Confocal single-molecule three-dimensional detection and tracking method and system

Abstract

The invention relates to a confocal single-molecule three-dimensional detection and tracking method and system, the system comprises an illumination system and a detection system, and the illumination system and the detection system of the system are conjugated; the phase modulation optical assembly comprises a first polaroid, a phase modulation module and a second polaroid; the illumination system and the detection system share a group of phase modulation optical components; the illumination point spread function and the detection point spread function can be modulated at the same time by adopting the common phase modulation optical assembly; wherein the phase modulation module is a modulation phase which is designed based on a phase combination method and has the property of a combined double-helix point spread function, and is used for converting a light beam into a light beam with the property of the combined double-helix point spread function; compared with the prior art, the method has the advantages that the axial detection depth of the optical system is enlarged, and meanwhile, the high signal-to-noise ratio and rapid tracking are guaranteed.

Description

Technical Field

[0001] The invention relates to the technical field of optical microscopic tracing, and in particular to a confocal single-molecule three-dimensional detection and tracing method and system. Background Art

[0002] Rapid and high-precision detection of the dynamic characteristics of intracellular macromolecules in three-dimensional space is crucial for studying viral infection mechanisms, DNA transcription, the interaction between nucleic acid macromolecules and physiological processes, and helps to better understand the pathogenesis of diseases and the intervention mechanisms of drugs, thereby accelerating the development of new drugs and vaccines. However, the movement of molecules in cells is often a relatively fast three-dimensional process, and its size is nanometer-scale. Conventional optical molecular detection technology needs to be combined with axial expansion imaging methods to obtain the three-dimensional dynamic process of molecules.

[0003] In the single-molecule tracking method, a commonly used method is to use a wide-field fluorescence microscope with a high-speed camera to image the movement of fluorescently labeled particles, but the information obtained is often the movement of particles in a two-dimensional plane, and the movement of particles in the axial direction cannot be obtained. Conventional laser confocal microscopy or multiphoton microscopy can achieve optical sectioning of tissues, three-dimensional reconstruction, and qualitative and quantitative analysis of certain specific components in the sample. However, this method requires point-by-point, line-by-line, and surface-by-surface scanning imaging, and has low temporal resolution. Super-resolution microscopy techniques, such as STED, STORM, PALM, etc., can achieve a resolution of tens of nanometers, but it often takes several minutes or even tens of minutes to form an image, which still cannot meet the requirements for rapid or even real-time three-dimensional tracking of nanoparticles in biological samples.

[0004] By adopting the method of point spread function engineering, the point spread function of the wide-field fluorescence microscope is modulated with a specially designed phase at the detection end to generate a point spread function with axial encoding capability, thereby enabling the wide-field fluorescence microscope to have the ability of three-dimensional positioning imaging. However, this method is generally used for fluorescence imaging. Modulating the fluorescence at the detection end will weaken the intensity of the fluorescence, resulting in a low signal-to-noise ratio or even the inability to image. In order to effectively detect fluorescent molecules, a strong laser is required to excite the fluorescence, which is easy to cause fluorescence bleaching, which is not conducive to long-term detection and imaging. Summary of the invention

[0005] The purpose of the present invention is to provide a confocal single-molecule three-dimensional detection and tracking method and system in order to ensure high signal-to-noise ratio and rapid tracking.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A confocal single-molecule three-dimensional detection and tracking system of a phase modulation optical component, the system comprising an illumination system and a detection system, the illumination system comprising a laser, a first beam expansion lens, a second beam expansion lens, a dichroic mirror, an XY scanning galvanometer, a third beam expansion lens, a fourth beam expansion lens, a phase modulation optical component, a reflector, a fifth beam expansion lens, a sixth beam expansion lens and an objective lens;

[0008] The detection system includes a detector, a detection lens, a dichroic mirror, an XY scanning galvanometer, a third beam expander lens, a fourth beam expander lens, a phase modulation optical component, a reflector, a fifth beam expander lens, a sixth beam expander lens and an objective lens;

[0009] The phase modulation optical component includes a phase modulation module, and the phase modulation module is a first phase modulation module or a second phase modulation module;

[0010] The phase modulation module is a phase mask designed based on a phase combination method, which is used to convert a light beam into a light beam having the properties of an Sπ double helix point spread function, and the combined double helix phase is the optical transfer function of the phase modulation module.

