A microscopic imaging system and method applied to non-fluorescent multi-particle resolution

Abstract

A kind of applied to non-fluorescent multi-particle resolution microscopic imaging system and method, including light source module, polarization modulation module, illumination module, imaging module, image processing module and displacement control module, the light source module emission end is provided with polarization modulation module, the polarization modulation module emission end is provided with illumination module, the illumination module emission end is provided with high-power objective and sample stage, the imaging module incidence end is provided with sample stage, the image processing module is connected with imaging module, the displacement control module is connected with sample stage.This application utilizes the polarization selection principle of interference scattering imaging, by the illumination light polarization state control system point spread function, realize the effective distinction of multiple particles, improve the resolution ability of imaging system to multiple similar particles.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging and detection technology for microparticles, and in particular to a microscopic imaging system and method for non-fluorescent multi-particle resolution. Background Technology

[0002] Super-resolution optical microscopy has become an important tool in cell biology, neuroscience, pharmaceuticals, and nanophotonics. Currently, the most widely used super-resolution nanotechnology is based on fluorescence microscopy, enabling selective labeling of samples. This fluorescence-labeled microscopy technique offers high image contrast; however, limitations in labeling methods restrict its application. In recent years, many label-free super-resolution imaging methods have been proposed. Interferometric scattering microscopy (iSCAT) and single-particle interferometric reflection microscopy (SPIR) exhibit high sensitivity to nanoscale objects, in which the scattered field from the object is superimposed with a coherent reference field. Rotating coherent scattering microscopy (ROCS) employs tilted, coherent illumination in dark-field mode to improve image resolution and contrast.

[0003] Improving the resolving power of optical microscopes is a key issue in microscopic imaging technology. Among fluorescence super-resolution techniques, typical examples include stimulated emission depletion fluorescence imaging (STED), fluorescence differential imaging (FED), and structured light illumination super-resolution optical microscopy (SIM), all based on point scanning imaging. In STED, the fluorescence is quenched by a hollow lossy light field, reducing the system's point spread function (PSF) and thus improving resolution. FED uses temporal differential imaging to reduce the optical system's PSF, thereby improving contrast. SIM encodes high-frequency information of the object into lower spatial frequencies to achieve improved resolution.

[0004] However, compared to fluorescence super-resolution nanomicroscopy, although label-free imaging techniques can achieve nanoscale detection scale and localization accuracy for single molecules, the detection resolution for two or more point sources remains limited. Studies have reported that the resolution of label-free scattering detection can be improved through substrate material design. Plasma materials have higher effective refractive indices in the visible light band; therefore, plasmonic materials such as surface plasmon polarization structures and hyperbolic metamaterials are used in scattering imaging to improve resolution. Label-free microscopes enhanced with special materials face certain limitations in application and fabrication. Another way to improve resolution is by manipulating the point spread function, adjusting its size or shape to enhance image resolution. Currently, label-free non-fluorescent imaging techniques still lack a method for improving the detection resolution of multiple particles on a sample surface under wide field-of-view imaging based on the manipulation of the system's point spread function. Summary of the Invention

[0005] To overcome the above problems, the present invention provides a microscopic imaging system and method for non-fluorescent multi-particle resolution.

[0006] The first aspect of the present invention provides a microscopic imaging system for non-fluorescent multi-particle resolution, including an illumination optical path, an imaging optical path, and a control and processing module. The emitting end of the illumination optical path and the incident end of the imaging optical path are jointly provided with a sample stage (12), and the sample to be detected is provided on the sample stage (12).

[0007] The illumination optical path includes a light source module, a polarization modulation module, and an illumination module arranged sequentially along the optical path. The light source module provides illumination light and modulates the light into parallel linearly polarized light. The polarization modulation module adjusts the incident parallel linearly polarized light to an S-polarization state. The illumination module introduces the adjusted S-polarized light into the sample surface to be tested.

[0008] The imaging optical path includes a high-power objective lens (11) and an imaging module arranged sequentially along the optical path. The scattered light from the sample particles on the sample surface to be detected and the reflected light from the substrate are collected together by the high-power objective lens (11), and the imaging module performs interference imaging between the scattered signal and the reflected light.

[0009] The control and processing module includes an image processing module (17) and a displacement control module (18). The image processing module (17) performs image processing on the interference imaging signal, and the displacement control module (18) controls the sample position in real time.

