An ultramicroscopic imaging method based on sub-spectrum demodulation inversion structural frequency
By using a subspectral demodulation-based method to invert structure frequencies and a small numerical aperture optical microscopy system, combined with spectral data processing, super-resolution and large field-of-view microscopic imaging was achieved without damaging the sample. This solved the problem of the mutual constraint between imaging resolution and field of view, and has high practical value and scientific significance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHONGQING UNIV
- Filing Date
- 2023-08-08
- Publication Date
- 2026-06-19
AI Technical Summary
In existing optical microscopy imaging techniques, there is a contradiction between imaging resolution and imaging field of view, and traditional label-free microscopy imaging methods have difficulty in achieving large field of view and super-resolution.
The super-resolution microscopy imaging method based on subspectral demodulation and inversion of structure frequencies utilizes a small numerical aperture optical microscopy imaging system. It employs a wide-spectrum visible light source and a large-bandwidth spectrometer, combined with a two-dimensional scanning galvanometer and imaging objective, to achieve denoising, background light removal, wavelength resampling, and frequency domain linearization of spectral data. It then calculates the temporal amplitude distribution of the subspectrum, inverts structure frequency information, and achieves high-precision microscopy imaging.
It achieves breakthroughs in imaging resolution and expands the imaging field of view at small numerical apertures, resolving the contradiction between imaging resolution and field of view. Furthermore, it eliminates the need for fluorescence staining, does not damage the sample, and reduces system costs.
Smart Images
Figure CN117110283B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of optical microscopy imaging technology, specifically relating to a super-resolution microscopy imaging method based on subspectral demodulation to invert structure frequencies. Background Technology
[0002] Optical microscopy is widely used in biomedicine, advanced manufacturing, and materials chemistry. With the rapid development of these industries, higher demands are being placed on the imaging resolution and market potential of optical microscopy. However, due to the limitations of optical diffraction, conventional optical microscopy struggles to achieve resolutions exceeding half a wavelength. Super-resolution optical microscopy, on the other hand, can achieve resolutions better than half a wavelength, enabling the resolution of even finer structures. For example, for subcellular structures within cells (such as lysosomes), whose structural dimensions are typically below 200 nm, conventional optical microscopy cannot identify their fine details. In such cases, super-resolution microscopy can clearly image these subcellular structures, providing reliable data for related biomedical and case studies.
[0003] Modern optical super-resolution microscopy techniques are mainly divided into two categories: fluorescently labeled and unlabeled microscopy. Fluorescently labeled microscopy observes fluorescently labeled molecules through fluorescence excitation, indirectly achieving imaging of biological samples. It mainly includes stimulated emission loss (STED) fluorescence microscopy, single-molecule localization-based super-resolution microscopy (SMLM), and structured light illumination-based super-resolution microscopy (SIM). However, fluorescently labeled microscopy suffers from problems such as phototoxicity to samples, bleaching of fluorescent labels, and crosstalk in multicolor fluorescent labels. Unlabeled super-resolution optical microscopy has developed rapidly due to its non-destructive nature. However, limited by current optical technologies, imaging resolution and imaging field of view are mutually restrictive in microscopy systems. Typically, to improve imaging resolution, the numerical aperture of the microscope objective needs to be increased, resulting in a reduced imaging field of view; similarly, to increase the imaging field of view, the numerical aperture of the microscope objective needs to be decreased, resulting in a decrease in imaging resolution. Therefore, the contradiction between imaging resolution and imaging field of view has always existed in microscopic imaging systems.
[0004] While existing optical super-resolution imaging techniques can achieve resolutions better than 100 nm, their imaging field of view is typically limited to single-cell microscopy, restricting the development of simultaneous observation of multiple cells. For example, label-free structured light illumination super-resolution microscopy uses a spatial structured light field to illuminate the sample, transferring high-frequency components to a spatial frequency range that can be propagated by the imaging system, thus achieving super-resolution imaging. However, this method has a small field of view, typically less than 25 μm × 25 μm. Another example is super-resolution Raman microscopy, which combines super-resolution optical microscopy with the "fingerprint" characteristics of Raman spectroscopy, achieving super-resolution imaging through the application of surface-enhanced Raman scattering (SERS). However, this method still has a limited imaging field of view, typically less than 30 μm × 30 μm. Therefore, expanding the imaging field of view and resolving the contradiction between imaging resolution and field of view, while achieving optical super-resolution imaging, is a pressing issue in the field of optical microscopy. Summary of the Invention
[0005] In view of this, the present invention provides a super-resolution microscopic imaging method based on subspectral demodulation and inversion of structure frequencies, which aims to solve the problem of mutual constraint between resolution and field of view in traditional label-free microscopic imaging, so that microscopic imaging can simultaneously have the characteristics of large field of view and super-resolution.