[0011] Further, the combined double helix phase is:

[0012]

[0013] in,

[0014]

[0015] in, Represents polar coordinates Phase modulation on Q1, Q2, Q3, ..., Q S is the total number of Fresnel zones, q1, q2, q3, ..., q S etc. represent the number of rotation periods, which are non-zero positive integers and can be equal or unequal, ζ1, ζ2, ζ3, ..., ζ S represents the defocus function, which is determined by Formula 2, W 20 is the defocus coefficient, which controls the defocus distance. This coefficient is determined by the effective axial detection range of the initial phase designed in Formula 1. S is the number of phase types used for stitching. N represents the fractional number of Fourier plane divisions. n represents the nth Fourier plane sector area. θ represents the central angle interval of each sector using the sector partitioning method. is a downward integer operator, m is the diffraction order of the wavefront scattering, and R is the radius of the grating aperture. According to the Abbe sine condition, the limiting radius R is given by f 4f ×NA / (G 2 -NA 2 ) 1 / 2 It is concluded that f 4fis the focal length of the 4f system, G is the system magnification, NA is the objective numerical aperture, λ is the wavelength of light, ρ is the polar coordinate,

[0016] The phase mask designed above is the initial phase, which is optimized by the phase inversion optimization algorithm to become the final phase mask. The phase mask designed by the above design method obtains different phases due to different parameter values, rather than one phase.

[0017] Furthermore, the phase modulation optical component consists of a first phase modulation module, a triangular prism, a first polarizer and a second polarizer.

[0018] Furthermore, when the phase modulation optical component is composed of a second phase modulation module, the second phase modulation module is a phase plate or a transmissive digital micromirror device DMD.

[0019] Another aspect of the present invention provides a confocal single-molecule three-dimensional detection and tracking method, the method comprising the above-mentioned system, the method comprising the following steps:

[0020] Furthermore, the laser of the illumination system emits an illumination light beam, which is expanded by a first beam expansion lens and a second beam expansion lens, reflected by a dichroic mirror and an XY scanning galvanometer, and adjusted in diameter by a third beam expansion lens and a fourth beam expansion lens, and modulated into a double helix light beam by a phase modulation optical component, and the propagation direction of the double helix light beam is changed by reflection by a reflector, and adjusted in diameter by a fifth beam expansion lens and a sixth beam expansion lens, and converged into parallel light, which is emitted to the excitation light focused by the objective lens and irradiated to the object to be measured, thereby stimulating the fluorescence of the object to be measured;

[0021] A detection light beam containing excitation light, fluorescence and other stray light is received by the objective lens of the detection system. The diameter of the detection light beam is adjusted by the fifth and sixth beam expansion lenses, and the fluorescence light beam is modulated into a double helix light beam by the phase modulation optical component through the reflector. The diameter of the fluorescence light beam is adjusted by the third and fourth beam expansion lenses, and the fluorescence light beam is reflected by the XY scanning galvanometer, and the excitation light and stray light are filtered out after filtering by the dichroic mirror, and the fluorescence light is focused on the imaging surface of the detector through the detection lens, forming a double helix image point on the imaging surface.

[0022] Furthermore, the phase modulation module converts the illumination beam and the fluorescent beam into an imaging beam having a combined double-helix point spread function property.

[0023] Furthermore, the phase modulation optical component is placed in the common optical path portion of the illumination optical path and the detection optical path to achieve simultaneous modulation of the illumination beam and the detection beam, that is, common phase mask modulation of the illumination end and the detection end.

[0024] Furthermore, when the phase modulation optical component is composed of the first phase modulation module, the triangular prism, the first polarizer and the second polarizer, it is a reflective spatial light modulator, and when the modulation optical component is composed of the second phase modulation module, it is a transmissive spatial light modulator.

[0025] Furthermore, when the phase modulation module is a reflective spatial light modulator, after the beam diameter is adjusted by the third beam expansion lens and the fourth beam expansion lens, the first polarizer modulates the polarization state of the beam, and outputs polarized light suitable for the first phase modulation module. After passing through the triangular prism, it is input into the first phase modulation module for modulation, and then passes through the triangular prism and the second polarizer to output an illumination beam modulated into a double helix beam.

