A confocal interferometric scattering-stimulated emission loss super-resolution compound microscope system and its application

Abstract

This invention discloses a confocal interference scattering-stimulated emission loss super-resolution compound microscope system and its application. The system includes an STED module, a confocal module, an iSCAT module, a sample and objective module, and a pinhole module. The STED module generates STED light and coaxially illuminates the sample with the laser from the confocal module. The confocal module generates fluorescence laser light and performs optical signal splitting and filtering on the sample. The iSCAT module generates laser light and detects interference scattering signals. The pinhole module is located on the conjugate image plane of the confocal module and has a continuously adjustable aperture size. This invention integrates the iSCAT and STED channels in the same confocal optical path, achieving for the first time the synchronous acquisition of label-free scattering signals and fluorescence super-resolution images under the same field of view, time, and spatial focal plane. Each channel shares a scanner, a pinhole structure, and a relay optical path, achieving a compact and functionally integrated high-performance imaging scheme.

Description

Technical Field

[0001] This invention relates to the field of optical microscopy systems, specifically to a confocal interference scattering-stimulated emission loss super-resolution composite microscope system and its applications. Background Technology

[0002] Super-resolution microscopy is a crucial technique for overcoming the diffraction limit of traditional optical microscopes and is widely used in life sciences, materials science, and nanotechnology. Among these techniques, stimulated emission depletion microscopy (STED) is a typical fluorescence super-resolution imaging method that achieves nanoscale resolution through spatially modulated fluorescence emission, making it invaluable for imaging the fine structures within living cells. However, STED technology is highly dependent on fluorescent labeling, making it difficult to reflect the original physical state of the sample. Furthermore, its application is limited under conditions of low labeling efficiency, fluorescence bleaching, or high phototoxicity.

[0003] Complementing this, interferometric scattering microscopy (iSCAT) is a highly sensitive, label-free imaging technique based on the principle of interference. Its signal originates from the interference between the sample's scattered light and the substrate's reflected light, and can be used to track the motion and positioning of particles or molecules as small as 10 nm or even smaller. The iSCAT signal intensity I can typically be expressed as: I = |Er| 2 + |Es| 2 + 2|Er||Es| cos(φ). Among them, |Er| 2 For the background reflected light term, |Es| 2 For scattering, 2|Er||Es| cos(φ) is an effective interferometric term. In actual imaging, since |Er| 2 When the intensity is much greater than the interference term, it often causes detector saturation or a decrease in the signal-to-noise ratio, thus affecting image quality. To suppress background reflection, previous studies have proposed introducing partial mirrors to selectively attenuate light in the illumination or reflection path, thereby reducing |Er|. 2 Interference with the signal. This method has a simple structure, but lacks flexible control capabilities and is difficult to integrate with existing multi-channel imaging systems.

[0004] CN114787609A discloses a method and apparatus for optimizing an interference scattering microscope, and an apparatus for imaging a sample using an interference scattering microscope. The method includes irradiating the sample with at least one coherent light source, holding the sample at a sample location including an interface with a refractive index change, irradiating the sample with irradiation radiation to generate a backpropagation signal from the sample, the backpropagation signal including light reflected at the interface and light scattered by the sample, splitting the backpropagation signal into a first signal and a second signal, modifying the second signal using a modification element such that the second signal is different from the first signal; directing the first signal and the second signal to a first detector and a second detector to generate a first image and a second image, respectively; and comparing the first image and the second image by a processor to determine one or more features of the sample.

[0005] In recent years, research has begun to explore integrating iSCAT technology into confocal microscopy platforms, using the size of the pinhole to control the ratio of background light to scattered signal entering the detector. For example, the paper "Nature Communications, 2023, 14: 2019" first reported a method of integrating iSCAT into a confocal structure and using a shared pinhole to adjust the signal background, initially demonstrating that the pinhole structure can be used to increase the relative proportion of interference signal, thereby optimizing the iSCAT image quality.

[0006] However, existing literature lacks a technical solution for integrating the iSCAT and STED imaging systems on the same platform, sharing pinhole modulation, and achieving synchronous image acquisition. The aforementioned studies are limited to signal modulation within traditional confocal structures, do not address the integration of fluorescence super-resolution capabilities, and have not established a systematic multimodal fusion platform.

