Confocal interference scattering-stimulated radiation loss super-resolution composite microscope system and application thereof

Abstract

The invention discloses a confocal interference scattering-stimulated radiation loss super-resolution composite microscope system and application thereof. The system comprises an STED module, a confocal module, an iSCAT module, a sample and objective lens module and a pinhole module. The STED module is used for generating STED light to coaxially irradiate a sample with the laser of the confocal module; the confocal module is used for generating fluorescent laser and generating light signal light splitting and filtering for a sample; the iSCAT module is used for generating laser and detecting interference scattering signals; and the pinhole module is arranged on a conjugate image surface of the confocal module and has a function of continuously adjusting the aperture size. According to the invention, by integrating the iSCAT and STED channels in the same confocal light path, synchronous acquisition of non-label scattering signals and fluorescent super-resolution images in the same visual field, the same time and the same space focal plane is realized for the first time, and the channels share a scanner, a pinhole structure and a relay optical path, so that the accuracy of the system is improved. And a high-performance imaging scheme with a compact structure and integrated functions is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical microscopy system, and particularly relates to a confocal interferometric scattering-stimulated emission depletion super-resolution compound microscope system and application thereof. BACKGROUND

[0002] Super-resolution microscopy is an important means to break through the diffraction limit of traditional optical microscopy, and is widely used in the fields of life science, material science and nanotechnology. Among them, stimulated emission depletion microscopy (STED) is a typical fluorescence super-resolution imaging method, which can achieve nanoscale resolution by spatially regulating fluorescence emission, and has important value in imaging fine structures in living cells. However, STED technology is highly dependent on fluorescence labeling, and it is difficult to reflect the original physical state of the sample, and its application is limited under conditions of low labeling efficiency, fluorescence bleaching or strong phototoxicity.

[0003] Complementarily, interferometric scattering microscopy (iSCAT) is a high-sensitivity non-labeling imaging technology based on interference principle. Its signal is derived from the interference between sample scattering light and substrate reflection light, and can be used to track the movement and positioning of particles or molecules with a size of 10 nm or even smaller. The signal intensity I of iSCAT can be generally expressed as: I = |Er| 2 + |Es| 2 + 2|Er||Es| cos(φ). Wherein, |Er| 2 is the background reflection light term, |Es| 2 is the scattering term, and 2|Er||Es| cos(φ) is the effective interference term. In actual imaging, due to the fact that |Er| 2 is much larger than the interference term, it often causes the saturation of the detector or the reduction of the signal-to-noise ratio, thereby affecting the image quality. In order to suppress the background reflection, some studies have proposed to introduce a partial mirror to selectively attenuate the light in the illumination or reflection path, thereby reducing the interference of |Er| 2 on the signal. This method is simple in structure, but does not have flexible control ability, and is difficult to be compatible with existing multi-channel imaging systems.

[0004] CN114787609A discloses a method and apparatus for an optimized interferometric scattering microscope, a method of imaging a sample by an interferometric scattering microscope, the method comprising illuminating the sample with at least one coherent light source, the sample being held at a sample location comprising an interface, the interface having a change in refractive index, illuminating the sample with the illuminating radiation to produce a back-propagating signal from the sample, the back-propagating signal comprising light reflected at the interface and light scattered by the sample, splitting the back-propagating signal into a first signal and a second signal, modifying the second signal using a modifying element such that the second signal is different from the first signal; directing the first signal and the second signal onto first and second detectors to produce first and second images respectively; and comparing the first and second images by a processor to determine one or more features of the sample.

[0005] In recent years, research has begun to explore the integration of iSCAT technology into a confocal microscopic platform by adjusting the size of the pinhole to regulate the ratio of background light and scattered signal entering the detector. For example, the document "Nature Communications, 2023, 14: 2019" first reported a method of integrating iSCAT into a confocal structure and adjusting the signal background using a shared pinhole, which preliminarily proved that the pinhole structure can be used to improve the relative proportion of interference signals, thereby optimizing the image quality of iSCAT.

[0006] However, there is no technical solution in the existing literature that integrates iSCAT and STED imaging systems on the same platform, uses a shared pinhole for regulation, and synchronously collects images. The above research is limited to signal modulation in the traditional confocal structure and does not involve the integration of fluorescence super-resolution capability. It also does not form a systematic multi-modal fusion platform.

