CN112230412B - Quick structured light illumination super-resolution microscope - Google Patents
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Abstract
The invention discloses a rapid structured light illumination super-resolution microscope, which comprises: the device comprises a structured light generating unit, an objective lens, a first relay lens, a filtering array, a second relay lens, a third relay lens and a camera which are sequentially arranged along a light path; if the structured light generates illumination stripes or lattice light spot arrays on the object plane, correspondingly, the light passing strips of the filter array correspond to the bright stripes one by one, or the light passing holes of the filter array correspond to the bright spots one by one; the fluorescent substance at the position of the bright line or the bright spot is excited to emit fluorescence by the structured light, and the fluorescence sequentially passes through the objective lens, the first relay lens, the filtering array, the second relay lens and the third relay lens and is converged to the camera; in an exposure period of the camera, when the structured light changes, the light-passing strip and the bright stripes move synchronously, or the light-passing holes and the bright spots move synchronously; the rapid structured light illumination super-resolution microscope provided by the invention can greatly improve the time resolution of the structured light illumination super-resolution microscope and improve the imaging speed of the structured light illumination super-resolution microscope.
Description
Technical Field
The invention relates to the technical field of microscopes, in particular to a rapid structured light illumination super-resolution microscope.
Background
A Structured light Illumination microscope (SIM) illuminates a sample by using specially modulated Structured light on the basis of a wide-field fluorescence microscope, extracts focal plane information from modulated image data by applying a specific algorithm, breaks through the limitation of diffraction limit, and reconstructs a three-dimensional image of a super-resolution slice layer. The structured light illumination technology is applied to the fluorescence microscope, has the advantages of simple light path structure, no special requirement on fluorescent molecules and capability of being applied to real-time dynamic three-dimensional imaging of living cells, thereby drawing wide attention in the field of biomedical imaging and being the super-resolution fluorescence microscopy technology with wide application prospect.
The essence of the structured light illumination ultrahigh resolution microscope is to link the degree of spatial structure variation with the frequency of the frequency domain space, the Point Spread Function (PSF) of the microscope corresponds to the Optical Transfer Function (OTF) of the frequency domain space, and the fine spatial structure corresponds to the high frequency signal of the frequency domain space. In essence, the structured light illumination super-resolution microscope moves the position of the light and dark stripes by changing the spatial structure of the illumination light to obtain a plurality of image photos, and then extracts a clear image through computer operation processing, namely the spatial resolution is improved by sacrificing the time resolution, so that the imaging speed of the structured light illumination super-resolution microscope is greatly limited.
Disclosure of Invention
The embodiment of the invention provides a rapid structured light illumination super-resolution microscope, which can greatly improve the time resolution of the structured light illumination super-resolution microscope and improve the imaging speed of the structured light illumination super-resolution microscope.
The embodiment of the invention provides a rapid structured light illumination super-resolution microscope, which comprises: the device comprises a structured light generating unit, an objective lens, a first relay lens, a filtering array, a second relay lens, a third relay lens and a camera which are sequentially arranged along a light path;
the structured light generated by the structured light generating unit generates an illumination stripe or a lattice light spot array on an object plane; the illumination stripes comprise bright stripes and dark stripes which are arranged at intervals; the lattice light spot array comprises uniformly arranged bright spots; if the structured light generates the illumination stripes on the object plane, the light filtering array comprises light blocking strips and light passing strips which are arranged at intervals, and the light passing strips correspond to the bright stripes one by one; if the structured light generates the lattice light spot array on the object plane, the light filtering array comprises a light baffle plate and light through holes, and the light through holes correspond to the bright spots one by one;
fluorescent substances at the positions of the bright lines or the bright spots are excited by the structured light to emit fluorescence, and the fluorescence sequentially passes through the objective lens and the first relay lens, the filtering array, the second relay lens and the third relay lens and is converged to the camera;
in an exposure period of the camera, when the structured light changes, the illumination stripe or the lattice light spot array moves, and accordingly, the light-passing stripe moves synchronously with the bright stripe or the light-passing hole moves synchronously with the bright point;
wherein the width of the light-passing strip or the aperture of the light-passing hole
M is a transverse magnification coefficient of imaging from the object plane to an image plane where the light through hole or the light through strip is located, lambda is the wavelength of the structured light, and NA is the numerical aperture of the objective lens; preferably, the width of the light-passing strip or the aperture of the light-passing hole
a is a proportionality coefficient, the value of a and the period T and diffraction limit of the bright grains or the bright spots
Is related to (1); preferably, the width of the light-passing strip or the aperture of the light-passing hole
When in use
When the temperature of the water is higher than the set temperature,
resolution of imaging
Exceeding the diffraction limit
When in use
When a is less than
Coefficient of (a), the imaging resolution
Exceeding the diffraction limit
Preferably, when
When the temperature of the water is higher than the set temperature,
the width of the light-passing strip or the aperture of the light-passing hole
The imaging resolution
In an exposure period of the camera, the synchronous moving distance L is more than or equal to M.T, so that the envelope of the moving filter array covers the light barrier strip or the light barrier of the filter array; preferably, when the width of the light-passing bar or the aperture of the light-passing hole
Then, the distance L of the synchronous movement is equal to M · T.
The invention provides a rapid structured light illumination super-resolution microscope, which adds a light filtering array in the prior structured light illumination super-resolution microscope system. The method comprises the steps that firstly, structured light generated by a structured light generating unit generates an illumination stripe or a lattice light spot array on an object plane, fluorescent substances at the position of a bright stripe or a bright spot are excited to emit fluorescence, then the fluorescence sequentially passes through an objective lens and a first relay lens and then reaches a filtering array, the filtering array is used for filtering out the edge part of a light strip or a light spot formed by the fluorescence through diffraction and diffusion, only the fluorescence at the central part passes through, finally, the fluorescence at the central part sequentially passes through a second relay lens and a third relay lens and is converged in front of a camera, and an image formed in front of the camera is a super-resolution image of the object plane at the position of the bright stripe or the bright spot. In an exposure period of a camera, the positions of illumination stripes or lattice light spot arrays on an object plane are moved rapidly, so that bright stripes or bright spots scan the whole object plane, all fluorescent substances on the object plane are illuminated rapidly, the light through strips of the filter array are arranged to correspond to the bright stripes one by one, or the light through holes correspond to the bright spots one by one, the light through strips are matched with the bright stripes to move rapidly synchronously, or the light through holes are matched with the bright spots to move rapidly synchronously, a large number of super-resolution images of the object plane with different bright stripes or different bright spot positions can be obtained, and the super-resolution clear images of the whole object plane can be obtained by splicing and integrating. The rapid structured light illumination super-resolution microscope provided by the embodiment of the invention omits the process of extracting a clear image according to the post-calculation processing of a plurality of images of the structured light illumination super-resolution microscope, and the camera directly stacks a plurality of different images in an exposure period to obtain the clear image by adding the light filtering array in front of the camera, so that the time resolution of the structured light illumination super-resolution microscope is greatly improved on the basis of keeping the spatial resolution, and the imaging speed of the structured light illumination super-resolution microscope is improved.