[0026] Furthermore, when the phase modulation module is a reflective spatial light modulator, after the fluorescent light beam is expanded by the fifth expanding lens and the sixth expanding lens, the second polarizer modulates the polarization state of the fluorescent light beam, and outputs polarized light suitable for the phase modulation module. After passing through the triangular prism, it is input into the first phase modulation module for modulation, and then passes through the triangular prism and the first polarizer to output the fluorescent light beam modulated into a double helix beam.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention creates a phase modulation optical component based on combined double-helix phase modulation with large depth of field and multi-cycle rotation, and creates a confocal single-molecule three-dimensional detection and tracking system of the phase modulation optical component with large depth of field based on the phase modulation optical component. After the phase modulation optical component simultaneously modulates the light beams of the illumination light path and the detection light path, a combined double-helix light beam with large depth of field and multi-cycle rotation is generated. The light beam has axial encoding capability, high positioning accuracy and a large depth of field. In addition, the design of the confocal optical path fully utilizes the advantages of the confocal high-resolution capability and can ensure high signal-to-noise ratio tracking. In addition, the method of common phase modulation of the illumination end and the detection end is adopted to effectively expand the axial detection depth of the confocal and enable it to have axial rapid positioning capability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the overall structure of the present invention;

[0030] Figure 2 is a comparison diagram of the combined double helix point spread function and the conventional point spread function at different depths, where Figure 2 (a) is the double helix point spread function at different depths with large depth of field. Figure 2 (b) is the conventional point spread function;

[0031] Figure 3 It is a curve diagram showing the relationship between the angle between the center of the side lobes of the combined double helix point spread function and the axial detection depth (Z-axis position);

[0032] Figure 4 is a schematic diagram of a combined double spiral phase mask;

[0033] Figure 5 is a schematic diagram of a phase modulation optical component for a confocal single-molecule three-dimensional detection and tracking system of a large depth-of-field phase modulation optical component provided by a second embodiment of the present invention, Figure 5 (a) is a phase modulation optical component of a transmissive phase modulation optical component, Figure 5 (b) is a phase modulation optical component of a reflective phase modulation optical component;

[0034] Figure 6 Schematic diagram of the illumination system of a confocal single-molecule three-dimensional detection and tracking system of a large depth of field phase modulation optical component of the present invention, wherein Figure 6 (a) is a schematic diagram of the illumination system of the transmissive phase modulation optical component. Figure 6 (b) is a schematic diagram of an illumination system of a reflective phase modulation optical component;

[0035] Figure 7 Schematic diagram of a detection system of a confocal single-molecule three-dimensional detection and tracking system of a large depth of field phase modulation optical component of the present invention, wherein Figure 7 (a) is a schematic diagram of the detection system of the transmission phase modulation optical component. Figure 7 (b) is a schematic diagram of a detection system of a reflective phase modulation optical component;

[0036] Figure 8 It is a schematic diagram of an imaging system of a confocal single-molecule three-dimensional detection and tracking system of a large depth-of-field phase modulation optical component of the present invention, Figure 8 (a) is a schematic diagram of an imaging system of a transmissive phase modulation optical component. Figure 8 (b) is a schematic diagram of an imaging system of a reflective phase modulation optical component;

[0037] In the figure, an illumination system 501, a detection system 502, a detector 402, a detection lens 401, a laser 301, a first beam expanding lens 302, a second beam expanding lens 303, a dichroic mirror 304, an XY scanning galvanometer 305, a third beam expanding lens 306, a fourth beam expanding lens 307, a phase modulation optical component 308, a reflector 309, a fifth beam expanding lens 310, a sixth beam expanding lens 311, an objective lens 312, a detection lens 401, a detector 402, a first polarizer 201, a triangular prism 202, a first phase modulation module 203, a second polarizer 204, and a second phase modulation module 205. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0039] The present invention provides a confocal single molecule three-dimensional detection and tracking method and system. The system comprises an illumination system 501 and a detection system 502 arranged in sequence along the transmission direction of the light path, such as Figure 8 Overall structure diagram Figure 1 shown.