[0010] Furthermore, the light source module includes a continuous laser (1) that provides continuous laser light, and a first low-power objective (2), a rotating scattering plate (3), and a second low-power objective (4) arranged sequentially along the laser light path; the first low-power objective (2) focuses the laser beam onto the rotating scattering plate (3), the rotating scattering plate (3) weakens the coherence of the incident light to eliminate interference fringes, and the second low-power objective (4) collects the light that has passed through the rotating scattering plate (3).

[0011] The polarization modulation module includes a polarizer (5), a first total reflection mirror (6), a half-wave plate (7), and a first analyzer (8) arranged sequentially along the optical path; the polarizer (5) adjusts the incident parallel light to linearly polarized light; the first total reflection mirror (6) adjusts the direction of the incident light; the half-wave plate (7) adjusts the polarization direction of the polarized light; and the first analyzer (8) adjusts the polarization direction of the incident linearly polarized light to S-polarized light.

[0012] Furthermore, the polarization modulation module includes a polarizer (5), a first total reflection mirror (6), a half-wave plate (7), a third total reflection mirror (19), and a second beam splitter (20) arranged sequentially along the optical path. A polarization analyzer (21) is provided at the output end of the second beam splitter (20).

[0013] The polarizer (5) adjusts the illumination light to be parallel linearly polarized light. After being reflected by the first total reflection mirror (6), the parallel linearly polarized light enters the half-wave plate (7) to adjust the polarization direction of the linearly polarized light. After being reflected by the third total reflection mirror (19), it is split into two beams by the second beam splitter (20). The transmitted light enters the polarization analyzer (21), and the reflected light is used as the illumination light. The half-wave plate (7) and the polarization analyzer (21) adjust the linear polarization direction in real time to make the polarization state of the illumination light change to the S-polarization state.

[0014] Furthermore, the illumination module includes a wide-field illumination lens (9), a beam splitter (10), and a high-magnification objective lens (11) arranged sequentially along the optical path. The wide-field illumination lens (9) converges the incident parallel linearly polarized light; the beam splitter (10) reflects the illumination light; and the high-magnification objective lens (11) collects the illumination light and emits it onto the surface of the sample to be tested.

[0015] Furthermore, the illumination module includes a polarizer (5), a first total reflection mirror (6), a half-wave plate (7), a first analyzer (8), an acousto-optic deflector (22), a 4f system first lens (23), a 4f system second lens (24), a beam splitter (10), and a high-magnification objective lens (11) arranged sequentially along the optical path;

[0016] The polarizer (5) adjusts the illumination light to parallel linearly polarized light. After being reflected by the first total reflection mirror (6), the parallel linearly polarized light enters the half-wave plate (7) to adjust the polarization direction of the linearly polarized light, and then enters the first analyzer (8) to adjust the linearly polarized light to S-polarized light. The parallel linearly polarized light is rapidly scanned in two dimensions by the acousto-optic deflector (22). The first lens (23) and the second lens (24) of the 4f system reflect the deflected beam through the beam splitter (10) and converge it to the back focal plane of the high-power objective lens (11). The converged illumination light forms a wide-field parallel light that illuminates the sample stage (12) through the high-power objective lens (11).

[0017] Furthermore, in the imaging optical path, after the sample particle scattered light and substrate reflected light are collected by the high-power objective lens (11) and then passed through the beam splitter (10), an imaging module is set thereafter; the incident end of the beam splitter (10) is connected to the high-power objective lens (11), and the emitting end of the beam splitter (10) is connected to the imaging module.

[0018] The imaging module includes a second total reflection mirror (13), a second analyzer (14), a telescope lens (15), and a detector (16). The second total reflection mirror (13) changes the direction of the probe light; the second analyzer (14) further adjusts the polarization component of the light used for interference imaging; the telescope lens (15) converges the scattered light and reflected light from the analyzed particles; and the detector (16) performs interference imaging of the scattered light and reflected light.

[0019] Furthermore, the control and processing module includes an image processing module (17) and a displacement control module (18). The image processing module (17) is used to process the signal obtained by the detector to obtain the particle interference signal; the displacement control module (18) controls the three-dimensional position of the sample in real time.

[0020] Furthermore, the first low-power objective (2) and the second low-power objective (4) have a magnification of 10 and a numerical aperture of 0.25, the high-power objective (11) has a magnification of 100 and a numerical aperture of 1.4; the beam splitter (10) has a transmission-reflection beam splitting ratio T:R = 50:50; and the second analyzer (14) has a polarization ratio S:P = 5:95.

