An ultramicroscope for single molecule localization

Abstract

The application discloses a kind of super-resolution fluorescence microscopes for single molecule positioning, belong to the technical field of super-resolution microscopic imaging, including illumination module, laser emission module, lock focus optical path module, objective system module, cylindrical lens module and imaging module;Laser emission module includes first optical fiber head, first lens, first filter, adjustable diaphragm, first mirror, second mirror, first focusing lens, third mirror and fourth mirror;Wherein, first mirror is used to reflect the parallel light beam passing through first filter and adjustable diaphragm to second mirror;Fourth mirror is used to reflect light beam to objective system module, and objective system module is used to irradiate received light beam on sample.Laser emission module can change the propagation direction of light beam by multiple reflections, so as to reduce the transmission volume of light beam, and then make the layout of super-resolution fluorescence microscope more compact, which can reduce the volume of super-resolution fluorescence microscopic system.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of super-resolution microscopic imaging, and particularly to a super-resolution fluorescence microscope for single molecule positioning. BACKGROUND

[0002] The fluorescence microscope is the most widely used technology for observing and analyzing biological cells due to its good molecular specificity and non-invasive imaging capability, and the dynamic range of observation is usually about 250nm. However, most of the biological molecules and molecular complex structures are smaller than 100nm in size, so it is usually necessary to use a super-resolution fluorescence microscope to observe the molecular structure.

[0003] The super-resolution fluorescence spectral imaging microscope usually includes a laser emitter, a dichroic mirror, a beam splitter, an image controller and a spectrometer. However, in order to increase the magnification of the super-resolution fluorescence microscope, the optical path of the super-resolution fluorescence microscope is usually long, resulting in a large size of the optical path components, so that the volume of the super-resolution fluorescence microscope system is usually large and the appearance shape is irregular. This not only occupies a large amount of research space, but also is difficult to move and transfer. SUMMARY

[0004] The present application provides a super-resolution fluorescence microscope for single molecule positioning to solve the above technical problems. The volume of the super-resolution fluorescence microscope system can be reduced, the research space can be effectively saved, and the use and transfer process of the super-resolution fluorescence microscope are more convenient.

[0005] The technical solution of the present application to solve the above technical problems is as follows:

[0006] A super-resolution fluorescence microscope for single molecule positioning includes an illumination module, a laser emission module, a focus locking optical path module, an objective system module, a cylindrical lens module and an imaging module.

[0007] The laser emission module includes a first optical fiber head, a first lens, a first filter, an adjustable diaphragm, a first mirror, a second mirror, a first focusing lens, a third mirror and a fourth mirror.

[0008] The first optical fiber head is connected to the illumination module. The emission port of the first optical fiber head is provided with a first lens, a first filter and an adjustable diaphragm in sequence and parallel to each other. The first lens is used to convert the divergent light beam emitted by the first optical fiber head into a parallel light beam. The first mirror is used to reflect the parallel light beam passing through the first filter and the adjustable diaphragm to the second mirror.

[0009] The first focusing lens is located between the second mirror and the third mirror, the parallel light beam reflected by the second mirror is focused by the first focusing lens and then reflected by the third mirror to the fourth mirror; the fourth mirror is used for reflecting the light beam to the objective lens system module, and the objective lens system module is used for irradiating the received light beam on the sample.

[0010] By adopting the above technical scheme, the light beam emitted by the illumination module is received by the laser emission module, and the light beam in the laser emission module is reflected by the first mirror, the second mirror, the third mirror and the fourth mirror and then reflected to the objective lens system module; the light path layout mode of the laser emission module can change the propagation direction of the light beam through multiple reflections, thereby obtaining a larger optical path in a limited space, and further making the layout of the super-resolution fluorescence microscope more compact, reducing the volume of the super-resolution fluorescence microscope system, effectively saving the research space, and making the use and transfer process of the super-resolution fluorescence microscope more convenient.

[0011] Further, the illumination module comprises a first optical fiber coupler, a first laser, a fifth mirror and a first dichroic mirror, the first optical fiber coupler is connected with the first optical fiber head through a first optical fiber, and the first optical fiber coupler is used for transmitting laser to the first optical fiber head through the first optical fiber;

[0012] The first laser is provided with a plurality of first lasers, and each first laser is provided with a fifth mirror corresponding to the position of the emission port, and each fifth mirror is provided with a first dichroic mirror on one side, and a plurality of first dichroic mirrors are sequentially arranged at the receiving port position of the first optical fiber coupler;

[0013] The fifth mirror is used for reflecting the light beam emitted by the first laser to the first dichroic mirror, the first dichroic mirror is used for reflecting the light beam reflected by the fifth mirror to the receiving port of the first optical fiber coupler, and the light beam reflected by the first dichroic mirror away from the first optical fiber coupler can pass through the first dichroic mirror close to the first optical fiber coupler.

