A super-resolution microscopic imaging system based on spot overlapping

Abstract

The application relates to the technical field of microscopic imaging, and discloses a microscopic imaging system for realizing super-resolution based on light spot overlapping, which comprises a laser module for generating a laser beam; a light spot adjusting module for generating a radio frequency driving signal according to a light spot adjusting instruction; the light spot adjusting instruction is used for indicating a light spot spacing; a heterodyne interference module for splitting the laser beam into a first laser beam and a second laser beam, shaping the first laser beam into a reference light source, modulating the second laser beam according to the radio frequency driving signal to form a light spot array, combining the reference light source and the light spot array to produce heterodyne interference and form a combined beam to irradiate a sample to be measured; a spectrum data receiving module for collecting light data generated after the sample to be measured reflects the combined beam; and an image inversion module for performing image inversion processing on the light data to generate imaging data of the sample to be measured. The system can realize super-resolution.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging technology, and more specifically to a microscopic imaging system that achieves super-resolution based on spot overlap. Background Technology

[0002] Laser scanning microscopy uses a focused laser beam to scan and probe a sample point by point, obtaining high-resolution spatial images.

[0003] In related technologies, to further enhance the molecular-specific detection capabilities of laser scanning microscopy, laser scanning is combined with radio frequency (RF) signal-based spatial labeling techniques to simultaneously retain the high spatial resolution of laser scanning and the molecular-specific analytical advantages of RF labeling. However, within this fusion framework, the final imaging resolution is determined by the diffraction limit, which is jointly determined by the numerical aperture of the optical system and the wavelength of the laser used. This means that, theoretically, the system's optimal spatial resolution cannot exceed the Airy disk diameter of its focused spot.

[0004] Therefore, there is an urgent need for a solution that can overcome the diffraction limit while maintaining the inherent advantages of laser scanning and spatial marking technologies. Summary of the Invention

[0005] This invention provides a super-resolution microscopic imaging system based on spot overlap to solve the problem that the spatial resolution of microscopic systems is limited by the diffraction limit in related technologies.

[0006] In a first aspect, the present invention provides a super-resolution microscopic imaging system based on spot overlap, comprising: Laser module, used to generate laser beam; A beam adjustment module is used to generate a radio frequency drive signal according to a beam adjustment command; the beam adjustment command is used to indicate the beam spacing. The heterodyne interference module is used to split the laser beam into a first laser beam and a second laser beam, and then shape the first laser beam into a reference light source; the spot adjustment module is also used to modulate the second laser beam according to the radio frequency driving signal to form a spot array; the heterodyne interference module is also used to combine the reference light source and the spot array to generate heterodyne interference, forming a combined beam that illuminates the sample to be tested; The spectral data receiving module is used to collect the optical data generated after the sample under test reflects the combined beam; The image inversion module is used to perform image inversion processing on the optical data to generate imaging data of the sample to be tested.

[0007] In one optional implementation, when the distance between adjacent light spots is less than the diameter of the light spot, the adjacent light spots overlap, and the higher the degree of overlap of the light spots, the higher the resolution of the system.

[0008] In one optional embodiment, the spot adjustment module includes an RF driving module and a spatial light modulation module; the RF driving module is used to receive a spot adjustment command and parse the RF frequency difference in the spot adjustment command to generate an RF driving signal based on the RF frequency difference and send it to the spatial light modulation module; the spatial light modulation module is disposed in the optical path of the second laser beam and is used to introduce a frequency shift into the second laser beam based on the received RF driving signal to generate a spot array with a target spot spacing.

[0009] In one optional implementation, the larger the radio frequency difference, the larger the target spot spacing; the smaller the radio frequency difference, the smaller the target spot spacing.

[0010] In one optional implementation, the system further includes a galvanometer scanning module; the galvanometer scanning module is used to move the combined beam in a horizontal / vertical direction to perform a two-dimensional scan of the sample under test.