[0021] A second aspect of the present invention provides a microscopic imaging method for non-fluorescent multi-particle resolution, comprising the following steps:

[0022] S1: The illumination light emitted by the continuous laser (1) is focused after passing through the first low-power objective lens (2), and after passing through the rotating scattering plate (3), it is collected and adjusted into parallel light by the second low-power objective lens (4);

[0023] S2: Parallel light passes through a polarizer (5) to adjust the illumination light into parallel linearly polarized light. After the parallel linearly polarized light is reflected by the first total reflection mirror (6), it enters a half-wave plate (7) to adjust the polarization direction of the linearly polarized light, and then enters the first analyzer (8) to adjust the linearly polarized light into S-polarized light.

[0024] Let θ1 be the angle between the transmission axis of the polarizer (5) and the x-axis, θ2 be the angle between the fast axis of the half-wave plate (7) and the x-axis, and θ3 be the angle between the transmission axis of the first analyzer and the x-axis. Combined with the following formula:

[0025]

[0026]

[0027] Set the included angle θ1 of the polarizer (5) and the included angle π / 2 of the first analyzer (8). By rotating the half-wave plate (7) by the angle θ2, satisfy A1=0, so that all the light energy after passing through the half-wave plate (7) passes through the first analyzer (8).

[0028] S3: The parallel linearly polarized light is converged at the back focal plane of the high-power objective lens (11) after passing through the wide-field illumination lens (9) and the beam splitter (10). The converged illumination light is then used to form a wide-field parallel light to illuminate the sample stage (12) through the high-power objective lens (11).

[0029] S4: The scattered light from the sample particles on the sample stage (12) and the reflected light from the sample substrate surface are collected by the high-power objective lens (11), and then pass through the beam splitter (10), the second total reflection mirror (13), and the second polarizer (14) to suppress the background reflected light before entering the lens barrel (15) to converge the light onto the detector (16) for interference imaging.

[0030] S5: The image processing module (17) processes a series of image matrices acquired by acquisition, obtains the image background by averaging or median filtering, obtains the signal by difference method and normalizes it.

[0031] S6: Calculate the point diffusion function (PSF) of the system under multiple particles and fit it with the particle spacing;

[0032] S7: Calculate the particle spacing based on the normalized image and fitted curve, and perform particle localization to obtain the position and spacing information of multiple particles.

[0033] The beneficial effects of this invention are:

[0034] 1. Based on the polarization dependence of interferometric scattering imaging, the ability to resolve aggregates of multiple scattering particles can be improved by controlling the PSF through polarization modulation.

[0035] 2. It adopts a reflective interferometric scattering imaging optical path, which is simple and compatible with multiple imaging modes, including dark field imaging, confocal imaging, and fluorescence imaging. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the structure of a non-fluorescent multi-particle resolution microscopic imaging system as described in Embodiment 1 of the present invention;

[0037] Figure 2 Here is a schematic diagram (a) of the light source module of the present invention and a partial structural diagram (b) of the rotating scattering plate;

[0038] Figure 3 This is a schematic diagram of the processing flow of the image processing module of the present invention;

[0039] Figure 4 This is a schematic diagram of the structure of a non-fluorescent multi-particle resolution microscopic imaging system as described in Embodiment 2 of the present invention;

[0040] Figure 5 This is a schematic diagram of the structure of a non-fluorescent multi-particle resolution microscopic imaging system as described in Embodiment 3 of the present invention;

[0041] Figure 6 The following is a comparison of simulation results of S-polarized light interferometric scattering microscopy (PSF) and P-polarized light interferometric scattering (PSF) under different particle spacings according to the present invention: along the y-direction;

[0042] Figure 7 The following is a simulation comparison of S-polarized light interferometric scattering microscopy (PSF) and P-polarized light interferometric scattering (PSF) under different particle spacings according to the present invention: along the x-direction;

[0043] Figure 8 This is a comparison of polarization interference scattering microscopy imaging (a) of multiple SiO2 particles with a diameter of 300 nm and interference scattering microscopy imaging (b) of conventional circularly polarized light illumination to detect P-polarized light.

