a three-dimensional super-resolution microscopic imaging system, including: an illumination unit for emitting excitation light of at least one wavelength in parallel; A phase modulation unit, disposed on a downstream optical path of the illumination unit, for performing phase modulation on the incident excitation light and forming a plurality of structured lights having different axial phases; The light conduction unit is arranged on a downstream light path of the phase modulation unit and used for projecting and converging incident multiple beams of the structural light to a sample to be detected; the imaging unit is arranged in a common path with the light conduction unit and is used for collecting and super-resolution reconstructing a fluorescent signal formed by exciting the sample to be detected so as to obtain a three-dimensional super-resolution image of the sample to be detected. Optionally, the plurality of structured light comprises at least 3 structured light beams having different axial phases. Optionally, the phase modulation unit at least includes a spatial light modulator, and a preset phase modulation pattern is loaded on the spatial light modulator, and is used for performing phase modulation on the incident excitation light and forming a plurality of beams of the structured light. Optionally, the phase modulation pattern loaded on the spatial light modulator is composed of a plurality of fresnel zone plate patterns distributed in an array, and the coordinates of the central points of different fresnel zone plate patterns on the spatial light modulator are different. Optionally, the phase modulation pattern loaded on the spatial light modulator at least comprises 3 fresnel zone plate patterns distributed in an array. Optionally, the phase modulation unit further comprises a first half-wave plate, a polarizing beam splitter and a second half-wave plate, wherein, The first half-wave plate is arranged on an upstream optical path of the polarization beam splitter, and the second half-wave plate is arranged on an optical path between the polarization beam splitter and the spatial light modulator. Optionally, the illumination unit comprises a light source, a first lens, a pinhole,