Fluorescence imaging system and method

Abstract

The invention relates to the technical field of optical microscopic imaging, and discloses a fluorescence imaging system and method. The system comprises a multi-color light source module used for emitting exciting light with different wavelengths, and the exciting light with different wavelengths is used for marking different components in a sample to be detected; the apochromatic illumination module is connected with the multi-color light source module and used for carrying out light beam processing on the exciting light based on a preset illumination mode to generate a target illumination light beam; the multi-channel imaging module is connected with the apochromatic illumination module and is used for collecting fluorescence signals emitted after the target illumination light beam irradiates the to-be-detected sample and separating the fluorescence signals according to the fluorescence spectrum of each component to obtain a plurality of imaging channels; and the image processing module is connected with the multi-channel imaging module and is used for receiving the imaging data from the plurality of imaging channels and fusing the imaging data to obtain a target fluorescence image. By implementing the technical scheme provided by the invention, efficient synchronous imaging of different components in a multicolor fluorescence labeled sample is realized.

Description

Technical Field

[0001] The present invention relates to the field of optical microscopy imaging technology, and particularly to a fluorescence imaging system and method. Background Art

[0002] Due to its high sensitivity and specificity, fluorescence imaging technology is widely used in the fields of biomedicine, materials science, and environmental monitoring for multi-component synchronous detection and dynamic process observation. Current fluorescence imaging systems usually use a single-wavelength or limited-bandwidth excitation light source, combined with a filter for spectral splitting to achieve multi-component identification.

[0003] However, with the increasing demand for multi-parameter analysis of complex samples, when the system needs to simultaneously excite multiple fluorescent markers, chromatic aberration is easily generated in the optical path for different-wavelength excitation lights, resulting in inconsistent spatial distribution of the illumination beam, and reducing the co-localization accuracy of multi-channel imaging. Summary of the Invention

[0004] In view of this, the present invention provides a fluorescence imaging system and method to solve the problem that when multi-wavelength excitation is performed, the spatial distribution of the illumination beam is uneven due to chromatic aberration in the optical path, thereby reducing the co-localization accuracy of multi-channel imaging.

[0005] In a first aspect, the present invention provides a fluorescence imaging system, including: a multi-color light source module for emitting excitation lights of different wavelengths, and the excitation lights of different wavelengths are used to label different components in a sample to be measured; an apochromatic illumination module optically connected to the multi-color light source module for performing beam processing on the excitation lights based on a preset illumination mode to generate a target illumination beam; a multi-channel imaging module optically connected to the apochromatic illumination module for collecting fluorescence signals emitted after the target illumination beam irradiates the sample to be measured, and separating the fluorescence signals according to the fluorescence spectra of each component to obtain a plurality of imaging channels; and an image processing module communicatively connected to the multi-channel imaging module for receiving imaging data from the plurality of imaging channels, and performing image fusion on the imaging data to obtain a target fluorescence image.

[0006] The fluorescence imaging system provided by the embodiments of the present invention realizes high-precision uniform illumination of excitation lights of different wavelengths through the synergistic effect of the multi-color light source module and the apochromatic illumination module, ensuring the stability and consistency of multi-component sample labeling. Combining with the spectral separation ability of the multi-channel imaging module, fluorescence signal crosstalk is effectively avoided, and the imaging resolution and specificity are significantly improved. At the same time, the image processing module finally generates a target fluorescence image with high signal-to-noise ratio and high contrast through intelligent fusion of multi-channel data, taking into account the accuracy and reliability of multi-component synchronous analysis while improving the detection efficiency.

[0007] In an alternative embodiment, the multi-color light source module includes: a first dichroic mirror for combining multiple excitation lights to obtain a combined light beam; a reflector disposed after the first dichroic mirror for guiding the combined light beam to an optical fiber; and an optical fiber disposed after the reflector for transmitting the combined light beam to an apochromatic illumination module.

[0008] The fluorescence imaging system provided by the embodiments of the present invention efficiently achieves precise beam combination of multi-wavelength excitation lights through the first dichroic mirror, simplifies the optical path structure and reduces the energy loss between multiple light sources. The collaborative design of the reflector and the optical fiber further optimizes the optical path transmission direction, enhancing the flexibility and stability of the excitation light guidance. The high-efficiency transmission characteristics of the optical fiber not only reduce the beam divergence and energy attenuation, but also effectively maintain the spectral characteristics and spatial uniformity of the excitation light, providing a high-quality and low-distortion excitation beam for the subsequent apochromatic illumination module.

[0009] In an alternative embodiment, the different components include cell structures and single particles; the multiple excitation lights include visible light bands and near-infrared light bands of different wavelengths, where the visible light bands are used for cell structure labeling and the near-infrared light bands are used for long-term single particle labeling.

[0010] The fluorescence imaging system provided by the embodiments of the present invention realizes efficient sorting and labeling of cell structures and single particles through the differential application of visible light and near-infrared light bands. The visible light band provides high-resolution labeling for cell structures, while the near-infrared light, due to its deep penetration and low phototoxicity, can support long-term stable tracking of single particles, significantly improving the synchronous observation ability of dynamic biological processes, while reducing the photobleaching effect and ensuring sample viability.

[0011] In an alternative embodiment, the preset illumination modes include at least one of a Kohler illumination optical path mode, a structured light illumination optical path mode, and a total internal reflection illumination mode; the Kohler illumination optical path mode is used for zero-order filtering of the excitation light; the structured light illumination optical path mode performs structured modulation processing on the excitation light; and the total internal reflection illumination mode is used for angular selective filtering of the excitation light.

[0012] The fluorescence imaging system provided by the embodiments of the present invention can flexibly switch the optical path strategy according to the sample characteristics by integrating the Kohler illumination, structured light illumination, and total internal reflection illumination modes. The Kohler mode ensures the uniformity of the excitation light and zero-order noise suppression, the structured light mode improves the imaging resolution through spatial modulation, and the total internal reflection mode achieves high signal-to-noise ratio imaging for surface particles. The multi-mode collaboration greatly expands the applicable scenarios of the system, taking into account the requirements of wide-field and super-resolution imaging.

[0013] In an alternative embodiment, the multi-channel imaging module includes: a microscope objective; a filter set including an excitation filter, a second dichroic mirror, and an emission filter, the second dichroic mirror being disposed behind the excitation filter, and the emission filter being disposed behind the second dichroic mirror; the excitation filter is configured to filter a target illumination beam to obtain a first illumination beam; the second dichroic mirror is configured to reflect the first illumination beam and irradiate the reflected first illumination beam onto a sample to be measured through the microscope objective to excite a corresponding fluorescence signal of the sample to be measured; the second dichroic mirror is further configured to receive the fluorescence signal collected by the microscope objective and project the fluorescence signal to obtain a transmitted signal; the emission filter is configured to filter the transmitted signal to obtain a target fluorescence signal with a preset fluorescence wavelength.

