STED light beam shaping system with self-adaptive alignment function

Abstract

The invention relates to an STED light beam shaping system with a self-adaptive alignment function. The STED light beam shaping system comprises an STED light generation module and a light path coaxial detection module, the STED light generation module comprises a dichroscope, a 4F system and an SLM; the light path coaxial detection module comprises a reflector, a pinhole filtering system, a focusing lens and a camera; a mixed light beam formed by coaxial exciting light and loss light is emitted to the dichroscope, the exciting light is reflected to the reflector through the dichroscope and then is irradiated to the SLM through the 4F system, the SLM is used for converting the loss light into vortex light, the vortex light enters the 4F system, then passes through the dichroscope and then coincides with the exciting light emitted to the reflector, and the vortex light is emitted outwards. The light path coaxial detection module is used for detecting the light spot coaxiality of the exciting light and the loss light; the zero-level reflection background caused by imperfect modulation of the SLM can be thoroughly eliminated, and the engineering pain point that displacement can be driven by angle adjustment is solved by introducing a 4F system.

Description

Technical Field

[0001] This invention relates to the field of super-resolution microscopy imaging technology, and in particular to an STED beam shaping system with adaptive alignment function. Background Technology

[0002] In stimulated emission loss (STED) microscopy, the spatially overlapping excitation spot and the annular loss spot are key to achieving super-resolution. Traditional physical phase plates (such as spiral phase plates) have the following limitations:

[0003] Static: It is impossible to dynamically adjust the size of the loss spot or correct the dynamic aberrations of the system.

[0004] Extinction ratio limitation: During the modulation process, reflective SLMs are limited by liquid crystal pixelation and diffraction efficiency, resulting in unmodulated 0th-order reflected background light. If the 0th-order light (solid spot) and the 1st-order light (ring spot) output by the SLM are coaxially output, the zero-intensity characteristic of the loss spot center will be directly destroyed, severely affecting the resolution.

[0005] Poor stability: Even a tiny temperature drift or mechanical vibration can cause nanometer-scale lateral and angular deviations in the excitation and loss light.

[0006] In summary, the existing STED microscopy system has unstable alignment accuracy between the excitation and loss beams. The purely mechanical components are more susceptible to environmental influences, which can cause the excitation and loss beams to deviate from their coaxial alignment, thus affecting the objective lens resolution. Therefore, improvements to the STED microscopy system are needed. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to overcome the problem that the existing STED microscopic imaging system has unstable alignment accuracy of excitation light and loss light, and that simple mechanical parts are more susceptible to environmental influences, which can cause the excitation light and loss light to deviate from the coaxiality, thus affecting the objective lens resolution.

[0008] To address the aforementioned technical problems, this invention provides an STED beam shaping system with adaptive alignment functionality, comprising:

[0009] STED light generation module and optical path coaxial detection module;

[0010] The STED light generation module includes a dichroic mirror, a 4F system, and an SLM arranged sequentially.

[0011] The optical path coaxial detection module includes a reflector, a pinhole filter system, a focusing lens, and a camera arranged in sequence.

[0012] When scanning and imaging an object is required, a mixed beam of excitation light and loss light on the same axis is emitted to the dichroic mirror. The excitation light is reflected by the dichroic mirror to the reflecting mirror. The loss light is transmitted through the dichroic mirror, then passes through the 4F system and illuminates the SLM. The SLM is used to convert the loss light into vortex light and then incident into the 4F system. After passing through the 4F system and the dichroic mirror, the vortex light coincides with the excitation light directed towards the reflecting mirror and is emitted outward.