Drawings
FIG. 1 is a schematic diagram of a prior art structured light illumination super-resolution imaging;
FIG. 2 is a schematic structural diagram of a fast structured-light illuminated super-resolution microscope provided by an embodiment of the present invention;
FIG. 3 is a schematic diagram of a structured light generating unit;
FIG. 4 is a schematic structural diagram of another fast structured-light illuminated super-resolution microscope provided by an embodiment of the present invention;
FIG. 5 is a schematic structural diagram of another fast structured-light illuminated super-resolution microscope provided by an embodiment of the present invention;
FIG. 6 is a schematic structural diagram of another fast structured-light illuminated super-resolution microscope according to an embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. It should be further noted that, for the convenience of description, only some of the structures related to the present invention are shown in the drawings, not all of the structures.
The resolution of a typical fluorescence microscope is determined by the wavelength of the emitted light (λ) and the numerical aperture of the objective lens (NA). According to Abbe's diffraction law, the limiting lateral resolution of a common fluorescence microscope is about 200nm, which causes the microscope to be difficult to observe biological structures smaller than the characteristic dimension of wavelength. In recent years, driven by the needs of biological research in nanoscale imaging, along with the development of light sources, detectors, novel fluorescent probes and novel imaging theories, various super-resolution fluorescent imaging technologies capable of breaking through diffraction limit are proposed, and the resolution of a fluorescent microscope is improved to 100nm or even less than 10 nm. According to different imaging principles, the super-resolution microscopy can be divided into three categories: the single molecule positioning super-resolution microscope based on light activation or light conversion fluorescent molecules, the stimulated emission loss super-resolution microscope based on the fact that fluorescence is emitted from a place except a central point of a point spread function is inhibited so that resolution is improved, and the structured light illumination super-resolution microscope based on optical transfer function expansion.
The essence of the structured light illumination ultrahigh resolution microscope is to link the degree of spatial structure variation with the frequency of the frequency domain space, the Point Spread Function (PSF) of the microscope corresponds to the Optical Transfer Function (OTF) of the frequency domain space, and the fine spatial structure corresponds to the high frequency signal of the frequency domain space. The principle of structured light illumination ultrahigh resolution imaging is that uniform illumination light is changed into structured light (comprising a sine wave mode with alternate light and shade) with a specific spatial structure, a sample is modulated on a spatial frequency domain, sample high-frequency information originally outside an optical transmission function OTF range is moved into the optical transmission function OTF range, the high-frequency information is restored through a specific algorithm, and the fineness of the structure of the illumination light, which is used for obtaining the high-frequency information beyond the optical transmission function OTF range, can be obtained.
The principle of the existing structured light illumination super-resolution microscopy is shown in fig. 1, and fig. 1 is a schematic diagram of the principle of the existing structured light illumination super-resolution microscopy. Referring to FIG. 1, when the structured light irradiates on the object plane, an illumination stripe or lattice light spot array is generated on the surface of the fluorescent material, and FIG. 1 shows that the structured light generates the illumination stripe on the object planeFor example, the illumination stripe includes a bright stripe and a dark stripe arranged at intervals. The fluorescent substance at the position of the bright pattern is illuminated by the structured light, the energy is absorbed to emit fluorescence, the fluorescence is diffracted in the light path, and when reaching the image plane, the fluorescence is expanded into a light spot, namely a diffraction spot, also called Airy spot (Airy Disk), the light intensity distribution of the diffraction spot is called as a point spread function PSF, the light intensity in the middle is very high, 84% of the light intensity is concentrated, and some side lobes are arranged around the light intensity distribution. If the distance between two adjacent bright stripes reaching the image surface is separated, namely the distance between two point spread functions PSF is separated, the two bright stripes can be easily identified and distinguished; if the distance between the two bright stripes is gradually reduced, when the peak value of the point spread function PSF of one bright stripe is superposed with the peak valley of the point spread function PSF of the adjacent bright stripe, the two bright stripes can be just distinguished; if the distance between the two bright lines is continuously reduced, the two bright lines cannot be distinguished. The limit at which the two bright-line positions can be resolved is called the diffraction limit, which is also the resolution limit of a fluorescence microscope, i.e. the diffraction limit is
The structured light illumination super-resolution microscope is a space structure for changing illumination light, obtains image photos of a plurality of different phases by moving the position of a stripe, extracts clear images through computer operation processing, namely improves the spatial resolution by sacrificing the time resolution, and greatly limits the imaging speed of the structured light illumination super-resolution microscope.
