Microimaging system and stimulated radiation loss microscope

Abstract

The invention belongs to the technical field of optical microscopic imaging, and discloses a microscopic imaging system and a stimulated radiation loss microscope. The microscopic imaging system comprises an illumination mechanism used for providing a first light beam and a second light beam which are coaxially incident; the phase modulation optical device comprises multiple layers of diffractive optical elements, and the multiple layers of diffractive optical elements are arranged at intervals in the transmission direction of the first light beam and the second light beam and used for conducting phase modulation and convergence on the first light beam and the second light beam in a matched mode. And the first light beam and the second light beam can form a first light spot and a second light spot at the focal plane of the stimulated radiation loss microscope, and the first light spot and the second light spot are in one-to-one correspondence and coincide in center. According to the technical scheme of the invention, the stimulated radiation depletion microscope can be normally matched with a commercial laser which has a large bandwidth and wavelength drift and is used as an excitation beam and a depletion beam.

Description

Technical Field

[0001] This application belongs to the field of optical microscopy imaging technology, specifically relating to a microscopy imaging system and a stimulated emission depletion microscope. Background Technology

[0002] Currently, about 80% of microscopic imaging research in life sciences requires the use of optical microscopes, and the progress of life sciences is accompanied by the development of optical microscopes.

[0003] Conventional optical microscopes are limited by spatial resolution, thus hindering biologists' detailed studies of subcellular structures. Stimulated Emission Depletion (STED) microscopy, however, uses a depleted beam of light modulated by a phase plate to form a depletion spot. Fluorescent molecules surrounding the diffracted spot are depleted through stimulated emission, converting them into a non-radiative state, achieving a spatial resolution better than 50 nm. Therefore, it is widely used in biological and biomedical research.

[0004] To obtain ultra-high resolution fluorescence images with high spatial resolution and good signal-to-noise ratio, the excitation beam focal point must be precisely spatially aligned with the ring-shaped spot formed by the depletion beam. Traditionally, a dichroic plate is used for alignment, but maintaining high-precision, long-term stable alignment (nanometer-scale) between the excitation and STED (strain-induced radiation depletion) spots is difficult. Furthermore, the substrate material of the phase plate in traditional stimulated emission depletion microscopy has dispersive properties, making the phase plate highly sensitive to wavelength. Even a slight deviation in the wavelength of the incident beam (e.g., ±10 picometers) can cause significant changes in wavefront modulation, resulting in a blurred or undesirable focused spot shape. However, commercially available single-frequency pulsed lasers typically provide beams with bandwidths of approximately 100 picometers and wavelength stability on the order of 10 picometers. Larger bandwidths and wavelength drift can lead to severe distortion of the resulting spot. Summary of the Invention

[0005] The purpose of this application is to at least address the limitations of conventional stimulated emission depletion microscopes in terms of coaxial stability and the wavelength of the incident beam. This purpose is achieved through the following means: In a first aspect, this application proposes a microscopic imaging system for stimulated emission depletion microscopy. The microscopic imaging system includes: an illumination mechanism for providing a first beam and a second beam coaxially incident; and a phase modulation optics device including multiple diffractive optical elements, the multiple diffractive optical elements being spaced apart along the transmission directions of the first beam and the second beam, the first beam and the second beam being incident perpendicularly to the diffractive optical elements, the multiple diffractive optical elements being used to cooperate in phase modulation and convergence of the first beam and the second beam, enabling the first beam and the second beam to form a first spot and a second spot at the focal plane of the stimulated emission depletion microscope, the first spot and the second spot being one-to-one corresponding and having their centers coincident; wherein the first beam and the second beam have an overlapping region, the overlapping region covering the phase modulation optics device.

[0006] According to the microscopic imaging system proposed in this application, the first and second beams provided by the illumination mechanism are coaxial and perpendicularly incident on the phase modulation optics. The first and second beams have an overlapping region that covers the phase modulation optics. When the first and second beams pass through the multilayer diffractive optical elements of the phase modulation optics, the multilayer diffractive optical elements can diffract the first and second beams respectively, and can synergistically modulate the phase of the first and second beams respectively. This ensures that the first and second light spots formed at the focal plane of the microscope can remain stable and strictly coincident at their centers for a long time. This solves the problem of wavelength sensitivity when using a single diffractive optical element in stimulated emission depletion microscopes, and also allows for normal adaptation when using commercial lasers with large bandwidth and wavelength drift as excitation and depletion beams.