[0013] The optical path coaxial detection module is used to detect the coaxiality of the excitation light and loss light spots. When detecting the excitation light spot, the excitation light is reflected by the dichroic mirror to the reflecting mirror. The excitation light after passing through the reflecting mirror enters the pinhole filter system to improve the spot quality of the excitation light. The excitation light with enhanced spot quality then passes through the focusing lens to map the first spot onto the camera, and the centroid position of the excitation light spot on the camera is calculated. When detecting the loss light spot, a preset phase map is loaded onto the SLM. The loss light is transmitted through the dichroic mirror to the reflecting mirror, then passes through the 4F system and illuminates the camera. The SLM is used to convert the loss light into loss light with adjusted light direction, and then incident it into the 4F system. The loss light with adjusted light direction passes sequentially through the 4F system, a dichroic mirror, and a reflector. The excitation light passing through the reflector enters the pinhole filter system to improve the spot quality of the loss light. The loss light with enhanced spot quality then passes through the focusing lens to map the second spot onto the camera. The centroid position of the loss light spot on the camera is calculated. Finally, the centroid positions of the excitation light spot and the loss light spot are compared and calculated to achieve coaxiality detection of the excitation light and loss light spots.

[0014] In one embodiment of the present invention, the SLM is used to convert the lossy light into vortex light, comprising:

[0015] A spiral phase map is applied to the SLM to generate vortex light, wherein the polar coordinate formula of the spiral phase map is:

[0016] ;

[0017] in, These represent the phase values ​​at different angles and radii. For radius, The topological charge number, It is the spatial azimuth angle. The initial phase constant;

[0018] The spiral phase diagram in polar coordinates is mapped to a rectangular coordinate system. This rectangular coordinate spiral phase diagram is then loaded onto the SLM. The formula for the rectangular coordinate spiral phase diagram is as follows:

[0019] ;

[0020] in, For different coordinates phase value, Let the coordinates be any point on the spiral phase diagram. To take the remainder, , These are the coordinates of the origin on the spiral phase diagram.

[0021] In one embodiment of the present invention, the SLM is used to convert the lossy light into vortex light, and further includes optimizing the vortex light, specifically:

[0022] A fork-shaped grating phase map is applied to the SLM to separate the 0-frequency component of the emitted light from the vortex light. The fork-shaped grating phase map is obtained by fusing a spiral phase map and a blazed grating phase map. The loss light is assumed to be incident parallel to the SLM, and the SLM and the incident loss light have an angle of θ. The angle difference is the angle between the first-order or -1st-order light emitted by the SLM and the 0th-order light. Represented as:

[0023] ;

[0024] in, The incident light wavelength, The period of the blazed grating loaded on the SLM and , For the grating period, The size of a pixel in an SLM;

[0025] The first-order or -1-order light emitted from the SLM is used as vortex light.

[0026] In one embodiment of the present invention, by setting the blazed grating period Different fork-shaped grating phase patterns will be generated by controlling the blazed grating period. This is used to control the emission angle of the vortex light.

[0027] In one embodiment of the present invention, the SLM has an angle of θ with the incident lossy light. The angle difference, where angle The range is 0 degrees to 5 degrees.

[0028] In one embodiment of the present invention, the pinhole filter system includes a first lens, a pinhole, and a second lens arranged in sequence. The distance between the first lens / second lens and the pinhole ranges from 30mm to 100mm, and the diameter of the pinhole ranges from 50um to 150um.

[0029] In one embodiment of the present invention, the formulas for calculating the centroid position of the excitation light spot on the camera and the formulas for calculating the centroid position of the vortex light spot on the camera are as follows:

[0030] ;

[0031] in, , The coordinates of the centroid of the light spot image are: , These are the pixel coordinates of the light spot image. Coordinates in the light spot image , The corresponding grayscale value.

[0032] In one embodiment of the present invention, the calculation of the centroid position of the excitation light spot on the camera and the calculation of the centroid position of the vortex light spot on the camera further includes calculating the size of the light spot on the camera, using the following formula:

[0033] ;

[0034] in, The diameter of the focused spot, For the focal length of the focusing lens, The wavelength of the light being lost or excited. The diameter of the beam is denoted as .