[0011] In one optional embodiment, the galvanometer scanning module includes a horizontal galvanometer and a vertical galvanometer; the system also includes a first lens, a second lens, and a first microscope objective; the combined beam is reflected sequentially by the horizontal galvanometer, the first lens (beam contraction), the second lens (beam expansion), and the vertical galvanometer, and then forms an outgoing beam through the first microscope objective to irradiate the sample to be tested.

[0012] In one optional embodiment, the galvanometer scanning module further includes a scanning control unit; the scanning control unit is used to drive the horizontal galvanometer and the vertical galvanometer to deflect respectively, so as to control the emitted beam to move in a two-dimensional plane according to a preset scanning mode; the preset scanning mode includes at least one of raster scanning, vector scanning or random point scanning.

[0013] In one alternative implementation, the spectral data receiving module includes a multi-pixel detector; the multi-pixel detector includes multiple rows and columns of pixels.

[0014] In one optional implementation, the multi-pixel detector is a single-pixel detector; the single-pixel detector is one of a photodiode, an avalanche photodiode, or a photomultiplier tube; or, the multi-pixel detector is an area array imaging detector; the area array imaging detector is one of a CCD detector, a CMOS detector, or a two-dimensional photoelectric sensor with multiple rows and columns of pixels.

[0015] In one optional implementation, the image inversion module includes a data acquisition card and a high-speed programmable logic device; the data acquisition card is used to sample and perform analog-to-digital conversion on the optical data to obtain processed optical data; the high-speed programmable logic device is used to convert the processed optical data to the frequency domain and then perform image reconstruction to generate imaging data of the sample under test.

[0016] The technical solution provided by this invention may include the following beneficial effects: The present invention provides a super-resolution microscopic imaging system based on beam overlap, comprising a laser module for generating a laser beam; a beam adjustment module for generating a radio frequency (RF) drive signal according to a beam adjustment command; the beam adjustment command is used to indicate the beam spacing; a heterodyne interference module for splitting the laser beam into a first laser beam and a second laser beam, and shaping the first laser beam into a reference light source; the beam adjustment module is also used to modulate the second laser beam according to the RF drive signal to form a beam array; the heterodyne interference module is also used to combine the reference light source and the beam array to generate heterodyne interference, forming a combined beam that illuminates the sample under test; a spectral data receiving module for acquiring the light data generated after the sample under test reflects the combined beam; and an image inversion module for performing image inversion processing on the light data to generate imaging data of the sample under test. The above scheme, by setting up the beam adjustment module and the heterodyne interference module, achieves dynamic adjustment of the beam spacing of the beam array by adjusting the RF drive signal, thereby adjusting the microscopic imaging resolution, avoiding limitations in microscopic imaging resolution, and ultimately achieving super-resolution. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of a microscopic imaging system based on spot overlap to achieve super-resolution according to an embodiment of the present invention; Figure 2 This is a schematic diagram of another microscopic imaging system based on spot overlap to achieve super-resolution according to an embodiment of the present invention; Figure 3 This is a schematic diagram of another microscopic imaging system based on spot overlap to achieve super-resolution according to an embodiment of the present invention; Figure 4 This is a schematic diagram of a light spot array according to an embodiment of the present invention; Figure 5This is a schematic diagram of an overlapping spot array according to an embodiment of the present invention; Figure 6 This is a schematic diagram of a light-blocking plate disposed in front of a light spot array according to an embodiment of the present invention; Figure 7 This is a schematic diagram of a light-blocking plate disposed in front of an overlapping light spot array according to an embodiment of the present invention; Figure 8 This is a schematic diagram of light intensity of a light-blocking plate disposed in front of a light spot array according to an embodiment of the present invention; Figure 9 This is a schematic diagram of light intensity where a light-blocking plate is disposed in front of an overlapping light spot array according to an embodiment of the present invention.

