A multimodal fluorescence microscopy system based on cage structure

Abstract

The present application discloses a multimodal fluorescence microscopy imaging system based on a cage structure, and relates to the field of optical imaging systems. The system comprises: a breadboard, a multi-axis cage structure support, a primary optical element, a secondary optical element, and an intermediate optical element; the multi-axis cage structure support is provided with mechanical through holes of different heights to provide optical paths of multiple different optical axes; the multi-axis cage structure support is mounted on the breadboard; the primary optical element is mounted on the breadboard in a modular manner to form a primary fluorescence microscopy imaging system; on the basis of the primary fluorescence microscopy imaging system, the secondary optical element and the intermediate optical element are mounted on the breadboard in a modular manner to form a secondary fluorescence microscopy imaging system, an intermediate fluorescence microscopy imaging system, or an advanced fluorescence microscopy imaging system. The present application can improve the flexibility and stability of the imaging system, and enable the same system to achieve multimodal fluorescence microscopy imaging without changing the main structure framework.

Description

Technical Field

[0001] The present application relates to the field of optical imaging systems, and in particular to a multimodal fluorescence microscopy imaging system based on a cage structure. Background Art

[0002] Fluorescence microscopy has a wide range of applications in the fields of subcellular structure and function due to its unique advantages such as high sensitivity, high specificity, and non-invasiveness. Various imaging techniques based on fluorescence microscopy, such as total internal reflection fluorescence microscopy (TIRFM) for observing the surface of samples at a depth of 100nm, breaking through the diffraction limit and achieving super-resolution structured illumination microscopy (SIM), have gradually become powerful tools in the scientific research process. With the rapid development of contemporary life sciences, mastering advanced instruments and technologies has become a necessary condition for scientific research, and corresponding experimental courses are urgently needed to guide students to master the working principles of high-precision instruments. However, current practical courses related to fluorescence imaging face the problem of lack of standardized imaging systems and difficulty in unifying teaching standards. Summary of the invention

[0003] The purpose of this application is to provide a multimodal fluorescence microscopy imaging system based on a cage structure, which can improve the flexibility and stability of the imaging system and enable the same system to achieve multimodal fluorescence microscopy imaging without changing the main structural framework.

[0004] To achieve the above objectives, this application provides the following solutions:

[0005] The present application provides a multi-modal fluorescence microscopy imaging system based on a cage structure, comprising: a breadboard, a multi-axis cage structure support, a primary optical element, a secondary optical element, and a secondary optical element;

[0006] The multi-axis cage structure support is provided with mechanical through holes of different heights to provide optical paths of multiple different optical axes; the multi-axis cage structure support is mounted on the breadboard; in the optical path, the primary optical element is mounted on the breadboard in a modular manner to form a primary fluorescence microscopy imaging system;

[0007] On the basis of the primary fluorescence microscopy imaging system, the secondary optical element is mounted on a breadboard in a modular manner to form a secondary fluorescence microscopy imaging system;

[0008] On the basis of the secondary fluorescence microscopy system, the intermediate optical elements are mounted on a breadboard in a modular manner to form an intermediate fluorescence microscopy system or an advanced fluorescence microscopy system.

[0009] Optionally, the primary optical elements include: a laser, a first reflector, a second reflector, a first lens, a second lens, a fifth reflector, a sixth reflector, a seventh reflector, an eighth reflector, a fifth lens, a ninth reflector, a first dichroic mirror, a second dichroic mirror and an objective lens arranged in sequence in the propagation direction of the optical path, and an emission filter, a tube lens and a camera arranged in sequence along the propagation direction of the fluorescence emission; the fluorescence is generated by the laser in the optical path by exciting fluorescent molecules in the sample.

[0010] Optionally, the primary fluorescence microscopy imaging system is a wide-field fluorescence microscope.

[0011] Optionally, the secondary optical element includes a third reflector, a digital micromirror array, a fourth reflector, a third lens, a spatial light filter hole and a fourth lens which are sequentially arranged in the propagation direction of the light path.

[0012] Optionally, the secondary fluorescence microscopy imaging system is a total internal reflection fluorescence microscope.

[0013] Optionally, the intermediate optical element comprises: a polarization adjustment element;

[0014] When the polarization adjustment element is a partitioned half-wave plate, on the basis of the secondary fluorescence microscopy imaging system, the intermediate optical element is mounted on a breadboard in a modular manner to form the intermediate fluorescence microscopy imaging system;

[0015] When the polarization adjustment element is a quarter wave plate, on the basis of the secondary fluorescence microscopy imaging system, the intermediate optical element is mounted on a breadboard in a modular manner to form the advanced fluorescence microscopy imaging system.