[0035] In one embodiment of the present invention, the step of comparing and calculating the centroid position of the excitation beam and the centroid position of the loss beam further includes: constructing a relationship between the deviation of the loss beam and the excitation beam spot positions and the beam angle, as shown in the formula:

[0036] ;

[0037] in, This refers to the deviation between the centroid position of the lost light and the excitation light spot on the camera. For the focal length of the focusing lens, This represents the beam angle deviation.

[0038] In one embodiment of the present invention, the distance between the dichroic mirror and the 4F system is in the range of 10-40 mm;

[0039] The distance between the 4F system and the SLM ranges from 10 to 40 mm;

[0040] The distance between the reflector and the pinhole filter system ranges from 10 to 40 mm;

[0041] The distance between the pinhole filter system and the focusing lens is in the range of 10-40mm;

[0042] The focal length of the focusing lens ranges from 50mm to 200mm.

[0043] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0044] The STED beam shaping system with adaptive alignment function described in this invention can completely eliminate the 0th order reflection background caused by the imperfect modulation of SLM by constructing an STED light generation module. By introducing a 4F system, it solves the engineering problem of "adjusting the angle will cause displacement". By constructing an optical path coaxial detection module, it can realize the detection of the coaxiality of the light spot. Attached Figure Description

[0045] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0046] Figure 1 This is a schematic diagram of the STED beam shaping system with adaptive alignment function in an embodiment of the present invention;

[0047] Figure 2 This is a schematic diagram of the specific optical path and structure of the STED light generation module in this embodiment of the invention;

[0048] Figure 3 This is a spiral phase diagram in an embodiment of the present invention;

[0049] Figure 4 This is a blazed grating phase diagram in an embodiment of the present invention;

[0050] Figure 5 This is the phase diagram of the fork-shaped grating in an embodiment of the present invention;

[0051] Figure 6 This is a schematic diagram of the angle of the emitted light after passing through the SLM in an embodiment of the present invention;

[0052] Figure 7 This is a schematic diagram of the specific optical path routing and structure of the optical path coaxial detection module in this embodiment of the invention. Detailed Implementation

[0053] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0054] Example 1

[0055] Reference Figure 1 , 2 As shown in Figure 7, this invention relates to an STED beam shaping system with adaptive alignment function, comprising:

[0056] STED light generation module and optical path coaxial detection module;

[0057] The STED light generation module includes a dichroic mirror, a 4F system (an optical Fourier transform system composed of two lenses with the same focal length) and an SLM (spatial light modulator) arranged in sequence.

[0058] The optical path coaxial detection module includes, in sequence, an electrically adjustable reflector, a pinhole filter system, a focusing lens, and a camera;

[0059] When scanning and imaging of an object is required, a mixed beam (polarization state P) formed by the coaxial excitation light and loss light is emitted to the dichroic mirror. The excitation light is reflected by the dichroic mirror to the reflecting mirror. The loss light is transmitted through the dichroic mirror, then passes through the 4F system and illuminates the SLM. The SLM is used to convert the loss light into vortex light and then incident into the 4F system. After passing through the 4F system and the dichroic mirror, the vortex light coincides with the excitation light incident on the reflecting mirror and is emitted outward (specifically, it is emitted outward to the scanning galvanometer system and reaches the objective lens to scan and image the object).

[0060] The optical path coaxial detection module is used to detect the coaxiality of the excitation light and loss light spots. When detecting the excitation light spot, the excitation light is reflected by the dichroic mirror to the reflecting mirror. The excitation light after passing through the reflecting mirror enters the pinhole filter system to improve the spot quality of the excitation light. The excitation light with enhanced spot quality then passes through the focusing lens to map the first spot onto the camera, and the centroid position of the excitation light spot on the camera is calculated. When detecting the loss light spot, a preset phase map (i.e., a blazed grating phase map) is loaded onto the SLM. The loss light is transmitted through the dichroic mirror to the reflecting mirror, and then passes through the 4F system. The excitation light is then irradiated by the SLM, which converts the loss light into direction-adjusted loss light and incident on the 4F system. The direction-adjusted loss light passes sequentially through the 4F system, a dichroic mirror, and a reflector. The excitation light from the reflector enters the pinhole filter system to improve the spot quality of the loss light. The amplified loss light then passes through the focusing lens to map a second spot onto the camera. The centroid position of the loss light spot on the camera is calculated. Finally, the centroid positions of the excitation light spot and the loss light spot are compared and calculated to achieve coaxiality detection of the excitation and loss light spots.

