A modular super-resolution fluorescence microscopy system

Abstract

The present invention discloses a modular super-resolution fluorescence microscopy system, which belongs to the field of microscopy imaging technology, and includes an excitation module for emitting excitation light and modulating excitation light parameters; a dual-light module for transmitting the excitation light to an objective module and transmitting fluorescence from the objective module to a dichroic mirror; an objective module for exciting a sample to emit fluorescence and transmitting the excited fluorescence to the dual-light module; a detection module for receiving fluorescence transmitted by the dichroic mirror; and a dichroic mirror for transmitting the excitation light to the dual-light module and transmitting fluorescence from the dual-light module to the detection module. The present invention adopts the above-mentioned modular super-resolution fluorescence microscopy system, adopts a modular design concept, and can expand a variety of super-resolution fluorescence microscopy devices in more subdivided fields in two imaging modes, point scanning and wide-field, respectively.

Description

Technical Field

[0001] The invention relates to the technical field of microscopic imaging, and in particular to a modular super-resolution fluorescence microscopic imaging system. Background Art

[0002] Fluorescence microscopy systems specifically refer to a type of microscopic imaging device that uses lasers to excite the target to emit fluorescence, and then performs microscopic imaging of the fluorescence. This patent discusses the situation of sharing a microscope objective, that is, the laser irradiates the sample through the microscope objective, and the fluorescence emitted by the sample is collected by the same objective. This is also the most commonly used and widely used type of optical path structure.

[0003] Super-resolution fluorescence microscopy is a technology that breaks through the optical diffraction limit (200nm) to observe microscopic targets. It is particularly widely used in the field of life sciences to observe the internal structure of cells. Since the internal structure of cells cannot actively emit light, fluorescent labeling technology is generally used to attach nanoscale fluorescent proteins to the target structure, laser illumination excites the fluorescent protein to emit light, and the super-resolution microscopy system is used to capture the luminescent position of the fluorescent protein to achieve the purpose of imaging the cell structure.

[0004] The super-resolution fluorescence microscopy system is realized through super-resolution fluorescence microscopy technology. Since the resolution of traditional microscopes is limited by the diffraction limit and cannot observe substances with smaller structures, scientists have proposed a variety of methods to break through the diffraction limit and observe smaller structures. These methods are all based on the optimization and adjustment of the structure of traditional fluorescence microscopy systems.

[0005] Super-resolution fluorescence microscopy technology can be divided into two imaging modes: point scanning imaging and wide-field imaging. The imaging technology based on point scanning illuminates and stimulates a very small area to emit fluorescence through excitation light, and receives it with a unit detector. Finally, it obtains global information through scanning and restores the image through the corresponding stitching algorithm. The wide-field imaging technology illuminates the entire sample area with excitation light, uses an array detector to receive the image of the entire area, and restores the image through the corresponding image processing algorithm.

[0006] The point scanning super-resolution fluorescence microscopy system is realized by point scanning imaging technology, which mainly adopts point-by-point excitation to obtain the whole image by scanning. That is, the illumination laser is converged after being emitted from the objective lens, and there is a point on the sample surface. The position of the illumination point is switched by the scanning device and the fluorescence information of the corresponding position is collected until the whole image is obtained. The detector used is a single-point detector.

[0007] The characteristic of the point scanning super-resolution fluorescence microscopy system is that the illumination area is extremely small, that is, the final illumination light needs to be collimated and incident on the microscope objective lens, and finally imaged by the microscope objective lens on the sample plane. The specific system principle is as follows Figure 1As shown, the solid line represents the illumination light, and the dotted line represents the fluorescence, wherein part of the illumination and fluorescence optical paths overlap. The light emitted by the laser 2 61 passes through a group of lenses 11 62 and 12 63, and then expands the beam and is incident on the dichroic mirror 2 64. The beam expansion system composed of the lens 11 62 and the lens 12 63 can collimate and expand the small-caliber light beam emitted by the laser 2 61 to the size required by the back-end system. The caliber varies according to different specific needs, and can be achieved by adjusting the focal length ratio of the lens 11 62 and the lens 12 63. The dichroic mirror 2 64 reflects the excitation light to the scanning galvanometer 2 651, which is used to scan the illumination spot of the sample plane. The scanning lens 2 66 and the tube lens 2 67 adjust the beam aperture to a size suitable for the objective lens 2 68, and the scanning lens 2 66 is suitable for receiving incident light with a large off-axis angle, which meets the conditions for use with the scanning galvanometer 2 651. The objective lens 2 68 converges the incident parallel light onto the sample stage 2 69 to excite the sample to emit fluorescence. Through the scanning of the scanning galvanometer 2 651, each point of the sample is stimulated to emit fluorescence in turn. The fluorescence emitted by each point passes through the objective lens 2 68, the tube lens 2 67, the scanning lens 2 66 and the scanning galvanometer 2 651 in turn, and then returns to the dichroic mirror 2 64 and is transmitted. The transmitted light continues to propagate along the direction of the optical axis, and after being converged by the lens 13 70, it is modulated by the small hole 71 to filter out the interfering light in the off-axis field of view. Finally, after passing through the lens 14 72 and the lens 15 73, it is received by the single-point detector 741. The single-point detector 741 generally selects devices such as avalanche diode APD or photomultiplier tube PMT.

