Optical beam expander, microscopic imaging system and stimulated radiation loss microscope

Abstract

The invention belongs to the technical field of optical microscopic imaging, and discloses an optical beam expander, a microscopic imaging system and a stimulated radiation loss microscope. Wherein the optical beam expander comprises a first diffractive optical element, a first pinhole filter and a first converging lens which are sequentially arranged in the light beam propagation direction, and the first diffractive optical element is used for receiving an incident light beam, carrying out wavefront modulation on the incident light beam and converging the incident light beam to form a converged light beam; the first pinhole filter is used for performing spatial filtering on the converged light beam, and the first converging lens is used for receiving the divergent light beam filtered by the first pinhole filter and expanding and collimating the divergent light beam into a parallel light beam. According to the technical scheme provided by the invention, by carrying out coherent processing of convergence, spatial filtering and beam expanding collimation on the incident light beam, stray light and broadband interference components in the light beam can be effectively eliminated, the monochromaticity and purity of the light beam are improved, and the wavelength stability of light beam propagation is favorably improved.

Description

Technical Field

[0001] This application belongs to the field of optical microscopy imaging technology, specifically relating to an optical beam expander, a microscopy imaging system, and a stimulated emission loss 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 annular spot formed by the loss beam must be extremely pure, and ultra-narrow band loss beam is a prerequisite for obtaining a pure annular spot. Therefore, bandwidth adjustment of the loss beam during beam expansion is a crucial step.

[0005] In related technologies, optical beam expanders mostly use traditional lens groups. However, traditional lens optical beam expanders only have the function of beam expansion and do not have the function of wavelength selection. Therefore, developing an optical beam expander that has both wavelength adaptability is of great significance for improving the imaging quality and application range of stimulated emission depletion microscopes. Summary of the Invention

[0006] The purpose of this application is to at least solve the problem of wavelength selection functionality in traditional beam expanders. This purpose is achieved through the following means: In a first aspect, this application proposes an optical beam expander for a microscopic imaging system. The optical beam expander includes a first diffractive optical element, a first pinhole filter, and a first converging lens arranged sequentially along the beam propagation direction. The first diffractive optical element is used to receive an incident beam of a specific wavelength and perform wavefront modulation on the incident beam, and to converge the incident beam to form a converging beam. The first pinhole filter is used to perform spatial filtering on the converging beam. The first converging lens is used to receive the diverging beam filtered by the first pinhole filter and to expand and collimate the diverging beam into a parallel beam.

[0007] According to the optical beam expander provided in this application, the optical beam expander includes an integral structure comprising a first diffractive optical element, a first pinhole filter, and a first converging lens arranged sequentially along the beam propagation direction. This integral structure can form a coherent processing flow of converging, spatial filtering, and beam expansion and collimation for incident beams of a specific wavelength. Specifically, after receiving an incident beam of a specific wavelength, the first diffractive optical element can impart a preset phase characteristic to the beam through wavefront modulation, and simultaneously converge it into a converged beam, providing conditions for subsequent precise filtering. For incident beams of non-designed wavelengths, the first diffractive optical element does not have a converging function. The first pinhole filter performs spatial filtering on the converged beam to filter out beams of non-designed wavelengths, effectively eliminating stray light and broadband interference components in the beam, and improving the monochromaticity and purity of the beam. The first converging lens receives the filtered diverging beam and completes beam expansion and collimation, outputting a parallel beam with good directionality, which helps to improve the wavelength stability of beam propagation.

[0008] In addition, the microscopic imaging system according to this application may also have the following additional technical features: In some embodiments of this application, the first diffractive optical element, the first pinhole filter, and the first converging lens are arranged coaxially.

[0009] In some embodiments of this application, the center of the pinhole of the first pinhole filter coincides with the focal point of the first diffractive optical element.

[0010] In some embodiments of this application, the first converging lens is a convex lens.