[0007] In addition, the microscopic imaging system according to this application may also have the following additional technical features: In some embodiments of this application, each layer of the diffractive optical element includes a substrate and a microstructure disposed on the substrate. The microstructure is etched on the substrate according to a preset phase distribution. The preset phase distribution is configured to enable the first beam and the second beam to form a first spot array and a second spot array at the focal plane of the stimulated emission depletion microscope after passing through the phase modulation optical device. The first spot array has a plurality of first spots, and the second spot array has a plurality of second spots. The first spots in the first spot array and the second spots in the second spot array correspond one-to-one and coincide in center.

[0008] In this array, the number of first light spots in the first light spot array can be one or more, the number of second light spots in the second light spot array is the same as the number of first light spots and corresponds one-to-one, and the center of the second light spot coincides with the center of the first light spot.

[0009] The light spot at the focal plane of a microscope is determined by the converging behavior of light, and the phase distribution can directly control the propagation trajectory of light. Therefore, by distributing microstructures on a substrate according to a preset phase, the first and second light beams, after passing through multiple layers of diffractive optical elements, can form a first spot array and a second spot array at the focal plane of the microscope. It can be understood that each spot unit in the array (a first spot and a corresponding matching second spot) is an independent super-resolution imaging unit. When scanning and imaging are performed after forming the first and second spot arrays, at least two spot units can be scanned simultaneously in parallel, instead of scanning each spot unit individually. This breaks through the bottleneck of only being able to scan a single spot, increases the scanning area per unit time, and thus improves imaging efficiency while ensuring super-resolution capability.

[0010] In some embodiments of this application, the first beam is an excitation beam, and the spots in the first spot array are solid excitation spots; the second beam is a loss beam relative to the first beam, and the spots in the second spot array are annular loss spots, the annular loss spots covering the edge region of the solid excitation spots.

[0011] In some embodiments of this application, the illumination mechanism includes a first laser, a first beam adjustment assembly, a second laser, and a second beam adjustment assembly. The first laser provides the first beam. The first beam adjustment assembly includes a first lens, a first filter, and a first reflector arranged sequentially along the transmission direction of the first beam. The second laser provides the second beam. The second beam adjustment assembly includes a second lens, a second filter, a dichroic element, a second reflector, and a quarter-wave plate arranged sequentially along the transmission direction of the second beam. The first lens expands the first beam into parallel light, the first filter adjusts the intensity of the first beam, the first reflector reflects the first beam to the dichroic element, the second lens expands the second beam into parallel light, the second filter adjusts the intensity of the second beam, the dichroic element makes the first beam and the second beam coaxial and perpendicularly incident on the quarter-wave plate and the phase modulation optics, and the quarter-wave plate converts the first beam and the second beam from linearly polarized light to circularly polarized light.

[0012] In some embodiments of this application, the first filter and the second filter are neutral density filters; and / or, the dichroic element is a dichroic sheet.

[0013] Secondly, this application also proposes a stimulated emission depletion microscope, comprising: a displacement stage for carrying and moving a sample containing a fluorescent substance; a microscopic imaging system according to any one of the embodiments of the first aspect, wherein the first beam and the second beam are capable of exciting the fluorescent substance in the sample to generate a fluorescent signal after being phase-modulated and converged by the phase-modulation optics; and a fluorescence detection system for collecting and detecting the fluorescence signal and converting the fluorescence signal into an electrical signal.

[0014] According to some embodiments of this application, the fluorescence detection system includes a signal collection component and a signal conversion element. The signal collection component is used to collect and converge the fluorescence signal, and the signal conversion element is used to convert the fluorescence signal into an electrical signal.

[0015] According to some embodiments of this application, the signal collection component includes a collection objective, a third filter, and a third lens arranged sequentially along the propagation direction of the fluorescence signal; wherein, the collection objective is used to collect and converge the fluorescence signal excited by the sample, the third filter is a fluorescence filter, and the third lens is used to converge the fluorescence signal.

[0016] According to some embodiments of this application, the signal collection component further includes a third mirror and a fourth mirror, which are used to reflect the fluorescence signal collected and converged by the collection objective lens to the third filter.

[0017] According to some embodiments of this application, the signal conversion element includes a photodetector.