[0007] Therefore, there is an urgent need to develop a novel microscopy system with a compact structure, unified optical path, and multi-channel high-resolution imaging capability, which can achieve simultaneous acquisition of label-free interference scattering imaging and fluorescence super-resolution imaging in the same field of view. It is particularly suitable for the fine analysis of complex biological processes such as single-particle analysis, living cell membrane dynamics, and subcellular structure recognition. Summary of the Invention

[0008] This invention aims to address the challenges in synergistic integration of interferometric scattering microscopy (iSCAT) and fluorescence super-resolution microscopy (such as STED) in terms of optical path integration, signal modulation, and imaging synchronization. It provides a confocal interferometric scattering-stimulated emission loss super-resolution composite microscope system. Through optical path integration and a shared pinhole modulation mechanism, it achieves the fusion and synchronous acquisition of structures from interferometric scattering and STED imaging, thereby improving system integration and imaging sensitivity and providing technical support for the precise analysis of living cells and nanoscale structures.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A confocal interference scattering-stimulated emission loss super-resolution composite microscope system includes an STED module, a confocal module, an iSCAT module, a sample and objective module, and a pinhole module;

[0011] The STED module is used to generate STED light, which is coupled into the main optical path after beam shaping and coaxially irradiates the sample with the laser from the confocal module.

[0012] The confocal module is used to generate a fluorescent laser and to generate a beam splitter and filter the optical signal of the sample; the fluorescent laser is coupled with the laser emitted by the iSCAT module and enters the main optical path to irradiate the sample; the fluorescent signal after beam splitting and filtering is imaged by the detector in the confocal module, and the interference scattering signal enters the detector in the iSCAT module for imaging.

[0013] The iSCAT module is used to generate laser light and detect interference scattering signals;

[0014] The sample and objective module is used to place the sample, focus the laser and STED light to irradiate the sample, and collect the light signal generated by the sample and return it to the confocal module;

[0015] The pinhole module is located on the conjugate image plane of the confocal module and has the function of continuously adjustable aperture size, which is used to filter off-focal plane signals and adjust the intensity of reflected background in iSCAT signals.

[0016] The main optical path includes an xy-axis scanner, and the sample and objective lens module includes a z-axis displacement device.

[0017] This invention integrates iSCAT (In-Surfural Acoustics-Computed Tomography) and STED (Surfural-Depleted Super-Resolution) channels within the same confocal optical path, achieving for the first time the simultaneous acquisition of label-free scattering signals and fluorescence super-resolution images within the same field of view, time, and spatial focal plane. The system employs a multi-channel detection architecture, enabling independent acquisition of fluorescence signals and interferometric scattering signals of different colors. Each channel shares a scanner, a pinhole structure, and a relay optical path, resulting in a compact, functionally integrated, high-performance imaging solution suitable for miniaturization and integration, and easily compatible with existing STED microscope platforms.

[0018] Another key innovation of this system lies in utilizing a shared pinhole structure to modulate the background light in the interference scattering signal. By reducing the pinhole diameter, off-focal plane signals and reflected background intensity can be effectively suppressed, making the interference term the dominant component. Reducing the aperture diameter lowers the background intensity, which greatly improves the signal-to-noise ratio of the iSCAT image, thereby increasing the intensity ratio of the interference component relative to the reflected background component in the interference scattering signal, making the interference term the dominant signal. Reducing the pinhole diameter effectively avoids detector saturation and enhances the imaging capability of nanoscale structures.

[0019] The iSCAT module generates a laser wavelength shorter than the fluorescence laser wavelength. This ensures that the interference scattering and fluorescence signals do not interfere with each other spectrally, improving channel independence, and the shorter wavelength also helps to improve iSCAT resolution.

[0020] The confocal module includes multiple sets of fluorescent lasers, each used to generate fluorescent lasers of different colors; preferably, three sets of fluorescent lasers are used, including red, green and blue lasers.

[0021] The confocal module includes a multi-component optical filtering system and a detector, which are used to separate fluorescence signals of different colors in the optical signal to achieve multi-channel detection and imaging.

[0022] The iSCAT module has an independent pinhole in front of the detector's optical path, which is used to further reduce background interference in the iSCAT signal and improve the clarity of the iSCAT image without affecting the fluorescence signal throughput.