Aiming at the problem of low imaging speed of a structured light illumination super-resolution microscope in the prior art, the embodiment of the invention provides a rapid structured light illumination super-resolution microscope, which comprises the following components: the device comprises a structured light generating unit, an objective lens, a first relay lens, a filtering array, a second relay lens, a third relay lens and a camera which are sequentially arranged along a light path;
the structured light generated by the structured light generating unit generates an illumination stripe or a lattice light spot array on an object plane; the lighting stripes comprise bright stripes and dark stripes which are arranged at intervals; the lattice light spot array comprises uniformly arranged bright spots; if the structured light generates illumination stripes on the object plane, the light filtering array comprises light blocking strips and light passing strips which are arranged at intervals, and the light passing strips correspond to the bright stripes one by one; if the structured light generates a lattice light spot array on the object plane, the filtering array comprises a light baffle plate and light through holes, and the light through holes correspond to the light spots one by one;
the fluorescent substance at the position of the bright line or the bright spot is excited to emit fluorescence by the structured light, and the fluorescence sequentially passes through the objective lens, the first relay lens, the filtering array, the second relay lens and the third relay lens and is converged to the camera;
in an exposure period of the camera, when the structured light changes, the illumination stripe or the lattice light spot array moves, and accordingly, the light-passing strip and the bright stripe move synchronously, or the light-passing hole and the bright point move synchronously;
wherein, the width of the light-passing strip or the aperture of the light-passing hole
M is a transverse magnification coefficient from an object plane to an image plane where a light through hole or a light through strip is located, lambda is the wavelength of structured light, and NA is the numerical aperture of the objective lens; preferably, the width of the light-transmitting strip or the aperture of the light-transmitting hole
a is a proportionality coefficient, the value of a and the period T and diffraction limit of bright lines or bright spots
Is related to (1); more preferably, the width of the light-transmitting strip or the aperture of the light-transmitting hole
When in use
When the temperature of the water is higher than the set temperature,
resolution of imaging
Exceeding the diffraction limit
When in use
When a is less than
Coefficient of (2), imaging resolution
Exceeding the diffraction limit
Preferably, when
When the utility model is used, the water is discharged,
width of light-passing strip or aperture of light-passing hole
Resolution of imaging
In an exposure period of the camera, the synchronous moving distance L is more than or equal to M.T, so that the envelope of the moving filter array covers the light barrier strip or the light barrier of the filter array; preferably, when the width of the light-passing strip or the aperture of the light-passing hole
Then, the distance L of the synchronous movement becomes M · T.
The embodiment of the invention provides a rapid structured light illumination super-resolution microscope, which is characterized in that a light filtering array is added in the conventional structured light illumination super-resolution microscope system. The method comprises the steps that firstly, structured light generated by a structured light generating unit generates an illumination stripe or a lattice light spot array on an object plane, fluorescent substances at the position of a bright stripe or a bright spot are excited to emit fluorescence, then the fluorescence sequentially passes through an objective lens and a first relay lens and then reaches a filtering array, the filtering array is used for filtering out the edge part of a light strip or a light spot formed by the fluorescence through diffraction and diffusion, only the fluorescence at the central part passes through, finally, the fluorescence at the central part sequentially passes through a second relay lens and a third relay lens and is converged in front of a camera, and an image formed in front of the camera is a super-resolution image of the object plane at the position of the bright stripe or the bright spot. In an exposure cycle of camera, through the position of illumination stripe or lattice light spot array on the quick travel object plane, make bright line or bright spot scan whole object plane, illuminate whole fluorescent substance on the object plane fast, and the logical light strip and the bright line one-to-one that set up filter array, or logical unthreaded hole and bright spot one-to-one, logical light strip cooperation bright line ground quick travel in step, or logical unthreaded hole cooperation bright spot ground quick travel in step, can obtain the super resolution image of the object plane of a large amount of different bright lines or different bright spot positions departments, the concatenation integration just can obtain the super resolution clear image of whole object plane. According to the rapid structured light illumination super-resolution microscope provided by the embodiment of the invention, the process of extracting a clear image according to the post-calculation processing of a plurality of images of the structured light illumination super-resolution microscope is omitted, and the filtering array is additionally arranged in front of the camera, so that the camera directly superposes a plurality of different images in an exposure period to obtain the clear image, the time resolution of the structured light illumination super-resolution microscope is greatly improved on the basis of keeping the spatial resolution, and the imaging speed of the structured light illumination super-resolution microscope is improved.
The above is the core idea of the present invention, and the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without any creative work belong to the protection scope of the present invention.
Fig. 2 is a schematic structural diagram of a fast structured light illumination super-resolution microscope according to an embodiment of the present invention, and as shown in fig. 2, a filter array 13, a second relay lens 14, and a third relay lens 15 are added at an image plane position in an existing structured light illumination super-resolution microscope system. Specifically, this quick structured light illumination super resolution microscope includes: the device comprises a structured light generating unit (marked in the figure), an objective lens 11, a first relay lens 12, a filter array 13, a second relay lens 14, a third relay lens 15 and a camera 16 which are arranged in sequence along an optical path.
Specifically, the structured light generating unit may be any optical path device generating structured light, and may generate structured light in a coaxial illumination manner, that is, structured light is generated by an imaging objective lens, may also be light sheet structured light vertically illuminated, and may also be structured light totally internally reflected for illumination, the rapid structured light illuminated super-resolution microscope provided by the embodiment of the present invention may be applicable to various structured light generating devices, a specific optical path of the structured light generating unit is not limited by the embodiment of the present invention, and exemplarily, fig. 3 is a schematic structural diagram of the structured light generating unit, and as shown in fig. 3, the structured light generating unit may include: a light source 34 for emitting an initial light; and a first lens 35, a spectroscope 36, a grating 37, a second lens 38, a mask 39, a third lens 40, a dichroic mirror 41, and a fourth lens 42, which are arranged in this order along the optical path; the mask 39 is used to pass + -1 st order diffracted light; the initial light passes through the first lens 35 to form collimated parallel light; the collimated parallel light passes through the beam splitter 36 to form polarized light; the polarized light is diffracted by the grating 37 to generate diffracted light; the diffracted light sequentially passes through a second lens 38, a mask 39 and a third lens 40 to obtain structured light; the structured light is reflected by the dichroic mirror 41, passes through the fourth lens 42 and the objective lens 11 in this order, and reaches the object plane 17 from the objective lens 11.