[0014] Further, the objective lens system module comprises a slide, an objective lens, a second dichroic mirror, a sixth mirror and a third dichroic mirror;

[0015] The slide is arranged at one end of the objective lens, the second dichroic mirror and the sixth mirror are sequentially arranged at the end of the objective lens away from the slide, and the third dichroic mirror is located on one side of the sixth mirror;

[0016] The second dichroic mirror is used for reflecting the light beam reflected by the fourth mirror to the sample, and the sample generates a fluorescent light beam after being irradiated by the light beam, and the fluorescent light beam passes through the slide, the objective lens and the second dichroic mirror, is reflected by the sixth mirror, passes through the third dichroic mirror, enters the cylindrical lens module and the imaging module.

[0017] Further, the lock focus optical path module is connected with the objective lens system module, and comprises a second laser, a second fiber coupler, a second optical fiber, a second optical fiber head, a second focusing lens, a seventh mirror, a beam splitting prism and a quarter wave plate.

[0018] The light beam emitted by the second laser is coupled into the second optical fiber through the second fiber coupler, and the second optical fiber is used for transmitting the light beam to the second optical fiber head, and the light beam emitted by the second optical fiber head passes through the second focusing lens to generate a focused light beam.

[0019] The seventh mirror is located on one side of the second focusing lens and is used for reflecting the focused light beam, the beam splitting prism is located on one side of the seventh mirror and is used for reflecting or transmitting the focused light beam, the beam splitting prism is provided with the quarter wave plate on the side close to the third dichroic mirror, and the beam splitting prism is provided with a photodiode on the side away from the third dichroic mirror.

[0020] The quarter wave plate is used for transmitting the light beam reflected between the beam splitting prism and the third dichroic mirror, and the photodiode is used for receiving the light beam passing through the quarter wave plate and the beam splitting prism.

[0021] Further, the cylindrical lens module comprises a space and a first four-way filter, the rear focal surface lens or the cylindrical lens is detachably connected in the space, and the first four-way filter is located on the side of the space close to the imaging module; when the rear focal surface lens is located in the space, the laser emission module emits the light beam, and the rear focal surface of the objective lens can be observed.

[0022] Further, the imaging module comprises a barrel lens, an eighth mirror, a fourth dichroic mirror, a twelfth mirror, a ninth mirror, a thirteenth mirror, a second filter, a tenth mirror, an eleventh mirror, a third filter and a camera.

[0023] The barrel lens is located on one side of the first four-way filter, and the fourth dichroic mirror, the twelfth mirror, the thirteenth mirror, the second filter, the third filter and the camera are sequentially arranged on the side of the barrel lens away from the first four-way filter.

[0024] The eighth mirror and the ninth mirror are located on one side of the thirteenth mirror, and the tenth mirror and the eleventh mirror are located on the other side of the thirteenth mirror; the fourth dichroic mirror is used to split the light beam transmitted through the barrel mirror into a transmitted light beam and a reflected light beam;

[0025] The transmitted light beam passes through the fourth dichroic mirror, is refracted by the twelfth mirror, the tenth mirror, the eleventh mirror and the short edge surface on one side of the thirteenth mirror, and then passes through the third filter to reach the camera.

[0026] The reflected light beam is reflected by the fourth dichroic mirror, is refracted by the eighth mirror, the ninth mirror and the short edge surface on the other side of the thirteenth mirror, and then passes through the second filter to reach the camera.

[0027] Further, the eighth mirror and the ninth mirror are adjustably arranged along the length direction of the second filter, for adjusting the optical path of the reflected light beam.

[0028] In summary, compared with the prior art, the technical scheme has the following beneficial effects:

[0029] The super-resolution fluorescence microscope for single molecule positioning disclosed in the application can observe the structure of a molecule. A laser emission module receives a light beam emitted by an illumination module. The light beam in the laser emission module is reflected by a first mirror, a second mirror, a third mirror and a fourth mirror, and then is reflected to an objective lens system module. The light path layout mode of the laser emission module can change the propagation direction of the light beam through multiple reflections, thereby reducing the transmission volume of the light beam, and further making the layout inside the super-resolution fluorescence microscope more compact, reducing the volume of the super-resolution fluorescence microscope system, effectively saving research space, and making the use and transfer process of the super-resolution fluorescence microscope more convenient. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The figure is a schematic diagram of the overall structure of the embodiment of the application;

[0031] Figure 2 The figure is a schematic diagram of the structure of the second adjustable mirror holder and the first mounting plate in the embodiment of the application;

[0032] Figure 3 The figure is a schematic diagram of the structure of the adjusting cover plate and the electric control junction box in the embodiment of the application;

[0033] Figure 4 The figure is a schematic diagram of the structure of the first optical fiber head part and the adjustable diaphragm part in the embodiment of the application;

[0034] Figure 5 The figure is a schematic diagram of the structure of the back focal plane lens part and the second four-way filter in the embodiment of the application;

[0035] Figure 6 Figure 1 is a structural schematic diagram of a displacement table and a coarse adjustment support in an embodiment of the present application.