[0044] Explanation of reference numerals in the attached figures: 1-Continuous laser, 2-First low-power objective lens, 3-Rotating scattering plate, 4-Second low-power objective lens, 5-Polarizer, 6-First total reflection mirror, 7-Half-wave plate, 8-First analyzer, 9-Wide field illumination lens, 10-Beam splitter, 11-High-power objective lens, 12-Sample stage, 13-Second total reflection mirror, 14-Second analyzer, 15-Tube lens, 16-Detector, 17-Image processing module, 18-Displacement control module, 19-Third total reflection mirror, 20-Second beam splitter, 21-Polarization analyzer, 22-Acousto-optic deflector, 23-First lens of the 4f system, 24-Second lens of the 4f system. Detailed Implementation

[0045] The technical solution of this invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0046] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings and are used only for the convenience of describing the invention and simplifying the description. They do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as limiting the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0047] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "joining" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0048] Example 1

[0049] See Figure 1 A microscopic imaging system for non-fluorescent multi-particle resolution includes an illumination optical path, an imaging optical path, and a control and processing module. The illumination optical path is sequentially connected in the following order: a continuous laser 1, a first low-power objective lens 2, a rotating scattering plate 3, a second low-power objective lens 4, a polarizer 5, a first total reflection mirror 6, a half-wave plate 7, a first analyzer 8, and a wide-field illumination lens 9. The imaging optical path is sequentially connected in the following order: a high-power objective lens 11, a beam splitter 10, a second total reflection mirror 13, a second analyzer 14, a tube lens 15, and a detector 16. The control and processing module consists of an image processing module 17 (see image processing module 17). Figure 3 It consists of a displacement control module 18 and a displacement control module 18.

[0050] In this embodiment, as Figure 2 As shown, the continuous laser 1 in the light source module outputs a Gaussian beam with a center wavelength of 445 nm. The laser beam is adjusted into parallel light by the first low-magnification objective lens 2 and the second low-magnification objective lens 4. A rotating scattering plate 3, consisting of a stepper motor 3-1 and a scattering plate 3-2, is placed at the focal point of the first low-magnification objective lens 2 to reduce the correlation of the laser beam and remove additional interference fringes caused by stray light in the interference system. The output beam size, laser energy, and coherence of the light source module can all be freely adjusted.

[0051] In this embodiment, the background reflected light from the sample particles is suppressed through the design of the polarization modulation module and the analyzer in the detection path. The polarization-modulated illumination light is in an S-polarization state, and the S-polarized reflected light on the sample surface is orthogonally polarized and suppressed by the second analyzer 14 in the detection optical path, thereby improving the image contrast.

[0052] Based on the above embodiments, such as Figure 6 As shown, for different y-direction spacings D y The following simulations were performed using the interference scattering PSF of the next two particles. Simulation parameters were set as follows: illumination laser wavelength 455 nm, high-magnification objective lens numerical aperture 1.4, magnification 100. Particle spacing D in the y-direction was also simulated. yAs the diameter increases from 0.2 μm to 0.4 μm, two particles cannot be distinguished in the central circular PSF of the P-polarized state, while two scattering particles can be distinguished in the four-lobed PSF of the S-polarized state.

[0053] Based on the above embodiments, such as Figure 7 As shown, the distance D in the y-direction between the two particles y At a spacing of 0.08 μm, for different x-direction spacings D x The interference scattering PSF of the next two particles was simulated. Simulation parameters were set as follows: illumination laser wavelength of 455 nm, high-magnification objective lens numerical aperture of 1.4, and magnification of 100. The particle x-axis spacing D was also simulated. y Increasing the size from 0.2 μm to 0.4 μm also showed that two particles could not be distinguished in the central circular PSF of the P-polarized state, while two scattering particles and their relative positions could be distinguished in the four-lobed PSF of the S-polarized state.

[0054] Based on the above embodiments, taking the interference scattering imaging of SiO2 with a particle size of 300nm as an example, a laser wavelength of 445nm is selected. S-polarized light illumination is achieved through a polarization modulation module, and the signal light and background light in the S-polarized state are detected by a polarizer and collected by detector 16. Figure 8 As shown in (a), a quarter-wave plate is placed between the beam splitter 10 and the high-power objective lens 11 for circularly polarized illumination. The S-polarized light from the reflecting end of the polarizing beam splitter 9 is used to form circularly polarized light that illuminates the sample surface via the quarter-wave plate. The azimuth angle of the second analyzer 14 is adjusted to 0°. ° The interference light between the P-polarized background light and the signal interference light is detected. After passing through the imaging module, the interference scattering image is acquired by detector 16, such as... Figure 8 As shown in (b). Compared to traditional imaging methods that use circularly polarized light illumination and detect the P-polarization component, the polarization-selective interferometric scattering microscopy system and method provided by this invention improves the resolution of multiple particles through a four-lobed PSF with polarization modulation, and enhances the signal-to-noise ratio by combining orthogonal polarization to suppress background light. It possesses advantages such as high sensitivity, high resolution, long observation time, and wide field of view.