When the structured light generated by the structured light generating unit is irradiated on the object plane 17, an illumination stripe or a lattice light spot array is formed on the object plane 17, wherein the illumination stripe comprises bright stripes and dark stripes which are arranged at intervals, and the lattice light spot array comprises bright spots which are uniformly arranged. The object plane 17 is uniformly laid with a common fluorescent substance, which can be excited and emit fluorescence under the irradiation of the structured light, and the fluorescent substance at the position of the bright line or the bright point on the object plane is excited by the structured light to emit fluorescence. The fluorescence passes through the objective lens 11 and the first relay lens 12, and a diffraction phenomenon occurs; if the structured light generates illumination stripes on the object surface 17, the bright stripes can be expanded into a light strip on the plane where the light filtering array 13 is located, the light filtering array 13 is additionally arranged at the position, the light filtering array 13 comprises light blocking strips and light passing strips which are arranged at intervals, the light passing strips correspond to the bright stripes one by one, the light blocking strips are used for filtering out edge parts of the light strip formed by the fact that the fluorescence is subjected to diffraction diffusion, namely, side lobes around the point spread function PSF, the light passing strips only allow the fluorescence in the central part to pass through, and then the clear images can be shot by the camera 16 through the collection of the second relay lens 14 and the third relay lens 15, namely, the clear images of the object surface 17 at the position where the bright stripes are located only; if the structured light generates a lattice light spot array on the object plane 17, the bright spot can be expanded into a light spot on the plane where the filter array 13 is located, the filter array 13 is additionally arranged at the position, the filter array 13 comprises a light barrier and light through holes, the light through holes correspond to the bright spot one by one, the light barrier is used for filtering out the edge part of the light spot formed by the diffraction and diffusion of the fluorescence, namely the peripheral side lobe of the point spread function PSF, the light through holes only allow the fluorescence at the central part to pass through, and then the clear image can be shot by the camera 16 through the collection of the second relay lens 14 and the third relay lens 15, namely the clear image of the object plane 17 at the position of the bright spot is only obtained.
In an exposure period of the camera 16, the structured light irradiated on the object plane 17 is changed rapidly, the position of the illumination stripe or the lattice light spot array is changed rapidly, so that the bright stripe or the bright spot can illuminate the whole object plane 17 in a rapid scanning manner in one exposure period of the camera 16, and the light-passing strip and the bright stripe move synchronously or the light-passing hole and the bright spot move synchronously, that is, the center of the light-passing strip of the filter array 13 is always coincided with the center of the light strip formed after the fluorescence emitted by the fluorescent substance at the bright stripe position is diffracted by the objective lens 11 and the first relay mirror 12, or the center of the light-passing hole of the filter array 13 is always coincided with the center of the light spot formed after the fluorescence emitted by the fluorescent substance at the bright spot position is diffracted by the objective lens 11 and the first relay mirror 12, so that a large number of super-resolution images of different bright stripes or different bright spot positions of the object plane 17 can be obtained, so that a super-resolution sharp image of the entire object plane 17 can be obtained.
The filter array 13 may be any array having filteringIn the functional optical device, if the structured light generates illumination stripes on the object plane 17, the filter array 13 includes light blocking strips and light passing strips which are arranged at intervals, and the light passing strips correspond to the bright stripes one by one; if the structured light generates a lattice light spot array on the object plane 17, the filter array 13 comprises a light barrier and light through holes, and the light through holes correspond to the bright spots one by one; the light blocking bars or plates are used to filter the edge portions of the light bars or light spots formed due to the diffraction phenomenon when the fluorescence passes through the filter array 13, and the light passing bars or holes are used to pass only the fluorescence of the central portions of the light bars or light spots. Width of light-passing strip or aperture of light-passing hole
M is a transverse magnification coefficient of imaging from an object plane 17 to an image plane where a light through hole or a light through strip is located, lambda is the wavelength of structured light, and NA is the numerical aperture of the objective lens 11; preferably, the width of the light-transmitting strip or the aperture of the light-transmitting hole
a is a proportionality coefficient, the value of a and the period T and diffraction limit of bright lines or bright spots
Is related to (1); more preferably, the width of the light-transmitting strip or the aperture of the light-transmitting hole
When in use
When the proportionality coefficient a takes on the value
The final imaging resolution R ranges from
Exceed the diffraction limit
When in use
When the value of the proportionality coefficient a is a smaller than
In a range of final imaging resolution R
Also exceeding the diffraction limit
Preferably, when
When the proportionality coefficient a takes on the value
The final imaging resolution R takes the value of
Compared with the diffraction limit, the imaging resolution is reduced by half, and is improved by 2 times; at this time, the width of the light-passing strip or the aperture D of the light-passing hole has a value range of
The range of the distance L of the synchronous movement is L ≧ M.T within one exposure period of the camera 16, so that the envelope of each movement of the filter array 13 can cover the light-blocking bars or plates of the filter array 13. Preferably, when the width of the light-passing strip or the aperture of the light-passing hole
Then, the distance of synchronous movement is L · T.
Optionally, the numerical aperture of the second relay lens 14 is larger than the numerical aperture of the first relay lens 12, so that all the fluorescence output by the filter array 13 falls into the second relay lens 14, so that all the fluorescence passing through the filter array 13 can be collected and can be smoothly imaged on the camera 16.
Alternatively, the filter array 13 may be a virtual filter array formed by a spatial light modulator; the spatial light modulator is used for adjusting the light passing strip and the bright stripes to synchronously move or the light passing holes and the bright points to synchronously move by opening the light passing strip or the light passing holes on the virtual filtering array and closing the light passing strips or the light passing holes at other positions on the virtual filtering array.
Specifically, the spatial light modulator may be a virtual filter array, for example, a liquid crystal panel including a plurality of switching tubes, and the opening and closing of the corresponding light-passing bar or light-passing hole may be controlled by a programmed program. When the spatial structure of the structured light changes, the position of the bright stripe or the bright spot on the object plane moves, and in order to enable the central part of the light stripe or the light spot to smoothly pass through the filtering array 13, a computer program is required to accurately control the virtual filtering array formed by the spatial light modulator to synchronously close the current light passing stripe or the light passing hole and synchronously open the light passing stripe or the light passing hole corresponding to the position of the moved bright stripe or the bright spot. In an exposure period of the camera 16, by rapidly moving the position of the illumination stripe or the lattice light spot array and using a computer programming program to control the virtual filter array formed by the spatial modulator to rapidly move synchronously in coordination with the illumination stripe or the lattice light spot array, the light passing stripe and the bright stripe of the filter array 13 move synchronously, or the light passing hole and the bright spot move synchronously, a large number of super-resolution images of the object plane 17 at different bright stripes or different bright spot positions can be obtained, and a super-resolution clear image of the whole object plane 17 can be obtained. The rapid structured light illumination super-resolution microscope provided by the embodiment of the invention is realized by adding the virtual filter array formed by the spatial light modulator in front of the camera 16 in the process of extracting the clear image by post-computation processing of obtaining a plurality of images of the conventional structured light illumination super-resolution microscope, thereby omitting the process of extracting the clear image by post-computation processing, greatly improving the time resolution of the structured light illumination super-resolution microscope on the basis of keeping the spatial resolution, and improving the imaging speed of the structured light illumination super-resolution microscope.