[0011] Secondly, this application also proposes a microscopic imaging system for stimulated emission depletion microscopy, comprising: The lighting mechanism includes a first laser and a second laser, the first laser being used to provide a first beam and the second laser being used to provide a second beam, the first beam and the second beam being coaxial; The first beam adjustment assembly includes a first filter, a second diffractive optical element, a second pinhole filter, a second converging lens, and a first reflector arranged sequentially along the transmission direction of the first beam. The first filter is used to adjust the intensity of the first beam. The second diffractive optical element is used to perform wavefront modulation on the first beam and converge the first beam to form a converged beam. The second pinhole filter is used to perform spatial filtering on the converged beam. The second converging lens is used to receive the divergent beam filtered by the second pinhole filter and expand and collimate the divergent beam into a parallel beam. The second beam adjustment assembly includes a second filter, an optical beam expander, a phase plate, a first dichroic element, a second dichroic element, and a quarter-wave plate arranged sequentially along the transmission direction of the second beam. The second filter is used to adjust the intensity of the second beam. The optical beam expander is used to filter and expand the second beam. The phase plate is used to perform wavefront modulation on the second beam. The first dichroic element is used to coaxially incident the second beam and the first beam reflected by the first mirror onto the second dichroic element. The second dichroic element is used to coaxially and perpendicularly reflect the first beam and the second beam onto the quarter-wave plate. The quarter-wave plate is used to convert the first beam and the second beam from linearly polarized light to circularly polarized light. A microscope objective lens is used to simultaneously converge 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 microscope. The optical beam expander is the optical beam expander described in any one of the first aspects of the embodiments.

[0012] In some embodiments of this application, the first beam is an 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, the annular loss spot covering the edge region of the solid excitation spot.

[0013] In some embodiments of this application, the first filter and the second filter are neutral density filters; and / or, the first dichroic element and the second dichroic element are dichroic sheets.

[0014] Secondly, this application also proposes a stimulated emission depletion microscope, comprising: A displacement stage for carrying and moving a sample containing a fluorescent substance; and As described in any one of the second aspect embodiments, the microscopic imaging system wherein the first beam and the second beam are capable of being converged by the microscope objective of the microscopic imaging system to excite a fluorescent substance in the sample to generate a fluorescence signal, the fluorescence signal being converged by the microscope objective and transmitted through the second dichroic element; and A fluorescence detection system is used to collect and detect the fluorescence signal and convert the fluorescence signal into an electrical signal.

[0015] In 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.

[0016] In some embodiments of this application, the signal collection component includes a second reflector, a third reflector, a third filter, and a third converging lens arranged sequentially along the propagation direction of the fluorescence signal. The second reflector is used to receive the fluorescence signal transmitted through the second dichroic element and reflect the fluorescence signal to the third reflector. The third reflector is used to reflect the fluorescence signal to the third filter, which is a fluorescence filter. The third converging lens is used to converge the fluorescence signal passing through the fluorescence filter.

[0017] 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

[0018] 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 1 This is a simplified structural diagram of an optical beam expander provided according to some embodiments of this application; Figure 2 This is a simplified structural diagram of a stimulated emission depletion microscope provided according to some embodiments of this application; Figure 3 A schematic diagram of the imaging results of an annular loss spot with a wavelength of 647.1 nm obtained by using the optical beam expander provided in some embodiments of this application for stimulated emission depletion microscopy.

[0019] The labels in the attached diagram are as follows: 100. Stimulated emission depletion microscope; 201. First beam; 202. Second beam; 203. Fluorescent beam; 10. Displacement stage; 20. Microscopic imaging system; 30. Fluorescence detection system; 21. Lighting mechanism; 211. First laser; 212. Second laser; 221. First filter; 222. Second diffractive optical element; 223. Second pinhole filter; 224. Second converging lens; 225. First reflector; 231. Second filter; 232. Optical beam expander; 2321. First diffractive optical element; 23211. Substrate; 23212. Microstructure; 2322. First pinhole filter; 2323. First converging lens; 233. Phase plate; 234. First dichroic element; 235. Second dichroic element; 236. Quarter wave plate; 24. Microscope objective; 31. Second reflector; 32. Third reflector; 33. Third filter; 34. Third converging lens; 35. Signal conversion element. Detailed Implementation

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] Please see Figure 1 This application provides an optical beam expander 232 for use in a microscopic imaging system 20. The optical beam expander 232 includes a first diffractive optical element 2321, a first pinhole filter 2322, and a first converging lens 2323 arranged sequentially along the beam propagation direction. The first diffractive optical element 2321 is used to receive the incident beam and perform wavefront modulation on the incident beam, and to converge the incident beam of the designed wavelength to form a converged beam. The first pinhole filter 2322 is used to perform spatial filtering on the converged beam. The first converging lens 2323 is used to receive the divergent beam filtered by the first pinhole filter 2322 and expand and collimate the divergent beam into a parallel beam.