[0016] Optionally, the intermediate fluorescence microscopy imaging system is a two-dimensional structured light illumination microscope.

[0017] Optionally, the advanced fluorescence microscopy imaging system is a three-dimensional structured light illumination microscope.

[0018] Optionally, the multi-axis cage structure support component includes a mechanical component; a plurality of groups of mechanical through holes with different heights are arranged on the center line of the mechanical component to provide optical paths of a plurality of different optical axes.

[0019] Optionally, it is characterized in that the different optical axes include one or more of a 40 mm optical axis, a 70 mm optical axis and a 100 mm optical axis.

[0020] According to the specific embodiments provided in this application, this application has the following technical effects:

[0021] The present application provides a multimodal fluorescence microscopy imaging system based on a cage structure. By providing mechanical through holes of different heights on a multi-axis cage structure support, the imaging system can be provided with a plurality of optical paths of different optical axes, thereby improving the flexibility of the imaging system. In addition, the stability of the imaging system can be improved by providing a multi-axis cage structure support. Further, on the basis of forming a plurality of optical paths of different optical axes, the primary optical elements, the secondary optical elements and the intermediate optical elements are mounted on a breadboard in a modular manner, and imaging systems of different complexity (i.e., primary fluorescence microscopy imaging system, intermediate fluorescence microscopy imaging system and advanced fluorescence microscopy imaging system) can be obtained in turn, so that the system can realize multimodal fluorescence microscopy imaging without changing the main structural framework, and can provide sufficient three-dimensional adjustment space, which is helpful to have a deeper understanding of the working principle behind each imaging system, and is of great significance for the cultivation of interdisciplinary talents and the scientific research application of related imaging technologies, thereby being able to solve the problem that the current practical courses related to fluorescence imaging are faced with the lack of standardized imaging systems and it is difficult to unify the teaching standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 A schematic diagram of the optical path of a primary fluorescence microscopy imaging system provided in one embodiment of the present application;

[0024] Figure 2 A schematic diagram of the optical path of a secondary fluorescence microscopy imaging system provided in one embodiment of the present application;

[0025] Figure 3 A schematic diagram of the optical path of an intermediate fluorescence microscopy imaging system provided in one embodiment of the present application;

[0026] Figure 4 A three-dimensional simulation diagram of a multimodal fluorescence microscopy imaging system based on a cage structure provided in one embodiment of the present application;

[0027] Figure 5 A schematic diagram of the optical path of an advanced fluorescence microscopy imaging system provided in one embodiment of the present application;

[0028] Figure 6 A schematic diagram of the three-dimensional structure of a multimodal fluorescence microscopic imaging system based on a cage structure provided in one embodiment of the present application;

[0029] Figure 7 A three-dimensional simulation diagram of a primary fluorescence microscopy imaging system provided in an embodiment of the present application;

[0030] Figure 8 A three-dimensional simulation diagram of the secondary, intermediate and advanced fluorescence microscopy imaging system provided in one embodiment of the present application. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0032] In order to make the above-mentioned objects, features and advantages of the present application more obvious and easy to understand, the present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0033] In an exemplary embodiment, the present application provides a multimodal fluorescence microscopy imaging system based on a cage structure, such as Figure 1 As shown, the system includes: a breadboard, a multi-axis cage structure support, a primary optical element, a secondary optical element, and a secondary optical element.

[0034] The multi-axis cage structure support is provided with mechanical through holes of different heights to provide optical paths of multiple different optical axes. The multi-axis cage structure support is mounted on a breadboard. In the optical path, primary optical components are mounted on the breadboard in a modular manner to form a primary fluorescence microscopy imaging system.

[0035] Based on the primary fluorescence microscopy system, the secondary optical components are mounted on a breadboard in a modular manner to form a secondary fluorescence microscopy system.

[0036] Based on the secondary fluorescence microscopy system, the intermediate optical components are mounted on a breadboard in a modular manner to form an intermediate fluorescence microscopy system or an advanced fluorescence microscopy system.

[0037] In another exemplary embodiment of the present application, the multi-axis cage structure support member includes a mechanical member. A plurality of groups of mechanical through holes at different heights are arranged on the center line of the mechanical member to provide optical paths of a plurality of different optical axes.

