Four-channel fluorescence detection light path module of super-resolution biological microscope

Abstract

The invention discloses a four-channel fluorescence detection light path module of a super-resolution biological microscope, which divides a fluorescence signal of an intermediate image plane (5) into four conjugate channels (7a-7d) through multi-stage dichroscope light splitting, reflector guiding and lens focal plane adjustment on the premise of not introducing additional aberration, and realizes real-time synchronous detection by using a single large-target-surface camera (8). By adjusting the distance between the reflectors, the inclination angle of the parallel plate glass or the position of the lens, the device can adapt to different camera target surface sizes and compensate chromatic aberration, and the problems of motor switching delay and dual-channel aberration in the prior art are solved.

Description

Technical Field

[0001] This application belongs to the technical field of bioimaging detection, and particularly relates to a four-channel fluorescence detection optical path module of a super-resolution biological microscope, which is applicable to multi-wavelength fluorescence real-time synchronous imaging. Background Technique

[0002] In the field of bioimaging detection, the design of fluorescence detection modules has long been limited by the physical limitations of spectroscopic techniques and the complexity of mechanical structures. The current mainstream technologies are mainly divided into two categories: single-channel filter wheel switching systems and dual-channel tilted optical path systems. The core components of the single-channel filter wheel system include a high-precision stepper motor, an aluminum alloy filter wheel, and an optical encoder. Its working process is as follows: The motor drives the filter wheel to rotate to the position of the target filter. After the encoder feeds back the in-place signal, the system delays for 50 - 100 ms to ensure the attenuation of mechanical vibration. However, the filter switching cycle of such a system is limited by the motor acceleration / deceleration curve, and the complete switching of four channels takes more than 200 ms, which cannot meet the real-time requirements of live cell dynamic imaging. In addition, when the temperature fluctuates by ±5 °C, the thermal expansion coefficient of the filter wheel material will cause a radial positioning error of the filter of ±5 μm, resulting in an optical axis shift, thereby reducing the signal-to-noise ratio of the fluorescence signal. The dual-channel tilted optical path system realizes two-channel synchronous detection by introducing an asymmetric optical path spectroscopy. However, the tilted optical path will introduce spherical aberration and coma, and the RMS value of its wavefront error can reach λ / 4 (λ = 532 nm), resulting in a 20% increase in the full width at half maximum (FWHM) of the point spread function (PSF), severely restricting the resolution of the super-resolution microscope. In addition, the assembly tolerance of the tilted lens group needs to be controlled within ±0.1°, posing extremely high requirements on mechanical processing and alignment processes. Although the above technologies can achieve multi-wavelength detection, they all have fundamental defects such as low time resolution, poor mechanical stability, and uncontrollable aberrations. Especially in high-throughput screening or the observation of dynamic biological processes, the existing solutions are difficult to meet the requirements of four-channel real-time and high-fidelity imaging.

[0003] In summary, the existing fluorescence detection technology has the following main defects: 1. Time delay and frame rate limitation: The switching time of the filter wheel system is limited by the motor response and mechanical damping. Taking four-channel imaging as an example, a single full-channel switch requires three filter position jumps (such as channel 1→2→3→4), and the total time is T=3×(acceleration time t1+deceleration time t2+stabilization time t3). For example, when using Sanyo Denki motor, t1=t2=5ms, t3=40ms, the total time is T=3×(5+5+40)=150ms, and the corresponding maximum frame rate is only 6.7fps, which cannot capture millisecond-level biological events such as calcium ion transients. 2. Mechanical vibration and long-term reliability issues: The high-speed start and stop of the filter wheel motor will cause periodic impact loads, resulting in bearing wear. After long-term use, the gear gap expands to ±5μm, further exacerbating the optical path deviation. Test data shows that after 100 hours of continuous operation, the system positioning repeatability drops from the initial ±1μm to ±5μm, requiring frequent calibration. 3. Uncontrollable aberration of the tilted optical path: The tilt angle design of the dual-channel system will destroy the symmetry of the optical path. Taking the lens group tilted 8° as an example, the spherical aberration introduced can be expressed as W 40 =(n 2 -1)sin 2 θ / (8n 3 )(n=1.5 is the lens refractive index, θ=8°), calculate W 40 = 0.12λ, resulting in the Strehl ratio of the PSF decreasing from 0.95 to 0.82, and the imaging contrast decreasing by 30%. In addition, the coma coefficient W 31 =3(n 2 -1)sinθ / (4n 3 )≈0.08λ, which causes obvious tailing of the point spread function in the edge area of ​​the image plane. 4. Wavelength drift caused by thermal expansion: The aluminum alloy material of the filter holder deforms when the temperature changes, causing the filter to tilt. This tilt changes the incident angle of the filter, thereby shifting its central wavelength, exceeding the wavelength range of the filter bandwidth, resulting in increased crosstalk between channels. 5. Insufficient scalability and flexibility: If the dual-channel system is to be expanded to four channels, two additional relay optical paths are required, such as an achromatic lens group with a focal length of f=100mm, which increases the total length of the system by more than 300mm, and the optical path difference of each channel needs to be strictly matched. This type of design is difficult to be compatible with camera targets of different sizes, limiting the versatility of the equipment.