[0036] Reference signs:

[0037] 0002, locking ring; 0003, coarse adjustment support; 0005, support plate; 0006, horizontal XY direction piezoelectric displacement table; 0007, sample support plate; 0008, displacement table; 0009, objective body;

[0038] 001, main mounting plate; 003, heat dissipation mounting plate; 006, coupler support; 007, first mounting plate; 010, second adjustable lens holder; 016, first adjustable lens holder; 017, support block;

[0039] 4, first dichroic mirror; 8, fifth mirror; 9, first fiber coupler; 10, first laser; 14, second laser; 15, first fiber head; 16, first lens; 17, first filter; 18, adjustable diaphragm; 19, first mirror; 20, second mirror; 21, first focusing lens; 22, fourth mirror; 23, third mirror; 24, second fiber head; 25, second focusing lens; 26, seventh mirror; 27, photodiode; 28, beam splitter prism; 29, quarter wave plate; 30, third dichroic mirror; 31, sixth mirror; 32, second dichroic mirror; 34, objective lens; 35, glass slide; 37, back focal plane lens; 38, empty space; 39, cylindrical lens; 40, first four-way filter; 41, barrel lens; 42, eighth mirror; 43, fourth dichroic mirror; 44, twelfth mirror; 45, ninth mirror; 46, thirteenth mirror; 47, second filter; 48, tenth mirror; 49, eleventh mirror; 50, third filter; 51, camera; 52, first fiber; 53, second fiber; 58, excitation beam; 62, transmitted beam; 63, reflected beam;

[0040] 100, first optical fiber head part; 101, excitation light bottom plate; 103, main dichroic mirror part; 104, adjustable diaphragm part; 106, first focusing lens part; 108, main bottom plate; 111, main mirror part; 113, cylindrical part; 114, fourth mirror part; 115, support; 116, fifth mirror part; 117, mirror part; 118, second optical fiber head part; 119, second focusing lens part; 120, upper mounting plate; 121, photodiode QPD part; 122, beam splitter prism part; 123, bottom plate; 124, sleeve lens part; 125, dichroic mirror part; 126, two-mirror part; 127, third mirror part; 130, camera part; 131, second mounting plate; 133, fixed support plate; 134, linear motor; 135, linear guide rail; 136, movable mounting plate; 137, second four-way filter; 138, rear focal plane lens part; 139, through hole; 140, cylindrical mirror part; 141, sample chamber movable part; 142, sample chamber fixed part; 143, main body shell; 144, adjustment cover plate; 145, camera housing; 146, electric control junction box. DETAILED DESCRIPTION

[0041] The principles and features of the present application are described below in conjunction with the accompanying drawings, which are only used to explain the present application and not intended to limit the scope of the present application.

[0042] The embodiments of the present application disclose an ultramicro fluorescent microscope for single molecule positioning.

[0043] Reference Figure 1 An ultramicro fluorescent microscope for single molecule positioning, comprising an illumination module II, a laser emission module I, a focus locking light path module III, an objective system module IV, a cylindrical mirror module and an imaging module V. The illumination module is connected with the laser emission module, the objective system module is connected with the laser emission module, the focus locking light path module and the cylindrical mirror module respectively, and the cylindrical mirror module is connected with the imaging module.

[0044] When observation is performed, the illumination module is used for emitting a light beam to illuminate a sample, the laser emission module receives the light beam emitted by the illumination module, and the transmission volume of the light beam is reduced through refraction and reflection; the refracted light beam enters the objective system module, the objective system module irradiates the received light beam on the sample, and accepts the fluorescence emitted by the sample after excitation and transmits the fluorescence to the cylindrical mirror module and the imaging module V, and the cylindrical mirror module and the imaging module V convert the optical signal into an electrical signal.

[0045] The focus locking light path module adopts a 785nm light source, emits a light beam to the objective system module, and is used for irradiating the light beam on the lower surface of the sample, and realizes real-time focus locking function by using the signal feedback of a photodiode.

[0046] The illumination module comprises a first fiber coupler 9, a first laser 10, a fifth mirror 8 and a first dichroic mirror 4, the first fiber coupler 9 is used for converging the light beams in the illumination module; the first laser 10 is provided with a plurality of, in the embodiment of the application, the first laser 10 is specifically provided with four, and the first laser 10 is specifically a 405nm laser, a 488nm laser, a 561nm laser and a 640nm laser.

[0047] Wherein, the emitting port position of each first laser 10 is correspondingly provided with an inclined fifth mirror 8, one side of each fifth mirror 8 is correspondingly provided with a first dichroic mirror 4, the fifth mirror 8 and the plane where the first dichroic mirror 4 is located are perpendicular to each other, and the four first dichroic mirrors 4 are sequentially arranged at the receiving port position of the first fiber coupler 9.

[0048] The fifth mirror 8 is used for reflecting the light beams emitted by the first laser 10 to the first dichroic mirror 4, due to the property of the first dichroic mirror 4 itself, it can transmit or reflect the light beams according to the wavelength of the light beams, therefore the first dichroic mirror 4 can reflect the light beams reflected by the fifth mirror 8 to the receiving port of the first fiber coupler 9, and the light beams reflected by the first dichroic mirror 4 away from the first fiber coupler 9 can pass through the first dichroic mirror 4 close to the first fiber coupler 9, thereby the light emitted by different lasers can be gathered at the receiving port of the first fiber coupler 9.