[0038] In the actual application process, this application adopts a multi-axis cage structure as a multi-axis cage support device. The serial use of multiple support devices can provide stable support for the overall optical path system, and is the best choice for building a reference system with an optical axis higher than 40mm. The specific structure of the multi-axis cage structure support is that multiple groups of through holes of different heights are arranged on the center line of the same mechanical part, and 4 standard φ6mm cage rod mounting holes with a center spacing of 30mm are distributed around the through hole. There are threaded fixing holes at the corresponding positions on the side of the cage rod mounting hole, which can be used to fix the relative position of the cage rod and the support device. The bottom of the multi-axis cage structure support is provided with a semi-open "U"-shaped through hole. When in use, the bottom surface of the multi-axis cage structure support is placed on the surface of the breadboard, and the position is moved so that the semi-open "U"-shaped through hole corresponds to the screw hole position on the breadboard, and then the M6 ​​cylindrical hexagon socket screw is used to connect the multi-axis cage structure support to the breadboard through the semi-open "U"-shaped through hole to provide stable support for the system and avoid the optical path deviation caused by the operation of replacing the device during the debugging process.

[0039] The present application relates to a multi-axis cage support device used in the main body of a multi-modal fluorescence microscopy imaging system based on a cage structure, which is a three-hole support device. The device also provides mechanical through holes with heights of 40mm, 70mm and 100mm from the bottom surface, and the optical axes used in the system are 40mm and 100mm. At the output end of the laser light source, the laser is installed on a breadboard so that the emitted laser is transmitted on the 40mm optical axis, and after being reflected by two reflectors, the light beam is raised from the 40mm optical axis to 100mm, and subsequent light beams are all transmitted on the 100mm optical axis.

[0040] In another exemplary embodiment of the present application, as the most basic fluorescence microscopy system, a widefield (WF) fluorescence microscope focuses the laser onto the rear focal plane of the objective lens, and after refraction by the objective lens, it is irradiated onto the sample surface in the form of parallel light. The structure of WF is very simple, but the sample volume it excites is large, and there are many defocus signals, which can only meet the most basic imaging requirements. In order to facilitate users to understand the working principle behind WF more deeply, in this embodiment, WF is used as a primary fluorescence microscopy imaging system built. Based on this, the primary optical elements include: a laser, a first reflector M1, a second reflector M2, a first lens L1, a second lens L2, a fifth reflector M5, a sixth reflector M6, a seventh reflector M7, an eighth reflector M8, a fifth lens L5, a ninth reflector M9, a first dichroic mirror DM1, a second dichroic mirror DM2 and an objective lens OB, and an emission filter EF, a tube lens TL and a camera (Camera) arranged in sequence along the propagation direction of the fluorescence emission. Fluorescence is generated by the laser in the light path by exciting fluorescent molecules in the sample.

[0041] Based on the above-mentioned primary optical elements, the following can be constructed: Figure 1 The WF shown in the figure. The workflow of this WF is mainly divided into the following steps:

[0042] S1: The fine laser beam generated by the laser is reflected by the first reflector M1 and the second reflector M2, and then enters the 4f system composed of the first lens L1 and the second lens L2 to be expanded. The expansion factor is determined by the focal lengths of the two lenses. The specific expansion factor is the ratio of the focal lengths of the two lenses. In the experiment, the appropriate expansion factor can be selected according to the field of view. Figure 1 LASER (Light Amplification by Stimulated Emission of Radiation).

[0043] S2: The expanded laser changes direction after being reflected by the fifth reflector M5, the sixth reflector M6, the seventh reflector M7, and the eighth reflector M8, and is incident on the fifth lens L5, while accurately moving along the optical axis formed by the multi-axis cage structure support.

[0044] S3: The parallel light incident on the fifth lens L5 is converged to the rear focal plane of the objective lens, and is reflected and redirected by the ninth reflector M9 and the first and second dichroic mirrors DM1 and DM2, which transmit long-wavelength light and reflect short-wavelength light.

[0045] S4: The laser light converged to the rear focal plane of the objective lens is refracted by the objective lens and irradiates the sample surface in the form of parallel light, exciting the fluorescent molecules in a large area of ​​the sample to emit fluorescence.

[0046] S5: The fluorescence emitted by the fluorescent molecules is collected by the objective lens, passes through the second dichroic mirror DM2, is filtered by the emission filter EF, and is finally focused by the tube lens TL onto the camera target surface to complete the imaging.