[0019] Explanation of reference numerals in the attached figures: 1. Laser module; 2. First glass slide; 3. Second glass slide; 4. Third glass slide; 5. Fourth glass slide; 6. Beam shaper; 7. Beam splitter; 8. Fifth glass slide; 9. Spatial light modulator; 10. Optical coupler; 11. Horizontal galvanometer; 12. First lens; 13. Second lens; 14. Vertical galvanometer; 15. Radio frequency drive module; 16. Main control system; 17. Image inversion module; 18. First microscope objective; 19. Sample to be tested; 20. Second microscope objective; 21. Third lens; 22. Unit point detector; 23. Dichroic mirror; 24. Filter. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0022] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0023] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] Laser scanning microscopy uses a focused laser beam to scan and probe a sample point by point, obtaining high-resolution spatial images.

[0025] In related technologies, to further enhance the molecular-specific detection capabilities of laser scanning microscopy, laser scanning is combined with radio frequency (RF) signal-based spatial labeling technology. This approach simultaneously retains the high spatial resolution of laser scanning and the molecular-specific analytical advantages of RF labeling, demonstrating significant application potential in the biomedical field. However, within this fusion technology framework, the final imaging resolution always faces a fundamental physical limitation: the diffraction limit, determined by both the numerical aperture of the optical system and the wavelength of the laser used. This means that no matter how precise the labeling and detection, the system's optimal spatial resolution theoretically cannot exceed the Airy disk diameter of its focused spot.

[0026] Therefore, there is an urgent need for a solution that can overcome the diffraction limit while maintaining the inherent advantages of laser scanning and spatial labeling technologies, so as to have the ability to analyze the finer structure of samples.

[0027] The following is combined Figures 1 to 9 The following describes embodiments of the present invention.

[0028] This embodiment provides a super-resolution microscopic imaging system based on spot overlap. Figure 1 This is a schematic diagram of a super-resolution microscopic imaging system based on spot overlap according to an embodiment of the present invention. The system includes a laser module, a spot adjustment module, a heterodyne interferometry module, a spectral data receiving module, and an image inversion module.

[0029] This laser module is used to generate a laser beam as a light source.

[0030] This spot adjustment module is used to generate radio frequency (RF) drive signals according to spot adjustment instructions. These instructions specify the spot spacing; that is, by adjusting the parameters related to spot spacing in the instruction, corresponding RF drive signals can be generated for subsequent adjustment of the spacing between spots in the spot array.

[0031] The heterodyne interference module is used to split the laser beam into a first laser beam and a second laser beam, and then shape the first laser beam into a reference light source. The spot adjustment module is also used to modulate the second laser beam according to the radio frequency drive signal to form a spot array. The heterodyne interference module is also used to combine the reference light source and the spot array to generate heterodyne interference, forming a combined beam that illuminates the sample under test. It should be noted that the spots in the spot array are uniformly distributed, that is, the spot spacing between any two adjacent spots is consistent. Different spots in the spot array correspond to different optical frequencies. After heterodyne interference, each spot in the spot array has a specific beat frequency envelope, and each beat frequency envelope has a specific beat frequency. This allows the spatial position of the spot to be marked using the beat frequency, and then the position of the spot on the sample under test can be marked using the beat frequency. The intensity of the beat frequency signal reflects the specific shape of the sample under test.

[0032] The spectral data receiving module is used to acquire the optical data generated after the sample under test reflects the combined beam; This image inversion module is used to perform image inversion processing on the optical data to generate imaging data of the sample under test. The image inversion module includes an image inversion algorithm, which can be selected as needed to perform image inversion processing on the optical data.

[0033] By adjusting the radio frequency drive signal, the spacing between the light spots in the light spot array can be dynamically adjusted, allowing adjacent light spots to switch between overlapping, closely adjacent, or separated states. In one optional embodiment, when the spacing between adjacent light spots is less than the spot diameter, the adjacent light spots overlap. Furthermore, assuming the adjacent light spots do not overlap, the higher the degree of overlap, the higher the resolution of the system. It should be noted that even when adjacent light spots do not overlap, a smaller spacing between the light spots also results in higher resolution.