[0014] In the fluorescence imaging system provided by the embodiments of the present invention, the hierarchical design of the filter set and the dichroic mirror realizes strict spectral separation of the excitation light and the fluorescence, effectively suppressing background noise and crosstalk between channels. The efficient cooperation between the microscope objective and the optical path reflection ensures high-throughput collection and accurate projection of the fluorescence signal, providing a low-distortion and high-fidelity optical signal input for multi-component parallel imaging.

[0015] In an alternative embodiment, the multi-channel imaging module further includes: a culture unit disposed on the sample side of the microscope objective for culturing a sample according to the physiological activity of the sample to be measured; a displacement component disposed inside the culture unit for adjusting the position of the sample to be measured.

[0016] In the fluorescence imaging system provided by the embodiments of the present invention, the integrated design of the culture unit and the displacement component can maintain the physiological activity environment (such as temperature, humidity) of the sample during the imaging process and support real-time position adjustment. This design not only avoids the loss of activity in traditional ex vivo imaging but also improves the observation accuracy and experimental repeatability in complex physiological scenarios (such as cell migration, particle movement).

[0017] In an alternative embodiment, the target fluorescence signal includes a cell fluorescence signal and a particle fluorescence signal, and the multiple imaging channels include a cell structure imaging channel and a particle imaging channel; the cell structure imaging channel is configured to generate a cell structure fluorescence image based on the cell fluorescence signal; the particle imaging channel is configured to generate a particle fluorescence image based on the particle fluorescence signal; a modulation device is disposed in the particle imaging channel, and the modulation device is configured to modulate the particle fluorescence signal and control the position information of the particle light spot corresponding to the particle fluorescence signal in the particle fluorescence image so that the position information presents an axial shape.

[0018] The fluorescence imaging system provided by the embodiments of the present invention realizes precise split-track imaging of cells and particles through the independent design of the cell structure imaging channel and the particle imaging channel, effectively avoiding signal cross-interference. The modulation device introduced in the particle channel extends the traditional two-dimensional imaging to three-dimensional spatial positioning by regulating the spot position information and encoding it as an axial shape feature, significantly improving the axial resolution ability of single particles. At the same time, the direct correlation between the spot morphology and the axial position simplifies the later data processing process, provides high-precision and high-sensitivity in-situ three-dimensional information for dynamic tracking and quantitative analysis, and takes into account the accuracy of multi-target synchronous observation and the depth of complex biological process analysis.

[0019] In an alternative embodiment, the modulation device is a cylindrical lens or a phase plate; the cylindrical lens is used to modulate the particle spot into an ellipse to determine the axial position of the particle based on the ratio of the major axis to the minor axis of the ellipse; the phase plate is used to modulate the particle spot into a double-helix structure to determine the axial position of the particle based on the distance and angle of the pair of spots.

[0020] The fluorescence imaging system provided by the embodiments of the present invention converts the axial position information of particles into spot morphology parameters (major axis / minor axis ratio or spot pair spacing / angle) through the modulation (ellipsoidization or double-helixization) of particle spots by a cylindrical lens or a phase plate, realizing three-dimensional positioning of single particles. This technology breaks through the limitations of traditional two-dimensional imaging, significantly improves the spatial resolution and axial positioning accuracy without complex layer scanning or interference optical paths, and is compatible with a variety of imaging modalities.

[0021] In an alternative embodiment, the image processing module is further configured to register the cell structure fluorescence image and the particle fluorescence image by using a pre-calibrated multi-color fluorescent microsphere sample, and fuse the registered particle fluorescence image into the cell structure fluorescence image to generate a target fluorescence image of the interaction between the particle and the cell.

[0022] The fluorescence imaging system provided by the embodiments of the present invention, based on the pre-calibration registration technology of multi-color fluorescent microspheres, can automatically correct the spatial offset between the cell and particle channels and generate an interaction image through algorithmic fusion, thus solving the common channel misalignment problem in multi-color imaging, ensuring the accuracy of cross-scale structure (such as organelles and nanoparticles) correlation analysis, and providing a reliable visualization tool for the study of cell-particle interaction mechanisms.

[0023] Second aspect, the present invention provides a fluorescence imaging method for the fluorescence imaging system of the first aspect or any corresponding embodiment thereof, including: emitting excitation lights of different wavelengths corresponding to different components based on the labeling information; performing apochromatic processing on the excitation lights to generate a target illumination beam that conforms to a preset illumination pattern; projecting the target illumination beam onto a sample to be measured, and collecting the fluorescence signals emitted by the sample to be measured upon excitation; performing spectral separation on the fluorescence signals based on the fluorescence spectral characteristics of different components to generate multi-channel imaging data; performing feature fusion processing on the multi-channel imaging data to generate a target fluorescence image.

[0024] Third aspect, the present invention provides a computer device, including: a memory and a processor, which are communicatively connected to each other. The memory stores computer instructions, and the processor executes the computer instructions to execute the fluorescence imaging method of the first aspect or any corresponding embodiment thereof.

[0025] Fourth aspect, the present invention provides a computer-readable storage medium, on which computer instructions are stored. The computer instructions are used to cause a computer to execute the fluorescence imaging method of the first aspect or any corresponding embodiment thereof.

[0026] Fifth aspect, the present invention provides a computer program product, including computer instructions, which are used to cause a computer to execute the fluorescence imaging method of the first aspect or any corresponding embodiment thereof. Description of the Drawings

[0027] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 is a schematic structural diagram of a fluorescence imaging system according to an embodiment of the present invention;

[0029] Figure 2 is a schematic structural diagram of another fluorescence imaging system according to an embodiment of the present invention;

[0030] Figure 3 is a schematic diagram of elliptical modulation of particle fluorescence according to an embodiment of the present invention;

[0031] Figure 4 is a schematic diagram of double helix modulation of particle fluorescence according to an embodiment of the present invention;

[0032] Figure 5Schematic diagram of the target fluorescence image showing the interaction between display particles and cells according to an embodiment of the present invention;

[0033] Figure 6 Flow schematic diagram of the fluorescence imaging method according to an embodiment of the present invention;

[0034] Figure 7 Hardware structure schematic diagram of the computer device according to an embodiment of the present invention. Detailed implementation manners

[0035] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0036] Biological processes at the subcellular scale, such as protein synthesis, cytoskeleton movement, and nucleolus division, etc., are of great significance for studying cell mechanisms and systems biology. Using a microscopy imaging system to localize and track single particles (such as molecules or particles) inside cells and analyze their kinetic characteristics and interactions with subcellular structures is a key means to reveal complex life activities such as cell communication, molecular transport, genetic information expression, and virus infection.

[0037] In terms of single-particle labeling, conventional fluorescent probes (such as organic fluorescent dyes, fluorescent proteins, etc.) have problems such as photobleaching, poor stability, and easy bleaching, and it is difficult to support long-term single-particle tracking. In recent years, emerging fluorescent probes such as carbon quantum dots, nanodiamonds, and lanthanide-doped upconversion nanoparticles (UCNPs) have become new choices for research due to their excellent photostability and anti-bleaching ability. However, the former two have limitations such as strong phototoxicity and large volume, and are not suitable for application in living cells. UCNPs can be excited by near-infrared light of 980 nm or 808 nm to emit visible light, and have advantages such as no photobleaching, no photoblinking, and low phototoxicity, and are very suitable for single-particle labeling and long-term observation.