[0007] Therefore, there is an urgent need to develop a new type of microscopic system that is compact in structure, unified in optical path, and has multi-channel high-resolution imaging capability. It can synchronously collect non-labeled interferometric scattering imaging and fluorescence super-resolution imaging in the same field of view, especially suitable for fine analysis of complex biological processes such as single-particle analysis, live cell membrane dynamics, and subcellular structure identification. SUMMARY

[0008] The present application aims to solve the problem that interferometric scattering microscopic imaging (iSCAT) and fluorescence super-resolution microscopic imaging (such as STED) cannot be integrated in the optical path, signal regulation, and imaging synchronization in the prior art. It provides a confocal interferometric scattering-stimulated emission depletion super-resolution compound microscope system. Through optical path integration and shared pinhole regulation mechanism, the structure of interferometric scattering and STED imaging is co-integrated and synchronously acquired, thereby improving the system integration and imaging sensitivity, and providing technical support for precise analysis of live cells and nanoscale structures.

[0009] To achieve the above object, the technical scheme adopted by the present application is: A confocal interference scattering-stimulated emission depletion super-resolution composite microscope system, comprising a STED module, a confocal module, an iSCAT module, a sample and objective module, and a pinhole module; The STED module is used to generate STED light, which is coupled into the main light path after beam shaping, and irradiates the sample coaxially with the laser of the confocal module; The confocal module is used to generate fluorescence laser and produce optical signal light filtering of the sample; the fluorescence laser is coupled into the main light path with the laser emitted by the iSCAT module to irradiate the sample; the fluorescence signal filtered by the optical signal light filtering is imaged by the detector in the confocal module, and the interference scattering signal is imaged by the detector in the iSCAT module; The iSCAT module is used to generate laser and detect interference scattering signal; The sample and objective module is used to place the sample, focus the laser and STED light to irradiate the sample, and collect the optical signal generated by the sample to return to the confocal module; The pinhole module is arranged at the conjugate image plane of the confocal module, has the function of continuously adjustable aperture size, and is used to filter non-focal plane signals and adjust the reflection background intensity in the iSCAT signal; The main light path comprises an x-y direction scanner, and the sample and objective module comprises a z-axis direction displacer.

[0010] The present application integrates interference scattering imaging (iSCAT) and stimulated emission depletion super-resolution imaging (STED) channels in the same confocal light path, and for the first time realizes the synchronous acquisition of non-labeled scattering signals and fluorescence super-resolution images in the same field of view, at the same time and in the same spatial focal plane. The system adopts a multi-channel detection architecture, which can realize independent acquisition of different color fluorescence signals and interference scattering signals, and each channel shares a scanner, a pinhole structure and a relay optical path, realizes a compact and integrated high-performance imaging scheme, and is suitable for miniaturization and integration, and is convenient for upgrading and integration with the existing STED microscope platform.

[0011] Another key innovation of the present system is to use a shared pinhole structure to regulate the background light in the interference scattering signal. By reducing the aperture size of the pinhole, non-focal plane signals and reflection background intensity can be effectively suppressed, so that the interference term becomes the dominant component. By reducing the aperture size, the background intensity can be reduced, which greatly improves the signal-to-noise ratio of the iSCAT image, so as to increase the intensity ratio of the interference component to the reflection background component in the interference scattering signal, so that the interference term becomes the dominant signal, the pinhole effectively avoids the saturation of the detector and enhances the imaging capability of the nanoscale structure.

[0012] The iSCAT module generates laser light with a wavelength shorter than the fluorescence laser light. This ensures that the interference scattering and fluorescence signals do not interfere with each other in the spectrum, improves channel independence, and the short wavelength helps to improve the resolution of iSCAT.

[0013] The confocal module includes multiple groups of fluorescence lasers, each for generating fluorescence laser light of a different color; preferably, three groups of fluorescence lasers are used, including red, green, and blue lasers.

[0014] The confocal module includes multiple groups of light splitting and filtering systems and detectors, each for separating fluorescence signals of different colors in the light signal to achieve multi-channel detection imaging.

[0015] The iSCAT module has an independent pinhole in front of the light path of the detector, which is used to further reduce background interference in the iSCAT signal without affecting the flux of the fluorescence signal, and to improve the clarity of the iSCAT image.

[0016] The STED module includes a 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 light path through the coupling mirror after passing through the beam shaping unit, achieving spatial compression of the fluorescence distribution, and coaxially irradiating the sample with the laser light of the confocal module.