[0023] The STED module includes an STED laser, a beam shaping unit, and a coupling mirror. The STED laser is used to generate STED light, which is coupled into the main optical path after passing through the beam shaping unit and the coupling mirror, thereby achieving spatial compression of the fluorescence distribution and coaxially irradiating the sample with the laser of the confocal module.

[0024] The confocal module includes a fluorescent laser, a beam combiner, a dichroic mirror, a filter, and a detector;

[0025] The fluorescent laser is used to generate fluorescent laser light, which is coupled to the laser light emitted by the iSCAT module through a beam combiner and enters the main optical path to illuminate the sample; the light signal generated by the sample is separated by a dichroic mirror, filtered by a filter, and enters the detector for imaging.

[0026] The iSCAT module includes an iSCAT laser source, a reflector, and a detector; the reflector reflects the laser emitted by the iSCAT laser source and couples it with the fluorescent laser into the main optical path;

[0027] The light signal generated by the sample is filtered by the confocal module, and the resulting interference scattering signal enters the detector through the reflector for imaging.

[0028] The sample and objective module includes a sample stage and an objective lens. The sample stage is a z-axis voltage generator used to achieve z-axis scanning and fix the sample to be observed. The objective lens is used to simultaneously focus the excitation light and the STED dissipative laser onto the sample and collect the light signal generated by the sample to the confocal module.

[0029] The detectors used for imaging in the composite microscope system are APD (avalanche photodiode) detectors, PMT (photomultiplier tube) detectors, or HyD (Hybrid PMT) detectors.

[0030] This invention also provides the application of the confocal interference scattering-stimulated emission depletion super-resolution composite microscopy system in the imaging of live cells, nanoparticles, or biological vesicles. For example, in the imaging of live cells, nanoparticles, or biological vesicles, unlabeled interference scattering images and fluorescence super-resolution images can be acquired simultaneously.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) In this invention, the structural fusion and synchronous acquisition of interference scattering and STED imaging are achieved through optical path integration and shared pinhole control mechanism. The system module has a compact structure and multiple channels are arranged in parallel, making it suitable for multimodal synchronous imaging tasks with high temporal and spatial resolution. By integrating with the STED laser channel, it has fluorescence super-resolution imaging capability and can achieve synchronous acquisition under the same field of view as the interference scattering imaging channel.

[0033] (2) The system of the present invention has a precision z-axis electric platform to realize interference scattering and STED signal slice scanning at different z heights. Combined with the xy scanner on the main optical path, the system has three-dimensional scanning capability and can realize the synchronous acquisition of iSCAT and STED images at different depths, meet the requirements of three-dimensional spatial imaging, and is suitable for modular upgrade of existing confocal or STED platforms. It has strong engineering feasibility and application prospects. Attached Figure Description

[0034] Figure 1This is a schematic diagram of the confocal interference scattering-stimulated emission loss super-resolution compound microscope system of the present invention. In the diagram, 1 is the STED module, 2 is the confocal module, 3 is the iSCAT module, 4 is the sample and objective lens module, and 5 is the pinhole module; 11 is the STED laser, 12 is the beam shaping unit, 13 is the mirror, 21 is the red excitation laser, 23 is the green excitation laser, 25 is the blue excitation laser, 22, 24, and 26 are beam combiners, 27 is the relay optics system, 28 is the dichroic mirror, 29, 212, and 215 are dichroic mirrors, 210, 213, and 216 are filters, and 211, 214, and 217 are APD detectors; 31 is the iSCAT excitation laser (violet laser), 32 and 33 are mirrors, and 34 is the APD detector; 41 is the sample, 42 is the objective lens, and 43 is the sample stage.

[0035] Figure 2 The image shown is an iSCAT scan of gold nanoparticles (AuNP) with a diameter of 10 nm, as shown in Example 1. The AuNPs are in an aqueous solution and adsorbed onto the surface of a glass substrate. The scale bar is 3 micrometers.

[0036] Figure 3 The image shows the detection results and signal intensity distribution of Example 2. In this image, A is the iSCAT, confocal and STED imaging of A549 extracellular vesicles (EVs), and the fluorescence is derived from the immunostaining of CD63 protein. B is the signal intensity distribution drawn along the dashed line in A.

