A large-field high-resolution near-field laminated microscopic imaging system and an imaging method thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- PEKING UNIV
- Filing Date
- 2025-07-23
- Publication Date
- 2026-07-21
Smart Images
Figure CN120870075B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to microscopic imaging technology, specifically to a large field-of-view, high-resolution near-field stacked microscopic imaging system and its imaging method. Background Technology
[0002] Currently, ptychography is an important microscopic imaging technique. By scanning a sample and recording multiple overlapping diffraction patterns, it reconstructs the sample's phase and amplitude information using iterative algorithms. It does not require a stable reference beam as in holography, and low-coherence light sources can be used for sample illumination. It has been widely applied in fields such as X-ray imaging and electron microscopy. In the field of bio-optical imaging, several mature ptychography techniques have been developed, such as conventional ptychography and coded ptychography. However, traditional ptychography techniques are limited by low resolution and low throughput, hindering their widespread application in biomedical optical imaging.
[0003] Combining synthetic aperture technology with layered imaging can effectively solve the above problems. Synthetic aperture technology is an imaging method that uses physical motion or array arrangement to superimpose signals from multiple small-aperture sensors in different orientations to synthesize an equivalent large-aperture image. Its core lies in overcoming the limitation of physical aperture size on resolution and achieving super-resolution imaging. Many layered imaging techniques, such as Fourier ptychography, now combine synthetic aperture and layered imaging, achieving both high-resolution and quantitative phase imaging, and have gained widespread attention in the field of biomedical optical imaging in recent years.
[0004] Because Fourier ptychographic microscopy (FPM) uses a far-field illumination architecture with light-emitting diodes (LEDs) as the illumination source, it presents two problems: first, compared to lasers, LED light sources are weaker, resulting in longer exposure times for the acquisition camera; second, the ability to synthesize apertures under far-field illumination is limited, making it difficult to further improve resolution. Summary of the Invention
[0005] To address the problems existing in the prior art, this invention proposes a large field-of-view, high-resolution near-field stacked microscopy imaging system and its imaging method, which reduces the exposure time of the acquisition camera and improves the synthetic aperture capability, realizing high spatiotemporal resolution stacked microscopy imaging with a simple architecture, and further promoting the application and development of stacked imaging technology.
[0006] One object of the present invention is to provide a large field of view, high resolution near-field stacked microscopy imaging system.
[0007] The large field-of-view, high-resolution near-field stacked microscopy imaging system of the present invention includes: a laser source device, a laser scanning device, an excitation illumination device, a fluorescence acquisition device, and an imaging device; wherein, the excitation illumination device includes an excitation objective and a fluorescent thin film; a sample is placed on the front side of the substrate, and a fluorescent thin film is formed on the back side of the substrate, the fluorescent thin film being located at the focal plane of the excitation objective;
[0008] A laser source emits a laser beam, which is then directed by a laser scanning device to achieve subsequent two-dimensional scanning excitation. The laser beam is relayed through a first 4-f system to an excitation objective lens, which focuses it as excitation light onto a fluorescent film, generating fluorescence. The laser scanning device controls the position of the excitation point on the fluorescent film, sequentially exciting fluorescence at different locations. Multiple excitation points form a group, and the area formed by each group is ring-shaped. The radius of the rings formed by multiple groups of excitation points gradually increases from the inside to the outside, i.e., a ring scan. Fluorescence penetrates the substrate and is incident on the sample from the back. The fluorescence is scattered through the sample, generating signal light carrying information about the sample's structure. Combined with the scanning module, this allows for sequential excitation of different locations on the fluorescent film, illuminating the sample and acquiring images from different positions. When the laser beam is incident on the fluorescent film as excitation light, electrons in the fluorescent film absorb the laser energy and transition from their ground state... The electron transitions to the excited state, and the excitation rate is positively correlated with the intensity of the incident laser light. Electrons radiatively transition back to the ground state from the excited state, releasing fluorescence. The fluorescence intensity is stronger, exceeding 5 mW, thus shortening the exposure time. The distance between the fluorescent film and the sample is on the order of millimeters, which allows the fluorescence generated by the excitation light to be close to the sample, increasing the upper limit of the synthesis aperture. This is beneficial for increasing the synthesis aperture, and the imaging resolution is related to the size of the synthesis aperture; the larger the synthesis aperture, the higher the resolution. Near-field illumination is achieved, making it easier to increase the synthesis aperture, thereby improving the imaging resolution. The fluorescence acquisition device collects the signal light and transmits it to the imaging device. The imaging device acquires low-resolution images, one low-resolution image for each excitation point. The computer processes and recovers the data, realizing synthesis aperture and stacked imaging, and combining multiple low-resolution images into a high-resolution image with high imaging resolution.