[0008] The wide-field super-resolution fluorescence microscopy system is realized by wide-field imaging technology, and the fluorescence image is obtained by plane field imaging. That is, the illumination laser is collimated after being emitted from the objective lens, which stimulates the entire field of view to emit fluorescence and collects the fluorescence of the entire field of view at the same time. The detector used is a plane array detector.

[0009] The characteristic of wide-field resolution fluorescence microscopy imaging system is to illuminate the entire field of view, that is, the final illumination light needs to be focused on the back focal plane of the objective lens, and the light finally emitted from the objective lens is collimated light, which evenly illuminates the entire field of view. The specific principle is as follows Figure 2As shown, the solid line represents the illumination light, and the dotted line represents the fluorescence. Compared with the point scanning system, devices with the same label can be reused, and devices with the same label but different suffixes are set in the same position or have the same function. The light emitted by the laser 2 61 passes through a group of lenses 11 62 and 12 63, and then expands the beam and is incident on the dichroic mirror 44. The beam expansion system composed of the lens 11 62 and the lens 12 63 can collimate and expand the small-caliber light beam emitted by the laser to the size required by the back-end system. According to different specific needs, the caliber is different, which can be achieved by adjusting the focal length ratio of the lens 11 62 and the lens 12 63. The dichroic mirror 2 64 reflects the excitation light to the reflector 2 652 for folding the light path. The tube lens 2 67 images the collimated light on the back focal plane of the objective lens 2 68, and finally the parallel light is incident on the sample stage 2 69 to excite the sample to emit fluorescence. The fluorescence emitted by the sample passes through the objective lens 2 68, the tube lens 2 67 and the reflector 2 652 in sequence, and then returns to the dichroic mirror 2 64 and is transmitted. The light passing through the dichroic mirror 2 64 is not parallel light, and needs to be transformed into parallel light by the lens 13 70. The distance between the lens 13 70 and the tube lens 2 67 must satisfy the object-image relationship. Finally, the parallel light is imaged on the array detector 742 through the lens 15 73. The array detector 742 is generally a device such as CCD or SCMOS.

[0010] In recent years, many super-resolution microscopy techniques have been derived from the two imaging modes of point scanning imaging and wide-field imaging. Representative methods of point scanning imaging modes include confocal microscopy and stimulated emission depletion microscopy (STED). Both use point scanning to achieve wide-band image acquisition. The former introduces a small hole in the optical path to filter the interference of off-axis information to improve the image signal-to-noise ratio, and has a high layer cutting effect, and the axial resolution is significantly improved; the latter nests the excitation light and the loss light, and uses the loss light to make the excited fluorescent molecules quickly fall back to the ground state without emitting fluorescence, so that only the fluorescent molecules in a very small area in the center can emit fluorescence normally, thereby significantly improving the resolution. In other words, the point scanning microscopy system can be expanded into STED, and it only needs to add a loss light generation module on the basis of the traditional point scanning optical path. Representative methods of wide-field imaging mode include structured illumination microscopy (SIM) and single-molecule microscopy (SMLM), both of which directly image the entire wide field of view. The former uses structured light to illuminate the sample, and from the frequency domain perspective, it moves the high-frequency information outside the optical transfer function of the optical system into the optical transfer function to achieve super-resolution imaging; the latter controls the fluorescent molecules to emit light sparsely in sequence, and determines its position coordinate information by fitting the imaging spot of a single molecule. The random sparse emission of a large number of single molecules is used to calculate the precise coordinates of each molecule in sequence to synthesize and restore the wide-field super-resolution image. In other words, the wide-field microscopy system can be expanded into SMLM and SIM, where SMLM does not require changing the optical path, and SIM needs to add a module to generate illumination stripes, generally using a spatial light modulator or interferometric illumination.