[0037] Figure 4 This is a z-slice image of A549 cells subjected to simultaneous iSCAT, confocal, and STED three-dimensional scanning in Example 3, with a scale bar of 10 micrometers.

[0038] Figure 5 The image shows iSCAT slices of A549 cells at a height of z = 1 μm for different pinhole sizes, with a scale bar of 10 μm. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.

[0040] A confocal interferometric scattering-stimulated emission loss super-resolution compound microscope system, such as Figure 1As shown, it includes STED module 1, confocal module 2, iSCAT module 3, sample and objective module 4, and pinhole module 5;

[0041] The STED module 1 includes an STED laser 11, a beam shaping unit 12, and a coupling mirror 13. The STED laser 11 is used to generate STED light. After passing through the beam shaping unit 12, the STED light is coupled into the main optical path through the coupling mirror 13 and coaxially irradiates the sample 41 with the laser of the confocal module 2.

[0042] The confocal module 2 is used to generate a fluorescent laser and to generate a light signal for the sample by optical signal splitting and filtering. The fluorescent laser is coupled with the laser emitted by the iSCAT module and enters the main optical path to irradiate the sample 41. The fluorescent signal after optical signal splitting and filtering is imaged by a detector in the confocal module 2, and the interference scattering signal is imaged by a detector in the iSCAT module 3. The iSCAT module 3 is used to generate laser and detect interference scattering signals.

[0043] The confocal module 2 includes three sets of fluorescent lasers: a red laser 21, a green excitation laser 23, and a blue excitation laser 25, which generate lasers with wavelengths of 640 nm, 561 nm, and 488 nm, respectively, to excite fluorescence signals in the corresponding wavelength bands. Three beamforming mirrors (22, 24, and 26) couple each fluorescent laser to the main optical path, and the lasers are focused onto the sample surface and scanned in the xy plane by a relay optical system 27 containing an xy-direction scanner.

[0044] The confocal module includes three sets of optical filtering systems and detectors. The sample return light signal first passes through the dichroic mirror 28 and then enters the detection channel. After passing through the pinhole module 5, it passes through three dichroic mirrors (29, 212, 215) in sequence to separate red, green and blue fluorescence signals. The fluorescence signals pass through filters (210, 213, 216) corresponding to the emission wavelengths and enter the corresponding APD detectors (211, 214, 217) to achieve multi-channel synchronous fluorescence imaging.

[0045] The iSCAT module 3 includes a laser 31 that generates a laser beam with a wavelength shorter than that of laser 25, such as 405 nm, as the excitation light for interference scattering. A mirror 32 couples this beam to the main optical path, and the beam is focused onto the sample surface and scanned in the xy plane by a relay optical system 27 containing an xy-direction scanner. The iSCAT module 3 also includes a detector; when the interference scattering signal from the sample returns, it first passes through a mirror 33 and then enters the iSCAT's APD detector 34 to obtain an iSCAT image.

[0046] The sample and objective lens module 4 includes a sample stage 43 and an objective lens 42. The sample stage 43 is a z-axis voltage generator used for z-axis scanning and fixing the sample 41 to be observed. The objective lens 42 is used to simultaneously focus the excitation light and the STED dissipative laser onto the sample 41, and collect the light signal generated by the sample 41 to the confocal module 2 and the iSCAT module 3. The sample stage 43 can be displaced in the z-axis direction, thereby achieving a comprehensive scan of the xyz three-dimensional angle.

[0047] All raw materials used in the specific implementation method were purchased from the market.

[0048] Example 1

[0049] Utilize Figure 1 The confocal interference scattering-stimulated emission depletion super-resolution microscopy system shown is used for iSCAT imaging of gold nanoparticles (AuNPs) with a diameter of 10 nm. The specific steps are as follows:

[0050] Step 1: Add an appropriate concentration (0.01 mg / mL) of 10 nm AuNP aqueous solution to the microscope coverslip placed on the sample stage 43, and let it stand for about 10 minutes to allow AuNP to be adsorbed on the surface of the slide.