[0017] The confocal module includes a fluorescence laser, a beam combiner, a dichroic mirror, a filter, and a detector. The fluorescence laser is used to generate fluorescence laser light, which is coupled into the main light path with the laser light emitted by the iSCAT module to irradiate the sample; the light signal generated by the sample is separated by the dichroic mirror, filtered by the filter, and imaged by the detector.

[0018] The iSCAT module includes an iSCAT laser source, a mirror, and a detector; the mirror reflects the laser light emitted by the iSCAT laser source and couples it into the main light path with the fluorescence laser light. The light signal generated by the sample is separated by the dichroic mirror, filtered by the filter, and imaged by the detector.

[0019] The sample and objective module includes a sample stage and an objective lens; the sample stage is a z-axis piezoelectric stage used for z-axis scanning and fixing the sample to be observed, and the objective lens is used to focus the excitation light and STED dissipation laser light on the sample at the same time, and collect the light signal generated by the sample to the confocal module.

[0020] The detector used for imaging in the compound microscope system is an APD (avalanche photodiode) detector, a PMT (photomultiplier tube) detector, or a HyD (Hybrid PMT) detector.

[0021] The application further provides application of the confocal interference scattering-stimulated emission depletion super-resolution composite microscope system in imaging of living cells, nanoparticles or biological vesicles.

[0022] Compared with the prior art, the application has the following beneficial effects: (1) In the application, the structure of interference scattering and STED imaging is realized by integrating and sharing the pinhole control mechanism, the system module structure is compact, multiple channels are arranged in parallel, and the application is suitable for multi-modal synchronous imaging tasks under high time resolution and high spatial resolution. By integrating with the STED laser channel, the application has the ability of fluorescence super-resolution imaging, and can realize synchronous acquisition under the same field of view with the interference scattering imaging channel.

[0023] (2) The system of the application has a precise z-axis electric platform, realizes interference scattering and STED signal slice scanning at different z heights, and simultaneously combines the xy scanner on the main light path, so that the system has three-dimensional scanning capability, can realize synchronous acquisition of iSCAT and STED images at different depths, meets the three-dimensional spatial imaging requirement, is suitable for modular upgrading of the existing confocal or STED platform, has strong engineering implementability and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 FIG. 1 is a structural schematic diagram of a confocal interference scattering-stimulated emission depletion super-resolution composite microscope system according to the application. In the figure, 1 is an STED module, 2 is a confocal module, 3 is an iSCAT module, 4 is a sample and objective module, and 5 is a pinhole module; 11 is an STED laser, 12 is a beam shaping unit, 13 is a mirror, 21 is a red excitation laser, 23 is a green excitation laser, 25 is a blue excitation laser, 22, 24 and 26 are beam combiners, 27 is a relay optical system, 28 is a dichroic mirror, 29, 212 and 215 are dichroic mirrors, 210, 213 and 216 are optical filters, 211, 214 and 217 are APD detectors; 31 is an iSCAT excitation laser (violet laser), 32 and 33 are mirrors, 34 is an APD detector; 41 is a sample, 42 is an objective, and 43 is a sample stage.

[0025] Figure 2 FIG. 2 is an iSCAT scanning image of gold nanoparticles (AuNP) with a diameter of 10 nm in Example 1, wherein the AuNP is adsorbed on the surface of a glass substrate in an aqueous solution; the scale is 3 microns.

[0026] Figure 3The 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.

[0027] 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.

[0028] 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

[0029] 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.

[0030] A confocal interferometric scattering-stimulated emission loss super-resolution compound microscope system, such as Figure 1 As shown, it includes STED module 1, confocal module 2, iSCAT module 3, sample and objective module 4, and pinhole module 5; 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] All raw materials used in the specific implementation method were purchased from the market.

[0037] Example 1 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: 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.