[0049] When observing the molecular structure, the 405nm laser, the 488nm laser, the 561nm laser and the 640nm laser can respectively provide light beams with different wavelengths for the sample, thereby different types of staining agents applied on the sample can be excited, and then the super-resolution fluorescence microscope can more conveniently observe and operate different types of samples, especially different staining agents are applied on two substances interacting with each other, and then excitation lights with different wavelengths are simultaneously irradiated, then the actions of the two substances and the interaction therebetween can be distinguished during observation.

[0050] The laser emission module comprises a first fiber head 15, a first lens 16, a first filter 17, an adjustable diaphragm 18, a first mirror 19, a second mirror 20, a first focusing lens 21, a third mirror 23 and a fourth mirror 22. Wherein, the first fiber head 15 is used for being connected with the illumination module, the first fiber coupler 9 is connected with the first fiber head 15 through a first fiber 52, and the first fiber coupler 9 is used for transmitting the laser to the first fiber head 15 through the first fiber 52.

[0051] The emitting port of the first optical fiber head 15 is sequentially provided with a first lens 16, a first filter 17 and an adjustable diaphragm 18 which are parallel to each other, wherein the first lens 16 is used to convert the divergent light beam emitted by the first optical fiber head 15 into a parallel light beam; the first reflecting mirror 19 is used to reflect the parallel light beam passing through the first filter 17 and the adjustable diaphragm 18 to the second reflecting mirror 20.

[0052] The first focusing lens 21 is located between the second reflecting mirror 20 and the third reflecting mirror 23, and the parallel light beam reflected by the second reflecting mirror 20 is focused by the first focusing lens 21 and then reflected by the third reflecting mirror 23 to the fourth reflecting mirror 22; the fourth reflecting mirror 22 is used to reflect the light beam to the objective lens system module, and the objective lens system module is used to irradiate the received light beam on the sample.

[0053] When observing the molecular structure, the laser emitting module receives the light beam emitted by the illumination module, and the light beam in the laser emitting module is reflected to the objective lens system module after passing through the first reflecting mirror 19, the second reflecting mirror 20, the third reflecting mirror 23 and the fourth reflecting mirror 22. The light path layout mode of the laser emitting module can change the propagation direction of the light beam by multiple reflections, thereby reducing the transmission volume of the light beam, and further making the layout of the super-resolution fluorescence microscope more compact, which can reduce the volume of the super-resolution fluorescence microscope system, effectively save the research space, and make the use and transfer process of the super-resolution fluorescence microscope more convenient.

[0054] The objective lens system module includes a slide 35, an objective lens 34, a second dichroic mirror 32, a sixth reflecting mirror 31 and a third dichroic mirror 30; the slide 35 is arranged at one end of the objective lens 34, and when observing the molecular structure, a detection sample parallel to the slide 35 is placed on the side of the slide 35 away from the objective lens 34; the second dichroic mirror 32 and the sixth reflecting mirror 31 are sequentially and obliquely arranged at the end of the objective lens 34 away from the slide 35, and the third dichroic mirror 30 is located on one side of the sixth reflecting mirror 31.

[0055] The second dichroic mirror 32 is used to reflect the light beam reflected by the fourth reflecting mirror 22 to the detection sample, and the sample generates a fluorescent light beam after being irradiated by the light beam; the fluorescent light beam passes through the slide 35, the objective lens 34 and the second dichroic mirror 32, is reflected by the sixth reflecting mirror 31 to pass through the third dichroic mirror 30, and then is incident into the cylindrical lens module and the imaging module.

[0056] When observing the molecular structure, the operator needs to adjust the sample observation position in advance, at this time the sample and the slide 35 are moved in the direction perpendicular to the axis of the objective lens 34 to collect different fields of view of the sample, and the distance of the sample relative to the objective lens 34 changes during the movement. At the same time, due to the uneven installation of the sample placed on the slide 35 during mechanical installation, the above displacement operation can adjust the installation position of the slide 35, thereby facilitating more accurate observation operation of the sample.

[0057] The focus light path module includes a second laser 14, a second fiber 53 coupler, a second fiber 53, a second fiber head, a second focusing lens 25, a seventh mirror 26, a beam splitting prism 28, and a quarter wave plate 29. The light beam emitted by the second laser 14 is coupled into the second fiber 53 through the second fiber 53 coupler, and the second fiber 53 is used to transmit the light beam to the second fiber head. The light beam emitted by the second fiber head is focused by the second focusing lens 25 to generate a focused light beam.

[0058] The seventh mirror 26 is obliquely arranged on one side of the second focusing lens 25 and is used to reflect the focused light beam. The beam splitting prism 28 is located on one side of the seventh mirror 26 and is used to reflect or transmit the focused light beam. The beam splitting prism 28 is provided with the quarter wave plate 29 on the side close to the third dichroic mirror 30, and the photodiode 27 is provided on the side away from the third dichroic mirror 30.

[0059] When observing the molecular structure, the quarter wave plate 29 is used to transmit the reflected light beam between the beam splitting prism 28 and the third dichroic mirror 30, and the photodiode 27 is used to receive the light beam passing through the quarter wave plate 29 and the beam splitting prism 28.