[0009] The laser source device includes an optical fiber, a collimating objective lens, a half-wave plate, and a polarizing beam splitter (PBS). After the laser beam exits the optical fiber, it passes through the collimating objective lens to convert the diverging laser beam into a parallel beam. The intensity of the transmitted laser beam is then adjusted by a combination of the half-wave plate and the polarizing beam splitter. The intensity of the laser beam transmitted through the PBS is adjusted by rotating the half-wave plate.
[0010] The laser scanning device includes a scanning module, a scanning lens, and a sleeve lens. After passing through the scanning module, the laser beam's emission direction is precisely controlled to achieve subsequent two-dimensional scanning excitation. To compensate for aberrations introduced by scanning, the laser beam exits the scanning module and enters the scanning lens and sleeve lens. The combination of the scanning lens and sleeve lens effectively corrects aberrations and ensures image quality. The scanning module employs a specially designed metal adapter at the sample placement area for precise adjustment of the sample plane's position. A gantry structure is also designed for the displacement stage, resulting in more rational component placement and significant space savings. The scanning module uses a scanning galvanometer. Because ordinary spherical lenses cause field curvature problems such as increased edge spot size during scanning, a scanning lens (SL) is used to achieve high-precision focusing and linear scanning, while suppressing various aberrations generated during dynamic scanning. The sleeve lens (TL) is used in conjunction with an infinity-correcting objective lens to construct an infinity-correcting optical system. In an infinity-corrected optical system, light rays passing through the objective lens from the object plane are not imaged by the objective lens, but instead enter the telescopic lens as a parallel beam at infinity. The telescopic lens forms an intermediate image, and the telescopic lens (TL) focuses on correcting chromatic aberration and residual aberration.
[0011] The scanning module is connected to the data acquisition card (DAQ), which is connected to the computer. The data acquisition card converts analog signals to digital signals and vice versa. The computer then accurately controls the excitation position of the laser spot on the fluorescent film.
[0012] The first 4-f system includes a first lens L1 and a second lens L2.
[0013] The fluorescent thin film is made of nanocrystals. Liquid nanocrystals are uniformly coated onto the back side of the substrate using a spin coater, and then a fluorescent thin film with a thickness of 1–100 nm is deposited on the back side of the substrate. The substrate is made of a transparent material with a thickness of 0.1–1 mm.
[0014] The fluorescence acquisition device includes a collecting objective, a third lens, and a filter. The sample is placed on a five-axis displacement stage and is located at the focal plane of the collecting objective. The collecting objective is responsible for collecting the signal light transmitted through the sample. The collecting objective and the third lens form a second 4-f system, which propagates the signal light completely backward. A filter is placed between the collecting objective and the third lens to filter out the excitation light and allow only the signal light carrying the sample's structural information to pass through.
[0015] The camera imaging device includes a third 4-f system and a camera; wherein, the fourth lens and the fifth lens form the third 4-f system, which together with the second 4-f system relays the sample surface to the photosensitive surface of the camera, and the camera surface is placed at the focal plane of the fifth lens to ensure that the camera can acquire complete imaging information.
[0016] In existing technologies, samples are fluorescently labeled, and the diameter of the excitation light spot limits the resolution; the smaller the spot, the higher the resolution. This invention places a fluorescent film on the back of the sample. The resolution is affected by the size of the synthesized aperture after the fluorescent sample is collected by the collecting objective. The resolution can be improved by increasing the maximum radius of the annular excitation fluorescence, thereby increasing the synthesized numerical aperture. The maximum radius of the annular excitation fluorescence is limited by the numerical aperture of the collecting objective.