[0040] The first beam expanding lens assembly is composed of a first beam expanding lens 302 and a second beam expanding lens 303 ; the second beam expanding lens assembly is composed of a third beam expanding lens 306 and a fourth beam expanding lens 307 ; the third beam expanding lens assembly is composed of a fifth beam expanding lens 310 and a sixth beam expanding lens 311 .

[0041] In the imaging system, the laser emitted by the laser 301 is expanded by the first beam expanding lens assembly 302 and 303 to expand the illumination beam, reflected by the dichroic mirror 304 and the XY scanning galvanometer 305, shaped by the second beam expanding lens assembly 306 and 307, modulated into a double helix beam by the phase modulation assembly 308, reflected by the reflector 309, focused by the third beam expanding lens assembly 310 and converged by 311 into parallel light and emitted to the objective lens 312, which is focused by the objective lens 312 to irradiate the object to be measured to stimulate the fluorescence of the object to be measured. The object to be tested can emit fluorescence after being excited by the excitation light. The light beam containing the excitation light, fluorescence and other stray light is received by the objective lens 312. The light beam is shaped by the third beam expanding lens components 311 and 310, and returned to the phase modulation component 308 through the reflector 309. The fluorescence light beam is modulated into a double helix light beam by the phase modulation component 308. The light beam is shaped by the second beam expanding lens components 306 and 307, and the excitation light and stray light are filtered out after the filtering effect of the dichroic mirror 304, and the fluorescence is transmitted. The fluorescence is focused on the imaging surface of the detector 402 through the detection lens 401, and a double helix image point is formed on the imaging surface.

[0042] The above-mentioned large depth of field confocal single-molecule three-dimensional detection and tracking technology performs large depth of field real-time tracking imaging through the confocal optical system provided by the present invention, and utilizes the high-precision axial positioning capability of the multi-cycle rotation combined double helix point spread function with high positioning accuracy three-dimensional encoding capability to achieve a molecular tracking imaging method with large depth of field, high signal-to-noise ratio, and long-term three-dimensional nano-positioning accuracy.

[0043] like Figure 7The detection system of the large depth of field confocal single molecule three-dimensional detection and tracking technology includes a detector 402, a detection lens 401, a dichroic mirror 304, an XY scanning galvanometer 305, a beam expander lens 306, a beam expander lens 307, a phase modulation component 308, a reflector 309, a beam expander lens 310, a beam expander lens 311, and an objective lens 312, which are sequentially arranged along the transmission direction of the optical path. The dichroic mirror 304, the XY scanning galvanometer 305, the beam expander lens 306, the beam expander lens 307, the phase modulation component 308, the reflector 309, the beam expander lens 310, the beam expander lens 311, and the objective lens 312 are shared with the illumination optical path.

[0044] In this detection system, the object to be tested can emit fluorescence after being excited by the excitation light. The light beam containing the excitation light, the fluorescence and other stray light is received by the objective lens 308. The light beam is modulated into a double-helix light beam with multiple-cycle rotation by the phase modulation component. After the filtering action of the dichroic mirror 304, the excitation light and the stray light are filtered out and the fluorescence is transmitted. The fluorescence is focused on the imaging surface of the detector 402 through the detection lens 401, forming a double-helix image point on the imaging surface.

[0045] Figure 6 A schematic diagram of an illumination system for a large depth of field confocal single-molecule three-dimensional detection and tracking technology is shown. For ease of explanation, only the parts related to this embodiment are shown.

[0046] In this embodiment, a laser output illumination method is adopted, such as Figure 6 As shown, the illumination system of the large depth of field confocal single-molecule three-dimensional detection and tracking technology includes a laser 301, a beam expander lens 302, a beam expander lens 303, a dichroic mirror 304, an XY scanning galvanometer 305, a beam expander lens 306, a beam expander lens 307, a phase modulation component 308, a reflector 309, a beam expander lens 310, a beam expander lens 311, and an objective lens 312, which are arranged in sequence along the transmission direction of the light path.