[0018] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0019] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of this application. Furthermore, the same reference numerals denote the same parts throughout the drawings. Wherein: Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of this application. Furthermore, the same reference numerals denote the same parts throughout the drawings. Wherein: Figure 1This is a simplified structural diagram of a stimulated emission depletion microscope provided according to some embodiments of this application; Figure 2 This is a schematic diagram of the structure of a phase modulation optical device; Figure 3 This is a schematic diagram of the structure of a diffractive optical element; Figure 4 This is an imaging schematic diagram of a first spot array provided according to some embodiments of this application; Figure 5 This is an imaging schematic diagram of a second spot array provided according to some embodiments of this application; Figure 6 The image is an image obtained by stimulated emission depletion microscopy of a fluorescent microsphere sample with a diameter of 40 nm according to some embodiments of this application.

[0020] The labels in the attached diagram are as follows: 100. Stimulated emission depletion microscope; 200. First spot array; 300. Second spot array; 10. Microscopic imaging system; 20. Displacement stage; 30. Fluorescence detection system; 11. Phase modulation optical devices; 111. Diffractive optical elements; 1111. Microstructures; 12. Illumination mechanism; 1211. First laser; 1212. First lens; 1213. First filter; 1214. First reflector; 1221. Second laser; 1222. Second lens; 1223. Second filter; 1224. Dichroic element; 1225. Second reflector; 1226. Quarter wave plate; 31. Collecting objective lens; 32. Third mirror; 33. Fourth mirror; 34. Third filter; 35. Third lens; 36. Signal conversion element. Detailed Implementation

[0021] Exemplary embodiments of this application will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of this application and to fully convey the scope of this application to those skilled in the art.

[0022] It should be understood that the terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” as used herein may also include the plural forms. The terms “comprising,” “including,” “containing,” and “having” are inclusive and therefore indicate the presence of the stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof. The method steps, processes, and operations described herein are not construed as requiring them to be performed in a particular order described or illustrated unless the order of performance is explicitly indicated. It should also be understood that additional or alternative steps may be used.

[0023] Although terms such as first, second, third, etc., may be used in this document to describe multiple elements, components, regions, layers, and / or segments, these elements, components, regions, layers, and / or segments should not be limited by these terms. These terms may be used only to distinguish one element, component, region, layer, or segment from another. Unless the context clearly indicates otherwise, terms such as "first," "second," and other numerical terms used herein do not imply order or sequence. Therefore, the first element, component, region, layer, or segment discussed below may be referred to as the second element, component, region, layer, or segment without departing from the teachings of the exemplary embodiments.

[0024] For ease of description, spatial relative terms may be used in the text to describe the relationship of one element or feature relative to another element or feature, as shown in the figure. These relative terms include, for example, "inside," "outside," "middle," "outer," "below," "below," "above," "over," etc. Such spatial relative terms are intended to include different orientations of the device in use or operation, other than those depicted in the figure. For example, if the device in the figure is flipped, an element described as "below other elements or features" or "below other elements or features" would subsequently be oriented as "above other elements or features" or "above other elements or features." Therefore, the example term "below" can include both upper and lower orientations. The device may be otherwise oriented (rotated 90 degrees or in other directions), and the spatial relative descriptors used in the text will be interpreted accordingly.

[0025] Please see Figure 1The first aspect of this application provides a microscopic imaging system 10 for a stimulated emission depletion microscope 100. The microscopic imaging system 10 includes an illumination mechanism 12 and a phase modulation optics device 11. The illumination mechanism 12 provides a first beam and a second beam that are coaxially incident. The phase modulation optics device 11 includes multilayer diffractive optical elements 111 arranged along the transmission directions of the first and second beams. The first and second beams are incident perpendicularly to the diffractive optical elements 111. The phase modulation optics device 11 modulates and converges the first and second beams, enabling them to form a first spot and a second spot at the focal plane of the stimulated emission depletion microscope 100. The first spot and the second spot correspond one-to-one and coincide in center. The first beam and the second beam have an overlapping region that covers the phase modulation optics device 11.

[0026] In some embodiments, see Figure 4 and Figure 5 The first beam is the excitation beam, and the first spot is a solid excitation spot; the second beam is a loss beam relative to the first beam, and the second spot is an annular loss spot, with one annular loss spot corresponding to the edge region of one solid excitation spot.

[0027] The excitation beam is usually a short-wavelength laser, such as 647nm, while the loss beam is a relatively long-wavelength beam, such as 775nm. The annular loss spot covers the edge region of the solid excitation spot, causing the fluorescent molecules at the edge of the excitation spot to be pulled back to the ground state due to stimulated emission depletion effect and no longer stimulated emission, so that only the fluorescence of the central region of the excitation spot is retained. Thus, a super-resolution image can be obtained after each excitation spot is scanned.