[0060] When observing the molecular structure, the focused light beam passes through the seventh mirror 26 and the beam splitting prism 28 after reflection, passes through the quarter wave plate 29, and then is reflected by the dichroic mirror and the sixth mirror 31. After passing through the dichroic mirror, the light beam is irradiated on the back focal plane of the objective lens 34, and then the light beam is irradiated on the lower surface of the slide 35. The light beam generates a reflected light beam 63 through the lower surface of the slide 35, and the reflected light beam 63 returns to the original path after being generated, passes through the objective lens 34 and the second dichroic mirror 32, is reflected by the sixth mirror 31 and the third dichroic mirror 30, passes through the quarter wave plate 29 and the beam splitting prism 28, and is irradiated on the photodiode 27.

[0061] At this time, the light spot of the reflected light beam 63 irradiated on the photodiode 27 can move along the setting direction of the moving photodiode 27, thereby deviating from the central position of the photodiode 27, at this time, the photodiode 27 converts the light signal into an electrical signal and transmits it to the objective lens system module, the objective lens system module can automatically adjust the position of the objective lens 34 relative to the sample, thereby moving the light signal to the central position of the photodiode 27, and automatically circulating the process can ensure that the distance of the objective lens 34 relative to the sample is unchanged, and the sample is locked at the focal position of the objective lens 34.

[0062] The cylindrical lens module comprises a vacant position 38 and a first four-way filter 40, the rear focal length lens 37 or the cylindrical lens 39 can be detachably connected in the vacant position 38, and the first four-way filter 40 is located on the side of the vacant position 38 close to the imaging module; when the rear focal length lens 37 is located in the vacant position 38, the light beam emitted by the laser emitting module can be observed to the rear focal plane of the objective lens 34, thereby the angle position of the excited photoelectric can be observed.

[0063] When the vacant position 38 is in an idle state, the fluorescence can pass through the cylindrical lens module without changing the shape of the light spot, thereby facilitating data collection work; when the cylindrical lens 39 is located in the vacant position 38, it is used for three-dimensional super-resolution positioning, and in the above process, the first four-way filter 40 can filter out stray light in the fluorescence.

[0064] The imaging module comprises a barrel lens 41, an eighth mirror 42, a fourth dichroic mirror 43, a twelfth mirror 44, a ninth mirror 45, a thirteenth mirror 46, a second filter 47, a tenth mirror 48, an eleventh mirror 49, a third filter 50 and a camera 51.

[0065] Among them, the barrel lens 41 is located on the side of the first four-way filter 40, and the fourth dichroic mirror 43, the twelfth mirror 44, the thirteenth mirror 46, the second filter 47, the third filter 50 and the camera 51 are sequentially arranged on the side of the barrel lens 41 away from the first four-way filter 40.

[0066] The eighth mirror 42 and the ninth mirror 45 are located on one side of the thirteenth mirror 46, and the tenth mirror 48 and the eleventh mirror 49 are located on the other side of the thirteenth mirror 46; the fourth dichroic mirror 43 is used to decompose the light beam transmitted through the barrel lens 41 into a transmitted light beam 62 and a reflected light beam 63.

[0067] Wherein, the transmitted light beam 62 passes through the fourth dichroic mirror 43, and then is refracted by the twelfth mirror 44, the tenth mirror 48, the eleventh mirror 49 and the short edge of the thirteenth mirror 46, and then reaches the camera 51 through the third filter 50. The reflected light beam 63 is reflected by the fourth dichroic mirror 43, and then is refracted by the eighth mirror 42, the ninth mirror 45 and the other short edge of the thirteenth mirror 46, and then reaches the camera 51 through the second filter 47.

[0068] When observing the molecular structure, the light beam is reflected by the mirror, which can effectively reduce the volume required for light beam reflection, so that the layout of the super-resolution fluorescence microscope is more compact, and the volume of the super-resolution fluorescence microscope system can be reduced.

[0069] The light beam is divided into the transmitted light beam 62 and the reflected light beam 63, and the transmitted light beam 62 and the reflected light beam 63 are branched to reach the camera 51, which can realize the dual-channel data acquisition of the super-resolution fluorescence microscope, so that the super-resolution fluorescence microscope can simultaneously collect data of two different wavelength dyeing structures, the data collection efficiency is high, and the repositioning accuracy of the two structures is high.

[0070] The eighth mirror 42 and the ninth mirror 45 are adjustable along the length direction of the second filter 47, which is used to adjust the optical path of the reflected light beam 63. So as to ensure that the focal points of the reflection side light path and the transmission side light path can be simultaneously irradiated on the imaging surface of the camera 51, and the data of different wavelength dyeing structures of the super-resolution fluorescence microscope is collected, and the relative position accuracy of the super-resolution fluorescence microscope when collecting data of two structures is improved.

[0071] Referring to Figure 2 Four first lasers 10 are respectively installed on two heat dissipation mounting plates 003, four fifth mirrors 8 are respectively installed on four first adjustable mirror holders 016, the first adjustable mirror holder 016 is installed on a support block 017, four first dichroic mirrors 4 are respectively installed on four second adjustable mirror holders 010, the second adjustable mirror holder 010 is installed on a first mounting plate 007, and the heat dissipation mounting plate 003, the first mounting plate 007 and the support block 017 are all installed on a main mounting plate 001.