[0034] In one optional embodiment, the spot adjustment module includes an RF driving module and a spatial light modulation module. The RF driving module is used to receive a spot adjustment command and parse the RF frequency difference in the spot adjustment command to generate an RF driving signal based on the RF frequency difference and send it to the spatial light modulation module. The spatial light modulation module is disposed in the optical path of the second laser beam and is used to introduce a frequency shift into the second laser beam based on the received RF driving signal to generate a spot array with a target spot spacing.

[0035] Optionally, the spatial light modulation module includes a spatial light modulator. Radio frequency (RF) driving signals of different frequencies drive the spatial light modulator to produce different modulation effects. By applying RF driving signals of different frequencies, the beam spacing can be adjusted. The beam spacing (the spatial distance between adjacent beams) and the RF frequency difference are key parameters. For example, in the one-dimensional beam array emitted after adjustment by the spatial light modulator, the zero-order beam is in the center, with ±1st and ±2nd order diffracted beams symmetrically distributed on both sides. The frequency of the zero-order beam is the same as the incident light frequency and is not affected by the RF driving signal; its position remains unchanged. The other diffracted beams are affected by the RF driving signal. Specifically, when the RF frequency difference decreases, the one-dimensional beam array tightens towards the center with the zero-order beam as the reference; when the RF frequency difference increases, the one-dimensional beam array diverges outwards with the zero-order beam as the reference. Since the applied RF frequency difference is the same between any two adjacent beams, the beam spacing between any two adjacent beams is the same. When the radio frequency difference decreases to a certain critical value, the light spots begin to transition from a side-by-side state to an overlapping state. A parameter for spot overlap can then be defined, indicating the ratio of the effective overlap area between adjacent spots to the effective area of ​​a single spot. It should be noted that system resolution does not increase indefinitely with increasing spot overlap. When the spots almost overlap, the illuminated areas are nearly identical, and the detector cannot distinguish such subtle differences, resulting in insufficient detector accuracy. Furthermore, excessive overlap leads to an excessively small scanning area, excessively long scanning time, and slow imaging. Therefore, there exists an optimal overlap range that balances resolution, accuracy, and imaging speed. Within this optimal overlap range, system resolution steadily improves with increasing spot overlap.

[0036] In one alternative implementation, the larger the radio frequency difference, the larger the target spot spacing; the smaller the radio frequency difference, the smaller the target spot spacing.

[0037] In one alternative implementation, the system further includes a galvanometer scanning module for moving the combined beam in a horizontal / vertical direction to perform a two-dimensional scan of the sample under test.

[0038] In one optional embodiment, the galvanometer scanning module includes a horizontal galvanometer and a vertical galvanometer; the system also includes a first lens, a second lens, and a first microscope objective; the combined beam is reflected sequentially by the horizontal galvanometer, the first lens (beam contraction), the second lens (beam expansion), and the vertical galvanometer, and then forms an outgoing beam through the first microscope objective to irradiate the sample to be tested.

[0039] In one optional embodiment, the galvanometer scanning module further includes a scanning control unit; the scanning control unit is used to control the horizontal galvanometer and the vertical galvanometer to work together. Specifically, the scanning control unit is used to drive the horizontal galvanometer and the vertical galvanometer to deflect respectively, so as to control the emitted beam to move in a two-dimensional plane according to a preset scanning mode; the preset scanning mode includes at least one of raster scanning, vector scanning or random point scanning.

[0040] In one alternative implementation, the spectral data receiving module includes a multi-pixel detector; the multi-pixel detector includes multiple rows and columns of pixels to achieve large-scale temporal optical signal acquisition.

[0041] In one alternative implementation, the multi-pixel detector is a single-pixel detector; the single-pixel detector is one of a photodiode, an avalanche photodiode, or a photomultiplier tube; or, the multi-pixel detector is an area array imaging detector; the area array imaging detector is one of a CCD (Charge-Coupled Device) detector, a CMOS (Complementary Metal Oxide Semiconductor) detector, or a two-dimensional photoelectric sensor with a multi-row, multi-column pixel distribution.