[0028] When the first and second beams, provided by the illumination mechanism 12 and coaxially and perpendicularly incident on the phase-modulated optics, pass through the multilayer diffractive optical elements 111 of the phase-modulated optics, the multilayer diffractive optical elements 111 can diffract the first and second beams respectively and perform phase modulation on the first and second beams respectively. Each layer of diffractive optical elements 111 is optimized for beams in different wavelength bands, and the first and second light spots formed at the focal plane of the microscope can remain stable and strictly coincident at the center for a long time. This solves the problem of wavelength sensitivity when using a single diffractive optical element 111 and can also be properly adapted to use commercial lasers with large bandwidth wavelength drift as excitation and loss beams.

[0029] According to some embodiments of this application, each diffractive optical element 111 includes a substrate and a microstructure 1111 disposed on the substrate. The microstructure 1111 is etched on the substrate according to a preset phase distribution. The preset phase distribution is configured to enable the first beam and the second beam to form a first spot array 200 and a second spot array 300 at the focal plane of a stimulated emission depletion microscope after passing through a phase modulation optical device. The first spot array 200 has a plurality of first spots, and the second spot array 300 has a plurality of second spots. The first spots in the first spot array 200 and the second spots in the second spot array 300 correspond one-to-one and their centers coincide.

[0030] The number of first light spots in the first light spot array 200 can be one or more, the number of second light spots in the second light spot array 300 is the same as the number of first light spots and corresponds one-to-one, and the center of the second light spot coincides with the center of the first light spot.

[0031] Among them, see Figure 4 and Figure 5 The first beam, after passing through the phase modulation optics 11 in this embodiment, can form a first spot array 200 at the focal plane of the microscope, and the first spot in the formed first spot array 200 can be a solid excitation spot. The second beam, after passing through the phase modulation optics 11 in this embodiment, can form a second spot array 300 at the focal plane of the microscope, and the first spot in the formed second spot array 300 can be a ring-shaped loss spot.

[0032] For example, please see Figure 2 The phase modulation optical device 11 includes three or five diffractive optical elements 111 arranged in sequence, and the specific number can be set according to actual needs.

[0033] For example, see Figure 3 Each diffractive optical element 111 is formed by photolithography of a microstructure with a preset phase distribution onto a substrate. The multilayer diffractive optical elements 111 work together to adjust and converge the phase of the first beam and the second beam, so that a first spot array 200 and a second spot array 300 can be formed at the focal plane of the microscope.

[0034] The light spot at the focal plane of a microscope is determined by the converging behavior of light, and the phase distribution can directly control the propagation trajectory of light. Therefore, by etching the microstructure 1111 onto the substrate according to a preset phase distribution, the first and second light beams, after passing through the multilayer diffractive optical elements 111, can form a first light spot array 200 and a second light spot array 300 at the focal plane of the microscope. It can be understood that each light spot unit in the array (a first light spot and a corresponding matching second light spot) is an independent super-resolution imaging unit. When scanning and imaging are performed after the first light spot array 200 and the second light spot array 300 are formed, at least two light spot units can be scanned simultaneously in parallel, instead of scanning and imaging each light spot unit one by one. This breaks through the bottleneck of only being able to scan a single light spot, increases the scanning area per unit time, and thus improves imaging efficiency while ensuring super-resolution capability.

[0035] Specifically, the microstructure 1111 is fabricated using micro-nano fabrication technology and photolithographically applied to a substrate. The core function of the microstructure 1111 is to precisely control the phase, amplitude, or polarization state of light, causing specific diffraction and interference of incident light, thereby achieving customized optical functions such as beam shaping, beam splitting, and focusing.

[0036] For example, the substrate can be a light-transmitting substrate such as quartz or glass. The surface oil is removed by ultrasonic cleaning, and the surface oxide layer is removed by plasma cleaning, so that the surface roughness of the substrate is less than 1 / 10 of the wavelength.

[0037] Understandably, the light spot at the focal plane of a microscope is determined by the convergence behavior of light, while the phase distribution directly regulates the propagation trajectory of light. By distributing the microstructure 1111 on the substrate according to a preset phase distribution, which can be obtained through simulation iteration training, the first beam and the second beam can form a first light spot array 200 and a second light spot array 300 at the focal plane of the microscope after passing through the multilayer diffractive optical element 111.

[0038] For example, the first light spot array 200 has two first light spots, and the second light spot array 300 also has two second light spots.