[0011] Regardless of the above methods, it is necessary to build a complex and expensive microscopic imaging system, in which the laser used to excite fluorescence, the objective lens used for microscopic imaging, and the microscope accessories are actually universal. In view of this, a modular super-resolution fluorescence microscopic imaging system design method with reusable accessories and reduced costs is proposed. Summary of the invention

[0012] The purpose of the present invention is to provide a modular super-resolution fluorescence microscopy system, which modularizes the system design, reuses some modules, and utilizes the specialized design of the remaining modules to realize the integrated universal design and construction of various super-resolution microscopy systems; by replacing some modules, the two imaging modes of point scanning and wide field can be quickly switched, which greatly reduces the hardware cost of scientific research and applied research, and the construction of two types of devices can be realized with a cost close to that of a set of devices.

[0013] To achieve the above object, the present invention provides a modular super-resolution fluorescence microscopy imaging system, comprising:

[0014] An excitation module, used for emitting excitation light and modulating excitation light parameters;

[0015] A dual light module, used for transmitting the excitation light to the objective lens module and transmitting the fluorescence from the objective lens module to the dichroic mirror;

[0016] The objective lens module is used to excite the sample to emit fluorescence and transmit the excited fluorescence to the dual-light module;

[0017] A detection module, used for receiving the fluorescence transmitted by the dichroic mirror;

[0018] A dichroic mirror is used to transmit the excitation light to the dual-light module and transmit the fluorescence from the dual-light module to the detection module.

[0019] Preferably, the modulatable parameters of the excitation light are aperture, polarization, intensity and spatial distribution.

[0020] Preferably, the excitation module is composed of a laser, a beam expansion system and a modulation system which are arranged in sequence.

[0021] Preferably, the modulation system includes lens one, lens two, a micro-mirror array, lens three and lens four which are arranged in sequence.

[0022] Preferably, the objective lens module is composed of a tube lens, an objective lens and a sample stage which are arranged in sequence, and the sample stage carries a sample.

[0023] Preferably, the structure of the dual light module is a lens five, a reflector and a lens six which are arranged in sequence.

[0024] Preferably, the structure of the detection module is lens seven, modulation module, lens nine and area array detector which are arranged in sequence.

[0025] Preferably, the structure of the dual-light module is a scanning galvanometer and a scanning lens arranged in sequence.

[0026] Preferably, the structure of the detection module is lens seven, lens eight, modulation module, lens nine and point detector which are arranged in sequence.

[0027] Therefore, the present invention adopts the above-mentioned modular super-resolution fluorescence microscopy imaging system, which has the following beneficial effects:

[0028] (1) The modular design enables switching between point scanning and wide-field imaging modes;

[0029] (2) Modular and scalable design, thus enabling the expansion of super-resolution fluorescence microscopy devices in more sub-fields;

[0030] (3) Cost reduction, high-value devices such as lasers and microscopes can be reused;

[0031] (4) It can realize the switching of two types of imaging devices: wide field and point scanning. The modular design makes the switching simple and does not require complicated assembly steps;

[0032] (5) A set of hardware equipment can be used to implement multiple types of super-resolution fluorescence microscopy methods, reuse the light source and microscopy components, and reduce the impact of differences in light source and microscopy components when comparing the performance of various solutions;

[0033] (6) By adopting a modular design approach and by adding or reducing modules and optimizing and modifying corresponding modules, it is possible to expand the super-resolution fluorescence microscopy imaging devices into a variety of more subdivided fields in both point scanning and wide-field imaging modes.