[0042] This single-pixel detector boasts extremely high detection sensitivity and response speed, making it particularly suitable for scenarios such as fluorescence lifetime measurement and detection of weak luminescent signals. The single-pixel detector is positioned at the focal point of the light spot in the light-collecting path. Its output is directly connected to a high-precision transimpedance amplifier, which converts the weak photocurrent generated by the single-pixel detector into a voltage signal. This voltage signal is then sent to the data acquisition card in the spectral data receiving module for analog-to-digital conversion. Excitation light (such as fluorescence or scattered light) from the sample is collected and focused by the objective lens, and then entirely incident on the photosensitive surface of the single-pixel detector. The single-pixel detector does not distinguish the spatial source of the light; instead, it converts the total luminous flux of all received wavelength components into a continuous analog electrical signal. This analog electrical signal is amplified and digitized to form a digital signal stream containing only information about the change in total light intensity over time.

[0043] This area array imaging detector, while acquiring the light intensity signal reflected from the combined beam by the sample under test, can also intelligently identify specific pixels illuminated by the light spot through its internally integrated addressing logic. It then combines the row and column address information of that pixel with the light intensity information into a composite digital time-domain signal output. In other words, the output signal of this area array imaging detector is a serial or parallel data stream. Each data unit in this data stream contains the address information of a pixel and the light intensity information acquired by that pixel, thereby converting the spatially distributed light spot signal into a time-domain signal sequence with positional markers. The area array imaging detector is placed on the image plane, and its pixel array directly receives the imaging light spot of the sample under test. The area array imaging detector integrates driving circuitry, signal processing circuitry, and a digital interface. The light emitted from the sample under test forms a spatial light intensity distribution image on the pixel array of the area array imaging detector. Specifically, each pixel in the pixel array independently converts the sensed light intensity into an electrical signal. The area array imaging detector reads out the electrical signals of all pixels and outputs a complete digital image frame sequence containing two-dimensional spatial information.

[0044] Optionally, when selecting a single-pixel detector as a multi-pixel detector, either a single unit point detector or a multi-channel unit point detector can be used. When using a single unit point detector, since all wavelength signals are received by the same detector, perfect inter-channel synchronization is ensured, while avoiding the calibration complexity associated with multiple detectors. When using a multi-channel unit point detector, each wavelength channel is acquired in parallel by an independent unit point detector, achieving true parallel detection. This not only significantly improves the signal acquisition rate, making it particularly suitable for high-speed, real-time dynamic imaging, but also avoids potential signal loss and delay during computational decomposition. Furthermore, because the optical path is physically split, it provides naturally high isolation for each wavelength channel, fundamentally avoiding crosstalk between different wavelength signals. Simultaneously, the unit point detector for each channel can be independently optimized for the characteristics of its corresponding wavelength, thereby obtaining an independent and optimal signal-to-noise ratio for each channel.

[0045] In one optional implementation, the image inversion module includes a data acquisition card and a high-speed programmable logic device. The data acquisition card samples and performs analog-to-digital conversion on the optical data, and performs signal conditioning (e.g., amplification, filtering, isolation, linearization, and signal conversion) to obtain processed optical data. The high-speed programmable logic device transforms the processed optical data from the time domain to the frequency domain and then performs image reconstruction to generate imaging data of the sample under test. Physically driven image reconstruction can be achieved by solving the transformation matrix, or data-driven image optimization can be performed using a pre-trained deep neural network model to suppress noise, enhance resolution, and ultimately achieve high-fidelity image reproduction.