[0072] The first fiber coupler 9 is arranged on a coupler support 006, the coupler support 006 is installed on the first mounting plate 007, the main mounting plate 001 is provided with an illumination housing, the illumination housing is provided with a heat dissipation hole and a handle. The heat dissipation mounting plate 003 and the bottom of the first mounting plate 007 are both provided with grooves, which are used to increase the heat dissipation area and reduce the weight. The main support plate 0005 adopts a breadboard structure, and the main materials used include stainless steel, aluminum alloy and structural steel.

[0073] Referring to Figure 3The super-resolution fluorescence microscope of the present application also includes a main housing 143, a camera housing, an adjustment cover 144, an electric control junction box 146, a sample chamber fixing part 142, and a sample chamber movable part 141. Among them, the sample chamber movable part 141 is rotatably connected to the main housing 143, and the sample chamber movable part 141 can be rotated around the hinge and flipped back to facilitate the operator to take and place samples. The electric control junction box 146 is used to place the integrated wiring port. After the adjustment cover 144 is opened, it is convenient for the operator to adjust the optical path of the imaging part. The material of the outer shell part includes steel plate and aluminum alloy.

[0074] Reference Figure 4 The super-resolution fluorescence microscope of the present application further includes a primary dichroic mirror 103, a first optical fiber head, a collimating lens, an adjustable aperture, a first focusing lens, and a reflector assembly. These components are all mounted on an excitation light base plate 101.

[0075] The primary dichroic mirror 103 is used to reflect the focal point of the excitation light beam 58 upward toward the rear focal plane of the objective lens 34. The primary dichroic mirror 103 comprises a dichroic mirror, a mounting bracket, a magnetic mounting block, and a support frame. Both the excitation light baseplate 101 and the primary dichroic mirror 103 are mounted on a main baseplate 108.

[0076] The first optical fiber head portion includes an optical fiber head, an optical fiber head holder, a motor, a slide rail, and a sensor, and the first optical fiber head portion can be moved by the motor. The collimating lens portion includes a lens, a lens holder, and a filter, and the collimating lens portion can be shifted and adjusted along the optical path axis.

[0077] The adjustable aperture part includes an adjustable aperture 18, an aperture bracket and a magnetic mounting block. When observing the molecular structure, different models of adjustable apertures 18 can be replaced in different working states. The first focusing lens part includes a focusing lens and a lens bracket. The focusing lens part as a whole can be adjusted along the axial direction of the optical path, thereby facilitating finding a suitable focal position; the reflector assembly is used to cooperate in adjusting the direction and position of the optical path.

[0078] The super-resolution fluorescence microscope of the present application also includes a second optical fiber head part, a second focusing lens part, a first reflector part, a beam splitting prism part, a photodiode part, a main reflector part 111, a sleeve lens part 124, a dichroic mirror part 125, a second reflector part, a third reflector part, a fourth reflector part, a fifth reflector part and a camera part. The second optical fiber head part, the second focusing lens part and the reflector part 117 are all mounted on the upper mounting plate 120, and the upper mounting plate 120 is mounted on the base plate 123 through four supporting columns.

[0079] The second optical fiber head part comprises an optical fiber head, an optical head mounting rack, and a support rack. The second focusing lens part comprises a lens, a lens mounting cylinder, a locking nut, and an anti-shaking guide support. The mirror part 117 comprises a mirror and an adjustable mounting rack. The main mirror part 111 comprises a main mirror, an adjustable support, a dichroic mirror, a dichroic mirror support, a magnetic mounting block, and an overall support block 017. The main mirror part 111 is mounted on the main bottom plate 108.

[0080] The beam splitter prism part comprises a beam splitter prism 28, a cage mounting rack, a mounting rack, an adjustable support, a support block 017, a quarter-wave plate 29, a wave plate mounting cylinder, and a locking support. The beam splitter prism part is mounted on the bottom plate 123. The photodiode QPD part comprises a QPD, a QPD mounting rack, an electric linear slide, and an adjustable support mechanism. The photodiode QPD part is mounted on the bottom plate 123, and the bottom plate 123 is mounted on the main bottom plate 108.

[0081] The sleeve lens part 124 comprises a sleeve lens and a sleeve lens support, which can be adjusted in two dimensions on the main bottom plate 108. The dichroic mirror part 125 comprises a dichroic mirror and a dichroic mirror mounting rack. The second mirror part, the third mirror part, the fourth mirror part, and the fifth mirror part each comprise a mirror and an adjustable support. The second mirror part and the third mirror part are mounted on the support block 127, and the support block 127 further comprises three mirrors.

[0082] The fourth mirror part and the fifth mirror part are mounted on the support 115, which is mounted on a manual slide, and the manual slide is mounted on the support block 017. An operator can adjust the positions of the fourth mirror part and the fifth mirror part by adjusting the manual slide knob to adjust the optical path. The camera part comprises a camera 51, a second mounting plate 131, and a positioning device.