[0047] As the simplest fluorescence imaging system, WF can help users understand the most basic principles of fluorescence microscopy. The collected images can be compared with subsequent imaging systems and serve as good control group data.

[0048] In actual application, when the WF function is realized, the light beam emitted by the laser will be converged on the back focal plane of the objective lens by the fifth lens L5 after a series of shaping and turning, and finally provide uniform illumination for the fluorescent sample in the field of view through the objective lens. The digital micro-mirror array (DMD) does not participate in the modulation of the illumination beam. Therefore, when the WF function is realized, the DMD will be isolated from the optical path. The specific operation process is as follows Figure 7As shown, the third reflector M3 and the fourth reflector M4 are taken out by loosening the fixing screws on the top of the third reflector M3 and the fourth reflector M4. At this time, the optical path will not pass through the DMD but directly reach the fifth reflector M5. Because the optical path does not need to be modulated, different components need to be installed inside the two two-dimensional translation adjustment frames (adjustment frame ① and adjustment frame ②) in front of the sixth reflector M6.

[0049] In another exemplary embodiment of the present application, TIRFM also uses parallel light to illuminate the sample, but the laser focused on the rear focal plane of the objective lens deviates from the optical axis, so that the refracted parallel light illuminates the junction of the sample and the coverslip at a larger angle. If this angle exceeds the critical angle of total reflection, total reflection will occur. At this time, the excitation light will produce an evanescent wave on the sample side that decays exponentially with the penetration depth. The effective excitation depth is only about 100 nm, so it is very suitable for observing samples close to the surface of the glass slide. Based on this, on the basis of using WF as a secondary fluorescence microscopy imaging system, in order to understand the working principle of TIRFM, in this embodiment, the secondary optical elements can include a third reflector M3, DMD, a fourth reflector M4, a third lens L3, a spatial light filter hole Mask and a fourth lens L4 arranged in sequence in the propagation direction of the light path. Based on this, combined with the following Figure 1 The WF structure shown in the figure can be combined with a new diffraction module formed by a third reflector M3, a DMD, a fourth reflector M4, a third lens L3, a spatial light filter hole Mask and a fourth lens L4, and set on a breadboard in a modular manner to obtain a structure as shown in FIG. Figure 2 TIRFM shown.

[0050] In this embodiment, based on WF, a new diffraction module is added to generate light that deviates from the optical axis. Based on this, the TIRFM workflow is mainly divided into the following steps:

[0051] S1: The expanded light is obtained in exactly the same manner as the WF laser beam expansion process in the above embodiment.

[0052] S2: The expanded light is reflected by the third reflector M3 and irradiated onto the surface of the digital micromirror array (DMD) at a blazing angle to generate multi-order diffraction light. The ±1-order diffraction light and the 0-order diffraction light are reflected by the fourth reflector M4 and the fifth reflector M5, and are refracted by the third lens L3 and focused onto the surface of the spatial light filter hole Mask.

[0053] S3: The spatial light filter hole Mask is blocked, and only the +1-order diffraction light is allowed to pass. The laser that passes smoothly is reflected by the sixth reflector M6, incident on the 4f system composed of the fourth lens L4 and the fifth lens L5, and is focused to the rear focal plane of the objective lens. As with WF, the seventh reflector M7, the eighth reflector M8, the ninth reflector M9 and the first dichroic mirror DM1 and the first dichroic mirror DM2 are used in the process to reflect and change the direction.

[0054] S4: The laser focused on the rear focal plane of the objective lens is refracted into parallel light by the objective lens. However, since the incident light deviates from the optical axis, this beam of parallel light will be incident on the sample surface at a certain angle. If the angle exceeds the critical angle, total reflection will occur. The incident angle is adjusted by adjusting the period of the loaded stripes on the DMD so that it finally enters at the critical angle.

[0055] S5: The fluorescent molecules in the sample are excited by the evanescent wave, and the emitted fluorescence is imaged by the same fluorescence acquisition module as WF.

[0056] TIRFM requires that the incident light deviates far enough from the optical axis. For example, using a system with immersion oil and cover glass with a refractive index of 1.518 and an objective lens numerical aperture of 1.4, the maximum angle of incidence is 67.3°. The sample refractive index is about 1.33, so the critical angle of incidence is 61.2°. The period of the stripes loaded on the DMD will change the diffraction angle of the diffracted light, so it can be adjusted to adjust the distance that the incident light deviates from the optical axis, and then select the spatial light filter hole Mask with a suitable position for the light hole, so that the final laser can be incident at a specific angle.