[0046] Figure 2 This is a schematic diagram of another microscopic imaging system based on spot overlap to achieve super-resolution according to an embodiment of the present invention. Figure 2 As shown, laser module 1 generates a laser beam as a light source. After being reflected sequentially by the first glass plate 2 and the second glass plate 3, the laser beam is incident on the beam splitter 7. The beam splitter 7 splits the laser beam into a first laser beam and a second laser beam. The first laser beam is incident on the reference arm optical path, and the second laser beam is incident on the measurement arm optical path. The first glass plate 2 can be a half-glass plate used to rotate the polarization direction of the incident linearly polarized light (laser beam). The polarization beam splitter (beam splitter 7) will then emit two laser beams (the first laser beam and the second laser beam) with controllable intensity according to the different polarization directions. By rotating the first glass plate 2, the ratio of the first laser beam to the second laser beam can be changed, thereby adjusting the light intensity of the first laser beam and the second laser beam. In the reference arm optical path, the first laser beam is reflected sequentially by the third glass plate 4 and the fourth glass plate 5 before entering the beam shaper 6. The beam shaper 6 shapes the first laser beam to form a reference light source. In the measurement arm optical path, the second laser beam is polarized by the fifth glass plate 8 to ensure the spatial light modulator 9 meets the polarization requirements of the light, and then enters the spatial light modulator 9. The spatial light modulator 9 introduces a frequency shift into the second laser beam according to the radio frequency drive signal emitted by the radio frequency drive module 15, modulating it into a spot array. This introduces a different frequency shift into each spot in the spot array to ensure that each spot has a different optical frequency. The number of spots and the spacing between spots in the spot array are related to the radio frequency drive signal. This spot array can be a one-dimensional or two-dimensional spot array. Next, the optical coupler 10 combines the reference light source with the spot array to obtain a combined beam. After beam combining, a beat frequency signal is generated to realize the heterodyne interference of the spot array. After heterodyne interference, each spot in the spot array has a specific beat frequency envelope, and each beat frequency envelope has a specific beat frequency, so as to mark the spatial position of the spot using the beat frequency.

[0047] Next, the combined beam is reflected sequentially by the horizontal galvanometer 11, narrowed by the first lens 12, expanded by the second lens 13, and reflected by the vertical galvanometer 14. It is then scaled to a preset size by the first microscope objective 18 to form an outgoing beam, which illuminates the sample 19 under test. The size of the spot in the scaled combined beam represents the spatial resolution of the image; the smaller the spot, the higher the spatial resolution. The first lens 12 and the second lens 13 work together to collimate the incident beam, preventing beam divergence. The light signal generated after the sample 19 reflects the combined beam passes sequentially through the second microscope objective 20 and the third lens 21, and is then collected by the unit point detector 22 to obtain light data, which is transmitted to the image inversion module 17. The second microscope objective 20 collects the forward scattered light from the incident beam after being scattered by the sample; the lens 21 focuses the incident light. The image inversion module 17 performs image inversion processing on the light data to generate imaging data of the sample under test and transmits it to the host control system 16. The host control system 16 can visualize the imaging data of the sample under test. The host control system 16 is also connected to the radio frequency drive module 15 to generate spot adjustment commands and send them to the radio frequency drive module 15.

[0048] The super-resolution microscopy system based on beam overlap shown in this embodiment uses only one laser source and generates only one measurement arm optical path. It requires fewer lenses, has a simple system structure, is easy to integrate, has a small finished product size, good flexibility, and is convenient to use. The overlap of the beam spots is changed by altering the radio frequency drive signal, resulting in high adjustment precision and low adjustment difficulty. Since all beam spots originate from the same source, they naturally possess good coherence, avoiding mutual interference problems caused by wavelength and phase instability between different lasers. Therefore, the super-resolution microscopy system based on beam overlap shown in this embodiment is suitable for scenarios requiring high system stability and compactness.