[0017] The excitation light intensity is 30–40 mW, and the fluorescence intensity is greater than 5 mW, approximately 5–10 mW. The distance between the conventional LED light source and the sample is much greater than the distance between the thin film and the sample in this invention, significantly reducing light loss during propagation in the air. The fluorescence intensity of this invention is positively correlated with the excitation light intensity, thus achieving stronger light intensity and reduced exposure time. Further increasing the excitation light intensity to enhance the fluorescence intensity further shortens the exposure time required by the camera in a single acquisition. Shorter exposure times effectively reduce image noise and, for imaging live cells, effectively reduce motion blur.
[0018] The wavelength of the excitation light is determined by the inherent energy level structure of the fluorescent thin film material itself.
[0019] Another objective of this invention is to propose a near-field stacked synthetic aperture super-resolution microscopy method based on fluorescent thin films.
[0020] The present invention provides a near-field stacked synthetic aperture super-resolution microscopy method based on fluorescent thin films, comprising the following steps:
[0021] 1) A fluorescent thin film is formed on the back side of the substrate, and the sample is placed on the front side of the substrate. The fluorescent thin film is located at the focal plane of the excitation objective.
[0022] 2) The laser source device emits laser light, and the laser light is controlled by the laser scanning device to achieve subsequent two-dimensional scanning excitation;
[0023] 3) The laser light is relayed to the excitation objective via the first 4-f system, and then focused by the excitation objective as excitation light onto the fluorescent film, generating fluorescence. When the laser light is incident on the fluorescent film as excitation light, electrons in the fluorescent film absorb the laser energy and transition from the ground state to the excited state. The excitation rate is positively correlated with the intensity of the incident laser light. Electrons radiate back from the excited state to the ground state, releasing fluorescence. The fluorescence intensity is stronger, thus shortening the exposure time. Furthermore, the imaging resolution is related to the size of the synthetic aperture. The larger the synthetic aperture, the higher the resolution. Near-field illumination is achieved, which is more conducive to increasing the size of the synthetic aperture, thereby improving the imaging resolution.
[0024] 4) The laser scanning device controls the position of the excitation point on the fluorescent film, and sequentially excites the fluorescence at different positions on the fluorescent film. Multiple excitation points form a group, and the area formed by a group of excitation points is ring-shaped. The radius of the ring formed by multiple groups of excitation points gradually increases from the inside to the outside.
[0025] 5) Fluorescence penetrates the substrate and is incident on the sample from the back. The fluorescence penetrates the sample and is scattered to generate signal light carrying the sample's structural information. Combined with the scanning module, different positions of the fluorescent film are excited accordingly, and the sample is illuminated and images are acquired from different positions. The fluorescence acquisition device collects the signal light and transmits it to the imaging device.
[0026] 6) The imaging device acquires low-resolution images. Each excitation point acquires a low-resolution image. The computer processes and recovers the data to achieve synthetic aperture and stacked imaging, combining multiple low-resolution images into a high-resolution image with high imaging resolution.
[0027] Advantages of this invention:
[0028] The imaging system constructed in this invention has two advantages for imaging biological samples: First, it uses laser-excited fluorescence to illuminate the sample, which provides higher light intensity and improves imaging quality compared to traditional LED light sources, while also shortening the exposure time required by the camera. Second, the imaging resolution is related to the size of the synthetic aperture; the larger the synthetic aperture, the higher the resolution. Compared to the far-field illumination of traditional LEDs, the fluorescence illumination scheme of this invention can achieve near-field illumination, making it easier to increase the size of the synthetic aperture. Near-field illumination can further expand the size of the synthetic aperture and improve imaging resolution compared to traditional far-field illumination methods. Attached Figure Description
[0029] Figure 1 This is a schematic diagram of an embodiment of the large field-of-view, high-resolution near-field stacked microscopy imaging system of the present invention;
[0030] Figure 2 This is a partial optical path diagram of fluorescence excitation illumination in an embodiment of the large field-of-view, high-resolution near-field stacked microscopy imaging system of the present invention. Detailed Implementation
[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.