[0038] In terms of imaging systems, to achieve real-time observation of single particles and cell structures, a microscopy imaging system needs to simultaneously possess the functions of precise single-particle positioning, tracking, and the ability to image cell structures with super-high resolution. In addition, the temporal resolution and spatial resolution of the imaging system are equally crucial. Current super-resolution microscopy imaging systems can meet the observation requirements of cells and subcellular structures, but there are still some limitations: First, related systems mainly operate in the visible light band, and have low transmittance and severe aberrations in the near-infrared band, making it impossible to adapt to the imaging requirements of UCNPs probes; Second, they lack the integrated function of three-dimensional single-particle positioning and cannot image particles and cell structures simultaneously.

[0039] In view of this, the technical solution of the present invention integrates a near-infrared band excitation light source through a multi-color light source module to adapt to the non-destructive long-term labeling requirements of UCNPs probes, and combines an apochromatic illumination module to correct the aberration of the near-infrared light path, significantly improving the light transmittance and illumination uniformity in the near-infrared band. The multi-channel imaging module uses spectral separation technology to independently capture the high-resolution visible light signals of cell structures and the near-infrared fluorescence signals of single particles, and encodes the particle spot morphology into axial position information through a modulation device to achieve three-dimensional positioning without layer scanning. At the same time, the image processing module fuses multi-channel spatio-temporal data to complete single-particle dynamic tracking and interaction analysis while ensuring super-high resolution cell structure imaging, ultimately breaking through the bottlenecks of traditional systems in near-infrared compatibility, three-dimensional positioning, and multi-target synchronous observation, and providing an integrated solution with high precision and low phototoxicity for subcellular scale life activity research.

[0040] In this embodiment, a fluorescence imaging system is provided, as Figure 1 shown, the fluorescence imaging system includes: a multi-color light source module 100, an apochromatic illumination module 200, a multi-channel imaging module 300, and an image processing module 400.

[0041] The multi-color light source module 100 is used to emit excitation light of different wavelengths, and the excitation light of different wavelengths is used to label different components in the sample to be tested.

[0042] The excitation light of different wavelengths refers to multiple specific bands of light emitted by the multi-color light source module, such as visible light (e.g., 488 nm, 561 nm) and near-infrared light (e.g., 808 nm, 980 nm). Specifically, different wavelengths correspond to the excitation requirements of different fluorescent markers. For example, visible light is used to excite traditional fluorescent probes (such as the green fluorescent protein GFP for labeling cell structures), and near-infrared light is adapted to new probes (such as lanthanide-doped upconversion nanoparticles UCNPs) to achieve low-phototoxicity and non-bleaching labeling of single particles.

[0043] The different components in the sample to be measured refer to multiple structures or targets that need to be observed simultaneously in a biological sample, such as cell structures and single particles. The cell structures include sub-organelles such as cell nuclei, mitochondria, and cytoskeletons (labeled with visible light probes), and the single particles include virus particles, nano-drug carriers, signal molecules, etc. (labeled with near-infrared probes such as UCNPs). Through multi-wavelength excitation light sorting and labeling, multi-target parallel imaging is achieved.

[0044] Specifically, the multi-color light source module 100 emits excitation light of different wavelengths (such as 488 nm visible light and 980 nm near-infrared light) by integrating multiple independent light sources (such as lasers or LED arrays) and beam splitting elements (such as dichroic mirrors, acousto-optic modulators). Different wavelengths correspond to the excitation spectra of specific fluorescent probes (such as 488 nm to excite GFP-labeled cell nuclei and 980 nm to excite UCNPs-labeled single particles). Through optical path beam combining and time-sharing / space-sharing control, precise sorting and synchronous excitation of multi-component labeling are achieved.

[0045] The apochromatic illumination module 200 is optically connected to the multi-color light source module 100 and is used to perform beam processing on the excitation light based on a preset illumination mode to generate a target illumination beam.

[0046] The preset illumination mode refers to the optical path mode configured by the apochromatic illumination module according to experimental requirements, such as the Kohler illumination optical path mode, the total internal reflection illumination mode, etc. The target illumination beam refers to the optimized excitation light processed by the apochromatic module. Specifically, the apochromatic module 200 uses an apochromatic lens group and an adjustable aperture to perform aberration correction (especially in the near-infrared band) and beam shaping on the excitation light in combination with the preset illumination mode. By dynamically adjusting the lens spacing and optical path parameters, chromatic aberration and spherical aberration are eliminated, and a uniform and low-distortion target illumination beam is generated to adapt to different imaging requirements (such as wide-field uniform illumination or super-resolution structured light field).

[0047] The multi-channel imaging module 300 is optically connected to the apochromatic illumination module 200 and is used to collect the fluorescence signals emitted by the target illumination beam irradiating the sample to be measured, and separate the fluorescence signals according to the fluorescence spectra of each component to obtain multiple imaging channels.

[0048] The fluorescence signal refers to the specific wavelength optical signal emitted by the fluorescent probe after the sample to be measured is excited, such as cell fluorescence signals and particle fluorescence signals. The cell fluorescence signals come from probes such as GFP (emission peak 510 nm), RFP (emission peak 610 nm), etc.; the particle fluorescence signals come from probes such as UCNPs (excited by 980 nm and emitting 540 nm or 660 nm visible light), etc.

[0049] Fluorescence spectrum refers to the intensity distribution characteristics of the light emitted by fluorescent substances in the wavelength dimension. For example, GFP mainly emits green light at 500 - 550 nm, and UCNPs can be designed to emit red light (~650 nm) or blue light (~475 nm). The imaging channel refers to an independent imaging path separated based on the fluorescence spectrum, and each channel corresponds to a component. For example, it can be divided into a cell structure channel and a particle imaging channel. The cell structure channel collects signals in the visible light band (such as the GFP channel) to generate high-resolution cell images; the particle imaging channel collects visible fluorescence signals excited by near-infrared light (such as the UCNPs channel) to encode the three-dimensional position information of the particles.

[0050] Specifically, the multi-channel imaging module 300 uses specific optical elements (such as beam splitters, filters, etc.) to collect the fluorescence signals emitted from the sample to be measured. After the optimized excitation beam generated by the apochromatic illumination module irradiates the sample, different components in the sample will emit fluorescence signals of different wavelengths. The multi-channel imaging module separates the fluorescence signals of different wavelengths through spectroscopic processing according to the fluorescence emission spectrum of each component, and finally obtains multiple independent imaging channels, which correspond to different components or markers respectively.

[0051] The image processing module 400, which is communicatively connected to the multi-channel imaging module 300, is used to receive the imaging data from the multiple imaging channels, perform image fusion on the imaging data, and obtain the target fluorescence image.