[0047] In the illumination system, a laser is emitted by a laser 301, which is expanded by the first beam expanding lens assembly 302 and 303, reflected by the dichroic mirror 304 to the XY scanning galvanometer 305, scanned and reflected by the XY scanning galvanometer 305, expanded by the second beam expanding lens assembly 306 and 307, and modulated by the phase modulation optical assembly 308 to generate a double helix beam, which is output to the reflector 309, shaped by the third beam expanding lens assembly 310 and 311, and finally focused and irradiated to the object to be measured by the objective lens 312 to excite the fluorescence of the object to be measured.

[0048] Figure 5 A schematic diagram of a phase modulation optical component for large depth of field confocal single-molecule three-dimensional detection and tracking technology of the present invention is shown. For ease of explanation, only the part related to this embodiment is shown.

[0049] Based on the single-molecule three-dimensional positioning method provided in the above embodiment, this embodiment further provides a phase modulation optical component that can be used in a confocal single-molecule three-dimensional detection and tracking system of a large depth of field phase modulation optical component.

[0050] The optical component 308 mainly includes a first polarizer 201, a triangular prism 202, phase modulation modules 203 and 205, and a second polarizer 204, which are sequentially arranged along the transmission direction of the optical path. Among them, the phase modulation modules 203 and 205 have the property of a combined double-helix point spread function, and are designed based on the above method. They have the functions described in the above embodiment 1, and are not repeated here. In this system, the first polarizer 201 performs polarization state modulation on the illumination beam, and outputs polarized light suitable for the phase modulation module 203. The phase modulation module 203 outputs the laser beam after beam expansion into an imaging beam with the property of a combined double-helix point spread function. The second polarizer 204 performs polarization state modulation on the excited fluorescent beam, and outputs polarized light suitable for the phase modulation module 203. The phase modulation module 203 outputs the laser beam after beam expansion into an imaging beam with the property of a combined double-helix point spread function.

[0051] In this embodiment, there are two ways to implement the phase modulation module 308. One is to implement it through reflection modulation, which requires the use of a triangular prism 202, and the phase modulation module 203 is such as a reflective spatial light modulator; the other is to implement it through transmission modulation, and the phase modulation module 205 is such as a phase plate, a transmission digital micromirror device DMD, a transmission spatial light modulator and other optical modulation components, and a triangular prism is not required here.

[0052] The phase modulation module 203 / 205 of the present invention has the property of a combined double-helix point spread function. Specifically, through point spread function engineering, a multi-cycle rotation combined double-helix phase is used to modulate the point spread function of the optical system, generating a multi-cycle rotation combined double-helix point spread function with axial encoding capability, and realizing single-molecule positioning and trajectory tracking with three-dimensional nano positioning accuracy. The multi-cycle rotation combined double-helix point spread function is composed of two light spots symmetrical about the center. As the detection depth changes, the angle between the two light spots changes, thus having the ability of axial encoding. There are two ways to implement this optical component, one is to implement it by transmission modulation, such as optical modulation components such as phase plate, transmission digital micromirror device DMD, transmission spatial light modulator, etc.; the other is to implement it by reflection modulation, such as reflection spatial light modulator.

[0053] The specific design process is as follows:

[0054]

[0055] in,

[0056]

[0057] in, Represents polar coordinates Phase modulation on Q1, Q2, Q3, ..., Q S is the total number of Fresnel zones, q1, q2, q3, ..., q S etc. represent the number of rotation periods, which are non-zero positive integers and can be equal or unequal, ζ1, ζ2, ζ3, ..., ζ S represents the defocus function, which is determined by Formula 2, W 20 is the defocus coefficient, which controls the defocus distance. This coefficient is determined by the effective axial detection range of the initial phase designed in Formula 1. S is the number of phase types used for stitching. N represents the fractional number of Fourier plane divisions. n represents the nth Fourier plane sector area. θ represents the central angle interval of each sector using the sector partitioning method. is a downward integer operator, m is the diffraction order of the wavefront scattering, and R is the radius of the grating aperture. According to the Abbe sine condition, the limiting radius R is given by f 4f ×NA / (G 2 -NA 2 ) 1 / 2 It is concluded that f 4f is the focal length of the 4f system, G is the system magnification, NA represents the objective numerical aperture, λ represents the wavelength of light, and ρ represents the polar coordinates.