[0032] like Figure 1 As shown, the large field-of-view, high-resolution near-field stacked microscopy imaging system of this embodiment includes: a laser source device, a laser scanning device, an excitation illumination device, a fluorescence acquisition device, and an imaging device; wherein, the excitation illumination device includes an excitation objective lens O2 and a fluorescent thin film; a sample is placed on the front side of the substrate, and a fluorescent thin film is formed on the back side of the substrate, with the fluorescent thin film located at the focal plane of the excitation objective lens;
[0033] The laser source device includes an optical fiber F, a collimating objective lens O1, a half-wave plate P, and a polarizing beam splitter (PBS). After the laser beam is emitted from the optical fiber F, it passes through the collimating objective lens O1 to convert the diverging laser beam into a parallel laser beam. The combination of the half-wave plate P and the polarizing beam splitter PBS achieves arbitrary intensity ratios, and the transmitted laser beam achieves continuous intensity adjustment. The intensity of the laser beam transmitted through the polarizing beam splitter PBS is adjusted by rotating the half-wave plate.
[0034] The laser scanning device includes a scanning module S, a scanning lens SL, and a sleeve lens TL. After passing through the scanning module S, the laser beam's emission direction is precisely controlled by the module to achieve subsequent two-dimensional scanning excitation. To compensate for aberrations introduced by the scanning, the laser beam exiting the scanning module S enters the scanning lens SL and the sleeve lens. The combination of the scanning lens SL and the sleeve lens TL effectively corrects aberrations, suppresses various aberrations generated during dynamic scanning, and ensures image quality. The sleeve lens TL, along with the excitation objective lens O2 and the collection objective lens O3 of the infinity correction objective, forms an infinity correction optical system. In this system, light rays passing through the objective lens from the object surface are not imaged by the objective lens but instead enter the sleeve lens TL as a parallel beam at infinity, forming an intermediate image. The sleeve lens TL effectively corrects chromatic aberration and residual aberrations.
[0035] The scanning module is connected to the data acquisition card, which is connected to the computer. The data acquisition card converts analog signals to digital signals and vice versa. The computer then accurately controls the excitation position of the laser spot on the fluorescent film.
[0036] The first 4-f system includes a first lens L1 and a second lens L2;
[0037] The fluorescence acquisition device includes a collecting objective lens O3, a third lens L3, and a filter FI. The sample is placed on a five-axis displacement stage and is located at the focal plane of the collecting objective lens. The collecting objective lens O3 is responsible for collecting the signal light transmitted through the sample. The collecting objective lens and the third lens form a second 4-f system to propagate the signal light completely backward. The filter FI is placed between the collecting objective lens O3 and the third lens L3 to filter out the excitation light and allow only the signal light carrying the sample structural information to pass through.
[0038] The camera imaging device includes a third 4-f system and a camera C; wherein, the fourth lens L4 and the fifth lens L5 form the third 4-f system, which together with the second 4-f system relays the sample surface to the photosensitive surface of the camera C. The camera surface is located at the focal plane of the fifth lens L5, ensuring that the camera C can acquire complete imaging information. The camera C adopts a scientific-grade complementary metal-oxide-semiconductor (SCMOS) camera. Figure 1 M1 to M10 are the first to tenth reflecting mirrors, respectively.
[0039] The near-field stacked synthetic aperture super-resolution microscopy method based on fluorescent thin films in this embodiment includes the following steps:
[0040] 1) Prepare a glass slide that directly contacts the sample as a substrate. Use a spin coater to evenly coat the nanocrystalline liquid luminescent material onto the back of the glass slide. After drying, a fluorescent film is formed. Place the sample on the front of the substrate. The fluorescent film is located at the focal plane of the excitation objective lens. Place the sample on an electrically adjustable five-axis displacement stage to adjust its three-dimensional spatial position and pitch angle.
[0041] 2) The laser source device emits a continuous laser beam at 473nm. The excitation light is controlled by a laser scanning device to adjust the emission direction of the laser beam in order to achieve subsequent two-dimensional scanning excitation.
[0042] 3) The laser light is relayed to the excitation objective via the first 4-f system, and then focused by the excitation objective onto the nanocrystalline fluorescent film as excitation light, generating 561nm green fluorescence. When the laser light is incident on the fluorescent film as excitation light, electrons in the fluorescent film absorb the laser energy and transition from the ground state to the excited state. The excitation rate is affected by the intensity of the incident laser light. Electrons radiatively transition from the excited state back to the ground state, releasing fluorescence, which is stronger, thus shortening the exposure time.