[0025] Understandably, the first diffractive optical element 2321 includes a substrate 23211 and a microstructure 23212 fabricated on the substrate 23211 according to a preset phase distribution, used to focus an incident light beam of a designed wavelength. For example, the substrate 23211 can be a transparent substrate such as quartz or glass, with surface oil removed by ultrasonic cleaning and surface oxide layer removed by plasma cleaning, resulting in a surface roughness of less than 1 / 10 of the wavelength. Specifically, the microstructure 23212 is fabricated using micro / nano fabrication technology and photolithographically etched onto the substrate; the preset phase distribution can be obtained through simulation iteration training.

[0026] As an example, when the wavelength of the incident lossy beam is 647.1 nm, the first diffractive optical element 2321, in order to achieve the wavefront modulation function of beam convergence, has a microstructure phase distribution centered on the spherical convergence phase, which must satisfy the following formula:

[0027] In the formula, Pi Let be the wavelength of the incident beam, taken as 647.1 nm. The design focal length of the first diffractive optical element (which must satisfy the requirement that the focal point of the first diffractive optical element coincides with the center of the pinhole of the first pinhole filter). The radial distance from a point in the plane of the first diffractive optical element to the optical axis.

[0028] The above modulation wavefront This can be achieved by utilizing the dispersion properties of the substrate material; for the designed wavelength, this can be achieved by processing an appropriate relief depth. Satisfying the formula:

[0029] In the formula This refers to the refractive index of the substrate material at the designed wavelength. However, for non-designed wavelengths, the different refractive indices of the substrate cannot meet the modulation wavefront requirements. The spherical wave requirement means that it cannot be converged through a pinhole filter.

[0030] The first pinhole filter 2322 is a device that uses a pinhole to spatially filter a light beam, eliminating stray light and non-designed wavelength components.

[0031] The first converging lens 2323 is a device that can expand and collimate a diverging beam passing through a pinhole filter into a parallel beam.

[0032] According to the optical beam expander 232 provided in this application, the optical beam expander 232 includes an integral structure comprising a first diffractive optical element 2321, a first pinhole filter 2322, and a first converging lens 2323 arranged sequentially along the beam propagation direction. This integral structure can form a coherent processing flow for converging, spatial filtering, and beam expansion and collimation of an incident beam (such as a lossy beam) of a designed wavelength. Specifically, after receiving an incident beam of a specific wavelength, the first diffractive optical element 2321 can impart a preset phase characteristic to the beam through wavefront modulation, while converging it into a converged beam, providing conditions for subsequent precise filtering. The first pinhole filter 2322 performs spatial filtering on the converged beam to filter out beams of non-designed wavelengths, effectively eliminating stray light and broadband interference components in the beam, and improving the monochromaticity and purity of the beam. The first converging lens 2323 receives the filtered divergent beam and completes beam expansion and collimation, outputting a parallel beam with good directionality, which helps to improve the wavelength stability of beam propagation, thereby helping to improve the imaging resolution and image quality of the stimulated emission loss microscope 100.

[0033] According to some embodiments of this application, the first diffractive optical element 2321, the first pinhole filter 2322, and the first converging lens 2323 are arranged coaxially.

[0034] The first diffractive optical element 2321, the first pinhole filter 2322, and the first converging lens 2323 are arranged coaxially, meaning their central axes coincide and are aligned with the central path of the beam propagation. This allows the incident beam to sequentially converge, spatially filter, and expand and collimate along the same optical axis, reducing beam deflection and refraction during propagation and improving the wavelength stability of the beam.

[0035] According to some embodiments of this application, the center of the pinhole of the first pinhole filter 2322 coincides with the focal point of the first diffractive optical element 2321.

[0036] The first pinhole filter 2322 has a light-transmitting hole in the shape of a pinhole, and the center of the pinhole of the first pinhole filter 2322 is the geometric center point of the light-transmitting hole.