[0051] Step 2: Activate the iSCAT module laser to open the iSCAT channel, set the diameter of pinhole module 5 to 1 Airy unit (AU), and perform image acquisition. The iSCAT scan image obtained by the APD detector of the iSCAT module is as follows: Figure 2 As shown, multiple clear AuNPs are visible, indicating that this system has good interferometric scattering imaging sensitivity in nanoscale particle imaging.

[0052] Example 2

[0053] Utilize Figure 1 The confocal interference scattering-stimulated emission depletion super-resolution microscopy system shown performs simultaneous iSCAT, confocal, and STED imaging of extracellular vesicles (EVs) with an average diameter of 160 nm. The specific method includes the following steps:

[0054] Step 1, extract EV samples (concentration approximately 10) from A549 cells. 8 Add ( / mL) to the coverslip on the sample stage 43 and let stand for 10 minutes to allow the EV to be adsorbed onto the glass surface.

[0055] Step 2, Immunofluorescence staining of EVs: After incubating EVs with an appropriate concentration of Alexa Fluor 647-labeled CD63 antibody for 30 minutes, the free antibody was washed away with PBS buffer.

[0056] Step 3: Turn off the STED module, turn on the red light channel and iSCAT channel to photograph the sample, set the pinhole size to 1 AU, and obtain the results at APD detectors 34 and 211 respectively. Figure 3 The iSCAT and confocal imaging are shown; the STED module is turned on to obtain the STED image on the APD detector 211.

[0057] Figure 3 In Figure A, the iSCAT, red confocal image (i.e., imaging of the APD detector 211 with the STED channel closed), and STED image (imaging of the APD detector 211 with the STED channel open) of the same sample region are shown. Each dotted signal corresponds to a single EV particle. Plotting the intensity of the dashed line portion (spanning two EVs) in the figure yields... Figure 3 In the B image, because the EV size is smaller than the optical diffraction limit, the widths of the two signal peaks in the iSCAT and confocal images are close to the diffraction limit; while in the STED image, the two signal peaks are significantly narrower, revealing the true size of the EV and verifying that the STED channel of this system has super-resolution imaging capability.

[0058] Example 3

[0059] Utilize Figure 1 The system shown performs simultaneous z-axis slice scanning of A549 cells using iSCAT and STED. The specific method includes the following steps:

[0060] Step 1: Culture A549 cells adherently on a coverslip and replace the solution with PBS buffer before imaging.

[0061] Step 2: Stain the cell membrane with red light-excited CellMask dye, and then wash off excess dye from the solution with PBS buffer.

[0062] Step 3: Enable iSCAT and STED channels, and set the pinhole size to 1 AU. Perform a z-axis scan from z = 0 μm to z = 4 μm. The results are as follows: Figure 4 As shown.

[0063] Figure 4 The results show a comparison between iSCAT and STED images acquired at four different z-heights. The two images exhibit spatial structural similarity, validating the system's ability to achieve simultaneous imaging between the iSCAT and STED channels. Furthermore, the iSCAT images reveal some cellular structures that were not identified in the STED images, indicating that iSCAT can image structures unlabeled by fluorescent dyes, possessing a unique advantage in imaging unlabeled targets. Therefore, the two imaging modalities in this system are highly complementary and can be used for high-resolution, multi-dimensional imaging studies of complex biological structures.

[0064] Example 4

[0065] Utilize Figure 1 The confocal interferometric scattering-stimulated emission depletion super-resolution microscopy system shown was used to perform iSCAT z-axis section scanning of A549 cells to evaluate the changes in imaging quality under different pinhole sizes. The specific steps are as follows:

[0066] Step 1: Culture A549 cells adherently on a coverslip, and replace the original culture medium with PBS buffer before imaging.

[0067] Step 2: Enable the iSCAT channel and focus at a height of approximately 1 μm above the coverslip surface. Sequentially set the pinhole size to 3.7 AU, 1.6 AU, 1.1 AU, and 0.2 AU, and acquire iSCAT images under the corresponding conditions. The image results are shown below. Figure 5 As shown.

[0068] Figure 5 The results show that as the pinhole size decreases, the system's suppression of background light, especially off-focal light, is significantly enhanced, and the image details are more clearly presented. With a pinhole size of 0.2 AU, the microvilli structure on the cell membrane surface and the cell nucleus boundary (indicated by arrows in the figure) are distinguishable in the image. However, with larger pinholes, these structures are less distinct due to background interference. These experimental results validate the crucial role of the pinhole structure in this system. By adjusting the aperture size, the background interference term in iSCAT imaging can be effectively controlled, thereby enhancing the dominant component of the interference signal and achieving highly sensitive imaging of weak nanoscale structures.