[0034] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the structure of a point scanning super-resolution fluorescence microscopy imaging system in the background technology;

[0036] Figure 2 It is a structural principle diagram of a wide-field resolution fluorescence microscopy imaging system in the background technology;

[0037] Figure 3 A schematic structural diagram of an embodiment of a modular super-resolution fluorescence microscopy imaging system of the present invention;

[0038] Figure 4 A schematic diagram of the structure of an excitation module of an embodiment of a modular super-resolution fluorescence microscopy imaging system of the present invention;

[0039] Figure 5 A schematic diagram of the structure of a modulation system of an embodiment of a modular super-resolution fluorescence microscopy imaging system of the present invention;

[0040] Figure 6 This is a schematic diagram of the structure of a dual optical module according to Embodiment 1 of the present invention;

[0041] Figure 7 This is a schematic diagram of the structure of a dual optical module according to Embodiment 2 of the present invention;

[0042] Figure 8 Schematic diagram of the structure of an objective lens module according to an embodiment of the present invention;

[0043] Fig. 9 This is a schematic diagram of the structure of the detection module of Example 1 of the present invention;

[0044] Fig.10 It is a schematic diagram of the structure of the detection module of embodiment 2 of the present invention.

[0045] Reference numerals

[0046] 1. Excitation module; 11. Laser; 12. Beam expansion system; 13. Modulation system; 131. Lens one; 132. Lens two; 133. Micro-mirror array; 134. Lens three; 135. Lens four; 2. Dual-light module; 21. Lens five; 22. Reflector; 23. Lens six; 24. Scanning galvanometer; 25. Scanning lens; 3. Objective lens module; 31. Tube lens; 32. Objective lens; 33. Sample stage; 4. Detection module; 41. Lens seven; 42. Lens eight; 43. Modulation module; 44. Lens nine; 45. Array detector; 46. Point detector; 5. Dichroic mirror;

[0047] 61. Laser 2; 62. Lens 11; 63. Lens 12; 64. Dichroic mirror 2; 651. Scanning galvanometer 2; 652. Reflector 2; 66. Scanning lens 2; 67. Tube lens 2; 68. Objective lens 2; 69. Sample stage 2; 70. Lens 13; 71. Pinhole; 72. Lens 14; 73. Lens 15; 741. Single-point detector; 742. Area array detector. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. The components of the embodiments of the present invention generally described and shown in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0049] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0050] The present invention provides a modular super-resolution fluorescence microscopy imaging system, including an excitation module 1, a dual-light module 2, an objective lens module 3, a detection module 4 and a dichroic mirror 5. The four modules are arranged with a specific optical path interface to realize module assembly and switching. The above four modules can be divided into three categories: an objective lens module 3 without replacing components, an excitation module 1 with components replaced according to specific application requirements, and a dual-light module 2 and a detection module 4 with components replaced according to point scanning or wide-field imaging methods.

[0051] like Figure 3As shown, the solid line represents the excitation light, and the dotted line represents the fluorescence. The excitation module 1 is used to emit the excitation light and modulate the excitation light parameters, and the modulatable quantities include but are not limited to the caliber, polarization, intensity, spatial distribution, etc. The excitation light passes through the dichroic mirror 5 and is reflected to the dual light module 2. The dual light module 2 is a common optical path for the excitation light and the fluorescence, and is used to transmit the excitation light to the objective lens module 3 and transmit the fluorescence from the objective lens module 3 to the dichroic mirror 5. In addition, the module needs to control the light beam according to two types of imaging modes. The objective lens module 3 includes an objective lens 32, a tube lens 31 and a sample stage 33. The physical distance between the objective lens 32 and the tube lens 31 needs to satisfy the object-image relationship. Regardless of the imaging mode, the module does not need to be adjusted, and its setting is only related to the imaging magnification, field of view, and numerical aperture. The objective lens module 3 transmits the illumination light to the sample stage 33 to excite the sample to emit fluorescence, and transmits the excited fluorescence to the dual light module 2. The fluorescence transmitted by the dual light module 2 passes through the dichroic mirror 5 to the detection module 4. The detection module 4 is composed of an imaging system and a detector, and is used to receive the fluorescence transmitted by the dichroic mirror 5. For different imaging modes, different detectors are selected and the imaging system is slightly different. The dichroic mirror 5 is used to transmit the excitation light to the dual light module 2 and transmit the fluorescence from the dual light module 2 to the detection module 4.