[0052] The imaging data refers to the original image information captured by each channel, including spatial information, intensity information, and temporal information, etc. The target fluorescence image refers to the final output image after being fused by the image processing module. For example, it can be an image with the cell structure (such as green false color) and particle localization (such as red spots) superimposed, revealing the interaction between particles and organelles (such as the virus infection process). Specifically, the image processing module 400 is connected to the multi-channel imaging module 300 through a communication interface to receive the fluorescence signal data from each imaging channel. These data contain the fluorescence signals emitted by the sample under different wavelength excitations, representing different components. The image processing module 400 processes and fuses these imaging data, and synthesizes the image information of multiple channels into a unified target fluorescence image through a preset algorithm for comprehensive analysis and observation of the sample.

[0053] The fluorescence imaging system provided by the embodiments of the present invention achieves high-precision uniform illumination of excitation lights with different wavelengths through the synergistic effect of the multi-color light source module and the apochromatic illumination module, ensuring the stability and consistency of multi-component sample labeling. Combining with the spectral separation ability of the multi-channel imaging module, it effectively avoids fluorescence signal crosstalk and significantly improves the imaging resolution and specificity. At the same time, the image processing module finally generates a target fluorescence image with high signal-to-noise ratio and high contrast through the intelligent fusion of multi-channel data, taking into account the accuracy and reliability of multi-component synchronous analysis while improving the detection efficiency.

[0054] In an alternative embodiment, as Figure 2 shown, the multi-color light source module 100 includes a first dichroic mirror (not shown in Figure 2 ), a reflector (not shown in Figure 2 ), and an optical fiber 101.

[0055] The first dichroic mirror is used to combine multiple excitation lights to obtain a combined light beam;

[0056] The reflector is arranged behind the first dichroic mirror and is used to guide the combined light beam to the optical fiber;

[0057] The optical fiber 101 is arranged behind the reflector and is used to transmit the combined light beam to the apochromatic illumination module.

[0058] The combined light beam refers to a composite light beam formed by combining multiple excitation lights with different wavelengths (i.e., multiple excitation lights) into the same optical path through the first dichroic mirror, which is used to achieve multi-wavelength synchronous excitation. For example, visible light bands (such as 405nm, 488nm) are used to excite fluorescence probes (such as GFP) for cell structure labeling; near-infrared bands (such as 808nm, 980nm) are used to excite upconversion nanoparticles (UCNPs) for single-particle labeling. Specifically, the first dichroic mirror selectively reflects and transmits multiple excitation lights with different wavelengths (such as 405nm visible light and 808nm near-infrared light), combines multiple lights into the same path to form a combined light beam. Subsequently, the reflector adjusts the beam direction and accurately guides it into the input end of the optical fiber 101. The optical fiber 101 transmits the combined light beam through total internal reflection, maintaining the spectral characteristics and spatial uniformity, and finally delivers the excitation light with low distortion and multiple wavelengths to the apochromatic illumination module 200, providing a stable input for the aberration correction and mode modulation of the subsequent illumination optical path.

[0059] The fluorescence imaging system provided by the embodiments of the present invention efficiently realizes the precise beam combination of multi-wavelength excitation light through the first dichroic mirror, simplifies the optical path structure and reduces the energy loss between multiple light sources. The collaborative design of the mirror and the optical fiber further optimizes the optical path transmission direction, enhancing the flexibility and stability of the excitation light guidance. The high-efficiency transmission characteristics of the optical fiber not only reduce the beam divergence and energy attenuation, but also effectively maintain the spectral characteristics and spatial uniformity of the excitation light, providing a high-quality and low-distortion excitation beam for the subsequent apochromatic illumination module.

[0060] In an alternative embodiment, the different components include cell structures and single particles; the multiple excitation lights include visible light bands and near-infrared light bands with different wavelengths. The visible light band is used for cell structure labeling, and the near-infrared light band is used for long-term labeling of single particles.

[0061] The cell structure refers to sub-organelles or regions with specific functions in a biological sample, such as the nucleus, mitochondria, cytoskeleton, etc., which can be labeled with traditional fluorescent probes that can be excited by visible light (such as Hoechst 405 staining the nucleus and GFP labeling microtubules). A single particle refers to discrete objects in the nano- to micron-scale, such as virus particles, nano-drug carriers, signaling molecules, etc., which can be labeled with UCNPs probes excited by near-infrared light. Due to its non-photobleaching property, it is suitable for long-term tracking (such as 980nm excitation of UCNPs to label virus particles).

[0062] The range of the visible light band is 400-700nm, which is the electromagnetic spectrum band that can be perceived by the human eye. The visible light band is used to excite traditional fluorescent probes, such as: 405nm (DAPI staining the nucleus), 488nm (GFP labeling the cytoplasm), 561nm (RFP labeling mitochondria). The range of the near-infrared light band is 700-2500nm, such as specific wavelengths mentioned above, 808nm, 980nm, etc. The near-infrared light band is adapted to UCNPs probes (such as 980nm excitation, emitting 540nm green light or 660nm red light).

[0063] The fluorescence imaging system provided by the embodiments of the present invention realizes the efficient sorting and labeling of cell structures and single particles through the differential application of visible light and near-infrared light bands. The visible light band provides high-resolution labeling for cell structures, while the near-infrared light, due to its deep penetration and low phototoxicity, can support the long-term stable tracking of single particles, significantly improving the synchronous observation ability of dynamic biological processes, while reducing the photobleaching effect and ensuring the sample activity.

[0064] In an alternative embodiment, the preset illumination modes include at least one of Kohler illumination optical path mode, structured illumination optical path mode, and total internal reflection illumination mode; the Kohler illumination optical path mode is used for zero-order filtering of the excitation light; the structured illumination optical path mode performs structured modulation on the excitation light; the total internal reflection illumination mode is used for angular selective filtering of the excitation light.

[0065] The Kohler illumination optical path mode (which can also be called the wide-field mode) is used for microscopic imaging to provide uniform illumination. Specifically, the light stop in the Kohler illumination optical path allows the zero-order diffracted light to pass through the first filtering hole at the center of the spatial filter, while blocking all the outer second filtering holes. At this time, the excitation light is not structured and forms uniform illumination.

[0066] The structured illumination optical path mode includes two-dimensional structured illumination optical path mode and three-dimensional structured illumination optical path mode.

[0067] The two-dimensional structured illumination optical path mode improves the lateral resolution (about 2 times) through structured illumination light. Specifically, the light stop in the two-dimensional structured illumination optical path blocks the first filtering hole at the center but allows the ±1st-order diffracted light to pass through the second-ring filtering holes. The excitation light is modulated into a two-dimensional periodic grating structure (such as sine stripes), and a super-resolution image is reconstructed through multi-directional and multi-phase illumination combined with algorithms.

[0068] The three-dimensional structured illumination optical path mode refers to the symmetric three-beam three-dimensional structured illumination mode, which combines the zero-order and ±1st-order diffracted lights to achieve three-dimensional super-resolution. Specifically, the light stop in the three-dimensional structured illumination optical path allows the zero-order light (the first filtering hole) and the ±1st-order light (the second-ring filtering holes) to pass through simultaneously. The excitation light forms a symmetric three-beam interference (central beam + two symmetric side beams), generating an axially modulated structured light field, and the axial resolution is improved through three-dimensional reconstruction algorithms.