[0049] Figure 3 This is a schematic diagram of another microscopic imaging system based on spot overlap to achieve super-resolution according to an embodiment of the present invention. Figure 3 Examples and Figure 2The difference in this embodiment is that the laser module 1 in the system contains multiple lasers, which can generate laser beams of different wavelengths as input light sources, and supports the simultaneous input of one or more laser beams of different wavelengths, incident on the system along the same optical path. The spectral data receiving module is equipped with multi-channel unit point detectors 22. On the one hand, it collects multi-channel optical data generated after the sample reflects the combined beam, with different channels corresponding to specific wavelengths. A single unit point detector 22 can simulate a corresponding wavelength filter to achieve multi-channel spectral acquisition. On the other hand, it receives the light signal emitted by the sample after it is stimulated, and distributes it to one or more unit point detectors 22 of the corresponding fluorescence channel through the optical path. A specific wavelength optical filter 24 can be configured in front of the unit point detector 22 to obtain spectral data of the target wavelength. In addition, a dichroic mirror group is provided, which includes multiple dichroic mirrors 23. This group can split the combined beam from the second microscope objective 20 into multiple beams and guide them into different channels. After adjustment by a lens group (including multiple third lenses 21), the corresponding spectral signals are filtered out by a filter group. Finally, the signals are acquired by a multi-channel unit point detector to generate optical data corresponding to each wavelength of laser. One or more fluorescence channels can be provided, each channel processing information for a specific wavelength. Different wavelengths correspond to different fluorescent markers, thereby characterizing the optical information of multiple specific targets.

[0050] The super-resolution microscopic imaging system based on beam overlap shown in this embodiment uses multiple lasers only to generate laser beams of multiple wavelengths to meet the requirements of a multi-channel unit point detector, without changing the method of achieving super-resolution. This unit point detector has only one photosensitive element and does not have direct imaging capability, but it has high detection sensitivity and fast response speed, meeting the application scenarios of high-speed, high-throughput, and weak fluorescence detection. Each unit point detector can simulate filters of different wavelengths to achieve the acquisition of multi-channel spectral data.

[0051] For example, Figure 4 This is a schematic diagram of a light spot array according to an embodiment of the present invention. Taking a one-dimensional light spot array generated by the system as an example, each light spot in the one-dimensional light spot array has a different optical frequency, and the number and spacing of the light spots are related to the radio frequency driving signal. Radio frequency marking technology uses beat frequency to mark the position of the light spot on the sample under test, and uses the intensity of the beat frequency signal to reflect the specific shape of the object under test. Figure 5 This is a schematic diagram of an overlapping spot array according to an embodiment of the present invention, such as... Figure 5 As shown, after adjusting the beam spacing through the beam adjustment module, a one-dimensional beam array with overlapping beams can be formed. The beam spacing can be adjusted by changing the intensity of the radio frequency drive signal.

[0052] However, if the size of the sample to be tested is smaller than the diameter of a light spot, the specific shape of the sample cannot be determined. For example, Figure 6This is a schematic diagram of a light-blocking plate disposed in front of a light spot array according to an embodiment of the present invention; Figure 7 This is a schematic diagram of a light-blocking plate disposed in front of an overlapping light spot array according to an embodiment of the present invention; Figure 8 This is a schematic diagram of light intensity of a light-blocking plate disposed in front of a light spot array according to an embodiment of the present invention; Figure 9 This is a schematic diagram of light intensity showing a light-blocking plate positioned in front of an overlapping light spot array, according to an embodiment of the present invention. Figure 6 and Figure 8 As shown, when light-blocking plates smaller than one light spot diameter are placed in different relative positions, the results are the same. That is, when the light spots do not overlap, the limiting resolution is the size of one light spot diameter. Figure 7 and Figure 9 As shown, for a light-blocking plate with a diameter smaller than one light spot diameter, more optical information data can be obtained from an array of overlapping light spots with smaller spot spacing. This data can then be processed by an image inversion module to determine the specific shape of the sample under test, achieving a resolution smaller than one light spot diameter. The system resolution is related to the spot spacing; the smaller the spot spacing, the higher the system resolution. When the spacing between two light spots is smaller than the spot diameter, the spots will overlap. Under the premise of incomplete spot overlap, the higher the overlap degree, the higher the system resolution. Here, W / M^2 represents watts per square meter, and MHz represents megahertz.

[0053] The super-resolution microscopic imaging system based on spot overlap provided in this embodiment achieves super-resolution by setting up a spot adjustment module and a heterodyne interference module, thereby dynamically adjusting the spot spacing of the spot array by adjusting the radio frequency drive signal to adjust the microscopic imaging resolution, avoiding the limitation of microscopic imaging resolution, and thus achieving super-resolution.