[0055] Example 2

[0056] An imaging method based on the non-fluorescent multi-particle resolution microscopic imaging system described in Example 1 includes the following steps:

[0057] S1: The illumination light emitted by the continuous laser 1 is focused after passing through the first low-power objective lens 2, and after passing through the rotating scattering plate 3, it is collected and adjusted into parallel light by the second low-power objective lens 4.

[0058] S2: The parallel light is adjusted to parallel linearly polarized light by the polarizer 5. After being reflected by the first total reflection mirror 6, the parallel linearly polarized light enters the half-wave plate 7 to adjust the polarization direction of the linearly polarized light. Then, it enters the first analyzer 8 to adjust the linearly polarized light to S-linearly polarized light.

[0059] S3: The parallel linearly polarized light is converged at the back focal plane of the high-magnification objective lens 11 after passing through the wide-field illumination lens 9 and the beam splitter 10. The converged illumination light is then used by the high-magnification objective lens 11 to form a wide-field parallel light that illuminates the sample stage 12.

[0060] S4: The scattered light from the sample particles on the sample stage 12 and the reflected light from the sample substrate surface are collected by the high-power objective lens 11 and then pass through the beam splitter 10, the second total reflection mirror 13, and the second polarizer 14. After suppressing the background reflected light, the light enters the lens tube 15 and converges the light onto the detector 16 for interference imaging.

[0061] S5: The image processing module 17 processes a series of image matrices acquired by the acquisition, obtains the image background by averaging or median filtering, obtains the signal by the difference method and normalizes it;

[0062] S6: Calculate the point diffusion function (PSF) of the system under multiple particles and fit it with the particle spacing;

[0063] S7: Calculate the particle spacing based on the normalized image and fitted curve, and perform particle localization to obtain the position and spacing information of multiple particles.

[0064] Example 3

[0065] See Figure 4 A microscopic imaging system for non-fluorescent multi-particle resolution includes an illumination optical path, an imaging optical path, and a control and processing module. The illumination optical path is sequentially connected to a continuous laser 1, a first low-power objective lens 2, a rotating scattering plate 3, a second low-power objective lens 4, a polarizer 5, a first total reflection mirror 6, a half-wave plate 7, a third total reflection mirror 19, a second beam splitter 20, and a wide-field illumination lens 9. The imaging optical path is sequentially connected to a high-power objective lens 11, a beam splitter 10, a second total reflection mirror 13, a second analyzer 14, a tube lens 15, and a detector 16. The control and processing module consists of an image processing module 17 and a displacement control module 18. A polarization analyzer 21 is provided at the exit end of the second beam splitter 20.

[0066] Example 4

[0067] An imaging method based on the non-fluorescent multi-particle resolution microscopic imaging system described in Example 3 includes the following steps:

[0068] S1: The illumination light emitted by the continuous laser 1 is focused after passing through the first low-power objective lens 2, and after passing through the rotating scattering plate 3, it is adjusted into parallel light by the second low-power objective lens 4.

[0069] S2: The parallel light is adjusted to parallel linearly polarized light by the polarizer 5. After being reflected by the first total reflection mirror 6, the parallel linearly polarized light enters the half-wave plate 7 to adjust the polarization direction of the linearly polarized light. After being reflected by the third total reflection mirror 19, it is split into two beams by the second beam splitter 20. The transmitted light enters the polarization analyzer 21, and the reflected light is used as the illumination light.

[0070] S3: The linear polarization direction is adjusted in real time by using the half-wave plate 7 and the polarization analyzer 21 to change the polarization state of the illumination light to the S-polarization state;

[0071] S4: The reflected parallel linearly polarized light is converged to the back focal plane of the high-magnification objective lens 11 after passing through the wide-field illumination lens 9 and the beam splitter 10. The converged illumination light is then used by the high-magnification objective lens 11 to form a wide-field parallel light that illuminates the sample stage 12.

[0072] S5: The scattered light from the sample particles on the sample stage 12 and the reflected light from the sample substrate surface are collected by the high-power objective lens 11 and then pass through the beam splitter 10, the second total reflection mirror 13, and the second polarizer 14. After suppressing the background reflected light, the light enters the lens tube 15 and converges the light onto the detector 16 for interference imaging.