[0039] In one embodiment, the depth of each microstructure of the multilayer diffractive optical element is optimized using a deep learning algorithm. To construct the target image, the three electric field components of the first light spot can be calculated using the following formula:

[0040] The first light spot array is obtained by copying and splicing the first light spot.

[0041] Add to the incident field The spiral phase yields the following three electric field components for the second light spot:

[0042] The second light spot array is obtained by copying and splicing the second light spot.

[0043] in, , and These are the three components of the electric field vector at a point in space. The half-aperture angle at which the focal point opens relative to the focusing lens; θ represents the amplitude of the incident light; i is the imaginary unit used to describe the phase characteristics of the electric field; k represents the wave number in vacuum, defined as k = 2π / λ, where λ is the wavelength of the incident light, and θ represents the aperture angle, i.e., the angle between the optical axis of the focusing lens and the incident light ray. It represents the azimuth angle, which is the angle of rotation of the light ray in a plane perpendicular to the optical axis; The angle r is the angle between the direction of propagation of a ray of light on the aperture of the focusing lens and the radius vector from the focal point to the field point P. P The distance from the focal point to the field point P; Indicates the aperture pupil amplitude modulation factor; and Indicates the geometric projection factor; and cosθ+(1 cosθ)cos2 Indicates the polarization and component decomposition factor; It represents a minute change in the aperture angle; It represents a small change in azimuth angle; This indicates the corresponding angle range on the focusing lens aperture. and The phase information carried by the emitted wavelet from the tiny wavelet source when it propagates to the long point P, along with the angular weight of the wavelet source; exp(i ) represents the spiral phase factor.

[0044] The depth of each microstructure on the multilayer diffractive optical element is further optimized. The optimization targets for the first wavelength and the second wavelength are the first light spot and the second light spot mentioned above, respectively. When the algorithm converges, a one-to-one corresponding light spot pair that meets the requirements can be obtained.

[0045] In another embodiment, based on the microstructure phase distribution of each diffractive optical element, a composite mode of basic converging phase + dual-spot splitting phase + error compensation phase can be adopted with functional synergy as the core. The three types of phases have clear division of labor and work together to ultimately achieve the technical effect of precise matching of dual-spot array.

[0046] The final phase distribution of the microstructure 1111 on each layer of diffractive optical element 111 is a superposition of three types of phases, and the core expression is: φ(x,y)=φfocus(x,y)+φsplit(x,y)+φcomp(x,y).

[0047] Here, φfocus(x,y) represents the basic focusing phase, φsplit(x,y) represents the dual-spot splitting phase, and φcomp(x,y) represents the error compensation phase. When setting the preset phase distribution, the phase distribution of each microstructure layer is uniformly a superposition of three terms to ensure concentric focusing of the two beams and their respective constraints.

[0048] Furthermore, the fundamental convergence phase is based on the precise phase of the spherical wave, ensuring that the first and second beams are precisely converged at the focal plane of the microscope. The expression is as follows: φfocus(x,y)= (2π / λ) ( - f) Where λ represents the wavelength of the incident beam, the wavelength of the first beam λ1 can be any wavelength in the range of 561nm-647.05nm, and the wavelength of the second beam λ2 can be any wavelength in the range of 750nm-775.05nm. f represents the equivalent focal length of the diffractive optical element, which can be 0.5nm-1nm, and is matched with the focal length of the microscope objective. x and y represent the two-dimensional coordinates of the diffractive optical element, with an effective aperture of 2mm. Taking 2mm as an example, x can be -1nm to 1nm, and y can be -1nm to 1nm.

[0049] The dual-spot phase splitting is achieved using a dual-period fork grating phase splitting, expressed as: φ split (x,y)= 2π (x / P x + y / P y )·sgn(y) + 2π round(y / P y ) Among them, P x The grating period in the x-direction can be 1.2 μm to 2 μm. P y The grating period in the y-direction can be 1.2μm-2μm, and P x and P y The values ​​are the same to ensure that the centers of the first and second light spots completely coincide. sgn(y) is a sign function, equal to 1 when y > 0, -1 when y < 0, and 0 when y = 0. round(y / P) y ) represents the relationship between y / P y The result is rounded to the nearest integer.