[0004] Application Contents

[0005] This application aims to achieve simultaneous and real-time detection of four-channel fluorescence signals through a static spectroscopic structure and an adjustable optical path design, without introducing additional aberrations. The specific objectives include: 1. Removing mechanical moving parts: A cascaded spectroscopic network of multilayer dichroic mirrors is used to physically separate the four-channel optical paths through the wavelength-selective reflection / transmission characteristics of the coating, eliminating the filter wheel and motor drive structure, and reducing the channel switching time to 0 ms. 2. Dynamic aberration compensation: An axially fine-tunable focusing lens (adjustment range ±5 mm, resolution 1 μm) is integrated into each spectroscopic channel. By independently adjusting the axial position of each lens, the focal plane shift (chromatic aberration) caused by wavelength differences is compensated to ensure that the four-channel image planes are conjugated to the same camera target plane (conjugation error <λ / 10, λ = 532 nm). 3. Adjustable optical path spacing design: The lateral position of the mirror is adjusted by a precision translation stage (adjustment accuracy ±0.1 mm), and combined with the lateral image shift effect of an inclined parallel flat glass (inclination angle continuously adjustable from 0° to 15°, thickness 2 mm, material BK7) (Δx = t·α·(n - 1) / n, where t is the glass thickness, α is the inclination angle, and n is the refractive index), a 2×2 array arrangement of the four-channel image planes on the camera target plane is achieved (spacing adjustable range ±5 mm) to adapt to different sizes of camera target planes (such as 12 mm×12 mm to 36 mm×24 mm). 4. Flexible multi-spectral configuration: A modular filter set quick-release structure design is adopted, supporting users to quickly replace filters with different central wavelengths (such as 405 nm, 488 nm, 561 nm, 640 nm) and bandwidths (±2 nm to ±10 nm) according to experimental requirements, while retaining the filter wheel upgrade interface and being compatible with both static spectroscopy and dynamic switching working modes.

[0006] The specific technical solutions and features are as follows: A four-channel fluorescence detection optical path module for a super-resolution biological microscope, including a relay optical path module (50) and a target surface camera (8). The relay optical path module (50) includes a hierarchical dichroic mirror beam splitting network, a high-precision image plane adjustment mechanism, and a camera adaptation component. The hierarchical dichroic mirror beam splitting network includes a three-stage beam splitting structure. The beam splitting structure includes a dichroic mirror and a filter. The hierarchical dichroic mirror beam splitting network performs beam splitting on the incident light of the intermediate image plane (5) through the dichroic mirror and the filter. The high-precision image plane adjustment mechanism includes a six-dimensional adjustment bracket for a mirror, an axial fine adjustment structure for a lens, and an inclination mechanism for a parallel flat glass. The six-dimensional adjustment bracket for a mirror includes a mirror and a six-dimensional adjustment bracket. The axial fine adjustment structure for a lens includes a focusing lens and a threaded fine adjustment base. The inclination mechanism for a parallel flat glass includes an inclined flat glass and a rotating table. The high-precision image plane adjustment mechanism receives the incident light from the hierarchical dichroic mirror beam splitting network and performs light adjustment. The camera adaptation component includes a mirror array and a confocal plane calibration component. The mirror array includes a mirror and a linear translation stage. The camera adaptation component receives the incident light from the high-precision image plane adjustment mechanism and performs conjugate processing. The outgoing light of the camera adaptation component is received by the target surface camera (8). Specifically, the light of the intermediate image plane (5) is split by the relay optical path module (50) to form four conjugate fluorescence channels (7a, 7b, 7c, 7d), and is finally received by the target surface camera (8);