[0069] The total internal reflection illumination mode (which can also be called the total internal reflection structured illumination mode) is used for surface super-resolution imaging, and the excitation light only undergoes total internal reflection on the sample surface. Specifically, the light stop in the total internal reflection illumination optical path allows the ±1st-order diffracted light to pass through the third-ring filtering holes (corresponding to beams with a larger incident angle). The excitation light is incident at the critical angle and undergoes total internal reflection at the cover glass-sample interface, only exciting the fluorescence within about 100 nm of the sample surface, significantly reducing the background noise.

[0070] The fluorescence imaging system provided by the embodiments of the present invention integrates Kohler illumination, structured light illumination, and total internal reflection illumination modes. The system can flexibly switch the optical path strategy according to the characteristics of the sample. The Kohler mode ensures the uniformity of the excitation light and the suppression of zero-order noise. The structured light mode improves the imaging resolution through spatial modulation. The total internal reflection mode enables high signal-to-noise ratio imaging for surface particles. The multi-mode collaboration greatly expands the applicable scenarios of the system, taking into account the wide-field and super-resolution imaging requirements.

[0071] In an alternative embodiment, as Figure 2 shown, the multi-channel imaging module 200 includes:

[0072] a microscope objective 302;

[0073] a filter set 301, including an excitation light filter, a second dichroic mirror, and an emission light filter. The second dichroic mirror is arranged behind the excitation light filter, and the emission light filter is arranged behind the second dichroic mirror. The excitation light filter is used to filter the target illumination beam to obtain a first illumination beam. The second dichroic mirror is used to reflect the first illumination beam and irradiate the first illumination beam reflected through the microscope objective onto the sample to be measured, so as to excite the corresponding fluorescence signal of the sample to be measured. The second dichroic mirror is also used to receive the fluorescence signal collected by the microscope objective and project the fluorescence signal to obtain a transmitted signal. The emission light filter is used to filter the transmitted signal to obtain a target fluorescence signal with a preset fluorescence wavelength.

[0074] The excitation light filter refers to a high-pass or band-pass filter used to filter out stray wavelengths in the excitation light. Its transmission band strictly matches the wavelength of the excitation light source (such as allowing 405 nm or 808 nm to pass), and only the light in the target band (such as 405 nm for nuclear labeling) is retained to avoid interference from other wavelengths to sample labeling.

[0075] The second dichroic mirror is a wavelength-selective beam-splitting element coated with a special film layer to achieve the separation of the reflection and transmission bands. For example, in the excitation stage, it reflects the excitation light (such as 405 nm) to the microscope objective 302 for focused illumination of the sample. In the fluorescence collection stage, it transmits the fluorescence signal emitted by the sample (such as 425 - 470 nm) and reflects the residual excitation light (blocking reverse interference).

[0076] The emission light filter refers to a narrow-band filter located at the end of the fluorescence signal path, which only allows the target fluorescence wavelength to pass, further eliminating background noise (such as autofluorescence or scattered light) to ensure signal specificity.

[0077] The first illumination beam refers to the pure excitation light filtered by the excitation light filter. The transmitted signal refers to the optical signal after the fluorescence signal is transmitted through the second dichroic mirror. The target fluorescence signal refers to the fluorescence signal strictly screened by the emission light filter. Specifically, after the micro objective lens 302 collects the target illumination beam 201 output by the apochromatic module 200, the excitation light filter first filters out the stray light of non-target wavelengths to generate a pure first illumination beam. The second dichroic mirror reflects this beam to the micro objective lens 302 to focus and irradiate the sample to excite fluorescence. Subsequently, the micro objective lens 302 collects the fluorescence signal emitted by the sample. The second dichroic mirror then transmits the fluorescence and blocks the residual excitation light to form a transmitted signal. Finally, the emission light filter screens the transmitted signal according to the preset fluorescence wavelength and outputs the target fluorescence signal containing only the target fluorescence. Through the cascading effect of the filter and the dichroic mirror, precise excitation of the excitation light and specific separation of the fluorescence signal are achieved, providing a high signal-to-noise ratio input for multi-channel imaging.

[0078] In the fluorescence imaging system provided by the embodiment of the present invention, the hierarchical design of the filter group and the dichroic mirror realizes strict spectral separation of the excitation light and the fluorescence, effectively suppressing background noise and crosstalk between channels. The efficient cooperation of the micro objective lens and the optical path reflection ensures high-throughput collection and precise projection of the fluorescence signal, providing a low-distortion and high-fidelity optical signal input for multi-component parallel imaging.

[0079] In an alternative embodiment, as Figure 2 shown, the multi-channel imaging module 200 further includes:

[0080] A culture unit 308, disposed on the sample side of the micro objective lens 302, for culturing the sample according to the physiological activity of the sample to be tested;

[0081] A displacement assembly 309, disposed inside the culture unit 308, for adjusting the position of the sample to be tested.

[0082] The culture unit 308 refers to an incubator for live cells integrated on the sample side of the micro objective lens, which is used to provide environmental conditions for maintaining the physiological activity of the sample to be tested (such as live cells or tissues), including constant temperature (such as 37 °C), humidity (such as 95%), CO2 concentration (such as 5%) and nutrient supply, to ensure that the cells are in a normal metabolic state during the observation process. It is directly located below the micro objective lens 302, facilitating real-time dynamic observation of the behavior of live cells.

[0083] The displacement component 309 refers to a three-dimensional displacement stage embedded inside the culture unit 308, which is used to adjust the spatial position of the sample with micron-level precision. Its specific functions include: lateral movement (X / Y axes): scanning different field-of-view regions to locate targets of interest (such as specific cells or particles); axial movement (Z axis): achieving multi-layer focusing and cooperating with the microscope objective to obtain clear images. The displacement component 309 can precisely control the sample position through electric drive or manual fine-tuning without disrupting the culture environment, meeting the requirements of long-term tracking and multi-dimensional imaging.

[0084] Specifically, the culture unit 308 maintains a stable cell survival environment through a sealed design. Meanwhile, it integrates the displacement component 309, enabling real-time adjustment of the XYZ three-dimensional coordinates of the sample under the condition of constant temperature and humidity through software control or manual operation. For example, when tracking the interaction between virus particles and the cytoskeleton, the displacement stage can finely adjust the sample position to follow the particle movement trajectory, while the incubator continuously supplies oxygen and nutrients to ensure the biological authenticity of the experiment.

[0085] In the fluorescence imaging system provided by the embodiments of the present invention, the integrated design of the culture unit and the displacement component can maintain the physiological active environment (such as temperature, humidity) of the sample during the imaging process and support real-time position adjustment. This design not only avoids the loss of activity in traditional ex vivo imaging but also improves the observation accuracy and experimental repeatability in complex physiological scenarios (such as cell migration, particle movement).

[0086] In an optional implementation manner, the target fluorescence signals include cell fluorescence signals and particle fluorescence signals, and the multiple imaging channels include a cell structure imaging channel and a particle imaging channel; the cell structure imaging channel is used to generate a cell structure fluorescence image based on the cell fluorescence signal; the particle imaging channel is used to generate a particle fluorescence image based on the particle fluorescence signal.