[0050] The error compensation phase corrects the system bias using polynomial fitting, and the expression is: φ comp (x,y)= a0+ a1x + a2y + a3x²+ a4xy + a5y² Wherein, a0 is a constant term, which can be -0.1π to 0.1π, used to correct the overall phase shift caused by the non-uniform refractive index of the substrate material. a1 is the x-direction linearity coefficient (x-direction offset correction), which can be -0.05 rad / mm to -0.05 rad / mm, used to correct the parallelism error along the x-direction in the assembly of the multilayer diffractive optical element. a2 is the y-direction linearity coefficient (y-direction offset correction), which can be -0.05 rad / mm to -0.05 rad / mm, used to correct the parallelism error along the y-direction in the assembly of the multilayer diffractive optical element. a3 is the x² term coefficient (x-direction wavefront distortion correction), which can be -0.02 rad / mm² to -0.02 rad / mm², used to compensate for the spherical aberration in the x-direction after the beam passes through the multilayer diffractive optical element. a4 is the xy term coefficient (cross distortion correction), which can be -0.01 rad / mm² to -0.01 rad / mm², used to correct the cross error in the xy direction during the etching process of the diffractive optical element. a5 is the coefficient of the y² term (y-direction wavefront distortion correction), which has the same value as a3 to ensure that the wavefront distortion correction effect in the y direction is symmetrical with that in the x direction.

[0051] Furthermore, the specific values ​​of a0-a5 can be determined through optimization using optical simulation software.

[0052] In some embodiments, the phase modulation optics 11 are millimeter-sized. Replacing the illumination objective and a conventional phase plate, it allows the excitation beam and loss beam to be phase-modulated and converged in a millimeter-scale coaxial configuration. Furthermore, it enables corresponding beams in the first and second beam arrays 200 and 300, after phase modulation and convergence by the phase modulation optics 11, to remain stable and strictly coincident for extended periods. In addition, it solves the wavelength sensitivity problem encountered when using a single diffractive optical element 111, and is well-suited for use as excitation and loss beams with commercially available lasers exhibiting large bandwidth and wavelength drift.

[0053] Please see Figure 1 According to some embodiments of this application, the lighting mechanism 12 includes a first laser 1211, a first beam adjustment assembly, a second laser 1221, and a second beam adjustment assembly. The first laser 1211 is used to provide a first beam. The first beam adjustment assembly includes a first lens 1212, a first filter 1213, and a first reflector 1214 arranged sequentially along the transmission direction of the first beam. The second laser 1221 is used to provide a second beam. The second beam adjustment assembly includes a second lens 1222, a second filter 1223, a dichroic element 1224, a second reflector 1225, and a quarter-wave plate 1226 arranged sequentially along the transmission direction of the second beam.

[0054] The first lens 1212 is used to expand the first beam into parallel light; the first filter 1213 is used to adjust the intensity of the first beam; the first reflector 1214 is used to reflect the first beam to the dichroic element 1224; the second lens 1222 is used to expand the second beam into parallel light; the second filter 1223 is used to adjust the intensity of the second beam; the dichroic element 1224 is used to make the first beam and the second beam coaxial and perpendicularly incident on the quarter-wave plate 1226 and the phase modulation optical device; the second reflector... 1225 is used to reflect the first and second beams, which are coaxially incident after passing through the dichroic element 1224, to the quarter-wave plate 1226. The quarter-wave plate 1226 is used to convert the first and second beams from linearly polarized light to circularly polarized light, ensuring that the intensity distribution of the second beam in the second beam array 300 is uniform and symmetrical. This allows the annular loss beam to uniformly and efficiently extinguish the edge fluorescence of the solid excitation beam, thereby compressing the size of the effective light-emitting area, ensuring super-resolution, and helping to improve the uniformity of imaging.

[0055] Furthermore, the illumination mechanism 12 provided in this application optimizes the transmission path of the light path, reduces optical components, reduces the influence of the physical adjustment of the geometric relationship between unit devices and the inherent temperature and vibration instability of the mechanical adjustment mechanism, and reduces the risk of phenomena such as the deviation of the excitation spot and the loss spot, which helps the stimulated emission loss microscope 100 to work reliably for a long time in various environments.

[0056] According to some embodiments of this application, the first filter 1213 and the second filter 1223 are neutral density filters; and / or, the dichroic element 1224 is a dichroic sheet.

[0057] The first filter 1213 is a neutral density filter. A neutral density filter can adjust the light intensity of the first beam and maintain a consistent attenuation level without altering the color balance of the first beam, thus reducing the risk of color shift in imaging or signal detection. Similarly, when the first filter 1213 is a neutral density filter, it can adjust the light intensity of the second beam and maintain a consistent attenuation level without altering the color balance of the second beam, thus reducing the risk of color shift in imaging or signal detection.