[0058] The phase mask designed above is the initial phase, which is optimized by the phase inversion optimization algorithm to become the final phase mask. The phase mask designed by the above design method obtains different phases due to different parameter values, rather than one phase.

[0059] The combined double helix phase can be obtained by using formula 1. If this function is applied to an optical system as an optical transfer function, the point spread function of the optical system is modulated into a combined double helix point spread function. Figure 2 is a case when S is 2. The final phase mask can be obtained by optimizing the designed combined double spiral phase by the phase inversion method, as shown in Figure 4 shown.

[0060] A combined double helix point spread function optical system is to add a specially designed optical module to the Fourier plane of the standard imaging system. This optical module makes its transmittance function form a combined double helix point spread function in the focal area of ​​the Fourier change. The optical component created above has this characteristic. The image formed by this optical component is two main lobes located in the imaging plane and symmetrical about the center. As the axial detection depth changes, the angle between the two main lobes changes. When using a multi-cycle rotating combined double helix point spread function for three-dimensional positioning, the lateral position coordinates of the molecule are estimated by the midpoint of the line connecting the centers of the two main lobes, and its axial position is obtained according to the angle between the two main lobes. For details, please refer to Figure 3 The relationship curve between the two main lobe angles of the multi-period rotation combined double helix point spread function and the axial detection depth is shown.

[0061] The present invention creates an optical phase modulation optical component based on multi-cycle rotation combined double helix phase modulation, and places the optical phase modulation optical component in the common optical path portion of the illumination end and the detection end. After the phase modulation optical component simultaneously modulates the light beam of the illumination light path and the light beam of the detection light path, a large depth of field multi-cycle rotation angle combined double helix light beam is generated, and the light beam has axial encoding capability, high positioning accuracy and a large depth of field. In addition, the illumination end and the detection end are used to simultaneously modulate the laser beam, making full use of the advantages of confocal microscopy imaging and single molecule tracking imaging. When the numerical aperture of the objective lens is 1.4, the imaging depth of the present invention can reach more than ten microns, and the depth of field can be adjusted by changing the design parameters of the combined double helix phase, which can be used for dynamic tracking imaging of multiple moving molecules with a large depth of field in living cells, which helps to better understand the occurrence mechanism of nervous system diseases and the intervention mechanism of drugs, thereby accelerating the research and development progress of new drugs.

[0062] The overall steps of the construction system of the present invention are:

[0063] Creating a phase-modulated optical component for combined dual-helix beam modulation;

[0064] The phase modulation optical component is used to simultaneously modulate the illumination point spread function and the detection point spread function of the confocal microscope system in a common optical path at the illumination end and the detection end to obtain an illumination combined double helix point spread function and a detection combined double helix point spread function with large axial size;

[0065] Creation of a detection system for a confocal single-molecule three-dimensional detection and tracking system with phase-modulated optical components;

[0066] The shape of the main lobe of the double helix point spread function in the image of the molecule to be detected determines the spatial position of the molecule to be detected, and reconstructs the three-dimensional motion trajectory and three-dimensional image of the molecule to be detected.

[0067] The phase modulation optical component includes various optical elements arranged in sequence along the transmission direction of the optical path:

[0068] A first polarizer, used to modulate the polarization state of the laser beam after beam expansion into linearly polarized light that can be modulated by the optical module;

[0069] An optical module, used for performing combined double-helix phase modulation on the laser to generate an imaging beam with combined double-helix point spread function properties;

[0070] The second polarizer is used to modulate the polarization state of the excited fluorescent light beam into linear polarized light that can be modulated by the optical module.

[0071] The lighting system comprises the following components arranged in sequence along the light transmission direction:

[0072] A laser for generating an illumination beam;

[0073] A first beam expansion lens assembly, used for expanding the laser beam emitted by the laser;

[0074] A dichroic mirror, used for reflecting the illumination light beam;

[0075] XY scanning galvanometer, a two-dimensional scanning galvanometer, is used to change the propagation direction of the illumination beam for scanning imaging;

[0076] A second beam expansion lens assembly, used to expand the laser beam emitted by the laser to match the aperture of the modulation optical assembly;

[0077] A modulation optical component, using the above-mentioned phase modulation optical component, is used to convert the illumination light beam into a combined double helix light beam with axial encoding capability;