[0087] The cell fluorescence signal refers to the fluorescence emitted after the fluorescence probe (such as GFP, RFP) that labels the cell structure is excited, and the particle fluorescence signal refers to the fluorescence emitted after the fluorescence probe (such as UCNPs) that labels a single particle is excited by near-infrared light.

[0088] The cell structure imaging channel is a dedicated optical path for capturing cell fluorescence signals, which is used to generate a high-resolution cell structure fluorescence image to display the spatial distribution and dynamic changes of subcellular organelles. The particle imaging channel is a dedicated optical path for capturing particle fluorescence signals, which is used to generate a particle fluorescence image and realize three-dimensional particle positioning in combination with the modulation device 306.

[0089] A fluorescence image of cell structure refers to an image output through a cell structure imaging channel, which can clearly show the morphology and position of structures such as cell nuclei and mitochondria, and is used to analyze the interaction between organelles or structural dynamics (such as division and migration). A particle fluorescence image refers to an image output through a particle imaging channel, which is used to display the spatial distribution and movement trajectory of single particles.

[0090] For example, as Figure 2 shown, the multi-channel imaging module 300 reflects the illumination beam 201 using the filter set 301, and then passes through the microscope objective 302 to reach the sample to be measured for illumination. The excited fluorescence 303 is collected by the microscope objective 302, passes through the filter set 301 again to reach the imaging tube lens 304, and then the filter set 301 is set according to the fluorescence spectrum of the marker to separate different cell structure and particle fluorescences, and image them on the channel cameras respectively. The number of imaging channels is related to the application requirements. At least one channel needs to be set for subcellular structure imaging, and one channel for single particle imaging. As Figure 2 shown, the number of imaging channels is 3, one of which is for cell structure imaging, and the other two are for single particle imaging. The imaging bands of the three channels are separated by the filter sets 3051 and 3052. 3071 is the cell structure imaging camera (cell structure imaging channel), and 3072 and 3073 are single particle imaging cameras (particle imaging channels). The two single particle imaging channels can be used for fluorescence ratio imaging through pre-correction.

[0091] In an alternative embodiment, as Figure 2 shown, a modulation device 306 is provided in the particle imaging channel. The modulation device 306 is used to modulate the particle fluorescence signal and control the position information of the particle spot corresponding to the particle fluorescence signal in the particle fluorescence image, so that the position information presents an axial shape.

[0092] The modulation device 306 refers to an optical element used to modulate the shape of the particle spot. Specifically, in the particle imaging channel, the modulation device 306 forms an association between the spot morphology of the particle in the two-dimensional image and its axial (Z-axis) position by changing the propagation characteristics of the fluorescence signal. The modulation device 306 uses a preset optical modulation (such as phase adjustment or diffraction effect) so that when the particle is at different depths, its fluorescence signal forms a spot with a specific shape (such as asymmetric distribution, bifurcated stripes or gradient morphology) on the imaging plane. For example, when the particle is close to the focal plane, the spot presents a compact circle; as the particle moves away from the focal plane, the spot gradually stretches or differentiates into a specific pattern. By analyzing the shape parameters of the spot (such as aspect ratio, symmetry or stripe spacing), the system can directly resolve the three-dimensional position information of the particle from a single two-dimensional image without layer-by-layer scanning, thereby improving the imaging efficiency and spatio-temporal resolution.

[0093] The fluorescence imaging system provided by the embodiments of the present invention realizes precise split-track imaging of cells and particles through the independent design of the cell structure imaging channel and the particle imaging channel, effectively avoiding signal cross-interference. The modulation device introduced in the particle channel extends the traditional two-dimensional imaging to three-dimensional spatial positioning by regulating the spot position information and encoding it into axial shape features, significantly improving the axial resolution ability of single particles. At the same time, the direct correlation between the spot morphology and the axial position simplifies the later data processing process, provides high-precision and high-sensitivity in-situ three-dimensional information for dynamic tracking and quantitative analysis, and takes into account the accuracy of multi-target synchronous observation and the depth of complex biological process analysis.

[0094] In an alternative embodiment, the modulation device 306 is a cylindrical lens or a phase plate; the cylindrical lens is used to modulate the particle spot into an ellipse to determine the axial position of the particle based on the ratio of the major axis to the minor axis of the ellipse; the phase plate is used to modulate the particle spot into a double-helix structure to determine the axial position of the particle based on the distance and angle between the spot pairs.

[0095] When the modulation device 306 is a cylindrical lens, the cylindrical lens modulates the particle fluorescence spot into an ellipse, and directly correlates the axial (Z-axis) position of the particle by calculating the ratio of the major axis to the minor axis of the ellipse (for example, the larger the ratio of the major axis to the minor axis, the farther the particle is from the defocus plane), as Figure 3 shown.

[0096] When the modulation device 306 is a phase plate, the phase plate modulates the spot into a double-helix structure, and analyzes the axial (Z-axis) position of the particle by measuring the spacing and rotation angle of the helical spot pairs (for example, the spacing changes linearly with depth, and the angle is proportional to the depth), as Figure 4 shown.

[0097] The fluorescence imaging system provided by the embodiments of the present invention converts the axial position information of particles into spot morphology parameters (ratio of major axis to minor axis or spacing / angle between spot pairs) through the modulation (ellipsoidization or double-helixization) of particle spots by a cylindrical lens or a phase plate, realizing three-dimensional positioning of single particles. This technology breaks through the limitations of traditional two-dimensional imaging, significantly improves the spatial resolution and axial positioning accuracy without complex layer scanning or interference optical paths, and is compatible with multiple imaging modalities.

[0098] In an alternative embodiment, the image processing module 400 is further configured to register the cell structure fluorescence image and the particle fluorescence image by using a pre-calibrated multi-color fluorescent microsphere sample, and fuse the registered particle fluorescence image into the cell structure fluorescence image to generate a target fluorescence image of the interaction between the particle and the cell.

[0099] The image processing module 400 realizes cross-channel image registration and fusion through a pre-calibrated multi-color fluorescent microsphere sample. Specifically, in the system calibration stage, the same microspheres are used to label multiple fluorescences (for example, green light corresponds to the cell channel, and red light corresponds to the particle channel), the microsphere images of each channel are obtained, and their coordinate features are extracted. Subsequently, based on the microsphere coordinates, the spatial transformation matrix between channels (such as translation and rotation parameters) is calculated to correct the image misalignment caused by the optical path difference. Finally, the registered particle trajectory image (such as a red light spot) and the cell structure image (such as a green contour) are superimposed and fused to generate a target fluorescence image showing the interaction between particles and cells (such as the association between the virus infection path and the organelle position), such as Figure 5 shown.

[0100] For example, the labeled cell structure is the cytoskeleton, its excitation light is 405nm, and the emission light band is 425 - 470nm. The labeled single particle is a specific protein, the excitation light is 808nm, and the emission light is 650 ± 10nm. Through long-term observation of the two channels, the three-dimensional trajectories of the two protein particles and the activities of the cytoskeleton are obtained to explore the interaction relationship between the cytoskeleton and the specific protein.