[0057] Among them, when light propagates from a denser medium to a less dense medium, when the incident angle is greater than the critical angle, total internal reflection will occur. Here, making the light deviate from the center of the optical axis is mainly used to provide different incident angles. The farther the light beam on the rear focal plane of the objective lens deviates from the optical axis, the greater the incident angle of the light beam refracted by the objective lens to the sample plane. When the incident angle is greater than the critical angle, total internal reflection can occur in the sample area close to the cover glass surface, thereby effectively removing the interference of background fluorescence.

[0058] Therefore, in the process of adjusting TIRFM imaging, the critical angle of incidence is achieved by adjusting the distance that the light deviates from the optical axis.

[0059] Furthermore, a light beam deviating from the center of the optical axis is usually generated by scanning the light with a galvanometer. Here, in order to be compatible with the subsequent structured light super-resolution imaging, the present application adopts a diffraction module, that is, an electronic grating DMD, to achieve this. By adjusting the period of the stripe pattern loaded on the electronic grating DMD, the distance that the first-order diffraction point deviates from the center of the optical axis can be changed, thereby changing the angle of incidence on the sample plane and realizing total internal reflection illumination.

[0060] Two-Dimensional Structured Illumination Microscopy (2D-SIM) applies sinusoidal illumination to the sample through the interference of two parallel beams of light to produce moiré fringes, allowing the camera to collect high-frequency information in the sample that breaks the optical diffraction limit. In actual operation, it is necessary to collect three sinusoidal structured light illumination patterns with a phase difference of 2π / 3 in three directions with an angle of 60° to each other, a total of 9 patterns. In combination with the existing SIM (Structured Illumination Microscopy) reconstruction algorithms, isotropic lateral double spectrum spreading can be achieved to achieve a resolution of about 120 nm. In addition, using three structured light illumination images in one direction, the reconstruction algorithm can also be used to obtain optical layer cutting images, and the out-of-focus low-frequency signals can be filtered out to improve the system's axial layer cutting capability.

[0061] Unlike WF and TIRFM, the images collected by 2D-SIM need to be reconstructed through algorithms to achieve optical sectioning or super-resolution. For optical sectioning, three images with a phase difference of 2π / 3 in one direction are required. The high-frequency components of the defocused signal decay rapidly, and the interference of the defocused signal on the image is reduced by filtering the low-frequency components, thereby improving the axial sectioning force. For super-resolution, it is necessary to collect three sinusoidal structured light illumination patterns with a phase difference of 2π / 3 in three directions with an angle of 60° to each other, for a total of 9 images, for reconstruction.

[0062] The data acquisition of 2D-SIM is also slightly different from the previous two. In order to quickly obtain 9 images for reconstruction, this application makes full use of the linkage between DMD and camera, uses DMD output trigger to access the camera input trigger hardware interface, and completes the synchronization of camera sampling and DMD fringe loading.

[0063] Three-Dimensional Structured Illumination Microscopy (3D-SIM) is very similar to two-dimensional structured light microscopy (2D-SIM), but it requires structured light generated by the interference of three parallel light beams to achieve axial super-resolution. At this time, the spectrum of the structured light will have a double frequency component, and it is necessary to collect phase differences of 2π / 5 in three directions with an angle of 60° to each other. A total of 15 structured light illumination patterns can complete the reconstruction.

[0064] The optical path diagram of 3D-SIM is as follows Figure 5As shown in the figure, the structured light in 3D-SIM is generated by the interference of three beams of light. On the basis of 2D-SIM, the intermediate zero-order light must pass through the spatial light filter hole Mask, and the polarization adjustment element must replace the partitioned half-wave plate (Pizza Half-Wave Plate, Pizza-HWP) with a quarter-wave plate (Quarter-Wave Plate, QWP). The workflow is mainly divided into the following steps:

[0065] S1: For the laser beam expansion method, please refer to the above content.

[0066] S2: The diffracted light is focused by the third lens L3 onto the surface of the spatial light filter hole Mask. After the ±1-order light and the 0-order light pass through the spatial light filter hole Mask, QWP is used to uniformly adjust the three beams of light into circularly polarized light. It should be noted that in order to reduce the hardware cost of the imaging system, the fringe structure light is generated by using circularly polarized light interference, and the fringe contrast will decrease to a certain extent. Subsequent upgrades to scientific research imaging systems can pass through an electro-optic modulator (EOM) and QWP after laser beam expansion, adjust the polarization direction to be consistent with the fringe direction, and then enter the DMD for diffraction to ensure the maximum interference fringe contrast.