[0061] I. STED Light Generation Module

[0062] It should be noted that this embodiment introduces a 4F system (relay function) into the STED light generation module, which can ensure that the vortex light emitted from the 4F system and the excitation light remain coaxial.

[0063] Furthermore, the SLM is used to convert the lost light into vortex light, including:

[0064] A spiral phase map is applied to the SLM to generate vortex light, wherein the polar coordinate formula of the spiral phase map is:

[0065]

[0066] in, These represent the phase values ​​at different angles and radii. For radius, This is the topological charge, which is typically taken as [value missing] in STED microscopy. or A hollow light spot with a zero-order central singularity; It is the spatial azimuth angle. is the initial phase constant.

[0067] The spiral phase diagram in polar coordinates is mapped to a rectangular coordinate system. This rectangular coordinate spiral phase diagram is then loaded onto the SLM. The formula for the rectangular coordinate spiral phase diagram is as follows:

[0068]

[0069] in, For different coordinates phase value, Let the coordinates be any point on the spiral phase diagram. To take the remainder, , These are the coordinates of the origin on the spiral phase diagram.

[0070] Furthermore, considering that the vortex light generated by simply loading a spiral phase is not perfect, its central intensity is theoretically 0. However, since SLM itself cannot achieve perfect phase modulation, it will lead to the generation of a 0-frequency component, resulting in the vortex light center not being dark enough. Therefore, this embodiment adds a blazed grating phase map to the vortex phase map to form a fork-shaped grating phase map, thereby separating the 0-frequency component from the vortex light. Specifically:

[0071] This embodiment loads a fork-shaped grating phase map onto the SLM, thereby separating the 0-frequency component from the vortex light in the spiral phase map. The fork-shaped grating phase map (see details...) Figure 5 (See spiral phase diagram for details) Figure 3 (This is a first-order spiral phase diagram) and a blazed grating phase diagram (see details). Figure 4 (This is obtained through fusion.)

[0072] Please see Figure 6 Suppose that the loss beam is incident parallel to the SLM, and the SLM and the incident loss beam have an angle of θ. The angle difference determines the angle at which the 0th order light emitted from the SLM exits. Represented as:

[0073] ;

[0074] The 0th order light is solid, and this embodiment does not require a 0th order light.

[0075] The angle between the -1st order light (or 1st order light) emitted by the SLM and the 0th order light Represented as:

[0076]

[0077] in, The incident light wavelength (referring to the loss light). The period of the blazed grating loaded on the SLM and , For the grating period, The size of a pixel in an SLM. The goal is to separate the -1 order light (or 1 order light) from the 0 order light.

[0078] Because the lossy light incident parallel to the SLM will simultaneously emit 0th-order, -1st-order, and 1st-order light, the -1st-order or 1st-order light can directly enter the 4F system (the specific choice between -1st-order and 1st-order light entering the 4F system depends on the user's selection), while the 0th-order light cannot enter the 4F system. In short, this embodiment uses the -1st-order or 1st-order light (hollow ring shape) emitted by the SLM as vortex light.

[0079] Furthermore, by setting the blazed grating period Different fork-shaped grating phase patterns will be generated by controlling the blazed grating period. This is used to control the emission angle of the vortex light.

[0080] Furthermore, in this embodiment, the SLM has an angle with the incident lossy light. The angle difference, where angle The range is 0 degrees to 5 degrees. Preferably, in this embodiment... The theoretical position is 0 degrees.

[0081] The STED light generation module designed in this embodiment can not only eliminate the 0-frequency component and improve the quality of vortex light, but also control the beam's exit angle by loading different phase diagrams onto the SLM via a computer.