Optionally, the fast structured light illuminated super-resolution microscope may further include: at least one galvanometer; at least one galvanometer is arranged on the light path between the objective lens 11 and the first relay lens 12; the position of the light-passing strip or the light-passing hole of the filter array 13 is fixed; and the at least one galvanometer is used for rotating to a position corresponding to the bright pattern position or the bright spot position along the rotating shaft so as to enable the light-passing strip and the bright pattern to synchronously move or the light-passing hole and the bright spot to synchronously move. In particular, the galvanometer may be any scanning mirror that rotates about an in-plane axis of rotation. In the embodiment of the present invention, a fast-rotating galvanometer may be used to cooperate with the fixed filter array 13 with the position of the light-passing strip or the light-passing hole unchanged, so as to achieve fast movement of the filter array 13 in synchronization with the illumination stripe or the lattice light spot array, and the galvanometer may cooperate with the bright stripe or the bright spot to rotate synchronously and fast, so as to enable the light-passing strip and the bright stripe of the filter array 13 to move synchronously, or the light-passing hole and the bright spot to move synchronously, so as to obtain a large number of super-resolution images of the object plane 17 at different bright spots, and further obtain a super-resolution clear image of the whole object plane 17 within one exposure period of the camera 16.
Fig. 4 is a schematic structural diagram of another fast structured-light illuminated super-resolution microscope provided by the embodiment of the present invention, as shown in fig. 4, optionally, the fast structured-light illuminated super-resolution microscope may further include a first galvanometer 18; the fast structured light illuminated super resolution microscope may further comprise: a first mirror 19, a second mirror 20, and a third mirror 21; the fluorescence emitted from the objective lens 11 is reflected by the first side of the first galvanometer 18, then sequentially passes through the first relay mirror 12, the first reflector 19, the filter array 13, the second reflector 20, the third reflector 21 and the second relay mirror 14 to the second side of the first galvanometer 18, and is reflected to the third relay mirror 15 at the second side of the first galvanometer 18; the first galvanometer 18 is rotatable about a first axis of rotation and a second axis of rotation; the first and second axes of rotation are both in the plane of the first galvanometer 18 and are perpendicular to each other.
Specifically, the first galvanometer 18 may be any scanning mirror that rotates along a first rotation axis and a second rotation axis that are perpendicular to each other in a plane, and in an exposure period of the camera 16, the first galvanometer 18 rotates rapidly along the first rotation axis and the second rotation axis in coordination with movement of a bright stripe or a bright spot position, so that a central position of a light stripe or a light spot of fluorescence emitted by a fluorescent substance at the bright stripe or the bright spot position coincides with a central position of a light through stripe or a light through hole of which a fixed position is not moved, so that central part fluorescence of the light stripe or the light spot smoothly passes through the filter array 13 and edge part fluorescence of the light stripe or the light spot is filtered out. The first side of the first galvanometer 18 is used for fast rotation to match the movement of the illumination stripes or the lattice spot array, the second side of the first galvanometer 18 is used for counteracting the optical path vibration, and when the first galvanometer 18 rotates fast, the filter array 13 vibrates fast relative to the object plane 17, but the object plane image collected on the camera 16 does not vibrate. The first mirror 19, the second mirror 20 and the third mirror 21 are used to reflect light. The fluorescent substance at the position of the bright line or the bright spot is excited to emit fluorescence, the fluorescence passes through the objective lens 11 to reach the first side of the first galvanometer 18, is reflected at the first side of the first galvanometer 18, is reflected to the filter array 13 by the first reflector 19, is filtered out of the fluorescence at the edge part when passing through the light-passing strip or the light-passing hole, only the fluorescence at the central part is reflected to the second relay mirror 14 by the second reflector 20 and the third reflector 21, is collected by the second relay mirror 14, reaches the second side of the first galvanometer 18 to counteract the vibration of the light path, is reflected to the third relay mirror 15 at the second side of the first galvanometer 18, and is finally imaged on the camera 16. In an exposure period of the camera 16, the position of the illumination stripe or the lattice light spot array is rapidly moved, and the first galvanometer 18 is rapidly rotated along the first rotating shaft and the second rotating shaft which are perpendicular to each other in the galvanometer plane to synchronously and rapidly move, so that the position of the light passing stripe or the light passing hole of the filter array 13 and the position of the moved bright stripe or bright spot synchronously move, a large number of super-resolution images of the object plane 17 at different bright stripes or different bright spot positions can be obtained, and the super-resolution clear image of the whole object plane 17 can be obtained. The rapid structured light illumination super-resolution microscope provided by the embodiment of the invention is realized by adding the first vibrating mirror 18 which can rapidly rotate along the mutually vertical direction between the objective lens 11 and the first relay lens 12 and matching with the light filter array 13 with the fixed light transmission strip or the unchanged light transmission hole position, in the process of extracting a clear image by post-calculation processing of obtaining a plurality of images of the conventional structured light illumination super-resolution microscope, so that the time resolution of the structured light illumination super-resolution microscope is greatly improved on the basis of keeping the spatial resolution, and the imaging speed of the structured light illumination super-resolution microscope is improved.
Fig. 5 is a schematic structural diagram of another fast structured-light illuminated super-resolution microscope provided in the embodiment of the present invention, as shown in fig. 5, optionally, the fast structured-light illuminated super-resolution microscope may further include a second galvanometer 22 and a third galvanometer 23; the fast structured light illuminated super resolution microscope may further comprise: a fourth relay mirror 24, a fifth relay mirror 25, a sixth relay mirror 26, a seventh relay mirror 27, a fourth mirror 28, a fifth mirror 29, a sixth mirror 30, a seventh mirror 31, an eighth mirror 32, and a ninth mirror 33; the fluorescence emitted from the objective lens 11 is emitted through the first side of the second galvanometer 22, then sequentially passes through the fourth relay lens 24, the seventh reflector 31, the eighth reflector 32, the ninth reflector 33 and the fifth relay lens 25 to the second side of the second galvanometer 22, and is reflected to the sixth relay lens 26 at the second side of the second galvanometer 22; the second galvanometer 22 is rotatable along a first axis of rotation; the first axis of rotation is in the plane of the second galvanometer 22; the fluorescence is emitted from the sixth relay lens 26, passes through the seventh relay lens 27 to the first side of the third galvanometer 23, is reflected by the first side of the third galvanometer 23, sequentially passes through the first relay lens 12, the filter array 13, the fourth mirror 28, the fifth mirror 29, the sixth mirror 30, the second relay lens 14 to the second side of the third galvanometer 23, and is reflected to the third relay lens 15 at the second side of the third galvanometer 23; the third galvanometer 23 is rotatable along a second axis of rotation; the second rotation axis is located on the plane of the third galvanometer 23; the first rotation axis and the second rotation axis are perpendicular to each other.