[0037] The focal point of the first diffractive optical element 2321 is the center point of the smallest spot formed by the convergence of the light beam after wavefront modulation by the element.

[0038] The center of the pinhole of the first pinhole filter 2322 coincides with the focal point of the first diffractive optical element 2321, thereby accurately capturing the core region of the beam formed by the convergence of the first diffractive optical element 2321, efficiently filtering stray light and non-designed wavelength components in the beam, optimizing the spatial filtering effect, making the filtered beam more in line with the requirements of beam expansion and collimation, and helping to improve the purity of the output parallel beam.

[0039] According to some embodiments of this application, the converging lens is a convex lens.

[0040] As an example, a convex lens can be selected as a plano-convex lens, a biconvex lens, or a meniscus lens, depending on the need.

[0041] A convex lens is a transparent optical element with a thickness greater in the middle than at the edges. It can efficiently receive the diverging beam after being filtered by the first pinhole filter 2322 and realize the beam expansion and collimation conversion. The output parallel beam has good directionality and can meet the control requirements of the stimulated emission loss microscope 100 for the propagation direction of the loss beam. Moreover, the convex lens has a mature structure and strong adaptability, which is conducive to the industrial production and assembly of the equipment.

[0042] Please see Figure 1 and Figure 2 According to some embodiments of this application, this application also proposes a microscopic imaging system 20 for a stimulated radiation depletion microscope 100, including: an illumination mechanism 21, a first beam 201 adjustment assembly, a second beam 202 adjustment assembly, and a microscope objective 24.

[0043] Specifically, the lighting mechanism 21 includes a first laser 211 and a second laser 212. The first laser 211 is used to provide a first beam 201, and the second laser 212 is used to provide a second beam 202. The first beam 201 and the second beam 202 are coaxial.

[0044] The first beam 201 adjustment assembly includes a first filter 221, a second diffractive optical element 222, a second pinhole filter 223, a second converging lens 224, and a first reflector 225 arranged sequentially along the transmission direction of the first beam 201. The first filter 221 is used to adjust the intensity of the first beam 201. The second diffractive optical element 222 is used to perform wavefront modulation on the first beam 201 and to converge the first beam 201 to form a converged beam. The second pinhole filter 223 is used to perform spatial filtering on the converged beam. The second converging lens 224 is used to receive the divergent beam filtered by the second pinhole filter 223 and to expand and collimate the divergent beam into a parallel beam.

[0045] The second beam 202 adjustment assembly includes a second filter 231, an optical beam expander 232, a phase plate, a first dichroic element 234, a second dichroic element 235, and a quarter-wave plate 236 arranged sequentially along the transmission direction of the second beam 202. The second filter 231 is used to adjust the intensity of the second beam 202, the optical beam expander 232 is used to filter and expand and collimate the second beam 202, the phase plate 234 is used to perform wavefront modulation on the second beam 202, and the first dichroic element... 234 is used to coaxially incident the second beam 202 and the first beam 201 reflected by the first reflector 225 onto the second dichroic element 235. The second dichroic element 235 is used to coaxially and perpendicularly reflect the first beam 201 and the second beam 202 onto the quarter-wave plate 236. The quarter-wave plate 236 is used to convert the first beam 201 and the second beam 202 from linearly polarized light to circularly polarized light. The optical beam expander 232 is the optical beam expander 232 of any one of the first aspect embodiments.

[0046] The microscope objective 24 is used to simultaneously converge the first beam 201 and the second beam 202 so that the first beam 201 and the second beam 202 can form a first light spot and a second light spot at the focal plane of the microscope.

[0047] Specifically, the first beam 201 is the excitation beam, and the first beam spot is a solid excitation beam spot. The second beam 202 is a loss beam relative to the first beam 201, and the second beam spot is an annular loss beam spot, which covers the edge region of the solid excitation beam spot.

[0048] As an example, the excitation beam is typically a shorter wavelength beam, such as 532 nm, while the loss beam is a relatively longer wavelength beam, such as 647.1 nm. See also... Figure 3 , Figure 3 A schematic diagram of the imaging results of a ring-shaped lossy spot with a wavelength of 647.1 nm obtained by using the optical beam expander 232 provided in some embodiments of this application for stimulated emission depletion microscope 100. Clearly, Figure 3 The annular loss spot formed in the process is relatively clear and pure.