[0006] To achieve the above objectives, the technical solution of the present invention is as follows:
[0007] A super-resolution microscopic imaging method based on subspectral demodulation and inversion of structure frequencies includes a microscopic imaging system. The microscopic imaging system includes an optical fiber circulator connected to a visible light broadband light source, a spectrometer, and a scanning end. The scanning end has a first collimator, a two-dimensional scanning galvanometer, and an imaging objective arranged sequentially along the optical path. The key feature is that the super-resolution microscopic imaging method includes the following steps:
[0008] S1: Acquisition of Spectral Data
[0009] Illumination is achieved using the aforementioned broadband visible light source. The light field scattering signals of two different structures on the same object under test are collected through the scanning end, and broadband data of the two different structures are obtained using a spectrometer.
[0010] S2: Subspectral demodulation and inversion of structure space frequency information
[0011] First, spectral denoising and background light removal are performed on the two original broadband data with different structures to improve the signal-to-noise ratio. Then, wavelength resampling and frequency domain linearization are performed to divide the spectrum into N segments. The temporal amplitude distribution of each sub-spectrum is calculated by two-dimensional discrete Fourier transform to achieve ultra-high sensitivity difference analysis of the longitudinal structure.
[0012] Then, the longitudinal structural differences between two different structures within the same depth range are used to reflect the lateral structural morphology. By analyzing the dominant spatial frequency differences between two adjacent points on the focal plane, the lateral structure is located with high precision, thereby achieving super-resolution microscopic imaging of the object under test.
[0013] Preferably, in step S1, a broad-spectrum visible light source (405nm-640nm) is used for illumination, and a scanning optical microscopy system is constructed by focusing through a small numerical aperture microscope objective. A wide-bandwidth spectrometer is used to collect the backscattered signal of the sample under test to obtain the corresponding broad-spectrum data. A two-dimensional scanning galvanometer is used to control the scanning step size, and the sample under test is scanned point by point.
[0014] Preferably, in step S2, based on the backscattered broadband signals within the focal depth range above and below the focal plane acquired at various points within the objective lens focal plane, the original broadband data is first subjected to spectral denoising and background light removal to improve the signal-to-noise ratio; then, wavelength resampling and frequency domain linearization are performed to improve the Fourier spectrum resolution accuracy; to improve the resolution sensitivity of the longitudinal structure, the entire spectrum is divided into N sub-spectrums; Fourier transform is performed on each sub-spectrum to obtain the longitudinal structural information at the focal plane; for the focal depth range, the longitudinal position corresponds to N amplitude data, and by finding the maximum value among the N amplitudes and its corresponding dominant spatial frequency of the sub-spectrum; by determining the difference in dominant spatial frequencies between two adjacent points on the focal plane, high-precision positioning of the lateral structure can be achieved, that is, the dominant spatial frequency is characterized on the two-dimensional imaging surface to realize microscopic reconstruction.
[0015] Compared with the prior art, the beneficial effects of the present invention are:
[0016] 1. This invention, based on a small numerical aperture optical microscopy imaging system, utilizes a super-resolution microscopy imaging method based on subspectral demodulation and inversion of structure frequencies to simultaneously achieve super-resolution and large field-of-view imaging. Compared to traditional optical microscopy imaging, this method can overcome the half-wavelength imaging resolution limit, achieving super-resolution microscopy imaging through small numerical aperture microscope objectives, thus significantly expanding the imaging field of view. It resolves the contradiction between imaging resolution and field of view, possessing high practical value and scientific significance for biomedicine, especially in fields requiring large-area, real-time, high-resolution observation.
[0017] 2. In this invention, label-free super-resolution microscopy can be achieved without complex fluorescence staining of the sample, causing no damage to the sample; thus, it belongs to label-free super-resolution microscopy. Furthermore, the system does not require expensive large numerical aperture objectives to achieve super-resolution microscopy, reducing system costs. Attached Figure Description
[0018] Figure 1This is a system structure diagram of a super-resolution microscopic imaging method based on subspectral demodulation and inversion of structure frequencies;
[0019] Figure 2 This is a flowchart of super-resolution microscopy imaging based on subspectral demodulation and inversion of structure frequencies. Detailed Implementation
[0020] The present invention will be further described below with reference to the embodiments and accompanying drawings.