[0078] Reflectors, which change the propagation direction of the illumination beam;

[0079] A third beam expander lens assembly is used to shape the modulated double helix to match the aperture of the objective lens and correct aberrations;

[0080] The objective lens is used to focus the illumination beam and output it to stimulate the fluorescence of the molecule to be tested;

[0081] The detection system includes the following components arranged in sequence along the optical transmission direction:

[0082] An objective lens, which shares the same objective lens with the illumination system and is used to receive the fluorescence emitted by the molecules to be measured;

[0083] A third beam expansion lens assembly, which shares the same beam expansion lens with the illumination system, is used to expand the excited fluorescence;

[0084] A reflector, which shares the same reflector with the illumination system and is used to change the transmission direction of the fluorescence;

[0085] A modulation optical component, which shares the same modulation optical component with the illumination system, and uses the above-mentioned phase modulation optical component to convert the fluorescent light beam into a combined double helix light beam with axial encoding capability;

[0086] A second beam expansion lens assembly, which shares the same beam expansion lens with the illumination system, is used to expand the excited fluorescence;

[0087] XY scanning galvanometer, which shares the same XY scanning galvanometer with the illumination system and is used to change the propagation direction of the detection beam;

[0088] A dichroic mirror, shared with the dichroic mirror of the illumination system, for filtering the detection light beam and outputting the fluorescence;

[0089] A lens for focusing the reflected fluorescence onto a detector for imaging;

[0090] The detector is used to receive the detection light beam and perform molecular tracking imaging.

[0091] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A confocal single-molecule three-dimensional detection and tracking system with phase modulation optical components, characterized in that: The system comprises an illumination system (501) and a detection system (502), wherein the illumination system (501) comprises a laser (301), a first beam expanding lens (302), a second beam expanding lens (303), a dichroic mirror (304), an XY scanning galvanometer (305), a third beam expanding lens (306), a fourth beam expanding lens (307), a phase modulation optical component (308), a reflector (309), a fifth beam expanding lens (310), a sixth beam expanding lens (311) and an objective lens (312); The detection system (502) comprises a detector (402), a detection lens (401), a dichroic mirror (304), an XY scanning galvanometer (305), a third beam expansion lens (306), a fourth beam expansion lens (307), a phase modulation optical component (308), a reflector (309), a fifth beam expansion lens (310), a sixth beam expansion lens (311) and an objective lens (312); The phase modulation optical component (308) comprises a phase modulation module, and the phase modulation module is a first phase modulation module (203) or a second phase modulation module (205); The phase modulation module is a phase mask designed based on a phase combination method, which is used to convert a light beam into a light beam having the properties of an Sπ double helix point spread function, and the combined double helix phase is the optical transfer function of the phase modulation module.

2. The confocal single-molecule three-dimensional detection and tracking system of a phase modulation optical component according to claim 1, characterized in that: The combined double helix phase is: in, in, Represents polar coordinates Phase modulation on Q1, Q2, Q3, ..., Q S is the total number of Fresnel zones, q1, q2, q3, ..., q S etc. represent the number of rotation periods, which are non-zero positive integers and can be equal or unequal, ζ1, ζ2, ζ3, ..., ζ S represents the defocus function, which is determined by Formula 2, W 20 is the defocus coefficient, which controls the defocus distance. This coefficient is determined by the effective axial detection range of the initial phase designed in Formula 1. S is the number of phase types used for stitching. N represents the fractional number of Fourier plane divisions. n represents the nth Fourier plane sector area. θ represents the central angle interval of each sector using the sector partitioning method. is a downward integer operator, m is the diffraction order of the wavefront scattering, and R is the radius of the grating aperture. According to the Abbe sine condition, the limiting radius R is given by f 4f ×NA / (G 2 -NA 2 ) 1 / 2 It is concluded that f 4f is the focal length of the 4f system, G is the system magnification, NA is the objective numerical aperture, λ is the wavelength of light, ρ is the polar coordinate, The phase mask designed above is the initial phase, which is optimized by the phase inversion optimization algorithm to become the final phase mask. The phase mask designed by the above design method obtains different phases due to different parameter values, rather than one phase.