[0052] like Figure 4 As shown, the excitation module 1 is composed of a laser 11, a beam expansion system 12 and a modulation system 13. The beam expansion system 12 includes a lens group. The number of lenses in the lens group is determined according to the required beam aperture. The light emitted by the laser 11 is expanded to the required aperture through the beam expansion system 12. Different imaging modes, different subdivisions of super-resolution technologies, and different modulation systems 13 will all have different requirements for the aperture. The control of the beam aperture can be achieved by reasonably configuring the focal length of the lens group. The traditional microscopic imaging system does not need to be configured with the modulation system 13. The modulation system 13 is only for the super-resolution microscopic imaging system and is generally designed specifically according to different super-resolution technologies.

[0053] For example, for a structured illumination microscopy imaging system (SIM) based on a micromirror array (DMD), the modulation system 13 can be designed as follows: Figure 5In the structure shown, the modulation system 13 includes lens 1 131, lens 2 132, micro-mirror array 133, lens 3 134 and lens 4 135 arranged in sequence. The parallel light passes through lens 1 131 and lens 2 132 and is incident on the micro-mirror array 133 and then passes through lens 3 134 and lens 4 135 to be emitted. The first group of lens combinations (lens 1 131 and lens 2 132) is used to adjust the beam aperture to meet the requirements of DMD, and the second group of lens combinations (lens 3 134 and lens 4 135) is used to adjust the DMD output beam aperture to meet the requirements of the back-end dual-light module 2. The design point is that each group of lenses must strictly meet the object-image relationship and strictly comply with the magnification design. The output light of the excitation module 1 is collimated light, which is incident on the dichroic mirror 5 and reflected to the dual-light module 2.

[0054] Example 1

[0055] like Figure 6 As shown, the solid line represents the excitation light, and the dotted line represents the fluorescence. In the wide-field imaging mode, the dual-light module 2 includes a lens five 21, a reflector 22, and a lens six 23, which are arranged in sequence. The collimated incident excitation light passes through the lens five 21 and is incident on the reflector 22, and then passes through the lens six 23 to be emitted as parallel light; the divergent fluorescence is incident on the dual-light module 2, and after passing through two lenses (lens five 21 and lens six 23) and the reflector 22, it is a convergent light and is emitted from the module. The excitation light is collimated and incident, and the fluorescence is divergent and incident, and convergent; the core components of the dual-light module 2 in the wide-field imaging mode are the reflector 22, and the key lenses are the lens six 23 (traditional lenses), whose focal lengths and positions are the key lenses of the objective lens module 3 to meet the object-image relationship. For the dual-light module 2, the wide-field imaging mode has one more lens than the point scanning imaging mode, the purpose of which is to ensure that wide-field illumination is achieved after passing through the objective lens module 3, and the focal lengths and positions of the lens five 21 and the lens six 23 meet the object-image relationship and system magnification requirements.

[0056] like Figure 8 As shown, the solid line represents the excitation light, the dotted line represents the fluorescence, and the left side is the wide-field imaging mode. The objective lens module 3 is composed of a tube lens 31, an objective lens 32 and a sample stage 33 arranged in sequence, and a sample is carried on the sample stage 33. For the wide-field imaging mode, the collimated light is incident on the tube lens 31 and converges on the back focal plane of the objective lens 32. Finally, the collimated light is emitted to illuminate the entire wide-field field of view. The fluorescence is collimated after passing through the objective lens 32 and is incident on the tube lens 31 and converged by the tube lens 31. In summary: collimated light is incident on the module, wide-field illumination, and fluorescence converges and exits from the module. The setting of the objective lens 32 and the tube lens 31 meets the requirements of the object-image relationship and the system magnification, and the optical distance between the objective lens module 3 and the dual-light module 2 meets the object-image relationship between the tube lens 31 and the lens 623 of the dual-light module 2.

[0057] like Fig. 9As shown, the dotted line represents fluorescence. The detection module 4 includes a lens 7 41, a modulation module 43, a lens 9 44 and an array detector 45 arranged in sequence. The divergent light is incident, collimated by the lens 7 41 and enters the modulation module 43. The focal length of the lens 7 41 cooperates with the lens 6 23 and the scanning lens 25 of the preceding dual-light module 2 to adjust the beam aperture so that it meets the requirements of the modulation module 43, and then passes through the lens 9 44 to form an image on the array detector 45. The modulation module 43 is used to meet the requirements of different types of super-resolution microscopic imaging systems for the detection end, and can be expanded to subdivided super-resolution microscopic imaging systems through the design and replacement of the modulation module 43. The detection module 4 receives the fluorescence transmitted by the dichroic mirror 5, and the optical distance between the detection module 4 and the dual-light module 2 meets the object-image relationship between the lens of the detection module 4 and the lens 6 23 of the dual-light module 2.