[0082] II. Optical Path Coaxial Detection Module

[0083] Furthermore, in this embodiment, the formulas for calculating the centroid position of the excitation light spot on the camera and the centroid position of the vortex light spot on the camera are as follows:

[0084]

[0085] in, , The coordinates of the centroid of the light spot image are: , These are the pixel coordinates of the light spot image. Coordinates in the light spot image , The corresponding grayscale value.

[0086] Furthermore, in this embodiment, when calculating the centroid position of the excitation light spot on the camera and the centroid position of the vortex light spot on the camera, the calculation also includes calculating the size of the light spot on the camera, using the following formula:

[0087]

[0088] in, The diameter of the focused spot, For the focal length of the focusing lens, The wavelength of the light being lost or excited. The diameter of the beam is denoted as .

[0089] Furthermore, the calculation of the centroid position of the excitation beam and the loss beam also includes: constructing the relationship between the deviation of the excitation beam and the excitation beam spot positions and the beam angle, as shown in the formula:

[0090]

[0091] in, This refers to the deviation between the centroid position of the lost light and the excitation light spot on the camera. For the focal length of the focusing lens, This represents the beam angle deviation.

[0092] In this embodiment, the distance between the dichroic mirror and the 4F system ranges from 10 to 40 mm.

[0093] In this embodiment, the distance between the 4F system and the SLM ranges from 10 to 40 mm.

[0094] In this embodiment, the distance between the reflector and the pinhole filter system ranges from 10 to 40 mm.

[0095] In this embodiment, the distance between the pinhole filter system and the focusing lens ranges from 10 to 40 mm.

[0096] In this embodiment, the focal length of the focusing lens ranges from 50mm to 200mm.

[0097] In this embodiment, the pinhole filtering system includes a first lens (a focusing lens), a pinhole, and a second lens (a collimating lens) arranged sequentially. The distance between the first / second lens and the pinhole ranges from 30mm to 100mm, and the pinhole diameter ranges from 50um to 150um. See details... Figure 7 .

[0098] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A STED beam shaping system with adaptive alignment function, characterized in that, include: STED light generation module and optical path coaxial detection module; The STED light generation module includes a dichroic mirror, a 4F system, and an SLM arranged sequentially. The optical path coaxial detection module includes a reflector, a pinhole filter system, a focusing lens, and a camera arranged in sequence. When scanning and imaging an object is required, a mixed beam of excitation light and loss light on the same axis is emitted to the dichroic mirror. The excitation light is reflected by the dichroic mirror to the reflecting mirror. The loss light is transmitted through the dichroic mirror, then passes through the 4F system and illuminates the SLM. The SLM is used to convert the loss light into vortex light and then incident into the 4F system. After passing through the 4F system and the dichroic mirror, the vortex light coincides with the excitation light directed towards the reflecting mirror and is emitted outward. The optical path coaxial detection module is used to detect the coaxiality of the excitation light and loss light spots. When detecting the excitation light spot, the excitation light is reflected by the dichroic mirror to the reflecting mirror. The excitation light after passing through the reflecting mirror enters the pinhole filter system to improve the spot quality of the excitation light. The excitation light with enhanced spot quality then passes through the focusing lens to map the first spot onto the camera, and the centroid position of the excitation light spot on the camera is calculated. When detecting the loss light spot, a preset phase map is loaded onto the SLM. The loss light is transmitted through the dichroic mirror to the reflecting mirror, then passes through the 4F system and illuminates the camera. The SLM is used to convert the loss light into loss light with adjusted light direction, and then incident it into the 4F system. The loss light with adjusted light direction passes sequentially through the 4F system, a dichroic mirror, and a reflector. The excitation light passing through the reflector enters the pinhole filter system to improve the spot quality of the loss light. The loss light with enhanced spot quality then passes through the focusing lens to map the second spot onto the camera. The centroid position of the loss light spot on the camera is calculated. Finally, the centroid positions of the excitation light spot and the loss light spot are compared and calculated to achieve coaxiality detection of the excitation light and loss light spots.