[0043] The distance between the fluorescent film and the sample is on the order of millimeters in thickness, which makes the fluorescence generated by the excitation light on the fluorescent film close to the sample, thereby improving the imaging resolution;
[0044] 4) The laser scanning device controls the position of the excitation point on the fluorescent film, and sequentially excites the fluorescence at different positions on the fluorescent film. Multiple excitation points form a group, and the area formed by a group of excitation points is ring-shaped. The radius of the ring formed by multiple groups of excitation points gradually increases from the inside to the outside.
[0045] 5) Fluorescence penetrates the substrate and is incident on the sample from the back. The fluorescence penetrates the sample and is scattered to generate signal light carrying the sample's structural information. Combined with the scanning module, different positions of the fluorescent film are excited accordingly, and the sample is illuminated and images are acquired from different positions. The fluorescence acquisition device collects the signal light and transmits it to the imaging device.
[0046] 6) The imaging device acquires low-resolution images. Each excitation point acquires a low-resolution image. The computer processes and recovers the data to achieve synthetic aperture and stacked imaging, combining multiple low-resolution images into a high-resolution image with short exposure time and high imaging resolution.
[0047] In the field of optical imaging research, achieving super-resolution and further improving resolution are unavoidable issues.
[0048] According to Rayleigh's criterion, resolution d RayleighThe calculation formula is Where λ is the wavelength of light, NA is the numerical aperture of the objective lens, nsinθ is the numerical aperture, n is the ambient refractive index, and θ is the maximum angle of incidence of the objective lens.
[0049] The synthetic aperture method involves combining multiple sub-apertures into a larger aperture through circular scanning, thereby further expanding the numerical aperture (NA) of the objective lens and improving imaging resolution.
[0050] A mature imaging method is Fourier Transform Imaging (FPM). The problem with this architecture is that if an objective lens with a larger numerical aperture (NA) is selected, the maximum illumination radius to achieve the effective synthetic aperture of the ring scan will be larger as the LED illumination position is farther from the sample. In addition, the light source will be farther from the sample, and the light intensity will be weakened.
[0051] like Figure 2 As shown, the laser light is focused by the excitation objective O2 onto the fluorescent film, emitting fluorescence. The fluorescence is projected onto the platen, and the collection objective O3 collects the signal light transmitted through the sample. This invention employs a method of depositing a fluorescent film beneath the sample, significantly shortening the distance between the illumination source and the imaging sample. This solves the problem in the original architecture where further increasing the numerical aperture (NA) requires a substantial increase in the lamp plate area, leading to a longer illumination distance and reduced light intensity, which in turn prevents imaging due to insufficient light intensity. The numerical aperture of the collection objective in this invention is 0.13. By replacing the objective with a larger numerical aperture (NA) that was unusable in the original architecture, this invention achieves a higher resolution through the synthesis of the aperture.
[0052] Finally, it should be noted that the purpose of disclosing the embodiments is to help further understand the present invention. However, those skilled in the art will understand that various substitutions and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the present invention should not be limited to the content disclosed in the embodiments, and the scope of protection of the present invention is defined by the claims.
Claims
1. A large field-of-view, high-resolution near-field stacked microscopy imaging system, characterized in that, The near-field stacked microscopy imaging system includes: a laser source device, a laser scanning device, an excitation illumination device, a fluorescence acquisition device, and an imaging device; wherein, the excitation illumination device includes an excitation objective and a fluorescent thin film; the sample is placed on the front side of the substrate, and a fluorescent thin film is formed on the back side of the substrate, with the fluorescent thin film located at the focal plane of the excitation objective; A laser source emits a laser beam, which is then directed by a laser scanning device to achieve subsequent two-dimensional scanning excitation. The laser beam is relayed through a first 4-f system to an excitation objective lens, which focuses it onto a fluorescent thin film as excitation light, generating fluorescence. The laser scanning device controls the position of the excitation point on the fluorescent thin film, sequentially exciting fluorescence at different locations. Multiple excitation points form a group, and the area formed by each group is ring-shaped. The radius of the rings formed by multiple groups of excitation points gradually increases from the inside to the outside, i.e., a ring scan. Fluorescence penetrates the substrate and is incident on the sample from the back. The fluorescence is scattered through the sample, generating signal light carrying information about the sample's structure. Combined with the scanning module, this signal light is used to monitor the fluorescence. The optical thin film is excited at different locations, illuminating the sample and acquiring images from different positions. When the laser is used as the excitation light and incident on the fluorescent thin film, it releases fluorescence. The intensity of the fluorescence light is high, thus shortening the exposure time. The distance between the fluorescent thin film and the sample is on the order of millimeters, which makes the fluorescence generated by the excitation light on the fluorescent thin film close to the sample, increasing the synthesis aperture and thus improving the imaging resolution. The fluorescence acquisition device collects the signal light and transmits it to the imaging device. The imaging device acquires low-resolution images, one low-resolution image for each excitation point. The computer processes and restores the data to achieve synthesis aperture and stacked imaging, combining multiple low-resolution images into a high-resolution image with high imaging resolution.