[0007] Further, the specific structure of the hierarchical dichroic mirror beam splitting network is as follows: First-stage beam splitting: Using a fused silica substrate dichroic mirror (10), the incident fluorescence is divided into a reflection channel λ 1 and a transmission channel λ 2 . Second-stage beam splitting: The transmitted light λ 2 enters the second-layer dichroic mirror (12) and is further separated into a reflection channel λ 3 and a transmission channel λ 4 . Third-stage beam splitting: The transmitted light λ 4 enters the third-layer dichroic mirror (13) and is finally separated into a reflection channel λ 5 and a transmission channel λ 6 . Fourth-stage light: The transmitted light λ 6 is reflected by the mirrors (14, 15). Filters (16) are installed at the exits of the reflection channels.

[0008] Further, the size of the dichroic mirror (10) is 25 mm × 36 mm × 3 mm, and the surface coating is designed to have high reflectivity (reflectivity R > 95%) for λ 1 ≤ 480 nm and high transmittance (transmittance T > 90%) for λ 2 ≥ 480 nm.

[0009] Further, the dichroic mirror (12) has dimensions of 25 mm × 36 mm × 3 mm, and the surface coating is designed such that for λ 1 ≤ 580 nm, it has high reflectivity (reflectivity R > 95%), and for λ 2 > 580 nm, it has high transmittance (transmittance T > 90%).

[0010] Further, the dichroic mirror (13) has dimensions of 25 mm × 36 mm × 3 mm, and the surface coating is designed such that for λ 1 ≤ 610 nm, it has high reflectivity (reflectivity R > 95%), and for λ 2 ≥ 610 nm, it has high transmittance (transmittance T > 90%).

[0011] Further, the center wavelength tolerance of the filter (16) is ±1 nm, the bandwidth

[0012] is ±5 nm, the transmittance > 90%, and the crosstalk suppression between channels Crosstalk < 1%.

[0013] Further, the specific structure of the high-precision image plane adjustment mechanism is as follows: The mirrors (11, 14, 15) are mounted on the six-axis adjustment mount. The six-axis adjustment mount has six degrees of freedom and is used to finely adjust the pointing of the optical axes of each channel to ensure that the four image planes are strictly conjugate. In the axial fine adjustment structure of the lens, the focusing lens (17) is mounted on a threaded fine adjustment base, and the rotation of the adjustment ring compensates for the focal plane shift caused by different wavelengths. In the tilting mechanism of the parallel flat glass, the tilting flat glass (19) is mounted on the rotating table. The relationship between the tilt angle α of the tilting flat glass (19) and the lateral image shift Δx is Δx = t·α·(n - 1) / n. For example, when α = 10° (0.1745 rad), Δx = 2 × 0.1745 × (1.517 - 1) / 1.517 ≈ 0.23 mm, which is used to adapt to the target plane spacing.

[0014] Further, the six-axis adjustment mount is driven by piezoelectric ceramics.

[0015] Further, the translation stroke of the six-axis adjustment mount is ±5 mm, and the resolution is

[0016] 0.1 μm. The adjustment range of the pitch / yaw / rotation angle is ±2°, and the resolution is 0.001°.

[0017] Further, the focal length f of the focusing lens (17) is 50 mm, and NA = 0.3.