[0101] The fluorescence imaging system provided by the embodiment of the present invention, based on the pre-calibration registration technology of multi-color fluorescent microspheres, can automatically correct the spatial offset between the cell and particle channels, and generate an interaction image through algorithm fusion, thus solving the common channel misalignment problem in multi-color imaging, ensuring the accuracy of the correlation analysis of cross-scale structures (such as organelles and nanoparticles), and providing a reliable visualization tool for the study of cell-particle interaction mechanisms.

[0102] According to the embodiment of the present invention, an embodiment of a fluorescence imaging method is provided. It should be noted that the steps shown in the flowchart of the accompanying drawings can be executed in a computer system such as a set of computer-executable instructions, and although the logical order is shown in the flowchart, in some cases, the steps shown or described can be executed in a different order than here.

[0103] In this embodiment, a fluorescence imaging method is provided, which can be used for the above fluorescence imaging system, Figure 6 is a flowchart of the fluorescence imaging method according to the embodiment of the present invention, as Figure 6 shown, and the process includes the following steps:

[0104] Step S101, obtain the labeling information of different components in the sample to be tested.

[0105] Marker information refers to the excitation wavelength and emission spectral characteristics of fluorescence probes used for different components (such as cell structures, single particles) in the sample to be measured. Specifically, fluorescence probes are pre-selected through experimental design (for example, GFP is used to label the cytoskeleton and is excited by 488 nm, and UCNPs are used to label virus particles and are excited by 808 nm), and chemical labeling is performed during the sample preparation stage to ensure that each component corresponds to a specific excitation light and fluorescence signal.

[0106] Step S102: Emit excitation lights with different wavelengths corresponding to different components based on the marker information.

[0107] The multi-color light source module (such as a laser or LED array) emits excitation lights with different wavelengths in a time-sharing or space-sharing manner according to the marker information. For example, the visible light band (405 nm, 488 nm) is used to excite the cell structure probe; the near-infrared band (808 nm) is used to excite the single particle probe. Specifically, the light source is switched according to the preset parameters to ensure that each component is only excited by its corresponding wavelength.

[0108] Step S103: Perform apochromatic correction on the excitation light to generate a target illumination beam that conforms to the preset illumination mode.

[0109] The apochromatic illumination module corrects the chromatic aberration and spherical aberration of excitation lights with different wavelengths to ensure that the positions and intensities of lights with all wavelengths on the object-side focal plane are consistent. The preset illumination mode (such as Kohler illumination, structured light illumination) is achieved by adjusting beam expansion, shaping, and modulation to generate a uniform or structured target illumination beam.

[0110] Step S104: Project the target illumination beam onto the sample to be measured and collect the fluorescence signals emitted by the sample to be measured under excitation.

[0111] The target illumination beam is focused onto the sample to be measured through a microscope objective to excite the fluorescence probe. Subsequently, the same microscope objective collects the fluorescence signals emitted by the sample to be measured. After filtering out stray light by a filter set (such as a dichroic mirror, emission filter), the signals are received by a camera and converted into fluorescence signals (electrical signals).

[0112] Step S105: Based on the fluorescence spectral characteristics of different components, perform spectral separation on the fluorescence signals to generate multi-channel imaging data.

[0113] The filter set and spectral splitting element (dichroic mirror) are used to separate the fluorescence signals according to the emission wavelength. For example, the cell structure signal (such as 425 - 470 nm blue light) is reflected to the cell imaging channel, and the particle signal (such as 650 ± 10 nm red light) is transmitted to the particle imaging channel to form independent multi-channel imaging data.

[0114] Step S106: Perform feature fusion processing on the multi-channel imaging data to generate a target fluorescence image.

[0115] The image processing module uses multi-color fluorescent microspheres to calibrate the spatial offset of each channel, calculates the transformation matrix to align the images, and performs feature fusion processing on the aligned multi-channel imaging data to generate the target fluorescence image. For example, if it is necessary to generate a target fluorescence image of the interaction between particles and cell structures, a modulation device is used to determine the three-dimensional position and movement path of the particles, and the particle trajectory (such as a red light spot) is superimposed on the cell structure image (such as a green contour) to generate a target fluorescence image showing the interaction.

[0116] The fluorescence imaging method provided by the embodiments of the present invention, through the collaborative design of the multi-color light source module and the apochromatic illumination module, is compatible with the visible light and near-infrared bands, and adapts to new fluorescent probes with no photobleaching and low toxicity, solving the aberration and band limitations in near-infrared imaging of traditional systems. The multi-channel imaging module uses spectral splitting technology to independently capture cell structure and single-particle signals, and combines a modulation device to encode the particle spot morphology into axial position information, and only a single two-dimensional image is required to complete the three-dimensional rapid positioning of the particles. The image processing module accurately fuses the particle trajectory and the high-resolution cell structure image through multi-color fluorescent microsphere calibration and spatio-temporal registration algorithms to generate a dynamic interaction visualization result. Therefore, this method has the capabilities of long-term live cell observation, multi-target synchronous tracking efficiency, and high-precision three-dimensional analysis performance, providing a reliable tool for the study of life activities at the subcellular scale.

[0117] The embodiments of the present invention also provide a computer device, such as an imaging device for capturing, recording, and displaying images of an object or sample, please refer to Figure 7 .

[0118] Figure 7 is a schematic structural diagram of a computer device provided by an optional embodiment of the present invention, as Figure 7 shown. The computer device includes: one or more processors 10, a memory 20, and interfaces for connecting various components, including high-speed interfaces and low-speed interfaces. Each component communicates with each other using different buses and can be installed on a common motherboard or installed in other ways as needed. The processor can process instructions executed within the computer device, including instructions stored in the memory or on the memory to display graphical information of the GUI on an external input / output device (such as a display device coupled to the interface). In some optional embodiments, if necessary, multiple processors and / or multiple buses can be used together with multiple memories and multiple memories. Similarly, multiple computer devices can be connected, and each device provides some necessary operations (such as a server array, a set of blade servers, or a multi-processor system). Figure 7 One processor 10 is taken as an example in

[0119] The processor 10 may be a central processing unit, a network processor, or a combination thereof. Among them, the processor 10 may further include a hardware chip. The above-mentioned hardware chip may be an application-specific integrated circuit, a programmable logic device, or a combination thereof. The above-mentioned programmable logic device may be a complex programmable logic device, a field programmable gate array, a generic array logic, or any combination thereof.

[0120] Among them, the memory 20 stores instructions executable by at least one processor 10, so that the at least one processor 10 executes the fluorescence imaging method shown in the above embodiments.

[0121] The memory 20 may include a program storage area and a data storage area. Among them, the program storage area may store an operating system and application programs required for at least one function; the data storage area may store data created according to the use of the computer device, etc. In addition, the memory 20 may include a high-speed random access memory, and may also include a non-transitory memory, such as at least one disk storage device, a flash memory device, or other non-transitory solid-state storage devices. In some alternative embodiments, the memory 20 may optionally include a memory remotely provided with respect to the processor 10, and these remote memories may be connected to the computer device through a network. Examples of the above network include but are not limited to the Internet, an enterprise intranet, a local area network, a mobile communication network, and combinations thereof.