[0058] This embodiment shows the specific structure of the dual light module 2, the objective lens module 3 and the detection module 4 in the wide-field imaging mode. Through the setting of the objective lens module 3, the excitation light can illuminate the entire sample stage 33, and then the image of the entire area is received by the area array detector 45, thereby realizing wide-field super-resolution fluorescence microscopy imaging.

[0059] Example 2

[0060] like Figure 7 As shown, the solid line represents the excitation light, and the dotted line represents the fluorescence. The dual light module 2 includes a scanning galvanometer 24 and a scanning lens 25 arranged in sequence. The collimated light is incident on the scanning galvanometer 24, and is emitted through the scanning lens 25. The emitted light is convergent light, and the fluorescence light path completely overlaps with the excitation light path and is in the opposite direction. The excitation light is incident in a collimated manner and is emitted in a convergent manner, while the fluorescence is incident in a divergent manner and is collimated. The core component of the dual light module 2 in the point scanning imaging mode is the scanning galvanometer 24, and the key lens is the scanning lens 25, whose focal length and position both satisfy the object-image relationship with the key of the objective lens module 3.

[0061] like Figure 8 As shown, the solid line represents the excitation light, the dotted line represents the fluorescence, and the right side is the point scanning imaging mode. The objective lens module 3 is composed of a tube lens 31, an objective lens 32 and a sample stage 33 arranged in sequence. The sample stage 33 carries a sample. The components of the objective lens module 3 do not need to be replaced for the wide field and point scanning imaging modes. For the point scanning imaging mode, the divergent light is incident on the tube lens 31, and the collimated light is emitted to the back focal plane of the objective lens 32. The final image is a point on the sample surface. The fluorescence is collimated after the objective lens 32 and incident on the tube lens 31, and is converged by the tube lens 31. The excitation light and the fluorescence light path completely overlap in opposite directions. In summary: the divergent light is incident on the module, illuminating a single point, and the fluorescence converges and exits from the module. The setting of the objective lens 32 and the tube lens 31 meets the object-image relationship and the system magnification requirements, and the optical distance between the objective lens module 3 and the dual light module 2 meets the object-image relationship between the tube lens 31 and the scanning lens 25 of the dual light module 2.

[0062] like Fig.10 As shown, the dotted line represents fluorescence, and the detection module 4 includes lens seven 41, lens eight 42, modulation module 43, lens nine 44 and point detector 46 arranged in sequence. For the point scanning imaging mode, collimated light is incident, and the beam aperture is adjusted through lens seven 41 and lens eight 42 to meet the requirements of the modulation module 43, and then imaged on the point detector 46 through lens nine 44. For two different imaging modes, the detection module 4 selects different detectors. The modulation module 43 is used to meet the requirements of different types of super-resolution microscopy imaging systems for the detection end, and can be expanded to subdivided super-resolution microscopy imaging systems through the design and replacement of the modulation module 43. The detection module 4 receives the fluorescence transmitted by the dichroic mirror 5, and the optical distance between the detection module 4 and the dual-light module 2 meets the object-image relationship between the lens of the detection module 4 and the scanning lens 25 of the dual-light module 2.

[0063] This embodiment shows the specific structure of the dual light module 2, the objective lens module 3 and the detection module 4 in the point scanning imaging mode. Through the setting of the objective lens module 3, the excitation light illuminates and excites a very small area to emit fluorescence, and then the sample image is received by the point detector 46, thereby realizing point scanning super-resolution fluorescence microscopy imaging.