[0018] Further, the pitch of the threaded fine adjustment base is 0.5 mm, the rotation angle resolution is 1°, and the corresponding axial displacement Δz has a value range of 1.2 μm - 1.6 μm.

[0019] Furthermore, the specific structure of the camera adapter assembly is as follows: the reflector array is arranged by the reflectors (18) of the four channels arranged in a 2×2 array, each reflector is mounted on the linear translation stage, and the linear translation stage controls the image plane spacing and matches the target surface size by adjusting the lateral position of the reflector. The confocal plane calibration uses a 633nm helium-neon laser as the calibration light source, detects the wavefront of each channel through an interferometer, and adjusts the axial position of the lens (17) so that the four-channel focal plane coincidence error is <1μm, that is, the corresponding wavefront difference λ / 633. The light of the intermediate image plane 5 passes through the relay optical path module 50 to form four different fluorescence wavelength images 7a, 7b, 7c, 7d conjugated with the intermediate image plane 5, and a large target surface camera 8 can be used to realize the simultaneous detection of images of the four channels.

[0020] Furthermore, the linear translation stage has a travel range of ±10 mm and a resolution of 1 μm.

[0021] In summary, the present application replaces mechanical switching with hierarchical spectrometry, adopts cascade spectrometry of three layers of dichroic mirrors, and physically separates four-wavelength fluorescence signals into independent optical paths, completely eliminating the motion delay and vibration noise of the filter wheel. And through precise optical path adjustment to suppress aberrations, the combination of the six-dimensional adjustment frame and the lens fine-tuning mechanism can control the RMS value of the wavefront error of spherical aberration and coma within λ / 20 (measured value: RMS = 26.5nm when λ = 532nm), and the PSF half-width (FWHM) difference of the four channels is <5%, which meets the requirements of super-resolution imaging. The introduction of thermal stability design allows the fused quartz dichroic mirror and the Invar support structure to reduce the thermal drift of the system to the sub-micron level (0.55μm / 10℃), and cooperates with the environmental temperature control module (±0.1℃) to ensure the stability of long-term continuous imaging. Improve the compatibility of fast wavelength switching. In the filter wheel compatible mode, the stepper motor drives the filter switching time <10ms (compared to 50ms of the traditional filter wheel), and there is no mechanical shock, which is suitable for high-speed multi-wavelength time-series imaging. Enhance user configurability, the filter quick-release structure and the manual adjustment function of the parallel plate tilt angle enable the system to complete the configuration conversion from four-channel static imaging to eight-channel dynamic switching within 5 minutes, greatly improving the utilization rate of the equipment and overcoming the defects of the prior art. The four-channel fluorescence detection optical path module proposed in this application has many significant advantages and positive effects, and the beneficial effects achieved are as follows:

[0022] 1. Four-channel real-time imaging capability: Through hierarchical splitting and static optical path design, four-channel signals can reach the camera target surface at the same time. The time resolution is only limited by the camera exposure time, which is several orders of magnitude higher than the filter wheel solution.

[0023] 2. Sub-micron optical path stability: By using a fused silica dichroic mirror (thermal expansion coefficient of 0.55×10 -6 / °C) and an invar mirror mount, the thermal drift ΔL of the system at ΔT = 10°C is ΔL = α·L·ΔT = 0.55×10 -6 ×100 mm × 10 = 0.55 μm, which is 98% lower than the aluminum alloy solution.

[0024] 3. Active aberration compensation: The axial fine-tuning function of the lens (17) can dynamically correct chromatic aberration. The four-channel focal plane coincidence error < 1 μm (corresponding to a wavefront error of λ / 633), making the MTF curve consistency of the multi-channel images > 95%.

[0025] 4. Modular expandability: By adding dichroic mirror levels (such as the fifth-level beam splitter), the system can be expanded to six channels, only requiring corresponding addition of mirror and filter modules without modifying the main optical path structure.