[0067] S3: The three light beams are focused by the fourth lens L4 and the fifth lens L5 onto the rear focal plane of the objective lens, refracted into parallel light by the objective lens, and interfere on the sample surface to produce structured light.

[0068] S4: The fluorescence emitted by the fluorescent molecules in the sample is collected by the collection light path to complete the imaging.

[0069] The 3D-SIM optical path can further realize three-dimensional super-resolution fluorescence imaging based on 2D-SIM, and can be used as an exercise to deepen the understanding of structured light illumination technology after building 2D-SIM.

[0070] Based on the above description, the intermediate optical element includes: a polarization adjustment element.

[0071] When the polarization adjustment element is a partitioned half-wave plate, on the basis of the secondary fluorescence microscopy imaging system, the intermediate optical elements are mounted on the breadboard in a modular manner to form a Figure 3 The mid-level fluorescence microscopy imaging system (i.e., 2D-SIM) is shown.

[0072] When the polarization adjustment element is a quarter wave plate, on the basis of the secondary fluorescence microscopy imaging system, the secondary optical elements are mounted on the breadboard in a modular manner to form a Figure 5 The Advanced Fluorescence Microscopy Imaging System (i.e., 3D-SIM) is shown.

[0073] Based on the above description, the functions of TIRFM, 2D-SIM and 3D-SIM can all be realized on the basis of the WF optical path, and these system functions all require the introduction of DMD into the illumination optical path for light beam modulation, so first install the third reflector M3 and the fourth reflector M4 into the system and fix them with screws, and adjust the rotation and pitch angles of the reflectors to correct the optical axis. Then, according to the functions to be realized, install the corresponding optical components into the adjustment frame ① and the adjustment frame ② respectively. Figure 8 As shown, combined Figure 2 The TIRFM function to be realized by the optical path requires a beam of parallel light to illuminate the junction of the sample and the cover glass at a large angle. After the beam is expanded, it generates multiple diffraction lights after passing through the DMD surface. Only the +1st order diffraction light is allowed to pass through by blocking. At this time, the corresponding Mask plate needs to be installed on the adjustment frame ① and no optical components are installed in the adjustment frame ②.

[0074] Based on TIRFM, the 2D-SIM system allows the spatial light filter hole Mask to pass ±1-order diffraction light at the same time. The two beams of light are focused by the fourth lens L4 and the fifth lens L5 to the rear focal plane of the objective lens, refracted by the objective lens into parallel light, and interfere on the sample surface to form sinusoidal structured light. In order to make the polarization direction consistent with the stripe direction, Pizza-HWP is added after the Mask. Therefore, the spatial light filter hole Mask that allows ±1-order diffraction light to pass through is installed in the adjustment frame ①, and the Pizza-HWP is installed on the adjustment frame ② and aligned to the position through the adjustment mechanism to realize the 2D-SIM function.

[0075] like Figure 3 and Figure 5 As shown, the optical path of 3D-SIM is similar to that of 2D-SIM, except that the structured light needs to be generated by the interference of three beams of light, and the intermediate zero-order light and ±1-order light need to pass through the spatial light filter hole Mask accordingly, and the polarization adjustment element needs to replace Pizza-HWP with QWP. Therefore, the Mask that allows the intermediate zero-order light and ±1-order light to pass through the spatial light filter hole is installed in the adjustment frame ①, and the QWP is installed in the adjustment frame ② to realize the function of 3D-SIM.

[0076] In summary, the design idea of ​​the multimodal fluorescence microscopy imaging system based on the cage structure provided by the present application is: the WF, TIRFM, 2D-SIM and 3D-SIM systems share a set of basic optical devices (laser LASER, first reflector M1, second reflector M2, first lens L1, second lens L2, fifth reflector M5, third lens L3, sixth reflector M6, fourth lens L4, seventh reflector M7, eighth reflector M8, fifth lens L5, ninth reflector M9, first dichroic mirror DM1, second dichroic mirror DM2, objective lens OB, emission filter EF, tube lens TL and camera), and there is no need to replace or adjust these devices when switching system functions. To realize the function of multimodal fluorescence microscopy system, it is necessary to set mechanical devices that are easy to disassemble and replace at specific positions of the system structure. Taking the 2D-SIM optical path as an example, the multimodal fluorescence microscopy system structure is equipped with a detachable mirror fixture with pitch angle and rotation adjustment functions at the third reflector M3 and the fourth reflector M4, and a detachable and replaceable two-dimensional translation and rotation adjustment card holder is set at the Pizza-HWP and Mask positions. When teaching multimodal fluorescence microscopy, different devices can be replaced according to the optical path to realize the progressive functions of WF, TIRFM, 2D-SIM and 3D-SIM. The specific operations are as follows:

[0077] WF functions (such as Figure 7 As shown in Figure 2, WF is the most basic fluorescence microscope system. It focuses the laser onto the rear focal plane of the objective lens, and after refraction by the objective lens, it irradiates the sample surface in the form of parallel light. There is no need to modulate the light beam, so the light beam does not need to pass through the DMD. Figure 7 As shown, the third reflector M3 and the fourth reflector M4 are taken out by the quick disassembly method of the third reflector M3 and the fourth reflector M4. Meanwhile, no components are loaded in the adjustment frame ① and the adjustment frame ②.

[0078] TIRFM function is realized based on WF: TIRFM also uses parallel light to illuminate the sample, but the laser focused on the rear focal plane of the objective lens deviates from the optical axis, so the refracted parallel light illuminates the junction between the sample and the cover glass at a larger angle, so the light beam must be modulated. Figure 8 As shown, by installing the third reflector M3 and the fourth reflector M4 into the system and adjusting the position so that the light beam is transmitted along the center of the cage, and then loading the stripe modulation information on the DMD so that the light beam generates the first-order diffraction light that deviates from the center of the optical axis, the diffraction light will be incident on the sample surface along the optical path. In order to avoid the interference of ±1-order diffraction light and 0-order diffraction light on the sample surface, it is necessary to place a Mask on the adjustment frame ①, and the Mask is located on the rear focal plane of the third lens L3. At the same time, no device is installed in the adjustment frame ②.

[0079] The 2D-SIM function is realized on the basis of TIRFM: 2D-SIM is to produce sinusoidal structured light illumination on the sample through the interference of two parallel beams of light. The illumination structured light and the sample are superimposed to produce moiré fringes with a lower frequency than the sample frequency. The camera can collect low-frequency moiré fringes information, and then restore the high-frequency information in the sample through calculation to achieve super-resolution. Therefore, when switching from TIRFM to 2D-SIM function, it is necessary to replace the Mask on the adjustment frame ① with a type that can simultaneously pass ±1-order diffraction light, blocking the zero-order light and other unnecessary diffraction-order light. At the same time, the sinusoidal structured light with the strongest interference contrast will only be generated when the polarization direction of the illumination light is consistent with the direction of the stripes. Therefore, in order to make the polarization direction consistent with the direction of the stripes, it is necessary to install Pizza-HWP in the adjustment frame ② and rotate it to the position corresponding to the Mask light hole. The Pizza-HWP half-wave plate is a partitioned spliced ​​half-wave plate that can adjust the polarization direction of the six first-order lights corresponding to the three directions to S-polarized light, thereby generating sinusoidal structured light with higher contrast on the sample plane.

[0080] Realize 3D-SIM function based on 2D-SIM: 3D-SIM has similar optical paths to 2D-SIM, and requires three-dimensional structured light generated by the interference of three parallel beams (0th order plus ±1st order) to achieve axial super-resolution. Therefore, the Mask on the adjustment frame ① is replaced with a type that can pass ±1st order and 0th order diffraction light at the same time, and at the same time, a quarter-wave plate QWP is installed in the adjustment frame ②. If you need to generate three-dimensional structured light with high contrast, the requirements are the same as 2D-SIM. The polarization direction of the illumination light should be consistent with the direction of the interference fringes. The polarization directions of the three beams of light can be uniformly modulated by polarization modulation devices such as electro-optical modulators before the light is irradiated on the DMD for diffraction. However, due to the high cost of electro-optical modulation devices, the cost of the entire system will be significantly increased. Because a quarter-wave plate is used in this application to adjust the polarization direction of the three beams of light to circularly polarized light, the experimental verification that the decrease in the contrast of the fringes is within an acceptable range can meet the principle verification during the application process.