[0058] The dichroic element 1224 is a dichroic sheet that can reflect the first beam and project the second beam, thereby making the first beam and the second beam coaxial and perpendicularly incident on the phase modulation optical device 11.

[0059] Please see Figure 1 and Figure 6The second aspect of this application also provides a stimulated emission depletion microscope 100, comprising: a stage 20 for carrying and moving a sample containing fluorescent material; a microscopic imaging system 10 according to any one of the first aspects, wherein a first beam and a second beam are capable of exciting the fluorescent material in the sample to generate a fluorescent signal after being phase-modulated and converged by a phase-modulated optics device; and a fluorescence detection system 30 for collecting and detecting the fluorescence signal and converting the fluorescence signal into an electrical signal.

[0060] For example, fluorescent substances can be fluorescent molecules, which are organic or inorganic compounds that exhibit characteristic fluorescence emission in the ultraviolet, visible to near-infrared spectral range (100-1700 nm). Their fluorescence properties (such as emission wavelength and quantum yield) are sensitively altered by changes in environmental polarity, viscosity, refractive index, and biomarker concentration. Therefore, after the first and second light beams irradiate the sample, the fluorescent molecules within the sample are excited to generate a fluorescence signal. The fluorescence detection system 30 collects and detects the fluorescence signal, converts it into an electrical signal, and then converts the electrical signal into an image to achieve microscopic imaging.

[0061] The stimulated emission depletion microscope 100 proposed in the second aspect of this application includes the microscopic imaging system 10 of any one of the first aspect embodiments, and therefore has the technical effects of any of the above embodiments, which will not be repeated here.

[0062] According to some embodiments of this application, the fluorescence detection system 30 includes a signal collection component and a signal conversion element 36. The signal collection component is used to collect and converge fluorescence signals, and the signal conversion element 36 is used to convert fluorescence signals into electrical signals.

[0063] The signal collection assembly may include a collection objective lens 31 and an optical filter. The signal conversion element 36 may be a photodetector.

[0064] The signal collection component is used to collect and converge as many fluorescence signals as possible and transmit them to the subsequent signal conversion element 36. The signal conversion element 36 captures and detects the fluorescence signals and converts them into electrical signals, so that the electrical signals can be output as images by the signal processing element.

[0065] Please see Figure 1 According to some embodiments of this application, the signal collection component includes a collection objective lens 31, a third filter 34, and a third lens 35 arranged sequentially along the propagation direction of the fluorescence signal; wherein, the collection objective lens 31 is used to collect and converge the fluorescence signal excited by the sample, the third filter 34 is a fluorescence filter, and the third lens 35 is used to converge the fluorescence signal.

[0066] The collecting objective lens 31 is used to capture, collect, and converge the fluorescence signal. The third filter 34 is a fluorescence filter that filters the fluorescence signal and allows fluorescence to pass through, while filtering out other stray light to reduce interference from other light signals and help improve the clarity of the image. The third lens 35 is used to converge the fluorescence signal that has passed through the fluorescence filter and transmit it to the signal conversion element 36.

[0067] Please see Figure 1 According to some embodiments of this application, the signal collection component further includes a third reflector 32 and a fourth reflector 33, which are disposed between the collecting objective lens 31 and the third filter 34, and are used to reflect the fluorescence collected and focused by the collecting objective lens 31 to the third filter 34.

[0068] For example, the mirror surfaces of the third mirror 32 and the fourth mirror 33 are arranged opposite each other and are both angled relative to the direction of fluorescence transmission. The fluorescence signal collected by the objective lens 31 can be reflected by the third mirror 32 to the fourth mirror 33, and the fourth mirror 33 then reflects the fluorescence signal to the third filter 34, so that the fluorescence signal can be incident perpendicularly to the third filter 34, so that other stray light other than fluorescence can be filtered out by the third filter 34.

[0069] According to some embodiments of this application, the signal conversion element 36 includes a photodetector.

[0070] Photodetectors can detect and convert fluorescence signals into measurable electrical signals. They are characterized by high sensitivity, low noise, and fast response. Essentially, they utilize the photoelectric effect to allow photons to interact with electrons within the detector, generating flowing electrons that form electrical signals. These electrical signals can then be converted into images for display by signal processing elements or computers.

[0071] According to some embodiments of this application, the fluorescence detection system 30 further includes an image conversion element, which is electrically connected to the signal conversion element 36 and is used to convert the electrical signal converted by the signal conversion element 36 back into an image.