[0026] 5. Cross-platform compatibility: Supports mainstream scientific research cameras, with the target surface adaptation range covering from 1 / 2 inch to full frame, and seamless switching is achieved by adjusting the tilt angle of the parallel plate and the distance between the mirrors. Description of the Drawings

[0027] Figure 1 It is a general structural diagram of the fluorescence optical path of a microscope;

[0028] Figure 2 It is an overall diagram of the four-channel fluorescence relay optical path module of the biological fluorescence microscope of this application;

[0029] Figure 3 It is a design diagram of the reflection optical path structure of this application;

[0030] Figure 4 It is a design diagram of the refraction optical path structure of this application;

[0031] In the figure: 1. Sample to be measured; 2. Objective lens; 3. Dichroic mirror; 4. Tube lens; 5. Image plane; 6. Optical path box; 7. Image plane; 8. Target surface camera; 9. Lens; 10. Dichroic mirror; 11. Mirror; 12. Dichroic mirror; 13. Dichroic mirror; 14. Mirror; 15. Mirror; 16. Filter; 17. Lens; 18. Mirror; 19. Flat glass; 50. Relay optical path module. Detailed Description of the Invention

[0032] Here, the specific term "embodiment" does not necessarily mean that any embodiment described as "exemplary" is better than or superior to other embodiments. For the performance index tests in the embodiments of this application, unless otherwise specified, conventional test methods in the art are used. It should be understood that the terms described in this application are only for describing specific embodiments and are not used to limit the content disclosed in this application.

[0033] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the technical field to which this application pertains; other test methods and technical means not specifically noted in this application refer to the experimental methods and technical means commonly adopted by those of ordinary skill in the art.

[0034] The terms "substantially" and "about" as used herein are used to describe minor fluctuations. For example, they can refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%. Numerical data presented or represented in a range format herein is used only for convenience and brevity and should therefore be flexibly interpreted as including not only the values explicitly recited as the bounds of the range, but also all individual values or sub-ranges subsumed within that range. For example, the numerical range of "1 to 5%" should be interpreted as including not only the explicitly recited values of 1% to 5%, but also the individual values and sub-ranges within the indicated range. Thus, within this numerical range, individual values such as 2%, 3.5%, and 4% are included, as well as sub-ranges such as 1% to 3%, 2% to 4%, and 3% to 5%, etc. This principle also applies to ranges that list only one numerical value. In addition, such an interpretation applies regardless of the width of the range or the characteristics described.

[0035] In this document, including in the claims, conjunctions such as "comprising", "including", "carrying", "having", "containing", "involving", "accommodating", etc. are understood to be open-ended, that is, meaning "including but not limited to". Only the conjunctions "consisting of" and "composed of" are closed conjunctions.

[0036] For a better illustration of the content of this application, numerous specific details are given in the following specific embodiments. Those skilled in the art should understand that this application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, devices, etc. well-known to those skilled in the art are not described in detail in order to highlight the gist of this application.

[0037] On the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the obtained technical solutions belong to the content disclosed in the embodiments of the present application. It should be noted that the terms "center", "longitudinal", "lateral", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. mentioned in the present application indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings. They are only for the convenience of describing technical features and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation to the present application, unless it conflicts with the context. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance, unless it conflicts with the context.

[0038] The following describes the specific embodiments of the present application to facilitate those skilled in the art of the present technology to understand the present application. However, it should be clear that the present application is not limited to the scope of the specific embodiments. For those of ordinary skill in the art of the present technology, as long as various changes are within the spirit and scope of the present application defined and determined by the appended claims, these changes are obvious, and all application creations using the concept of the present application are within the scope of protection.