[0073] S5: The image processing module 17 processes a series of image matrices acquired by the acquisition, obtains the image background by averaging or median filtering, obtains the signal by the difference method and normalizes it;

[0074] S6: Calculate the point diffusion function (PSF) of the system under multiple particles and fit it with the particle spacing;

[0075] S7: Calculate the particle spacing based on the normalized image and fitted curve, and perform particle localization to obtain the position and spacing information of multiple particles.

[0076] Example 5

[0077] See Figure 5 A microscopic imaging system for non-fluorescent multi-particle resolution includes an illumination optical path, an imaging optical path, and a control and processing module. The illumination optical path is sequentially connected in the optical path direction to a continuous laser 1, a first low-power objective lens 2, a rotating scattering plate 3, a second beam expander collimating lens 4, a polarizer 5, a first total reflection mirror 6, a half-wave plate 7, a first analyzer 8, an acousto-optic deflector 22, a 4f system first lens 23, and a 4f system second lens 24. The imaging optical path is sequentially connected in the optical path direction to a high-power objective lens 11, a beam splitter 10, a second total reflection mirror 13, a second analyzer 14, a tube lens 15, and a detector 16. The control and processing module consists of an image processing module 17 and a displacement control module 18.

[0078] Example 6

[0079] A microscopic imaging method for non-fluorescent multi-particle resolution includes the following steps:

[0080] S1: The illumination light emitted by the continuous laser 1 is focused after passing through the first low-power objective lens 2, and after passing through the rotating scattering plate 3, it is adjusted into parallel light by the second low-power objective lens 4.

[0081] S2: The parallel light is adjusted to parallel linearly polarized light by the polarizer 5. After being reflected by the first total reflection mirror 6, the parallel linearly polarized light enters the half-wave plate 7 to adjust the polarization direction of the linearly polarized light. Then, it enters the first analyzer 8 to adjust the linearly polarized light to S-linearly polarized light.

[0082] S3: The parallel linearly polarized light is rapidly scanned in two dimensions by the acousto-optic deflector 22. The first lens 23 and the second lens 24 of the 4f system reflect the deflected beam through the beam splitter 10 and converge it to the back focal plane of the high-magnification objective lens 11. The converged illumination light forms a wide-field parallel light that illuminates the sample stage 12 through the high-magnification objective lens 11.

[0083] S4: The scattered light from the sample particles on the sample stage 12 and the reflected light from the sample substrate surface are collected by the high-power objective lens 11 and then pass through the beam splitter 10, the second total reflection mirror 13, and the second polarizer 14. After suppressing the background reflected light, the light enters the lens tube 15 and converges the light onto the detector 16 for interference imaging.

[0084] S5: The image processing module 17 processes a series of image matrices acquired by the acquisition, obtains the image background by averaging or median filtering, obtains the signal by the difference method and normalizes it;

[0085] S6: Calculate the point diffusion function (PSF) of the system under multiple particles and fit it with the particle spacing;

[0086] S7: Calculate the particle spacing based on the normalized image and fitted curve, and perform particle localization to obtain the position and spacing information of multiple particles.

[0087] The principle of this invention is as follows: Polarization-modulated interferometric scattering microscopy is based on the polarization selectivity of the point spread function (PSF). A combination of a polarizer, a half-wave plate, and a first analyzer is used to achieve S-polarized illumination of the sample particles. Under S-polarized illumination and detection conditions, the system's PSF exhibits a highly characteristic four-lobed distribution. When multiple particles form interferometric scattering, the four-lobed PSF can achieve high-sensitivity resolution of the interparticle spacing.

[0088] For a specific polarization modulation module, the required azimuth angles of the polarizer, half-wave plate, and first analyzer are given by the polarization relationship. Let θ1 be the angle between the polarizer's transmission axis and the x-axis, θ2 be the angle between the fast axis of the half-wave plate and the x-axis, and θ3 be the angle between the first analyzer's transmission axis and the x-axis, using the following formula:

[0089]

[0090]

[0091] Then, by setting the polarizer angle θ1 and the first analyzer angle π / 2, and by rotating the half-wave plate by the angle θ2, A1=0 is satisfied, so that all the light energy after passing through the half-wave plate passes through the first analyzer, thus satisfying the polarization modulation condition.

[0092] The principle behind polarization modulation for improving the resolution of multi-particle interference scattering is that interference scattering systems exhibit different polarization states and polarities (PSFs). The PSF of traditional P-beam interference scattering is a central circular interference ring with an outer circular interference ring, while the PSF of S-beam interference scattering is a central four-lobed interference ring with an outer four-lobed interference ring. By introducing an analyzer into the polarization modulation module and the detection path, a four-lobed PSF can be acquired, thereby improving the resolution of multi-particle interference scattering.