[0083] The structure has a compact layout and occupies a small volume. The mirror part 117 of the reflection side light path has a manual slide device for adjusting the optical path of the reflection side light path to achieve the same optical path as the transmission side, so that the focal points of the two fluorescence paths are simultaneously irradiated on the imaging surface of the camera 51. The imaging part only uses a sleeve lens to focus the light beam, which reduces the overall layout and saves costs.

[0084] The double-channel part and the camera part are each designed with a repeated positioning mechanism to ensure the positioning accuracy when disassembled and reassembled. The dichroic mirror assembly and the filter assembly have high repeated positioning accuracy, and multiple sets of dichroic mirror assemblies and filter assemblies with different wavelengths can be easily replaced. The adjustable mirror rack is designed with an external adjustment knob, which can be quickly and conveniently adjusted without using a wrench. The imaging effect is good, and the aberration is small.

[0085] Referring to Figure 5 The super-resolution fluorescence microscope of the present application further comprises a cylindrical portion 113, which comprises a linear motor 134, the fixed end of the linear motor 134 being mounted on a fixed support plate 133, and the driving end of the linear motor 134 being connected to a movable mounting plate 136, which is connected to the fixed second mounting plate 133 through a linear guide rail 135.

[0086] A back focal plane lens portion is mounted on the movable mounting plate 136, which comprises a lens, a lens mounting cylinder and a nut, and the back focal plane lens portion can be finely adjusted along the lens axis. A cylindrical mirror portion is also mounted on the movable mounting plate 136, which comprises a cylindrical mirror 39 and a pressing plate. A through hole 139 is provided between the back focal plane lens portion and the cylindrical mirror portion. The back focal plane lens portion, the cylindrical mirror portion and the through hole 139 can be controlled by the linear motor 134, so as to be automatically switched along the linear guide rail 135 to realize different functions. A second four-way filter 137 is fixedly mounted on the side of the movable mounting plate 136 away from the back focal plane lens portion, and the second four-way filter 137 is always in the working position.

[0087] Referring to Figure 6 The objective lens 34 body is mounted on a piezoelectric Z displacement stage 0008, which can move along the objective lens 34 axis. The displacement stage 0008 is mounted on a coarse adjustment support 0003. After the operator manually adjusts the coarse adjustment support 0003 to make the objective lens 34 body reach a suitable position relative to the sample, the coarse adjustment support 0003 is locked by a locking ring 0002. A horizontal XY piezoelectric displacement stage 0006 is mounted on a support plate 0005, and a sample support plate 0007 is mounted on the horizontal XY piezoelectric displacement stage 0006. The sample stage is placed on the sample support plate 0007.

[0088] The implementation principle of the super-resolution fluorescence microscope for single molecule localization in an embodiment of the present application is as follows: when observing the molecular structure, the laser emission module receives the light beam emitted by the illumination module. The light beam in the laser emission module is reflected by the first mirror 19, the second mirror 20, the third mirror 23 and the fourth mirror 22, and then reflected to the objective lens system module. The light path layout mode of the laser emission module can change the propagation direction of the light beam multiple times, thereby reducing the transmission volume of the light beam, and further making the layout inside the super-resolution fluorescence microscope more compact, which can reduce the volume of the super-resolution fluorescence microscope system, effectively save the research space, and make the use and transfer process of the super-resolution fluorescence microscope more convenient.

[0089] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A super-resolution fluorescence microscope for single-molecule localization, characterized in that include: Illumination module, laser emission module, focus-locking optical path module, objective lens system module, cylindrical lens module and imaging module; The laser emission module comprises a first optical fiber head (15), a first lens (16), a first filter (17), an adjustable diaphragm (18), a first reflector (19), a second reflector (20), a first focusing lens (21), a third reflector (23) and a fourth reflector (22); The first optical fiber head (15) is used to connect to the lighting module, and the emission port of the first optical fiber head (15) is sequentially provided with a first lens (16), a first filter (17), and an adjustable iris (18) which are parallel to each other, and the first lens (16) is used to convert the divergent light beam emitted by the first optical fiber head (15) into a parallel light beam; the first reflector (19) is used to reflect the parallel light beam passing through the first filter (17) and the adjustable iris (18) to the second reflector (20); The first focusing lens (21) is located between the second reflecting mirror (20) and the third reflecting mirror (23); the parallel light beam reflected by the second reflecting mirror (20) is focused by the first focusing lens (21) and then reflected by the third reflecting mirror (23) to the fourth reflecting mirror (22); the fourth reflecting mirror (22) is used to reflect the light beam to the objective lens system module, and the objective lens system module is used to irradiate the received light beam onto the sample; The objective lens system module includes a glass slide (35), an objective lens (34), a second dichroic mirror (32), a sixth reflecting mirror (31), and a third dichroic mirror (30); The focus locking optical path module is connected to the objective lens system module, and comprises a second laser (14), a second optical fiber coupler, a second optical fiber (53), a second optical fiber head, a second focusing lens (25), a seventh reflecting mirror (26), a beam splitting prism (28) and a quarter wave plate (29); The light beam emitted by the second laser (14) is coupled into the second optical fiber (53) through a second optical fiber coupler. The second optical fiber (53) is used to transmit the light beam to a second optical fiber head. The light beam emitted by the second optical fiber head is focused by a second focusing lens (25) to generate a focused light beam. The seventh reflecting mirror (26) is located on one side of the second focusing lens (25) and is used to reflect the focused light beam. The beam splitting prism (28) is located on one side of the seventh reflecting mirror (26) and is used to reflect or transmit the focused light beam. A quarter-wave plate (29) is provided on a side of the beam splitting prism (28) close to the third dichroic mirror (30), and a photodiode (27) is provided on a side of the beam splitting prism (28) away from the third dichroic mirror (30). The quarter-wave plate (29) is used to transmit the light beam reflected between the beam splitting prism (28) and the third dichroic mirror (30), and the photodiode (27) is used to receive the light beam passing through the quarter-wave plate (29) and the beam splitting prism (28); The cylindrical mirror module comprises a space (38) and a first four-pass filter (40), wherein a rear focal plane lens (37) or a cylindrical mirror (39) is detachably connected in the space (38), and the first four-pass filter (40) is located on a side of the space (38) close to the imaging module; when the rear focal plane lens (37) is located at the space (38), the laser emission module emits a light beam, and the rear focal plane of the objective lens (34) can be observed.