[0072] For example, the image conversion element may include a preamplifier, a data acquisition card, and image reconstruction software. The preamplifier amplifies the electrical signal converted by the signal conversion element 36, the data acquisition card converts the amplified electrical signal into a digital signal and stores it according to pixel position, and the image reconstruction software uses an algorithm to stitch together the corresponding "pixel grayscale values" of the digital signals into an image.

[0073] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A microscopic imaging system for stimulated emission depletion microscopy, characterized in that, include: An illumination mechanism for providing a first and second beam of coaxial incident light; A phase-modulated optical device includes multiple diffractive optical elements. The multiple diffractive optical elements are spaced apart along the transmission directions of the first beam and the second beam. The first beam and the second beam are incident perpendicularly to the diffractive optical elements. The multiple diffractive optical elements are used to cooperate in phase modulation and convergence of the first beam and the second beam, so that the first beam and the second beam can form a first spot and a second spot at the focal plane of the stimulated emission depletion microscope. The first spot and the second spot correspond one-to-one and coincide in center. The first beam and the second beam have an overlapping region, which covers the phase modulation optics.

2. The microscopic imaging system according to claim 1, characterized in that, Each layer of the diffractive optical element includes a substrate and a microstructure disposed on the substrate. The microstructure is etched on the substrate according to a preset phase distribution. The preset phase distribution is configured to enable the first beam and the second beam to form a first spot array and a second spot array at the focal plane of the stimulated emission depletion microscope after passing through the phase modulation optical device. The first spot array has a plurality of first spots, and the second spot array has a plurality of second spots. The first spots in the first spot array and the second spots in the second spot array correspond one-to-one and coincide in center.

3. The microscopic imaging system according to claim 1 or 2, characterized in that, The first beam is an excitation beam, and the first spot is a solid excitation spot; The second beam is a lossy beam relative to the first beam, and the second spot is an annular lossy spot that covers the edge region of the solid excitation spot.

4. The microscopic imaging system according to claim 1 or 2, characterized in that, The lighting mechanism includes a first laser, a first beam adjustment assembly, a second laser, and a second beam adjustment assembly. The first laser is used to provide the first beam. The first beam adjustment assembly includes a first lens, a first filter, and a first reflector arranged sequentially along the transmission direction of the first beam. The second laser is used to provide the second beam. The second beam adjustment assembly includes a second lens, a second filter, a dichroic element, a second reflector, and a quarter-wave plate arranged sequentially along the transmission direction of the second beam. Wherein, the first lens is used to expand the first beam into parallel light, the first filter is used to adjust the intensity of the first beam, the first reflector is used to reflect the first beam to the dichroic element, the second lens is used to expand the second beam into parallel light, the second filter is used to adjust the intensity of the second beam, the dichroic element is used to make the first beam and the second beam coaxial and perpendicularly incident on the quarter-wave plate and the phase modulation optical device, and the quarter-wave plate is used to convert the first beam and the second beam from linearly polarized light to circularly polarized light.

5. The microscopic imaging system according to claim 4, characterized in that, The first filter and the second filter are neutral density filters; And / or, the dichroic element is a dichroic sheet.

6. A stimulated emission depletion microscope, characterized in that, include: A displacement stage for carrying and moving a sample containing a fluorescent substance; The microscopic imaging system according to any one of claims 1-5, wherein the first beam and the second beam are capable of exciting the fluorescent material in the sample to generate a fluorescent signal after being phase-modulated and converged by the phase-modulation optics; A fluorescence detection system is used to collect and detect the fluorescence signal and convert the fluorescence signal into an electrical signal.

7. The stimulated emission depletion microscope according to claim 6, characterized in that, The fluorescence detection system includes a signal collection component and a signal conversion element. The signal collection component is used to collect and converge the fluorescence signal, and the signal conversion element is used to convert the fluorescence signal into an electrical signal.

8. The stimulated emission depletion microscope according to claim 7, characterized in that, The signal collection assembly includes a collection objective, a third filter, and a third lens arranged sequentially along the propagation direction of the fluorescence signal; The collecting objective is used to collect and converge the fluorescence signal excited by the sample, the third filter is a fluorescence filter, and the third lens is used to converge the fluorescence signal passing through the fluorescence filter.

9. The stimulated emission depletion microscope according to claim 8, characterized in that, The signal collection assembly further includes a third mirror and a fourth mirror, which are used to reflect the fluorescence signal collected and focused by the collection objective lens onto the third filter.

10. The stimulated emission depletion microscope according to claim 6, characterized in that, The signal conversion element includes a photodetector.

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