[0021] A super-resolution microscopic imaging method based on subspectral demodulation and inversion of structural frequencies is proposed. This method utilizes a broadband visible light source for illumination, focuses the light through a small numerical aperture microscope objective, constructs a scanning optical microscopy system, and acquires the backscattered signal of the sample using a high-bandwidth spectrometer. A three-dimensional scattering function of the object structure is constructed, and the light field scattering distribution formed by the object in each direction is analyzed to obtain its spatial frequency information. The high-sensitivity analysis of longitudinal structural differences among different sample structures within the same depth range is used to reflect the lateral structural morphology. By analyzing the dominant spatial frequency differences between adjacent points on the focal plane, high-precision localization of the lateral structure is achieved, thereby realizing super-resolution microscopic imaging.
[0022] Based on the principles of the above methods, super-resolution microscopy requires a microscopic imaging system as a carrier for implementation. Please refer to the attached document. Figure 1 The microscopic imaging system includes an optical fiber circulator 1, which is connected to a visible light broadband light source 2, a spectrometer 3, and a scanning end 4. The scanning end 4 has a first collimator 4a, a two-dimensional scanning galvanometer 4b, and an imaging objective lens 4c arranged sequentially along the optical path.
[0023] Based on the above system structure, the super-resolution microscopy imaging method includes the following steps:
[0024] S1: Illumination is achieved using a broadband visible light source 2, focusing is performed through a small numerical aperture microscope imaging objective 4c, and a scanning optical microscope system is constructed. A wide-bandwidth spectrometer 3 collects the backscattered signal of the sample to obtain the corresponding broadband data. During implementation, it is necessary to collect broadband data from two different structures on the same sample.
[0025] S2: Using a two-dimensional scanning galvanometer 4b, the scanning step size is controlled to scan two different structures of the object under test point by point, constructing the three-dimensional scattering functions of the two structures, analyzing the light field scattering distribution formed by the object in each direction, and inverting the spatial frequency information of the two structures. The specific process is as follows: First, the original broadband data of the two different structures are subjected to spectral denoising and background light removal to improve the signal-to-noise ratio. Then, wavelength resampling and frequency domain linearization are performed to divide the spectrum into N segments. Through two-dimensional discrete Fourier transform, the temporal amplitude distribution of each sub-spectrum is calculated to achieve ultra-high sensitivity difference analysis of the longitudinal structure. Then, the high sensitivity analysis of the longitudinal structural difference of the two different structures within the same depth range is used to reflect the lateral structural morphology. By analyzing the dominant spatial frequency difference between two adjacent points on the focal plane, high-precision positioning of the lateral structure is achieved, thereby realizing super-resolution microscopic imaging of the object under test.
[0026] For further details, please refer to the attached document. Figure 2 As shown, the super-resolution microscopy imaging process based on structure 1 and structure 2 within the sample under test is as follows:
[0027] Point-by-point using two-dimensional scanning galvanometer 4b Figure 2 Structures 1 and 2 within the sample under test are subjected to fine scanning. Their light field scattering signals U1(X1,Y1,T1) and U2(X2,Y2,T2) are collected using a small numerical aperture objective lens 4c, where X and Y represent the lateral dimensions of the two-dimensional plane of the sample under test, and T represents the longitudinal structural dimensions. Broad spectral information I1(X1,Y1,λ) and I2(X2,Y2,λ) are acquired using a spectrometer 3. The broad spectral distribution is modulated by the structural spatial frequencies of different sample points. Using the sub-spectral demodulation and inversion method in step S2, the sub-spectral amplitude distributions A(X1,Y1,Hz) and A(X2,Y2,Hz) of different structures can be extracted, thereby resolving the dominant longitudinal spatial frequency of the sample under test. By determining the difference in the dominant longitudinal spatial frequencies of sample points (x1,y1) and (x2,y2), ultra-high sensitivity difference analysis of the longitudinal structure is achieved. By utilizing the high-sensitivity resolution of the longitudinal structural differences between Structure 1 and Structure 2 within the same depth range, the transverse structural morphology can be reflected, ultimately achieving high-precision positioning of the spatial structure of the sample under test and realizing super-resolution microscopy.