[0122] The memory 20 may include a volatile memory, such as a random access memory; the memory may also include a non-volatile memory, such as a flash memory, a hard disk, or a solid-state drive; the memory 20 may also include a combination of the above types of memories.

[0123] The computer device further includes a communication interface 30 for the computer device to communicate with other devices or communication networks.

[0124] The embodiments of the present invention also provide a computer-readable storage medium. The method according to the embodiments of the present invention can be implemented in hardware, firmware, or be implemented as computer code that can be recorded on a storage medium, or be implemented as computer code originally stored in a remote storage medium or a non-temporary machine-readable storage medium and downloaded through a network and will be stored in a local storage medium, so that the method described herein can be stored in such software processing on a storage medium using a general-purpose computer, a dedicated processor, or programmable or dedicated hardware. Among them, the storage medium may be a magnetic disk, an optical disk, a read-only memory, a random access memory, a flash memory, a hard disk, or a solid-state drive, etc.; further, the storage medium may also include a combination of the above types of memories. It can be understood that a computer, a processor, a microprocessor controller, or programmable hardware includes a storage component that can store or receive software or computer code, and when the software or computer code is accessed and executed by the computer, the processor, or the hardware, the method shown in the above embodiments is implemented.

[0125] A part of the present invention can be applied as a computer program product, such as computer program instructions, which, when executed by a computer, can call or provide the methods and / or technical solutions according to the present invention through the operations of the computer. Those skilled in the art should understand that the forms in which computer program instructions exist in a computer-readable medium include but are not limited to source files, executable files, installation package files, etc. Correspondingly, the ways in which computer program instructions are executed by a computer include but are not limited to: the computer directly executes the instructions, or the computer compiles the instructions and then executes the corresponding compiled program, or the computer reads and executes the instructions, or the computer reads and installs the instructions and then executes the corresponding installed program. Herein, the computer-readable medium can be any available computer-readable storage medium or communication medium accessible to the computer.

[0126] Although embodiments of the present 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 present invention, and such modifications and variations all fall within the scope defined by the appended claims.

Claims

1. A fluorescence imaging system, characterized in that: The system comprises: A multicolor light source module, used to emit excitation light of different wavelengths, wherein the excitation light of different wavelengths is used to mark different components in the sample to be tested; an apochromatic illumination module, optically connected to the polychromatic light source module, and configured to perform beam processing on the excitation light based on a preset illumination mode to generate a target illumination beam; A multi-channel imaging module, optically connected to the apochromatic illumination module, for collecting the fluorescence signal emitted after the target illumination beam irradiates the sample to be tested, and separating the fluorescence signal according to the fluorescence spectrum of each component to obtain a plurality of imaging channels; The image processing module is communicatively connected with the multi-channel imaging module, and is used for receiving the imaging data from the multiple imaging channels, performing image fusion on the imaging data, and obtaining a target fluorescence image.

2. The system according to claim 1, characterized in that The multicolor light source module comprises: A first dichroic mirror, used for combining the multiple excitation lights to obtain a combined light beam; a reflector, disposed after the first dichroic mirror, for guiding the combined light beam to the optical fiber; The optical fiber is arranged after the reflector, and is used to transmit the combined light beam to the apochromatic illumination module.

3. The system according to claim 1, characterized in that The different components include cell structures and single particles; the multiple excitation lights include visible light bands and near-infrared light bands of different wavelengths, the visible light band is used for cell structure labeling, and the near-infrared light band is used for single particle long-term labeling.

4. The system according to claim 1, characterized in that The preset illumination mode includes at least one of a Kohler illumination light path mode, a structured light illumination light path mode, and a total internal reflection illumination mode; the Kohler illumination light path mode is used to perform zero-order filtering on the excitation light; The structured light illumination optical path mode performs structured modulation processing on the excitation light; The total internal reflection illumination mode is used to perform angle-selective filtering on the excitation light.

5. The system according to claim 1, characterized in that The multi-channel imaging module comprises: Microscope objective lens; A filter set, comprising an excitation light filter, a second dichroic mirror and an emission light filter, wherein the second dichroic mirror is arranged after the excitation light filter, and the emission light filter is arranged after the second dichroic mirror; The excitation light filter is used to filter the target illumination light beam to obtain a first illumination light beam; the second dichroic mirror is used to reflect the first illumination light beam, and irradiate the reflected first illumination light beam to the sample to be tested through the microscope objective lens to excite a fluorescence signal corresponding to the sample to be tested; The second dichroic mirror is also used to receive the fluorescent signal collected by the microscope objective lens, and project the fluorescent signal to obtain a transmission signal; The emission light filter is used to filter the transmission signal to obtain a target fluorescence signal with a preset fluorescence wavelength.

6. The system according to claim 5, characterized in that The multi-channel imaging module also includes: A culture part, arranged on the sample side of the microscope objective lens, for culturing the sample according to the physiological activity of the sample to be tested; The displacement component is arranged inside the culture part and is used to adjust the position of the sample to be tested.

7. The system according to claim 1, 5 or 6, characterized in that: The target fluorescence signal includes a cell fluorescence signal and a particle fluorescence signal, and the multiple imaging channels include a cell structure imaging channel and a particle imaging channel; the cell structure imaging channel is used to generate a cell structure fluorescence image based on the cell fluorescence signal; the particle imaging channel is used to generate a particle fluorescence image based on the particle fluorescence signal; The particle imaging channel is provided with a modulation device, which is used to modulate the particle fluorescence signal and control the position information of the particle spot corresponding to the particle fluorescence signal in the particle fluorescence image so that the position information presents an axial shape.

8. The system according to claim 7, characterized in that The modulation device is a cylindrical mirror or a phase plate; the cylindrical mirror is used to modulate the particle spot into an ellipse, so as to determine the axial position of the particle based on the ratio of the major axis to the minor axis of the ellipse; the phase plate is used to modulate the particle spot into a double helix structure, so as to determine the axial position of the particle based on the distance and angle of the spot pair.

9. The system according to claim 7, characterized in that The image processing module is also used to align the cell structure fluorescence image and the particle fluorescence image using a pre-calibrated multi-color fluorescent microsphere sample, and fuse the aligned particle fluorescence image into the cell structure fluorescence image to generate a target fluorescence image of the interaction between particles and cells.

10. A fluorescence imaging method, characterized in that: The fluorescence imaging system according to any one of claims 1 to 9, comprising: Obtaining labeling information of different components in the sample to be tested; emitting excitation light of different wavelengths corresponding to the different components based on the label information; Performing apochromatic processing on the excitation light to generate a target illumination light beam that conforms to a preset illumination mode; Projecting the target illumination light beam onto the sample to be tested, and collecting the fluorescence signal of the stimulated emission of the sample to be tested; Based on the fluorescence spectral characteristics of the different components, the fluorescence signals are spectrally separated to generate multi-channel imaging data; The multi-channel imaging data is subjected to feature fusion processing to generate a target fluorescence image.

Images