[0054] Although embodiments of the invention have been described in conjunction with the accompanying drawings, those skilled in the art can make various modifications and variations without departing from the spirit and scope of the invention, and such modifications and variations all fall within the scope defined by the invention.

Claims

1. A super-resolution microscopic imaging system based on spot overlap, characterized in that, include: Laser module, used to generate laser beam; A beam adjustment module is used to generate a radio frequency drive signal according to a beam adjustment command; the beam adjustment command is used to indicate the beam spacing. The heterodyne interference module is used to split the laser beam into a first laser beam and a second laser beam, and then shape the first laser beam into a reference light source; the spot adjustment module is also used to modulate the second laser beam according to the radio frequency driving signal to form a spot array; the heterodyne interference module is also used to combine the reference light source and the spot array to generate heterodyne interference, forming a combined beam that illuminates the sample to be tested; The spectral data receiving module is used to collect the optical data generated after the sample under test reflects the combined beam; The image inversion module is used to perform image inversion processing on the optical data to generate imaging data of the sample to be tested.

2. The system according to claim 1, characterized in that, When the distance between adjacent light spots is less than the diameter of the light spot, the adjacent light spots overlap, and the higher the degree of overlap of the light spots, the higher the resolution of the system.

3. The system according to claim 1, characterized in that, The spot adjustment module includes an RF drive module and a spatial light modulation module; the RF drive module is used to receive spot adjustment instructions and parse the RF frequency difference in the spot adjustment instructions, so as to generate an RF drive signal according to the RF frequency difference and send it to the spatial light modulation module. The spatial light modulation module is disposed in the optical path of the second laser beam and is used to introduce a frequency shift into the second laser beam according to the received radio frequency driving signal to generate a spot array with a target spot spacing.

4. The system according to claim 3, characterized in that, The greater the radio frequency difference, the larger the target spot spacing; the smaller the radio frequency difference, the smaller the target spot spacing.

5. The system according to any one of claims 1 to 4, characterized in that, The system also includes a galvanometer scanning module; the galvanometer scanning module is used to move the combined beam in the horizontal / vertical direction to perform a two-dimensional scan of the sample under test.

6. The system according to claim 5, characterized in that, The galvanometer scanning module includes a horizontal galvanometer and a vertical galvanometer; the system also includes a first lens, a second lens, and a first microscope objective; the combined beam is reflected sequentially by the horizontal galvanometer, the first lens (beam contraction), the second lens (beam expansion), and the vertical galvanometer, and then passes through the first microscope objective to form an outgoing beam that illuminates the sample to be tested.

7. The system according to claim 6, characterized in that, The galvanometer scanning module further includes a scanning control unit; the scanning control unit is used to drive the horizontal galvanometer and the vertical galvanometer to deflect respectively, so as to control the emitted beam to move in a two-dimensional plane according to a preset scanning mode; the preset scanning mode includes at least one of raster scanning, vector scanning or random point scanning.

8. The system according to any one of claims 1 to 4, characterized in that, The spectral data receiving module includes a multi-pixel detector; the multi-pixel detector includes multiple rows and columns of pixels.

9. The system according to claim 8, characterized in that, The multi-pixel detector is a single-pixel detector; the single-pixel detector is one of a photodiode, an avalanche photodiode, or a photomultiplier tube; or, the multi-pixel detector is an area array imaging detector; the area array imaging detector is one of a CCD detector, a CMOS detector, or a two-dimensional photoelectric sensor with multiple rows and columns of pixels.

10. The system according to any one of claims 1 to 4, characterized in that, The image inversion module includes a data acquisition card and a high-speed programmable logic device; the data acquisition card is used to sample and perform analog-to-digital conversion on the optical data to obtain processed optical data; the high-speed programmable logic device is used to convert the processed optical data to the frequency domain and then perform image reconstruction to generate imaging data of the sample under test.

Images