[0028] In this embodiment, the visible light broadband light source 2 has a wavelength range of 405nm-640nm, and the optimal light intensity can be adjusted according to requirements. The two-dimensional scanning galvanometer 4b has a beam diameter of up to 10mm, capable of covering all visible light wavelength ranges in this embodiment. The imaging objective 4c has an effective focal length of 20mm and a numerical aperture (NA) of 0.30. The spectrometer 3 is a wide-bandwidth spectrometer with 2048 pixels, covering a wavelength range of 185nm-920nm. (See attached diagram.) Figure 1As shown, the spectrometer 3 consists of a diffraction grating 3a, a focusing lens 3b, and a line camera 3c arranged sequentially along the optical path. The spectrometer 3 is also connected to a PC 5. A second collimator 6 is provided between the fiber optic circulator 1 and the spectrometer 3.
[0029] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention. Those skilled in the art, under the guidance of the present invention, can make various similar representations without departing from the spirit and claims of the present invention, and such modifications all fall within the protection scope of the present invention.
Claims
1. A super-resolution microscopic imaging method based on subspectral demodulation and inversion of structure frequencies, comprising a microscopic imaging system, wherein the microscopic imaging system includes an optical fiber circulator (1), the optical fiber circulator (1) being connected to a visible light broadband light source (2), a spectrometer (3), and a scanning end (4), wherein, The scanning end (4) has a first collimator (4a), a two-dimensional scanning galvanometer (4b), and an imaging objective (4c) arranged sequentially along the optical path direction. The super-resolution microscopy method comprises the following steps: S1: Acquisition of Spectral Data Illumination is provided by the visible light broadband light source (2), and light field scattering signals of two different structures on the same object are collected by the scanning end (4), and broadband data of the two different structures are obtained by the spectrometer (3). S2: Subspectral demodulation and inversion of structure space frequency information First, spectral denoising and background light removal are performed on the two original broadband data with different structures to improve the signal-to-noise ratio. Then, wavelength resampling and frequency domain linearization are performed to divide the spectrum into N segments. The temporal amplitude distribution of each sub-spectrum is calculated by two-dimensional discrete Fourier transform to realize the difference analysis of the longitudinal structure. Then, the longitudinal structural differences between two different structures within the same depth range are used to reflect the lateral structural morphology. Within the focal depth range, the longitudinal position corresponds to N amplitude data. By finding the maximum value among the N amplitudes and its corresponding dominant spatial frequency, and by analyzing the dominant spatial frequency difference between two adjacent points on the focal plane, high-precision positioning of the lateral structure is achieved, thereby realizing super-resolution microscopic imaging of the object under test.
2. The super-resolution microscopic imaging method based on subspectral demodulation and inversion of structure frequencies according to claim 1, characterized in that: The wavelength range of the visible light broadband light source (2) is 405 nm-640 nm.
3. The sub-spectral demodulation inversion structure frequency based super- resolution microscopic imaging method of claim 1, wherein: The spectrometer (3) is a wide-bandwidth spectrometer, consisting of a diffraction grating (3a), a focusing lens (3b), and a line camera (3c) arranged sequentially along the optical path. The wavelength range of the wide-bandwidth spectrometer is 185nm-920nm.
4. The super-resolution microscopic imaging method based on subspectral demodulation and inversion of structure frequencies according to claim 1, characterized in that: In step S1, the two different structures are scanned point by point by a two-dimensional scanning galvanometer (4b), and their light field scattering signals are collected by an imaging objective (4c).
5. The super-resolution microscopic imaging method based on subspectral demodulation and inversion of structure frequencies according to claim 1 or 4, characterized in that: The imaging objective (4c) has a focal length of 20 mm and an aperture value NA of 0.
30.
6. The super-resolution microscopic imaging method based on subspectral demodulation and inversion of structure frequencies according to claim 1, characterized in that: In step S1, the backscattered broadband signal within the focal depth range above and below the focal plane is obtained at each point in the focal plane of the imaging objective (4c).
7. The sub-spectral demodulation inversion structure frequency based super- resolution microscopic imaging method according to claim 1, characterized in that: The spectrometer (3) is connected to a PC (5).
8. The sub-spectral demodulation inversion structure frequency based super- resolution microscopic imaging method of claim 1, wherein: A second collimator (6) is provided between the fiber optic circulator (1) and the spectrometer (3).