[0039] Embodiment 1

[0040] Please refer to Figures 1 to 3 For the purpose of implementing a four-channel fluorescence detection optical path module of a super-resolution biological microscope proposed in the present application, the present application provides an embodiment, and the specific implementation is as follows: S1. Spectral splitting network assembly: Install the first dichroic mirror (10) at a 45° angle on the mirror frame (made of invar, with a thermal expansion coefficient of 1.2×10 -6 / °C) to ensure that the angle between the incident optical axis and the normal of the mirror surface is exactly 45° (error <0.1°). The light of the reflection channel λ 1 is deflected by 90° by the reflector (11) (surface silver film treatment or broadband dielectric film treatment, reflectivity >98%), enters the filter (16), and is focused by the lens (17) to the reflector (18), and finally imaged on the 7a area of the camera (8). The light of the transmission channel λ 2 is transmitted to the second dichroic mirror (12), and the light of its reflection channel λ 3 is imaged on the 7d area through the same filter (16) and lens (17); the transmitted light λ 4 continues to the third dichroic mirror (13), and the light of the reflection channel λ 5 is imaged on the 7c area, and the transmitted light λ 6Reflected by the mirrors (14, 15) to area 7b. S2. Image plane conjugate calibration: Place a resolution target at the sample position, and sequentially turn on the fluorescence simulation light sources of each channel, such as LED light sources with central wavelengths of 450 nm, 550 nm, 600 nm, and 650 nm. Monitor the clarity of the image plane of each channel in real time through the camera (8), and manually rotate the fine-tuning ring of the lens (17) (each 1° rotation corresponds to an axial movement of 1.4 μm) until the images of the four channels reach the best contrast simultaneously. S3. Spacing adjustment and matching with the target plane: Loosen the locking screw of the mirror (18), push the translation stage slider to adjust the image plane spacing between 7a and 7b from the initial value of 0 mm to the target value (such as 4 mm). Use a micrometer (accuracy ±1 μm) to measure the slider displacement to ensure that the four image planes are evenly distributed in a 2×2 array on the camera target plane. By combining different dichroic mirrors and filter combinations between the lenses, simultaneous detection of different fluorescence wavelengths in the four channels can be achieved. The filter wheel can also be used to replace the individual filter to achieve wavelength switching in the four channels. According to the size of the camera target plane, the channel spacing can be changed by reasonably adjusting the mirror spacing; the four-channel spacing can also be adjusted by adjusting the position of the mirror; due to the chromatic aberration of the system, the different fluorescence channels cannot be focused on the same plane, and by individually adjusting the position of the lens, the focal planes of different channels can be adjusted to the camera target plane.

[0041] Embodiment 2

[0042] Please refer to Figure 1 , Figure 2 , Figure 4, in order to implement a four-channel fluorescence detection optical path module for a super-resolution biological microscope proposed in this application, the present application provides a second embodiment, and the specific implementation is as follows: S1. Parallel flat glass integration: Insert a parallel flat glass (19) into the channel optical path, set the initial tilt angle to 0°, gradually increase the tilt angle to the target value (such as α = 8°) through a rotary stage, and at the same time observe the lateral offset Δx of the camera image plane, and verify that Δx = t·α·(n - 1) / n = 2×8°×(π / 180)×(0.517) / 1.517 ≈ 0.19 mm / °×8 ≈ 1.52 mm. Fine-tune α according to the target surface requirements. For example, when two image planes need to be accommodated in the horizontal direction of the target surface, set α = 10°, Δx ≈ 1.9 mm, and combine with the translation amount Δ = 3 mm of the mirror (18) to achieve a total spacing of 4.9 mm. S2. Filter wheel compatibility design: Remove the quick-release module of the filter (16) and replace it with a four-hole filter wheel (diameter 50 mm, including 405 nm, 488 nm, 561 nm, and 640 nm filters). The filter wheel is driven by a stepper motor, and a pulse signal is sent through a microcontroller (number of pulses = 200 steps / turn × 64 = 12,800 steps / full rotation). Each step corresponds to a filter wheel rotation angle of 360° / 12,800 ≈ 0.028°, and the switching time < 10 ms. By combining different dichroic mirrors and filter combinations between the lenses, simultaneous detection of different fluorescence wavelengths in four channels can be achieved. It is also possible to replace the individual filter with a filter wheel to achieve wavelength switching in four channels. According to the size of the camera target surface, the vertical spacing of the four channels can be adjusted by designing the tilt angle, thickness, and material of the parallel flat glass in different channels; the spacing of the four channels can also be adjusted by adjusting the position of the mirror; due to system chromatic aberration, different fluorescence channels cannot be focused on the same plane. By individually adjusting the position of the lens, the focal planes of different channels can be adjusted to the camera target surface.