3. The confocal single-molecule three-dimensional detection and tracking system of a phase-modulation optical component according to claim 2, characterized in that: The phase modulation optical component (308) is composed of a first phase modulation module (203), a triangular prism (202), a first polarizer (201) and a second polarizer (204).

4. The confocal single-molecule three-dimensional detection and tracking system of a phase modulation optical component according to claim 2, characterized in that: When the phase modulation optical component (308) is composed of a second phase modulation module (205), the second phase modulation module (205) is a phase plate or a transmission-type digital micromirror device (DMD).

5. A confocal single-molecule three-dimensional detection and tracking method, characterized in that: The method is based on the system according to any one of claims 1 to 4, and comprises the following steps: The laser (301) of the illumination system (501) emits an illumination light beam, which is expanded by a first beam expansion lens (302) and a second beam expansion lens (303), reflected by a dichroic mirror (304) and an XY scanning galvanometer (305), adjusted in diameter by a third beam expansion lens (306) and a fourth beam expansion lens (307), modulated into a double helix light beam by a phase modulation optical component (308), reflected by a reflector (309) to change the propagation direction of the double helix light beam, adjusted in diameter by a fifth beam expansion lens (310) and a sixth beam expansion lens (311), converged into parallel light and emitted to an objective lens (312), where the focused excitation light is irradiated onto an object to be tested, thereby exciting the fluorescence of the object to be tested; A detection light beam containing excitation light, fluorescence and other stray light is received by an objective lens (312) of a detection system (502). The detection light beam is adjusted in diameter by a fifth beam expansion lens (310) and a sixth beam expansion lens (311). The detection light beam is reflected by a reflector (309) to a phase modulation optical component (308) to modulate the fluorescence light beam into a double helix light beam. The fluorescence light beam is adjusted in diameter by a third beam expansion lens (306) and a fourth beam expansion lens (307). The fluorescence light beam is reflected by an XY scanning galvanometer (305). After filtering by a dichroic mirror (304), the excitation light and stray light are filtered out. The fluorescence light is transmitted. The fluorescence light is focused on an imaging surface of a detector (402) by a detection lens (401), and a double helix image point is formed on the imaging surface.

6. A confocal single-molecule three-dimensional detection and tracking method according to claim 5, characterized in that: The phase modulation module converts the illumination beam and the fluorescence beam into an imaging beam having a combined double-helix point spread function property.

7. A confocal single-molecule three-dimensional detection and tracking method according to claim 5, characterized in that: The phase modulation optical component (308) is placed in the common optical path portion of the illumination optical path and the detection optical path to achieve simultaneous modulation of the illumination light beam and the detection light beam, that is, common phase mask modulation of the illumination end and the detection end.

8. A confocal single-molecule three-dimensional detection and tracking method according to claim 5, characterized in that: When the phase modulation optical component (308) is composed of the first phase modulation module (203), the triangular prism (202), the first polarizer (201) and the second polarizer (204), it is a reflective spatial light modulator; when the modulation optical component (308) is composed of the second phase modulation module (205), it is a transmissive spatial light modulator.

9. A confocal single-molecule three-dimensional detection and tracking method according to claim 8, characterized in that: When the phase modulation module (308) is a reflective spatial light modulator, after the diameter of the light beam is adjusted by the third beam expansion lens (306) and the fourth beam expansion lens (307), the first polarizer (201) modulates the polarization state of the light beam, outputs polarized light suitable for the first phase modulation module (203), passes through the triangular prism (202), inputs the first phase modulation module (203) for modulation, and then passes through the triangular prism (202) and the second polarizer (204), outputs an illumination light beam modulated into a double helix light beam.

10. A confocal single-molecule three-dimensional detection and tracking method according to claim 9, characterized in that: When the phase modulation module (308) is a reflective spatial light modulator, after the fluorescent light beam is expanded by the fifth expanding lens (310) and the sixth expanding lens (311), the second polarizer (204) modulates the polarization state of the fluorescent light beam, outputs polarized light suitable for the phase modulation module (203), passes through the triangular prism (202), inputs the first phase modulation module (203) for modulation, and then passes through the triangular prism (202) and the first polarizer (201), outputting a fluorescent light beam modulated into a double helix light beam.

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