2. The near-field stacked microscopy imaging system as described in claim 1, characterized in that, The laser source device includes an optical fiber, a collimating objective lens, a half-wave plate, and a polarizing beam splitter. After the laser beam is emitted from the optical fiber, the collimating objective lens converts the diverging laser beam into a parallel laser beam, and the combination of the half-wave plate and the polarizing beam splitter adjusts the intensity of the transmitted laser beam.
3. The near-field stacked microscopy imaging system as described in claim 1, characterized in that, The laser scanning device includes a scanning module, a scanning lens, and a sleeve lens. The laser passes through the scanning module, which precisely controls the direction of the laser emission. After exiting the scanning module, the laser enters the scanning lens and the sleeve lens. The combination of the scanning lens and the sleeve lens effectively corrects aberrations.
4. The near-field stacked microscopy imaging system as described in claim 1, characterized in that, The fluorescent thin film is made of nanocrystals.
5. The near-field stacked microscopy imaging system as described in claim 1, characterized in that, The thickness of the fluorescent film is 1~100nm.
6. The near-field stacked microscopy imaging system as described in claim 1, characterized in that, The fluorescence acquisition device includes a collecting objective, a third lens, and a filter. The sample is placed on a five-axis displacement stage and is located at the focal plane of the collecting objective. The collecting objective collects the signal light transmitted through the sample. The collecting objective and the third lens form a second 4-f system to propagate the signal light completely backward. A filter is placed between the collecting objective and the third lens to filter out the excitation light and allow only the signal light carrying the sample's structural information to pass through.
7. The near-field stacked microscopy imaging system as described in claim 1, characterized in that, The imaging device includes a third 4-f system and a camera; wherein, the fourth lens and the fifth lens form the third 4-f system, which together with the second 4-f system relays the sample surface to the photosensitive surface of the camera, and the camera surface is located at the focal plane of the fifth lens.
8. An imaging method for the near-field stacked microscopy system as described in claim 1, characterized in that, The imaging method includes the following steps: 1) A fluorescent thin film is formed on the back side of the substrate, and the sample is placed on the front side of the substrate. The fluorescent thin film is located at the focal plane of the excitation objective lens. 2) The laser source device emits laser light, and the laser emission direction is controlled by the laser scanning device; 3) The laser light is relayed to the excitation objective lens through the first 4-f system. The excitation objective lens focuses the laser light onto the fluorescent film to generate fluorescence. The laser light is incident on the fluorescent film as the excitation light and releases fluorescence. The fluorescence intensity is strong, which shortens the exposure time. Furthermore, the distance between the fluorescent film and the sample is on the order of millimeters of the substrate thickness, which makes the fluorescence generated by the excitation light on the fluorescent film close to the sample, thereby improving the imaging resolution. 4) The laser scanning device controls the position of the excitation point on the fluorescent film, and sequentially excites the fluorescence at different positions on the fluorescent film. Multiple excitation points form a group, and the area formed by a group of excitation points is ring-shaped. The radius of the ring formed by multiple groups of excitation points gradually increases from the inside to the outside. 5) Fluorescence penetrates the substrate and is incident on the sample from the back. The fluorescence penetrates the sample and is scattered to generate signal light carrying the sample's structural information. Combined with the scanning module, different positions of the fluorescent film are excited accordingly, and the sample is illuminated and images are acquired from different positions. The fluorescence acquisition device collects the signal light and transmits it to the imaging device. 6) The imaging device acquires low-resolution images. Each excitation point acquires a low-resolution image. The computer processes and recovers the data to achieve synthetic aperture and stacked imaging, combining multiple low-resolution images into a high-resolution image with high imaging resolution.