[0043] In summary, in this application, hierarchical beam splitting is used to replace mechanical switching. The cascaded beam splitting of three-layer dichroic mirrors (beam splitting efficiency > 95%) is adopted to physically separate the four-wavelength fluorescence signals into independent optical paths, completely eliminating the motion delay and vibration noise of the filter wheel. Aberrations are suppressed through precise optical path adjustment. The combination of a six-axis adjustment mount and a lens fine-tuning mechanism can control the RMS value of the wavefront errors of spherical aberration and coma within λ / 20. The difference in the full width at half maximum (FWHM) of the point spread function (PSF) of the four channels is < 5%, meeting the requirements of super-resolution imaging. A thermal stability design is introduced, enabling the fused silica dichroic mirror and the invar support structure to reduce the system's thermal drift to the sub-micron level (0.55 μm / 10 °C). Together with the environmental temperature control module (±0.1 °C), it ensures the stability of long-term continuous imaging. The compatibility of fast wavelength switching is improved. In the filter wheel compatibility mode, the switching time of the filter driven by a stepper motor is < 10 ms (compared with 50 ms of a traditional filter wheel), and there is no mechanical impact, making it suitable for high-speed multi-wavelength time-sequential imaging. The user configurability is enhanced. The quick-release structure of the filter and the manual adjustment function of the tilt angle of the parallel plate enable the system to complete the configuration conversion from four-channel static imaging to eight-channel dynamic switching within 5 minutes, greatly improving the equipment utilization rate. It overcomes the defects of the prior art and achieves beneficial effects such as four-channel real-time imaging ability, sub-micron-level optical path stability, active aberration compensation, modular expandability, and cross-platform compatibility.

[0044] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0045] The technical solutions disclosed in this application and the technical details disclosed in the embodiments are only exemplary explanations of the application concept of this application and do not constitute limitations on the technical solutions of this application. Any conventional changes, replacements, or combinations made to the technical details disclosed in the embodiments of this application have the same application concept as this application and are within the protection scope of the claims of this application.

Claims

1. A four-channel fluorescence detection optical path module for a super-resolution biological microscope, comprising a relay optical path module (50) and a target surface camera (8). The relay optical path module (50) comprises three parts: a hierarchical dichroic mirror splitting network, a high-precision image plane adjustment mechanism and a camera adapter assembly. The hierarchical dichroic mirror splitting network comprises a three-level splitting structure, and the splitting structure comprises a dichroic mirror and a filter. The high-precision image plane adjustment mechanism comprises a six-dimensional adjustment frame for a reflector, a lens axial fine-tuning structure and a parallel flat glass tilting mechanism, the six-dimensional adjustment frame for the reflector comprises a reflector and a six-dimensional adjustment frame, the lens axial fine-tuning structure comprises a focusing lens and a threaded fine-tuning seat, and the parallel flat glass tilting mechanism comprises a tilted flat glass and a rotating table. The camera adapter assembly comprises a reflector array and a confocal plane calibration assembly, and the reflector array comprises a reflector and a linear translation stage. The light from the intermediate image plane (5) is split through the relay optical path module (50) to form four conjugate fluorescent channels (7a, 7b, 7c, 7d).

2. The four-channel fluorescence detection optical path module of a super-resolution biological microscope according to claim 1, characterized in that: The specific structure of the hierarchical dichroic mirror splitting network is as follows: First-level splitting: a fused silica substrate dichroic mirror (10) is used to split the incident fluorescence into a reflection channel λ1 and a transmission channel λ2. Second-level splitting: the transmitted light λ2 enters the second-level dichroic mirror (12) and is further split into a reflection channel λ3 and a transmission channel λ4. Third-level splitting: the transmitted light λ4 enters the third-level dichroic mirror (13) and is split into a reflection channel λ5 and a transmission channel λ6. Fourth-level light: the transmitted light λ6 is reflected by the reflectors (14, 15). Filters (16) are installed at the exits of the reflection channels.