Claims

1. A confocal interferometric scattering-stimulated emission loss super-resolution composite microscope system, characterized in that, Includes STED module, confocal module, iSCAT module, sample and objective module, and pinhole module; The STED module is used to generate STED light, which is coupled into the main optical path after beam shaping and coaxially irradiates the sample with the laser from the confocal module. The confocal module is used to generate a fluorescent laser and to generate a beam splitter and filter the optical signal of the sample; the fluorescent laser is coupled with the laser emitted by the iSCAT module and enters the main optical path to irradiate the sample; the fluorescent signal after beam splitting and filtering is imaged by the detector in the confocal module, and the interference scattering signal enters the detector in the iSCAT module for imaging. The iSCAT module is used to generate laser light and detect interference scattering signals; The sample and objective module is used to place the sample, focus the laser and STED light to irradiate the sample, and collect the light signal generated by the sample and return it to the confocal module; The pinhole module is located on the conjugate image plane of the confocal module and has the function of continuously adjustable aperture size; The main optical path includes an xy-axis scanner, and the sample and objective lens module includes a z-axis displacement device.

2. The confocal interferometric scattering-stimulated emission loss super-resolution composite microscope system according to claim 1, characterized in that, The iSCAT module generates a laser wavelength shorter than the fluorescent laser wavelength.

3. The confocal interferometric scattering-stimulated emission loss super-resolution composite microscope system according to claim 1, characterized in that, The confocal module includes multiple sets of fluorescent lasers, each used to generate fluorescent lasers of different colors; The confocal module includes a multi-component optical filtering system and a detector, which are used to separate fluorescence signals of different colors in the optical signal to achieve multi-channel detection and imaging.

4. The confocal interferometric scattering-stimulated emission loss super-resolution composite microscope system according to claim 1, characterized in that, The detector in the iSCAT module has an independent pinhole at the front of its optical path.

5. The confocal interferometric scattering-stimulated emission loss super-resolution composite microscope system according to claim 1, characterized in that, The STED module includes an STED laser, a beam shaping unit, and a coupling mirror. The STED laser is used to generate STED light, which is coupled into the main optical path after passing through the beam shaping unit and the coupling mirror, and coaxially irradiates the sample with the laser of the confocal module.

6. The confocal interferometric scattering-stimulated emission loss super-resolution composite microscope system according to claim 1, characterized in that, The confocal module includes a fluorescent laser, a beam combiner, a dichroic mirror, a filter, and a detector; The fluorescent laser is used to generate fluorescent laser light, which is coupled to the laser light emitted by the iSCAT module through a beam combiner and enters the main optical path to illuminate the sample; the light signal generated by the sample is separated by a dichroic mirror, filtered by a filter, and enters the detector for imaging.

7. The confocal interferometric scattering-stimulated emission loss super-resolution composite microscope system according to claim 1, characterized in that, The iSCAT module includes an iSCAT laser source, a reflector, and a detector; the reflector reflects the laser emitted by the iSCAT laser source and couples it with the fluorescent laser into the main optical path; The light signal generated by the sample is filtered by the confocal module, and the resulting interference scattering signal enters the detector through the reflector for imaging.

8. The confocal interferometric scattering-stimulated emission loss super-resolution composite microscope system according to claim 1, characterized in that, The sample and objective module includes a sample stage and an objective lens. The sample stage is a z-axis voltage generator used to achieve z-axis scanning and fix the sample to be observed. The objective lens is used to simultaneously focus the excitation light and the STED dissipative laser onto the sample and collect the light signal generated by the sample to the confocal module.

9. The confocal interferometric scattering-stimulated emission loss super-resolution composite microscope system according to claim 1, characterized in that, The detectors used for imaging in the compound microscope system are APD detectors, PMT detectors, or HyD detectors.

10. The application of the confocal interference scattering-stimulated emission loss super-resolution composite microscope system according to any one of claims 1-9 in the imaging process of living cells, nanoparticles or biological vesicles.

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