2. A super-resolution fluorescence microscope for single-molecule localization according to claim 1, characterized in that: The lighting module comprises a first fiber coupler (9), a first laser (10), a fifth reflector (8), and a first dichroic mirror (4); the first fiber coupler (9) is connected to the first fiber head (15) via a first optical fiber (52); the first fiber coupler (9) is used to transmit laser light to the first optical fiber head (15) via the first optical fiber (52); A plurality of the first lasers (10) are provided, a fifth reflector (8) is correspondingly provided at the emission port position of each first laser (10), a first dichroic mirror (4) is correspondingly provided on one side of each fifth reflector (8), and the plurality of first dichroic mirrors (4) are sequentially provided at the receiving port position of the first optical fiber coupler (9); The fifth reflector (8) is used to reflect the light beam emitted by the first laser (10) to the first dichroic mirror (4); the first dichroic mirror (4) is used to reflect the light beam reflected by the fifth reflector (8) to the receiving port of the first optical fiber coupler (9); the light beam reflected by the first dichroic mirror (4) far away from the first optical fiber coupler (9) can pass through the first dichroic mirror (4) close to the first optical fiber coupler (9).

3. The super-resolution fluorescence microscope for single-molecule localization according to claim 1, characterized in that: The glass slide (35) is arranged at one end of the objective lens (34), the second dichroic mirror (32) and the sixth reflector (31) are sequentially arranged at the end of the objective lens (34) away from the glass slide (35), and the third dichroic mirror (30) is located on one side of the sixth reflector (31); The second dichroic mirror (32) is used to reflect the light beam reflected by the fourth reflector (22) onto the detection sample. The sample is irradiated by the light beam to generate a fluorescent light beam. After the fluorescent light beam passes through the glass slide (35), the objective lens (34) and the second dichroic mirror (32), it is reflected by the sixth reflector (31) and passes through the third dichroic mirror (30) to be incident on the cylindrical mirror module and the imaging module.

4. A super-resolution fluorescence microscope for single-molecule localization according to claim 3, characterized in that: The imaging module comprises a cylindrical mirror (41), an eighth reflecting mirror (42), a fourth dichroic mirror (43), a twelfth reflecting mirror (44), a ninth reflecting mirror (45), a thirteenth reflecting mirror (46), a second optical filter (47), a tenth reflecting mirror (48), an eleventh reflecting mirror (49), a third optical filter (50), and a camera (51); The cylindrical lens (41) is located on one side of the first four-pass filter (40), and the fourth dichroic mirror (43), the twelfth reflector (44), the thirteenth reflector (46), the second filter (47), the third filter (50), and the camera (51) are sequentially arranged on a side of the cylindrical lens (41) away from the first four-pass filter (40); The eighth reflector (42) and the ninth reflector (45) are located on one side of the thirteenth reflector (46), and the tenth reflector (48) and the eleventh reflector (49) are located on the other side of the thirteenth reflector (46); the fourth dichroic mirror (43) is used to decompose the light beam passing through the tube mirror (41) into a transmitted light beam (62) and a reflected light beam (63); wherein the transmitted light beam (62) passes through the fourth dichroic mirror (43), is refracted by the twelfth reflecting mirror (44), the tenth reflecting mirror (48), the eleventh reflecting mirror (49), and the thirteenth reflecting mirror (46), and then passes through the third filter (50) to reach the camera (51); After being reflected by the fourth dichroic mirror (43), the reflected light beam (63) is refracted by the eighth reflecting mirror (42), the ninth reflecting mirror (45), and the thirteenth reflecting mirror (46), passes through the second optical filter (47), and reaches the camera (51).

5. The super-resolution fluorescence microscope for single-molecule localization according to claim 4, characterized in that: The eighth reflector (42) and the ninth reflector (45) are adjustable along the length direction of the second filter (47) and are used to adjust the optical path of the reflected light beam (63).

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