2. The STED beam shaping system with adaptive alignment function according to claim 1, characterized in that: The SLM is used to convert the lossy light into vortex light, including: A spiral phase map is applied to the SLM to generate vortex light, wherein the polar coordinate formula of the spiral phase map is: ； in, These represent the phase values ​​at different angles and radii. For radius, The topological charge number, It is the spatial azimuth angle. The initial phase constant; The spiral phase diagram in polar coordinates is mapped to a rectangular coordinate system. This rectangular coordinate spiral phase diagram is then loaded onto the SLM. The formula for the rectangular coordinate spiral phase diagram is as follows: ； in, For different coordinates phase value, Let the coordinates be any point on the spiral phase diagram. To take the remainder, , These are the coordinates of the origin on the spiral phase diagram.

3. The STED beam shaping system with adaptive alignment function according to claim 2, characterized in that: The SLM is used to convert the lossy light into vortex light, and also includes optimizing the vortex light, specifically: A fork-shaped grating phase map is applied to the SLM to separate the 0-frequency component of the emitted light from the vortex light. The fork-shaped grating phase map is obtained by fusing a spiral phase map and a blazed grating phase map. The loss light is assumed to be incident parallel to the SLM, and the SLM and the incident loss light have an angle of θ. The angle difference is the angle between the first-order or -1st-order light emitted by the SLM and the 0th-order light. Represented as: ； in, The incident light wavelength, The period of the blazed grating loaded on the SLM and , For the grating period, The size of a pixel in an SLM; The first-order or -1-order light emitted from the SLM is used as vortex light.

4. The STED beam shaping system with adaptive alignment function according to claim 3, characterized in that: By setting the blazed grating period Different fork-shaped grating phase patterns will be generated by controlling the blazed grating period. This is used to control the emission angle of the vortex light.

5. The STED beam shaping system with adaptive alignment function according to claim 3, characterized in that: The SLM has an angle with the incident lossy light. The angle difference, where angle The range is 0 degrees to 5 degrees.

6. The STED beam shaping system with adaptive alignment function according to claim 1, characterized in that: The pinhole filter system includes a first lens, a pinhole, and a second lens arranged in sequence. The distance between the first lens / second lens and the pinhole ranges from 30mm to 100mm, and the diameter of the pinhole ranges from 50um to 150um.

7. The STED beam shaping system with adaptive alignment function according to claim 1, characterized in that: The formulas for calculating the centroid position of the excitation light spot on the camera and the centroid position of the vortex light spot on the camera are as follows: ； in, , The coordinates of the centroid of the light spot image are: , The pixel coordinates of the light spot image. Coordinates in the light spot image , The corresponding grayscale value.

8. The STED beam shaping system with adaptive alignment function according to claim 1, characterized in that: The calculation of the centroid position of the excitation light spot on the camera, and the calculation of the centroid position of the vortex light spot on the camera, also includes calculating the size of the light spot on the camera, using the following formula: ； in, The diameter of the focused spot, For the focal length of the focusing lens, The wavelength of the light being lost or excited. The diameter of the beam is denoted as .

9. The STED beam shaping system with adaptive alignment function according to claim 1, characterized in that: The step of comparing and calculating the centroid position of the excitation beam spot with that of the loss beam spot also includes: constructing the relationship between the deviation of the loss beam and excitation beam spot positions and the beam angle, using the following formula: ； in, This refers to the deviation between the centroid position of the lost light and the excitation light spot on the camera. For the focal length of the focusing lens, This represents the beam angle deviation.

10. The STED beam shaping system with adaptive alignment function according to claim 1, characterized in that: The distance between the dichroic mirror and the 4F system is in the range of 10-40 mm; The distance between the 4F system and the SLM ranges from 10 to 40 mm. The distance between the reflector and the pinhole filter system ranges from 10 to 40 mm; The distance between the pinhole filter system and the focusing lens is in the range of 10-40mm; The focal length of the focusing lens ranges from 50mm to 200mm.

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