[0049] The first beam 201 is the excitation beam (forming a solid excitation spot), and the second beam 202 is the loss beam (forming a ring-shaped loss spot, covering the edge region of the solid excitation spot). The excitation beam is the beam that excites fluorescent substances in the sample to produce fluorescence signals; the loss beam is the beam that depletes the excited-state energy of fluorescent molecules through stimulated emission; the solid excitation spot is a circular spot with concentrated energy at the center and gradually changing edges, formed by converging excitation beams; the ring-shaped loss spot is a ring-shaped spot with extremely low energy at the center and concentrated energy at the edges, formed by modulating the loss beam. The ring-shaped loss spot can specifically target the fluorescent molecules at the edge of the solid excitation spot, depleting the fluorescence in non-core areas through stimulated emission, thus improving the spatial resolution of the fluorescence signal at the focal plane and contributing to obtaining higher-resolution super-resolution images.

[0050] In this embodiment, the coaxiality of the first beam 201 and the second beam 202 means that the propagation center paths of the two beams coincide.

[0051] The first laser 211 and the second laser 212 in the illumination mechanism 21 provide beams independently, and the wavelength and power parameters of the first beam 201 and the second beam 202 can be adjusted respectively, so as to adapt to the imaging requirements of different fluorescently labeled samples and improve the problem of limited adaptability of traditional equipment to the wavelength of the incident beam.

[0052] In the first beam 201 adjustment assembly, the first filter 221 can precisely control the intensity of the first beam 201, reducing damage to the sample caused by excessive intensity, while avoiding the problem of weak fluorescence signal caused by insufficient intensity. The cooperation of the second diffractive optical element 222, the second pinhole filter 223, and the second converging lens 224 can eliminate stray light and broadband interference in the excitation beam, improve the purity and directionality of the beam. The parallel beam after beam expansion and collimation is reflected by the first reflector 225 to the first dichroic element 234, and efficiently aligns with the second beam 202, providing a guarantee for the subsequent formation of a high-quality solid excitation spot.

[0053] In the second beam 202 adjustment assembly, the second filter 231 can precisely control the intensity of the second beam 202, and the optical beam expander 232 is used to expand and collimate the second beam 202. The phase plate can specifically modulate the phase of the second beam 202 to facilitate the formation of a ring-shaped loss spot that meets the requirements; the quarter-wave plate 236 can convert linearly polarized light into circularly polarized light, which can reduce energy loss and spot distortion caused by polarization state changes during the propagation and convergence of the second beam 202. The first dichroic element 234 can reflect the first beam 201 and transmit the second beam 202, so that the first beam 201 and the second beam 202 are efficiently coaxial and incident on the second dichroic element 235, which then coaxially and perpendicularly reflects the first beam 201 and the second beam 202 to the quarter-wave plate 236. The synergistic effect of multiple components can optimize the purity and shape of the annular second light spot, and improve the propagation stability of the first beam 201 and the second beam 202, thus providing support for the precise superposition of the first and second light spots formed at the focal plane of the microscope.

[0054] The microscope objective 24 is an optical element in a microscope used to converge a light beam and magnify sample imaging. It has a high numerical aperture and strong converging capability. The microscope objective 24 can simultaneously and efficiently converge the first beam 201 and the second beam 202 after adjustment, ensuring that the first beam 201 and the second beam 202 are accurately superimposed at the focal plane. This allows the annular loss spot to reliably cover the edge region of the solid excitation spot, giving full play to the stimulated emission loss effect, further optimizing the super-resolution imaging effect, while also meeting the convergence requirements of high-purity beams and reducing the risk of spot distortion.

[0055] According to some embodiments of this application, the first filter 221 and the second filter 231 are neutral density filters; the first dichroic element 234 and the second dichroic element 235 are dichroic sheets.