[0081] In summary, Figure 4 The overall optical path of the system is shown. The devices marked in the figure are all the optical components required for the 2D-SIM system. Figure 6 What is displayed is the three-dimensional structure of the multimodal fluorescence microscopy imaging system, which is a complete framework for realizing WF, TIRFM, 2D-SIM and 3D-SIM functions.

[0082] Since this system is designed to allow users to gradually understand and build more complex fluorescence imaging systems based on WF. This application achieves the overall flexibility of the system through the design of a cage structure, allowing users to add or change key components like building blocks according to their needs to achieve the switching of WF, TIRFM, 2D-SIM and 3D-SIM functions. In addition, after upgrading the key core components, this application can meet the needs of basic scientific research and realize the teaching concept of scientific research of imaging systems.

[0083] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0084] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. At the same time, for those skilled in the art, according to the ideas of this application, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting this application.

Claims

1. A multimodal fluorescence microscopy system based on a cage structure, characterized in that: The multimodal fluorescence microscopic imaging system based on a cage structure comprises: a breadboard, a multi-axis cage structure support, a primary optical element, a secondary optical element and a secondary optical element; The multi-axis cage structure support is provided with mechanical through holes of different heights to provide optical paths of multiple different optical axes; the multi-axis cage structure support is mounted on the breadboard; in the optical path, the primary optical element is mounted on the breadboard in a modular manner to form a primary fluorescence microscopy imaging system; On the basis of the primary fluorescence microscopy imaging system, the secondary optical element is mounted on a breadboard in a modular manner to form a secondary fluorescence microscopy imaging system; On the basis of the secondary fluorescence microscopy system, the intermediate optical elements are mounted on a breadboard in a modular manner to form an intermediate fluorescence microscopy system or an advanced fluorescence microscopy system.

2. The multimodal fluorescence microscopy system based on a cage structure according to claim 1, characterized in that: The primary optical elements include: a laser, a first reflector, a second reflector, a first lens, a second lens, a fifth reflector, a sixth reflector, a seventh reflector, an eighth reflector, a fifth lens, a ninth reflector, a first dichroic mirror, a second dichroic mirror and an objective lens arranged in sequence in the propagation direction of the optical path, and an emission filter, a tube lens and a camera arranged in sequence along the propagation direction of the fluorescence emission; the fluorescence is generated by the laser in the optical path by exciting the fluorescent molecules in the sample.

3. The multimodal fluorescence microscopy system based on a cage structure according to claim 2, characterized in that: The primary fluorescence microscopy imaging system is a wide-field fluorescence microscope.

4. The multimodal fluorescence microscopy imaging system based on a cage structure according to claim 1, characterized in that: The secondary optical element comprises a third reflector, a digital micromirror array, a fourth reflector, a third lens, a spatial light filter hole and a fourth lens which are sequentially arranged in the propagation direction of the light path.

5. The multimodal fluorescence microscopy system based on a cage structure according to claim 4, characterized in that: The secondary fluorescence microscopic imaging system is a total internal reflection fluorescence microscope.

6. The multimodal fluorescence microscopy system based on a cage structure according to claim 1, characterized in that: The intermediate optical element comprises: a polarization adjustment element; When the polarization adjustment element is a partitioned half-wave plate, on the basis of the secondary fluorescence microscopy imaging system, the intermediate optical element is mounted on a breadboard in a modular manner to form the intermediate fluorescence microscopy imaging system; When the polarization adjustment element is a quarter wave plate, on the basis of the secondary fluorescence microscopy imaging system, the intermediate optical element is mounted on a breadboard in a modular manner to form the advanced fluorescence microscopy imaging system.

7. The multimodal fluorescence microscopy imaging system based on a cage structure according to claim 6, characterized in that: The intermediate fluorescence microscopic imaging system is a two-dimensional structured light illumination microscope.

8. The multimodal fluorescence microscopy imaging system based on a cage structure according to claim 6, characterized in that: The advanced fluorescence microscopy imaging system is a three-dimensional structured light illumination microscope.

9. The multimodal fluorescence microscopy imaging system based on a cage structure according to claim 1, characterized in that: The multi-axis cage-type structural support component includes a mechanical component; a plurality of groups of mechanical through holes with different heights are arranged on the center line of the mechanical component to provide optical paths of a plurality of different optical axes.

10. The multimodal fluorescence microscopy imaging system based on a cage structure according to claim 1, characterized in that: The different optical axes include one or more of a 40 mm optical axis, a 70 mm optical axis and a 100 mm optical axis.

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