Specifically, the second galvanometer 22 may be any scanning mirror that rotates along a first axis of rotation in a plane, and the third galvanometer 23 may be any scanning mirror that rotates along a second axis of rotation in a plane, the first axis of rotation being perpendicular to the second axis of rotation. In an exposure period of the camera 16, the second galvanometer 22 rotates along a first rotation axis in a plane, the third galvanometer 23 rotates along a second rotation axis in the plane, and the two galvanometer rotate rapidly in cooperation with the movement of the position of the bright stripe or the bright spot, so that the central position of the light stripe or the light spot of which fluorescence is emitted by the fluorescent substance at the position of the bright stripe or the bright spot coincides with the central position of the light-passing stripe or the light-passing hole of which the fixed position is not fixed, and the fluorescence of the central part of the light stripe or the light spot smoothly passes through the filter array 13 and the fluorescence of the edge part of the light stripe or the light spot is filtered out. The first side of the second galvanometer 22 and the first side of the third galvanometer 23 are used for fast rotation to match the movement of the illumination stripes or the lattice light spot array, the second side of the second galvanometer 22 and the second side of the third galvanometer 23 are used for offsetting the light path vibration, and when the two galvanometers rotate fast, the filter array 13 vibrates fast relative to the object plane 17, but the object plane image collected on the camera 16 does not vibrate. The fluorescent substance at the position of the bright line or the bright spot is excited to emit fluorescence, the fluorescence is emitted into the first side of the second galvanometer 22 through the objective lens 11, is emitted by the first side of the second galvanometer 22, sequentially passes through the fourth relay lens 24, the seventh mirror 31, the eighth mirror 32, the ninth mirror 33 and the fifth relay lens 25 to the second side of the second galvanometer 22, cancels the optical path vibration at the second side of the second galvanometer 22, is reflected to the sixth relay lens 26 at the second side of the second galvanometer 22, is emitted by the sixth relay lens 26, passes through the seventh relay lens 27 to the first side of the third galvanometer 23, is reflected by the first side of the third galvanometer 23, sequentially passes through the first relay lens 12, the filter array 13, the fourth mirror 28, the fifth mirror 29, the sixth mirror 30 and the second relay lens 14 to the second side of the third galvanometer 23, cancels the optical path vibration at the second side of the third galvanometer 23, and is reflected at the second side of the third galvanometer 23 to the third relay lens 15 and finally converges on the camera 16. In an exposure period of the camera 16, by adjusting the illumination stripe or the lattice light spot array to move rapidly, and using the second galvanometer 22 to rotate along the first rotation axis in the plane, the third galvanometer 23 to rotate along the second rotation axis in the plane, the two galvanometers to move rapidly in synchronization with the illumination stripe or the lattice light spot array, the position of the light-passing stripe or the light-passing hole of the filter array 13 and the position of the moved bright stripe or bright spot move synchronously, a large number of super-resolution images of the object plane 17 at different bright spots can be obtained, and a super-resolution clear image of the whole object plane 17 can be obtained. The rapid structured light illumination super-resolution microscope provided by the embodiment of the invention is realized by adding two second vibrating mirrors 22 and three third vibrating mirrors 23 which can rapidly rotate along the mutually vertical direction between the objective lens 11 and the first relay lens 12 to cooperate with the light filtering array 13 with fixed light transmission strips or unchanged light transmission hole positions in the process of obtaining a plurality of images and extracting a clear image by post-calculation processing of the conventional structured light illumination super-resolution microscope, so that the time resolution of the structured light illumination super-resolution microscope is greatly improved on the basis of keeping the spatial resolution, and the imaging speed of the structured light illumination super-resolution microscope is improved.
FIG. 6 is a schematic structural diagram of another fast structured light illuminated super-resolution microscope provided by an embodiment of the present invention, as shown in FIG. 6, and optionally, the fast structured light illuminated super-resolution microscope may include a mechanical vibration device 43; the mechanical vibration device 43 is linked with the filter array 13 and is used for driving the light-passing strip and the bright stripe to move synchronously or driving the light-passing hole and the bright point to move synchronously.
Optionally, a light-activated fluorescent substance may be further disposed on the object plane 17; the structured light generation module can generate excitation light with a first wavelength, activation light with a second wavelength and inactivation light with a third wavelength which have the same structure; in the inactivated state, the light-activated fluorescent substance is excited by the excitation light of the first wavelength to emit fluorescent light of a first intensity; a light-activated fluorescent substance excited by the excitation light of the first wavelength in a state of having been activated by the activation light of the second wavelength, emitting fluorescence of a second intensity; in a state of having been activated by the activating light of the second wavelength, the light-activated fluorescent substance is irradiated with the inactivating light of the third wavelength, returning to an inactivated state; wherein the second intensity is greater than the first intensity, and the luminescence intensity of the fluorescence is proportional to the intensity of the excitation light of the first wavelength and proportional to the intensity of the activation light of the second wavelength; during one exposure period of the camera 16, the position of the illumination stripe or lattice light spot array is first fixed; secondly, executing an activation process, including: the structured light generating unit emits the activation light with the second wavelength to enable the light activated fluorescent substance to be in the activated state; a firing process is then performed, including: the structured light generating unit switches the excitation light emitting at the first wavelength to make the light-activated fluorescent substance emit fluorescence of the second intensity, and then performs an inactivation process, including: the structured light generating unit switches to emit the inactivation light with the third wavelength to make the light-activated fluorescent substance return to the unactivated state; finally, executing a moving process, comprising: moving the position of the illumination stripe or the lattice light spot array, and correspondingly enabling the light-passing stripe and the bright stripe to synchronously move, or the light-passing hole and the bright spot to synchronously move; the activation process, the excitation process, the inactivation process, and the movement process are repeatedly performed in order.