[0056] A neutral density filter is a type of optical filter that attenuates light of different wavelengths to a essentially uniform degree, reducing beam intensity without altering the beam's wavelength characteristics, polarization state, or propagation direction. It can smoothly adjust beam intensity without changing the beam's wavelength characteristics or polarization state, precisely controlling the energy of the excitation and attenuation beams according to the sample's fluorescence characteristics and imaging requirements. This adapts to imaging scenarios for different fluorescently labeled samples, reducing damage to the sample and imaging interference caused by inappropriate beam intensity.

[0057] A dichroic filter is a wavelength-selective optical element that can achieve high reflection of light in a specific wavelength range and high transmission of light in another wavelength range, thereby enabling the separation and synthesis of beams of different wavelengths. It can selectively reflect and transmit light based on the beam wavelength, reliably achieving coaxial synthesis and direction control of the excitation and loss beams. This ensures that the two beams propagate along a preset path and are precisely incident on the microscope objective 24, improving the consistency of beam alignment. Furthermore, its simple structure and low optical loss help preserve beam energy.

[0058] Please see Figure 2 According to some embodiments of this application, this application also proposes a stimulated emission depletion microscope 100, comprising: a displacement stage 10, a microscopic imaging system 20 as described in any of the above embodiments, and a fluorescence detection system 30. The displacement stage 10 is used to carry and move a sample containing fluorescent material; a first beam 201 and a second beam 202, after being converged by a microscope objective 24, can excite the fluorescent material in the sample to generate a fluorescence signal, which can be converged by the microscope objective 24 and transmitted through a second dichroic element 235; the fluorescence detection system 30 is used to collect and detect the fluorescence signal and convert it into an electrical signal.

[0059] For example, the fluorescent substance can be a fluorescent molecule, which is a class of organic or inorganic compounds that exhibit characteristic fluorescence emission in the ultraviolet, visible to near-infrared spectral range (100 nm - 1700 nm). Its 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 beam 201 and the second beam 202 irradiate the sample, the fluorescent molecules within the sample are excited to generate a fluorescence signal. The fluorescence signal (i.e., the fluorescence beam 203) can be focused by the microscope objective 24 and transmitted through the second dichroic element 235 to be collected by the fluorescence detection system 30. 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.

[0060] The stimulated radiation depletion microscope 100 proposed in this application includes the microscopic imaging system 20 of any of the above embodiments, and therefore has the technical effects of any of the above embodiments, which will not be repeated here.

[0061] According to some embodiments of this application, the fluorescence detection system 30 includes a signal collection component and a signal conversion element 35. The signal collection component is used to collect and converge fluorescence signals, and the signal conversion element 35 is used to convert fluorescence signals into electrical signals.

[0062] 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 35. The signal conversion element 35 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.

[0063] As an example, signal conversion element 35 is a photodetector. A photodetector can detect and convert fluorescence signals into measurable electrical signals. It features high sensitivity, low noise, and fast response. Essentially, it utilizes the photoelectric effect to allow photons to interact with electrons within the detector, generating flowing electrons, which in turn form an electrical signal. This electrical signal can then be converted into an image for display by signal processing elements or a computer.

[0064] In some embodiments, the fluorescence detection system 30 further includes an image conversion element electrically connected to the signal conversion element 35, for converting the electrical signal converted by the signal conversion element 35 back into an image.

[0065] 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 35, 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.

[0066] Please see Figure 2 According to some embodiments of this application, the signal collection component includes a second reflector 31, a third filter 33, and a third converging lens 34 arranged sequentially along the propagation direction of the fluorescence signal. The second reflector 31 is used to receive the fluorescence signal transmitted through the second dichroic element 235 and reflect the fluorescence signal to the third filter 33. The third filter 33 is a fluorescence filter. The third converging lens 34 is used to converge the fluorescence signal that has passed through the fluorescence filter.

[0067] The fluorescence signal transmitted through the second dichroic element 235 can be reflected by the second reflector 31 to the third filter 33. The third filter 33 is a fluorescence filter, which can filter the fluorescence signal and allow fluorescence to pass through, while filtering out other stray light to reduce interference from other light signals and improve the purity of the fluorescence signal, thereby helping to improve the clarity of the image. The third converging lens 34 is used to converge the fluorescence signal that has passed through the fluorescence filter for a second time and transmit it to the signal conversion element 35, which helps to enhance the signal intensity and reduce imaging noise.