[0038] Step 2: Turn on the iSCAT module laser to open the iSCAT channel, set the pinhole module 5 diameter to 1 Airy Unit (AU), and collect images. The iSCAT scanning image results obtained by the APD detector of the iSCAT module are shown in FIG. 8A, where multiple clear AuNPs can be seen, indicating that the system has good interferometric scattering imaging sensitivity in the imaging of nanoparticles. Figure 2

[0039] Example 2 The confocal interferometric scattering-stimulated emission depletion super-resolution microscope system as shown in FIG. 6 was used to perform iSCAT, confocal, and STED synchronous imaging of extracellular vesicles (EVs) with an average diameter of 160 nm. The specific method includes the following steps: Figure 1 Step 1: The EV sample (concentration about 10 8 / mL) extracted from A549 cells was added to the cover glass on the sample stage 43, and left to stand for 10 minutes to allow the EVs to adsorb to the glass surface. Step 2: Immunofluorescence staining of EVs: After incubation with an appropriate concentration of Alexa Fluor 647-labeled CD63 antibody for 30 minutes, the free antibody was washed away with PBS buffer.

[0040] Step 3: Turn off the STED module, turn on the red light channel and the iSCAT channel to take pictures of the sample, set the pinhole size to 1 AU, and obtain the results at the APD detectors 34 and 211, respectively, as shown in FIG. 8B, which are the iSCAT and confocal images; turn on the STED module to obtain the STED image at the APD detector 211.

[0041] Figure 3

[0042] Figure 3 In FIG. 8C, A is the iSCAT, red confocal (i.e., the imaging of the APD detector 211 under the closed STED channel), and STED images (the imaging of the APD detector 211 under the open STED channel) of the EVs obtained in the same sample area, and each point signal corresponds to a single EV particle. The intensity of the dashed line part (across two EVs) in the figure is plotted to obtain FIG. 8C, B. Since the size of the EVs is smaller than the optical diffraction limit, the two signal peak widths in the iSCAT and confocal images are close to the diffraction limit; in the STED image, the two signal peaks are significantly narrowed, revealing the true size of the EVs and verifying that the STED channel of the system has super-resolution imaging capability. Figure 3

[0043] Example 3 The confocal interferometric scattering-stimulated emission depletion super-resolution microscope system as shown in FIG. 6 was used to perform iSCAT, confocal, and STED synchronous imaging of extracellular vesicles (EVs) with an average diameter of 160 nm. The specific method includes the following steps: Figure 1 ​​​​The system shown performs iSCAT and STED synchronous z-axis section scanning on A549 cells, and the specific method includes the following steps: Step 1: A549 cells are adherently cultured on a cover glass, and the solution is replaced with PBS buffer before imaging.

[0044] Step 2: The cell membrane is stained with a red light excited CellMask dye, and then the excess dye in the solution is washed with PBS buffer.

[0045] Step 3: Enable iSCAT and STED channels, set the pinhole size to 1 AU. Perform z-axis scanning from z = 0 μm to z = 4 μm, and the results are shown in Figure 4 .

[0046] Figure 4 The comparison results of iSCAT images and STED images obtained at four different z-heights are shown. Both images have similarities in spatial structure, verifying the ability of the system to achieve synchronous imaging between the iSCAT and STED channels. At the same time, the iSCAT images also present some cell structures that are not identified in the STED images, indicating that iSCAT can image structures that are not labeled by fluorescent dyes, and has a unique advantage of imaging non-labeled targets. Therefore, the two imaging methods in the system have good complementarity, and can be used for high-resolution, multi-dimensional imaging research of complex biological structures.

[0047] Example 4 Using the confocal interference scattering-stimulated emission depletion super-resolution microscope system as shown in Figure 1 , z-axis section scanning of iSCAT is performed on A549 cells to evaluate the change in imaging quality under different pinhole size conditions. The specific steps are as follows: Step 1: A549 cells are adherently cultured on a cover glass, and the original culture solution is replaced with PBS buffer before imaging.

[0048] Step 2: Enable the iSCAT channel, and position the focus at a height of about 1 μm from the surface of the cover glass. Set the pinhole size to 3.7 AU, 1.6 AU, 1.1 AU and 0.2 AU in turn, and collect iSCAT images under the corresponding conditions. The image results are shown in Figure 5 .

[0049] Figure 5It is shown that the system has a significantly enhanced rejection of the background light, especially the non-focal plane light, as the pinhole size is reduced, 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 boundary of the cell nucleus (indicated by arrows in the figure) can be distinguished in the image, while these structures are not obvious in the case of a larger pinhole. The experimental results verify the key role of the pinhole structure in the system. By adjusting the aperture size, the background interference term in the iSCAT imaging can be effectively controlled, thereby enhancing the dominant component of the interference signal and realizing high-sensitivity imaging of weak nanoscale structures.

Claims

1. A confocal interference 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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