Specifically, the light-activated fluorescent substance may be any fluorescent protein, such as RS-GFP or PA-GFP, which can emit no light or only weak fluorescence when excited by the excitation light in a state of not being activated by the specific activation light, but can emit fluorescence much stronger than that in a state of not being activated when first activated by the specific activation light and then excited by the excitation light, and at this time can return to the state of not being activated when irradiated by the specific extinguishing light. When the fluorescent substance on the object plane 17 is a light-activated fluorescent substance, the position of the illumination stripe or the lattice light spot array is firstly fixed in one exposure period of the camera 16; secondly, the structured light generating unit emits activation light with a second wavelength to enable the light-activated fluorescent substance to be in an activated state; then the structured light generating unit switches to emit the excitation light with the first wavelength to enable the light-activated fluorescent substance to emit the fluorescent light with the second intensity; then the structure light generating unit switches to emit inactivation light with a third wavelength to enable the light-activated fluorescent substance to return to an unactivated state; finally, the position of the illumination stripe or the lattice light spot array is moved, and the light-passing stripe and the bright stripe or the light-passing hole and the bright spot are synchronously moved correspondingly; by repeating the above activation, excitation, inactivation and movement processes in sequence, a clear image of the entire object plane 17 can be obtained.
When the object plane 17 is provided with the light-activated fluorescent substance, the width of the light-passing strip or the aperture D of the light-passing hole is still equal
M is a transverse magnification coefficient of imaging from an object plane 17 to an image plane where a light through hole or a light through strip is located, lambda is the wavelength of structured light, and NA is the numerical aperture of the objective lens 11; preferably, the width of the light-passing strip or the aperture D of the light-passing hole may still be in the range
a is a proportionality coefficient, the value of a and the period T and diffraction limit of bright lines or bright spots
The relation (a) is related, but when a common fluorescent substance is arranged on the object plane 17, the value of a is different; more preferably, the width of the light-transmitting strip or the aperture of the light-transmitting hole
When in use
When the temperature of the water is higher than the set temperature,
resolution of imaging
Exceeding the diffraction limit
When in use
When a is less than
Coefficient of (2), imaging resolution
Exceeding diffraction limit
Preferably, when
When the temperature of the water is higher than the set temperature,
width of light-passing strip or aperture of light-passing hole
Resolution of imaging
Namely, on the basis that the resolution of the existing structured light illumination super-resolution microscope is improved by 2 times, the resolution of 2 times is further improved, namely, the diffraction limit is reduced to one fourth, and 4 times of super-resolution microscopic imaging is realized. The rapid structured light illumination super-resolution microscope provided by the embodiment of the invention is additionally provided with the filter array 13 at the image surface position of the conventional structured light illumination super-resolution microscope, so that rapid super-resolution imaging can be realized, and if the light-activated fluorescent substance is used on the object surface 17, rapid 4-time super-resolution microscopic imaging can be realized, namely, the spatial resolution is increased by two times, the time resolution is also increased, and the imaging speed of the structured light illumination super-resolution microscope is greatly increased.
It is to be noted that the foregoing is only illustrative of the preferred embodiments of the present invention and the technical principles employed. Those skilled in the art will appreciate that the present invention is not limited to the particular embodiments described herein, and that various obvious changes, rearrangements and substitutions will now be apparent to those skilled in the art without departing from the scope of the invention. Therefore, although the present invention has been described in greater detail by the above embodiments, the present invention is not limited to the above embodiments, and may include other equivalent embodiments without departing from the spirit of the present invention, and the scope of the present invention is determined by the scope of the appended claims.
Claims (13)
1. A rapid structured light illumination super-resolution microscope is characterized by comprising a structured light generation unit, an objective lens, a first relay lens, a light filtering array, a second relay lens, a third relay lens and a camera which are sequentially arranged along a light path;
the structured light generated by the structured light generating unit generates an illumination stripe or a lattice light spot array on an object plane; the illumination stripes comprise bright stripes and dark stripes which are arranged at intervals; the lattice light spot array comprises uniformly arranged bright spots; if the structured light generates the illumination stripes on the object plane, the light filtering array comprises light blocking strips and light passing strips which are arranged at intervals, and the light passing strips correspond to the bright stripes one by one; if the structured light generates the lattice light spot array on the object plane, the light filtering array comprises a light baffle plate and light through holes, and the light through holes correspond to the bright spots one by one;
fluorescent substances at the positions of the bright lines or the bright spots are excited by the structured light to emit fluorescence, and the fluorescence sequentially passes through the objective lens and the first relay lens, the filtering array, the second relay lens and the third relay lens and is converged to the camera;
in an exposure period of the camera, when the structured light changes, the illumination stripe or the lattice light spot array moves, and accordingly, the light-passing stripe moves synchronously with the bright stripe or the light-passing hole moves synchronously with the bright point;
wherein the width of the light-passing strip or the aperture of the light-passing hole
M is the transverse magnification coefficient of the image from the object plane to the image plane where the light through hole or the light through strip is located, lambda is the wavelength of the structured light, NA is the numerical aperture of the objective lens, a is a proportionality coefficient, the value of a and the period T and the diffraction limit of the bright line or the bright spot
Is related to (1); in an exposure period of the camera, the synchronous moving distance L is more than or equal to M.T, so that the envelope of the moving filter array covers the light blocking strips or the light blocking plates of the filter array, and T is the period of the bright fringes or the bright spots.
2. The fast structured-light illuminated super-resolution microscope of claim 1, wherein the fast structured-light illuminated super-resolution microscope is used when
When the temperature of the water is higher than the set temperature,
resolution of imaging
Exceeding the diffraction limit
When in use
When a is less than
The imaging resolution of
Exceeding the diffraction limit
3. The fast structured-light illuminated super-resolution microscope of claim 1, wherein the fast structured-light illuminated super-resolution microscope is used when
When the temperature of the water is higher than the set temperature,
the width of the light-passing strip or the aperture of the light-passing hole
Resolution of imaging
4. The rapid structured-light illuminated super-resolution microscope of claim 1, wherein the width of the light-passing bar or the aperture of the light-passing hole
Then, the distance L of the synchronous movement is equal to M · T.