[0068] Please see Figure 2In some embodiments, the signal-stimulated component further includes a third reflector 32. The third reflector 32 is arranged opposite to the mirror surface of the second reflector 31 and is angled relative to the fluorescence transmission direction. The fluorescence signal after passing through the second dichroic element 235 can be reflected by the second reflector 31 to the third reflector 32. The third reflector 32 then reflects the fluorescence signal to the third filter, so that the fluorescence signal can be incident perpendicularly to the third filter, so that other stray light other than fluorescence can be filtered out by the third filter 33.

[0069] 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. An optical beam expander for use in a microscopic imaging system, characterized in that, The optical beam expander includes a first diffractive optical element, a first pinhole filter, and a first converging lens arranged sequentially along the beam propagation direction. The first diffractive optical element is used to receive the incident beam and perform wavefront modulation on the incident beam, and to converge the incident beam to form a converging beam. The first pinhole filter is used to perform spatial filtering on the converging beam. The first converging lens is used to receive the diverging beam filtered by the first pinhole filter and to expand and collimate the diverging beam into a parallel beam.

2. The optical beam expander according to claim 1, characterized in that, The first diffractive optical element, the first pinhole filter, and the first converging lens are arranged coaxially.

3. The optical beam expander according to claim 1 or 2, characterized in that, The center of the pinhole of the first pinhole filter coincides with the focal point of the first diffractive optical element.

4. The optical beam expander according to claim 1 or 2, characterized in that, The first converging lens is a convex lens.

5. A microscopic imaging system for stimulated emission depletion microscopy, characterized in that, include: The lighting mechanism includes a first laser and a second laser, the first laser being used to provide a first beam and the second laser being used to provide a second beam, the first beam and the second beam being coaxial; The first beam adjustment assembly includes a first filter, a second diffractive optical element, a second pinhole filter, a second converging lens, and a first reflector arranged sequentially along the transmission direction of the first beam. The first filter is used to adjust the intensity of the first beam. The second diffractive optical element is used to perform wavefront modulation on the first beam and converge the first beam to form a converged beam. The second pinhole filter is used to perform spatial filtering on the converged beam. The second converging lens is used to receive the divergent beam filtered by the second pinhole filter and expand and collimate the divergent beam into a parallel beam. The second beam adjustment assembly includes a second filter, an optical beam expander, a phase plate, a first dichroic element, a second dichroic element, and a quarter-wave plate arranged sequentially along the transmission direction of the second beam. The second filter is used to adjust the intensity of the second beam. The optical beam expander is used to filter and expand the second beam. The phase plate is used to perform wavefront modulation on the second beam. The first dichroic element is used to coaxially incident the second beam and the first beam reflected by the first mirror onto the second dichroic element. The second dichroic element is used to coaxially and perpendicularly reflect the first beam and the second beam onto the quarter-wave plate. The quarter-wave plate is used to convert the first beam and the second beam from linearly polarized light to circularly polarized light. A microscope objective lens is used to simultaneously converge 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 microscope. The optical beam expander is the optical beam expander according to any one of claims 1-4.

6. The microscopic imaging system according to claim 5, 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.

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

8. A stimulated emission depletion microscope, characterized in that, include: A displacement stage for carrying and moving a sample containing a fluorescent substance; and The microscopic imaging system as described in any one of claims 5-7, wherein the first beam and the second beam, after being converged by the microscope objective of the microscopic imaging system, can excite the fluorescent substance in the sample to generate a fluorescence signal, the fluorescence signal being converged by the microscope objective and transmitted through the second dichroic element; and A fluorescence detection system is used to collect and detect the fluorescence signal and convert the fluorescence signal into an electrical signal.

9. The stimulated emission depletion microscope according to claim 8, 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.

10. The stimulated emission depletion microscope according to claim 9, characterized in that, The signal collection component includes a second reflector, a third reflector, a third filter, and a third converging lens arranged sequentially along the propagation direction of the fluorescence signal. The second reflector is used to receive the fluorescence signal transmitted through the second dichroic element and reflect the fluorescence signal to the third reflector. The third reflector is used to reflect the fluorescence signal to the third filter, which is a fluorescence filter. The third converging lens is used to converge the fluorescence signal passing through the fluorescence filter.

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