[0064] Therefore, the present invention adopts the above-mentioned modular super-resolution fluorescence microscopy imaging system, and can determine the objective lens module and the excitation module according to the system magnification, numerical aperture, field of view and other requirements, and switch the wide-field and point scanning imaging modes through the dual light module. Through the modulation system of the excitation module and the modulation module of the detection module, the development of different subdivision super-resolution fluorescence microscopy imaging systems under the wide-field and point scanning imaging modes is realized.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A modular super-resolution fluorescence microscopy system, characterized in that: include: An excitation module, used for emitting excitation light and modulating excitation light parameters; A dual light module, used for transmitting the excitation light to the objective lens module and transmitting the fluorescence from the objective lens module to the dichroic mirror; The objective lens module is used to excite the sample to emit fluorescence and transmit the excited fluorescence to the dual-light module; A detection module, used for receiving the fluorescence transmitted by the dichroic mirror; A dichroic mirror, used to transmit the excitation light to the dual-light module and transmit the fluorescence from the dual-light module to the detection module; The excitation light is reflected to the dual-light module after passing through the dichroic mirror. The objective lens module transmits the illumination light to the sample stage to excite the sample to emit fluorescence, and transmits the excited fluorescence to the dual-light module. The fluorescence transmitted by the dual-light module passes through the dichroic mirror to the detection module. The four modules have specific optical path interfaces agreed upon between them. The four modules are divided into three categories: objective lens module that does not require device replacement, excitation module that replaces devices according to specific application requirements, dual-light module and detection module that replace devices according to point scanning or wide-field imaging methods; The excitation module is composed of a laser, a beam expansion system and a modulation system arranged in sequence; The beam expansion system includes a lens group. The number of lenses in the lens group is determined by the required beam aperture. The light emitted by the laser is expanded to the required aperture through the beam expansion system. Different imaging modes, different subdivision super-resolution technologies, and different modulation systems will have different requirements for the aperture. The control of the beam aperture is achieved by reasonably configuring the focal length of the lens group. The modulation system comprises a lens 1, a lens 2, a micro-reflector array, a lens 3 and a lens 4 which are arranged in sequence; In the wide-field imaging mode, the dual-light module includes a lens five, a reflector, and a lens six arranged in sequence. The collimated incident excitation light passes through the lens five and is incident on the reflector, and then passes through the lens six and is still parallel light. The divergent fluorescence is incident on the dual-light module, and passes through the lens six, the reflector, and the lens five in sequence to become convergent light and is emitted from the module. In the wide-field imaging mode, the objective lens module is composed of a tube lens, an objective lens and a sample stage arranged in sequence. The sample stage carries a sample. The collimated light is incident on the tube lens and converges on the back focal plane of the objective lens. Finally, the collimated light is emitted to illuminate the entire wide-field field of view. The fluorescence is collimated after passing through the objective lens and is incident on the tube lens and converged by the tube lens. In the wide-field imaging mode, the detection module includes a lens seven, a first modulation module, a lens nine and an array detector arranged in sequence. The divergent light is incident, collimated by the lens seven, enters the first modulation module, and then is imaged on the array detector by the lens nine. The first modulation module is used to meet the requirements of different types of super-resolution microscopic imaging systems for the detection end, and is expanded to subdivided super-resolution microscopic imaging systems through the design and replacement of the first modulation module; In the point scanning imaging mode, the dual light module includes a scanning galvanometer and a scanning lens arranged in sequence. The collimated light is incident on the scanning galvanometer and is emitted through the scanning lens. The emitted light is convergent light. The fluorescence light path and the excitation light light path completely overlap and are in opposite directions. The components of the objective lens module do not need to be replaced for the wide field and point scanning imaging modes. In the point scanning imaging mode, the divergent light is incident on the tube lens, and the collimated light is emitted to the back focal plane of the objective lens. The final image is a point on the sample surface. The fluorescence is collimated after passing through the objective lens and incident on the tube lens, and is converged by the tube lens. The excitation light and the fluorescence light path completely overlap and are in opposite directions. In the point scanning imaging mode, the detection module includes lens seven, lens eight, the second modulation module, lens nine and a point detector arranged in sequence. Collimated light is incident, and the beam aperture is adjusted through lens seven and lens eight to meet the requirements of the second modulation module, and then imaged on the point detector through lens nine; The second modulation module is used to meet the requirements of different types of super-resolution microscopic imaging systems for the detection end, and is expanded to subdivided super-resolution microscopic imaging systems through the design and replacement of the second modulation module.

2. A modular super-resolution fluorescence microscopy system according to claim 1, characterized in that: The modulatable parameters of the excitation light are aperture, polarization, intensity and spatial distribution.

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