5. The rapid structured-light illuminated super-resolution microscope of claim 1, wherein the filter array is a virtual filter array formed by spatial light modulators;
the spatial light modulator closes the light passing strips or the light passing holes at other positions on the virtual filtering array by opening the light passing strips or the light passing holes on the virtual filtering array so as to adjust the light passing strips and the bright stripes to synchronously move or the light passing holes and the bright spots to synchronously move.
6. The fast structured light illuminated super resolution microscope of claim 1, further comprising: at least one galvanometer; the at least one galvanometer is arranged on an optical path between the objective lens and the first relay lens; the positions of the light-passing bars or the light-passing holes of the light filtering array are fixed;
the at least one vibrating mirror is used for rotating to a position corresponding to the bright pattern position or the bright point position along a rotating shaft, so that the light passing strip and the bright pattern move synchronously, or the light passing hole and the bright point move synchronously.
7. The rapid structured-light illuminated super-resolution microscope of claim 6, further comprising a first galvanometer; the structured light illuminated super-resolution microscope further comprises: the first reflector, the second reflector and the third reflector;
the fluorescence emitted by the objective lens is reflected by the first side of the first galvanometer, then sequentially passes through the first relay lens, the first reflector, the filtering array, the second reflector, the third reflector and the second relay lens to the second side of the first galvanometer, and is reflected to the third relay lens at the second side of the first galvanometer; the first galvanometer can rotate along a first rotating shaft and a second rotating shaft; the first rotating shaft and the second rotating shaft are both located on the plane where the first galvanometer is located, and the first rotating shaft and the second rotating shaft are perpendicular to each other.
8. The fast structured-light illuminated super-resolution microscope of claim 6, further comprising a second galvanometer and a third galvanometer; the structured light illuminated super-resolution microscope further comprises: a fourth relay mirror, a fifth relay mirror, a sixth relay mirror, a seventh relay mirror, a fourth reflector, a fifth reflector, a sixth reflector, a seventh reflector, an eighth reflector, and a ninth reflector;
the fluorescence emitted by the objective lens is emitted through the first side of the second galvanometer, then sequentially passes through the fourth relay lens, the seventh reflector, the eighth reflector, the ninth reflector and the fifth relay lens to the second side of the second galvanometer, and is reflected to the sixth relay lens at the second side of the second galvanometer; the second galvanometer can rotate along a first rotating shaft; the first rotating shaft is positioned on the plane where the second galvanometer is positioned;
the fluorescence is emitted by the sixth relay lens, passes through the seventh relay lens to the first side of the third vibrating mirror, is reflected by the first side of the third vibrating mirror, sequentially passes through the first relay lens, the filtering array, the fourth mirror, the fifth mirror, the sixth mirror and the second relay lens to the second side of the third vibrating mirror, and is reflected to the third relay lens at the second side of the third vibrating mirror; the third galvanometer can rotate along a second rotating shaft; the second rotating shaft is positioned on the plane where the third galvanometer is positioned; the first rotation axis and the second rotation axis are perpendicular to each other.
9. The rapid structured light illuminated super resolution microscope of claim 1, further comprising a mechanical vibration device;
the mechanical vibration device is linked with the filtering array and used for driving the light passing strip and the bright stripes to synchronously move or the light passing holes and the bright spots to synchronously move.
10. The fast structured-light illuminated super-resolution microscope of claim 1, wherein the numerical aperture of the second relay lens is larger than the numerical aperture of the first relay lens such that all of the fluorescence output by the filter array falls into the second relay lens.
11. The rapid structured-light illuminated super-resolution microscope according to claim 1, wherein the object plane is provided with a light-activated fluorescent substance;
the structured light generation unit generates excitation light with a first wavelength, activation light with a second wavelength and inactivation light with a third wavelength which have the same structure;
in the inactivated state, the light-activated fluorescent substance is excited by the excitation light of the first wavelength to emit the fluorescent light of a first intensity; in a state of having been activated by the activation light of the second wavelength, the light-activated fluorescent substance is excited by the excitation light of the first wavelength, emitting the fluorescence of the second intensity; in a state of having been activated by the second wavelength of the activating light, the light-activated fluorescent substance is irradiated with the third wavelength of the inactivating light, returning to the unactivated state; wherein the second intensity is greater than the first intensity, and the luminescence intensity of the fluorescence is proportional to the intensity of the excitation light of the first wavelength and proportional to the intensity of the activation light of the second wavelength;
during one exposure period of the camera, firstly fixing the position of the illumination stripe or the lattice light spot array; secondly, executing an activation process, including: the structured light generating unit emits the activation light of the second wavelength to make the light-activated fluorescent substance in an activated state; a firing process is then performed, including: the structured light generating unit switches to emit the excitation light of the first wavelength to cause the light-activated fluorescent substance to emit the fluorescent light of the second intensity; an inactivation process is then performed, including: the structured light generating unit switches to emit the inactivating light of the third wavelength to return the light-activated fluorescent substance to the unactivated state; finally, executing a moving process, comprising: moving the position of the illumination stripe or the lattice light spot array and correspondingly synchronously moving the light-passing stripe and the bright stripe or the light-passing hole and the bright spot; repeatedly performing the activation process, the excitation process, the inactivation process, and the movement process in order;
wherein the width of the light-passing strip or the aperture of the light-passing hole
M is the transverse magnification coefficient of the image from the object plane to the image plane where the light through hole or the light through strip is located, lambda is the wavelength of the structured light, NA is the numerical aperture of the objective lens, a is a proportionality coefficient, the value of a and the period T and the diffraction limit of the bright line or the bright spot
The relationship (2) is related.
12. The rapid structured-light illuminated super-resolution microscope of claim 11, wherein the rapid structured-light illuminated super-resolution microscope is used when
When the temperature of the water is higher than the set temperature,
resolution of imaging
Exceeding the diffraction limit
When in use
When a is less than
The imaging resolution of
Exceeding the diffraction limit
13. The rapid structured-light illuminated super-resolution microscope of claim 11, wherein the rapid structured-light illuminated super-resolution microscope is used when
When the temperature of the water is higher than the set temperature,
the width of the light-passing strip or the aperture of the light-passing hole
Resolution of imaging
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