3. The four-channel fluorescence detection optical path module of a super-resolution biological microscope according to claim 1 or 2, characterized in that: The dichroic mirror (10) has a size of 25mm×36mm×3mm, and its surface coating is designed to be highly reflective (reflectivity R>95%) at λ1<=480nm and highly transparent (transmittance T>90%) at λ2>=480nm. The dichroic mirror (12) has a size of 25mm×36mm×3mm, and its surface coating is designed to be highly reflective (reflectivity R>95%) at λ1<=580nm and highly transparent (transmittance T>90%) at λ2>580nm. The dichroic mirror (13) has a size of 25mm×36mm×3mm, and its surface coating is designed to be highly reflective (reflectivity R>95%) at λ1<=610nm and highly transparent (transmittance T>90%) at λ2>=610nm.

4. The four-channel fluorescence detection optical path module of a super-resolution biological microscope according to claim 1, characterized in that: The specific structure of the high-precision image plane adjustment mechanism is as follows: the reflectors (11, 14, 15) are mounted on the six-dimensional adjustment frame, and the six-dimensional adjustment frame has six degrees of freedom. In the lens axial fine-tuning structure, the focusing lens (17) is mounted on a threaded fine-tuning seat. In the parallel flat glass tilting mechanism, the tilted flat glass (19) is mounted on the rotating table, and the relationship between the tilt angle α of the tilted flat glass (19) and the lateral image shift Δx is Δx=t·α·(n-1) / n.

5. The four-channel fluorescence detection optical path module of a super-resolution biological microscope according to claim 1 or 4, characterized in that: The high-precision image plane adjustment mechanism can be optionally operated as follows: (1) the spacing between the reflectors (11, 14, 15) can be adjusted to change the channel spacing; (2) the position of each channel lens (17) can be individually adjusted to correct the focal plane to the camera target plane (8).

6. The four-channel fluorescence detection optical path module of a super-resolution biological microscope according to claim 1 or 4, characterized in that: The inclined parallel flat glass (19) has an adjustable inclination angle and thickness and is used to adjust the channel spacing by refraction.

7. The four-channel fluorescence detection optical path module of a super-resolution biological microscope according to claim 1 or 2, characterized in that: The filter (16) has a central wavelength tolerance of ±1nm, a bandwidth of ±5nm, a transmittance of >90%, and suppresses crosstalk between channels of <1%.

8. The four-channel fluorescence detection optical path module of a super-resolution biological microscope according to claim 1 or 4, characterized in that: The six-dimensional adjustment frame has a translation travel of ±5 mm, a resolution of 0.1 μm, a pitch / yaw / rotation angle adjustment range of ±2°, and a resolution of 0.001°.

9. A four-channel fluorescence detection optical path module for a super-resolution biological microscope according to claim 1, 4 or 7, characterized in that: The focusing lens (17) has a focal length of f=50 mm and NA=0.

3.

10. The four-channel fluorescence detection optical path module of a super-resolution biological microscope according to claim 1 or 4, characterized in that: The thread fine-tuning seat has a pitch of 0.5 mm, a rotation angle resolution of 1°, and a corresponding axial displacement Δz= in the range of 1.2 μm-1.6 μm.

11. The four-channel fluorescence detection optical path module of a super-resolution biological microscope according to claim 1 or 2, characterized in that: The optical filter (16) can be replaced by an optical filter wheel to achieve four-channel wavelength switching.

12. The four-channel fluorescence detection optical path module of a super-resolution biological microscope according to claim 1, characterized in that: The specific structure of the camera adapter assembly is as follows: the reflector array is arranged by four channels of reflectors (18) arranged in a 2×2 array, and each reflector is installed on the linear translation stage. The confocal plane calibration uses a 633nm helium-neon laser as a calibration light source.

13. The four-channel fluorescence detection optical path module of a super-resolution biological microscope according to claim 1 or 12, characterized in that: The linear translation stage has a travel range of ±10 mm and a resolution of 1 μm.

14. The four-channel fluorescence detection optical path module of a super-resolution biological microscope according to claim 1, characterized in that: The camera (8) is a single large-surface camera that simultaneously captures fluorescence images of four channels.

Images