[0093] The embodiments described in this specification are merely examples of implementations of the inventive concept. The scope of protection of this invention should not be considered as limited to the specific forms stated in the embodiments. The scope of protection of this invention also extends to equivalent technical means that can be conceived by those skilled in the art based on the inventive concept.

Claims

1. A microscopic imaging system for non-fluorescent multi-particle resolution, characterized in that: It includes an illumination optical path, an imaging optical path, and a control and processing module. The emitting end of the illumination optical path and the incident end of the imaging optical path are jointly provided with a sample stage (12), and the sample to be tested is placed on the sample stage (12). The illumination optical path includes a light source module, a polarization modulation module, and an illumination module arranged sequentially along the optical path. The light source module provides illumination light and modulates the light into parallel linearly polarized light. The polarization modulation module adjusts the incident parallel linearly polarized light to an S-polarization state. The illumination module introduces the adjusted S-polarized light into the sample surface to be tested. The illumination module includes a beam splitter (10). The imaging optical path includes a high-power objective lens (11) and an imaging module arranged sequentially along the optical path. The scattered light from the sample particles on the sample surface to be detected and the reflected light from the substrate are collected together by the high-power objective lens (11), and the imaging module performs interference imaging between the scattered signal and the reflected light. The imaging optical path is connected to the imaging module after the high-power objective lens (11) collects the scattered light from the sample particles and the reflected light from the substrate, and then passes through the beam splitter (10). The high-power objective lens (11) is connected to the incident end of the beam splitter (10), and the imaging module is connected to the emitting end of the beam splitter (10). The imaging module includes a second total reflection mirror (13), a second polarizer (14), a telescope lens (15), and a detector (16). The second total reflection mirror (13) changes the direction of the probe light; the second polarizer (14) further adjusts the polarization component of the light used for interference imaging; the telescope lens (15) converges the scattered light and reflected light from the analyzed particles; and the detector (16) performs interference imaging of the scattered light and reflected light. The control and processing module includes an image processing module (17) and a displacement control module (18). The image processing module (17) performs image processing on the interference imaging signal, and the displacement control module (18) controls the sample position in real time.

2. The microscopic imaging system for non-fluorescent multi-particle resolution as described in claim 1, characterized in that: The light source module includes a continuous laser (1) that provides continuous laser light, and a first low-power objective (2), a rotating scattering plate (3), and a second low-power objective (4) arranged sequentially along the laser light path; the first low-power objective (2) focuses the laser beam onto the rotating scattering plate (3), the rotating scattering plate (3) weakens the coherence of the incident light to eliminate interference fringes, and the second low-power objective (4) collects the light that has passed through the rotating scattering plate (3).

3. The microscopic imaging system for non-fluorescent multi-particle resolution as described in claim 2, characterized in that: The polarization modulation module includes a polarizer (5), a first total reflection mirror (6), a half-wave plate (7), and a first analyzer (8) arranged sequentially along the optical path; the polarizer (5) adjusts the incident parallel light to linearly polarized light; the first total reflection mirror (6) adjusts the direction of the incident light; the half-wave plate (7) adjusts the polarization direction of the polarized light; and the first analyzer (8) adjusts the polarization direction of the incident linearly polarized light to S-polarized light.

4. The microscopic imaging system for non-fluorescent multi-particle resolution as described in claim 2, characterized in that: The polarization modulation module includes a polarizer (5), a first total reflection mirror (6), a half-wave plate (7), a third total reflection mirror (19), and a second beam splitter (20) arranged sequentially along the optical path. A polarization analyzer (21) is provided at the output end of the second beam splitter (20). The polarizer (5) adjusts the illumination light to be parallel linearly polarized light. After the parallel linearly polarized light is reflected by the first total reflection mirror (6), it enters the half-wave plate 7 to adjust the polarization direction of the linearly polarized light. After being reflected by the third total reflection mirror (19), it is split into two beams by the second beam splitter (20). The transmitted light enters the polarization analyzer (21), and the reflected light is used as the illumination light. The half-wave plate (7) and the polarization analyzer (21) adjust the linear polarization direction in real time to make the polarization state of the illumination light change to the S-polarization state.

5. A microscopic imaging system for non-fluorescent multi-particle resolution as described in claim 3, characterized in that: The illumination module includes a wide-field illumination lens (9), a beam splitter (10), and a high-magnification objective lens (11) arranged sequentially along the optical path. The wide-field illumination lens (9) converges the incident parallel linearly polarized light; the beam splitter (10) reflects the illumination light; and the high-magnification objective lens (11) collects the illumination light and emits it onto the surface of the sample to be tested.

6. The microscopic imaging system for non-fluorescent multi-particle resolution as described in claim 4, characterized in that: The illumination module includes a polarizer (5), a first total reflection mirror (6), a half-wave plate (7), a first analyzer (8), an acousto-optic deflector (22), a 4f system first lens (23), a 4f system second lens (24), a beam splitter (10), and a high-power objective lens (11) arranged sequentially along the optical path. The polarizer (5) adjusts the illumination light to parallel linearly polarized light. After the parallel linearly polarized light is reflected by the first total reflection mirror (6), it enters the half-wave plate (7) to adjust the polarization direction of the linearly polarized light, and then enters the first analyzer (8) to adjust the linearly polarized light to S-polarized light. The parallel linearly polarized light is rapidly scanned in two dimensions by the acousto-optic deflector (22). The first lens (23) and the second lens (24) of the 4f system reflect the deflected beam through the beam splitter (10) and converge it to the back focal plane of the high-power objective lens (11). The converged illumination light forms a wide-field parallel light that illuminates the sample stage (12) through the high-power objective lens (11).

7. A microscopic imaging system for non-fluorescent multi-particle resolution as described in claim 6, characterized in that: The control and processing module includes an image processing module (17) and a displacement control module (18). The image processing module (17) is used to process the signal obtained by the detector to obtain the particle interference signal; the displacement control module (18) controls the three-dimensional position of the sample in real time.

8. A microscopic imaging system for non-fluorescent multi-particle resolution as described in claim 6, characterized in that: The first low-power objective (2) and the second low-power objective (4) have a magnification of 10 and a numerical aperture of 0.

25. The high-power objective (11) has a magnification of 100 and a numerical aperture of 1.

4. The beam splitter (10) has a transmission-to-reflection beam splitting ratio T:R=50:

50. The polarization ratio of the second analyzer (14) is S:P=5:

95.

9. An imaging method based on claim 5 for use in a non-fluorescent multi-particle resolution microscopic imaging system, characterized in that, Includes the following steps: S1: The illumination light emitted by the continuous laser (1) is focused after passing through the first low-power objective lens (2), and after passing through the rotating scattering plate (3), it is collected and adjusted into parallel light by the second low-power objective lens (4); S2: Parallel light passes through a polarizer (5) to adjust the illumination light into parallel linearly polarized light. After the parallel linearly polarized light is reflected by the first total reflection mirror (6), it enters a half-wave plate (7) to adjust the polarization direction of the linearly polarized light, and then enters the first analyzer (8) to adjust the linearly polarized light into S-polarized light. Let θ1 be the angle between the transmission axis of the polarizer (5) and the x-axis, θ2 be the angle between the fast axis of the half-wave plate (7) and the x-axis, and θ3 be the angle between the transmission axis of the first analyzer and the x-axis. Combined with the following formula: （1） （2） Set the included angle θ1 of the polarizer (5) and the included angle π / 2 of the first analyzer (8). By rotating the half-wave plate (7) by the angle θ2, A1=0 is satisfied, so that all the light energy after passing through the half-wave plate (7) passes through the first analyzer (8). S3: The parallel linearly polarized light converges to the back focal plane of the high-power objective lens (11) after passing through the wide-field illumination lens (9) and the beam splitter (10). The converged illumination light forms a wide-field parallel light that illuminates the sample stage (12) after passing through the high-power objective lens (11). S4: The scattered light from the sample particles on the sample stage (12) and the reflected light from the sample substrate surface are collected by the high-power objective lens (11), and then pass through the beam splitter (10), the second total reflection mirror (13), and the second polarizer (14) to suppress the background reflected light before entering the lens barrel (15) to focus the light onto the detector (16) for interference imaging. S5: The image processing module (17) processes a series of image matrices acquired by acquisition, obtains the image background by averaging or median filtering, obtains the signal by difference method and normalizes it. S6: Calculate the point diffusion function (PSF) of the system under multiple particles and fit it with the particle spacing; S7: Calculate the particle spacing based on the normalized image and fitted curve, and perform particle localization to obtain the position and spacing information of multiple particles.

Images