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 annular partition phase modulation element comprises a first phase modulation area and a second phase modulation area connected to the peripheral side of the first phase modulation area, the first phase modulation area is circular, and the second phase modulation area is annular; the first phase modulation area and the second phase modulation area are configured to perform phase modulation and convergence on a first light beam and a second light beam which are vertically incident respectively, so that a first light spot and a second light spot which are in one-to-one correspondence and coincide in center are formed on the focal plane of the stimulated radiation loss microscope respectively. According to the technical scheme, light energy loss caused when the microscope adopts a high-numerical-aperture illumination objective lens is improved, miniaturization of the system is facilitated, and the imaging quality is improved.

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 annular spot formed by the loss beam. Traditionally, a dichroic plate is used for alignment, but maintaining high-precision, long-term stable alignment (nanometer-scale) between the excitation and loss beams 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. Large bandwidths and wavelength drift can lead to severe distortion of the formed spot, rendering it unusable.

[0005] Furthermore, traditional stimulated emission depletion microscopes typically employ high numerical aperture illumination objectives, which not only result in significant light energy loss but also limit system miniaturization. Simultaneously, high numerical aperture collection objectives lead to substantial energy loss of fluorescence signals, affecting the image signal-to-noise ratio. Therefore, there is an urgent need for a microscopic imaging system and a stimulated emission depletion microscope that can address issues such as coaxial drift, wavelength sensitivity, high energy loss, and large system size. Summary of the Invention

[0006] The purpose of this application is to at least address the coaxial stability problem commonly encountered in conventional stimulated emission depletion microscopes, as well as the significant light energy loss resulting from the use of high numerical aperture illumination objectives, which limits system miniaturization and also affects image signal-to-noise ratio. This objective is achieved through the following methods: 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 an annular partitioned phase modulation element, including a first phase modulation region and a second phase modulation region. The first phase modulation region is circular, and the second phase modulation region is annular, with the second phase modulation region connected to the outer periphery of the first phase modulation region. The first beam and the second beam are incident perpendicularly to the annular partitioned phase modulation element. The first phase modulation region is configured to perform phase modulation and focusing on the first beam, forming a first spot at the focal plane of the stimulated emission depletion microscope. The second phase modulation region is configured to perform phase modulation and focusing on the second beam, forming 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 annular partitioned phase modulation element.

[0007] According to the microscopic imaging system proposed in this application, the illumination mechanism provides a first beam and a second beam that are coaxial and perpendicularly incident on a ring-shaped phase modulation element. The first beam and the second beam have an overlapping region that covers the ring-shaped phase modulation element. When the first beam and the second beam pass through the ring-shaped phase modulation element, the first phase modulation region of the ring-shaped phase modulation element can cause the first beam to diffract and perform phase modulation and convergence on the first beam to form a first spot at the focal plane of the microscope. The second phase modulation region of the ring-shaped phase modulation element can cause the second beam to diffract and perform phase modulation and convergence on the second beam to form a second spot at the focal plane of the microscope that corresponds to the first spot and coincides with its center. The first spot and the second spot formed at the focal plane of the microscope can remain stable and strictly coincide with their centers for a long time. The elimination of the high numerical aperture illumination objective used in traditional stimulated emission depletion microscopy not only improves the problem of large light energy loss caused by high numerical aperture illumination objectives in traditional stimulated emission depletion microscopy, which helps to achieve system miniaturization, but also allows the use of a relatively low numerical aperture objective for the collection objective, reducing energy loss of fluorescence signals and improving image quality. In addition, it also improves the wavelength sensitivity of traditional diffractive optical elements, enabling the microscopic imaging system to be properly adapted to use commercial lasers with large bandwidth and wavelength drift as excitation and depletion beams.

[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 annular partitioned phase modulation element includes a substrate and microstructures disposed on the substrate. The substrate has a first phase modulation region and a second phase modulation region. The first phase modulation region and the second phase modulation region are respectively etched with the microstructures distributed 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 and a second spot that correspond one-to-one and coincide in center at the focal plane of the stimulated emission depletion microscope after passing through the annular partitioned phase modulation element.

[0009] 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.

[0010] In some embodiments of this application, the incident surface of the first phase modulation region is coated with a first bandpass film, the first bandpass film being configured to transmit only the first light beam, and the incident surface of the second phase modulation region is coated with a second bandpass film, the second bandpass film being configured to transmit only the second light beam.

[0011] In some embodiments of this application, the diameter of the first phase modulation region is set in the range of 3mm-5mm; and / or, the outer diameter of the second phase modulation region is set in the range of 7mm-9mm.

[0012] 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. The second reflector reflects the first beam and the second beam, and causes the first beam and the second beam to be perpendicularly incident on the quarter-wave plate and the annular phase modulation element. The quarter-wave plate converts the first beam and the second beam from linearly polarized light to circularly polarized light.

[0013] 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.

[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; 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 annular partitioned phase modulation element; and a fluorescence detection system for collecting and detecting the fluorescence signal and converting the fluorescence signal into an electrical signal.

[0015] 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.

[0016] According to some embodiments of this application, the signal collection assembly includes a collection objective, a third reflector, a fourth reflector, a third filter, and a third lens arranged sequentially along the propagation direction of the fluorescence signal. The collection objective is used to collect and converge the fluorescence signal excited by the sample. The third reflector is used to reflect the fluorescence signal collected and converged by the collection objective to the fourth reflector. The fourth reflector is used to reflect the fluorescence signal to the third filter, which is a fluorescence filter. The third lens is used to converge the fluorescence signal.

[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 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 ring-shaped partitioned phase modulation element; Figure 3 This is a schematic diagram showing the focusing results of a ring-shaped partitioned phase modulation element according to some embodiments of this application for a first beam with different incident wavelengths of bandwidth from 646.94 nm to 647.05 nm. Figure 4 This is a schematic diagram showing the focusing results of a ring-shaped partitioned phase modulation element according to some embodiments of this application for a second beam with different incident wavelengths and bandwidths from 774.94 nm to 775.05 nm. Figure 5 The diagram illustrates a confocal image of an extracellular vesicle obtained using a conventional confocal microscope. Figure 6 This schematically illustrates the use of stimulated radiation depletion microscopy provided in this application to examine the same extracellular vesicles in... Figure 5The image obtained by stimulated emission depletion microscopy in the same region.

[0019] The labels in the attached diagram are as follows: 100. Stimulated emission depletion microscope; 200. First spot; 300. Second spot; 10. Microscopic imaging system; 20. Displacement stage; 30. Fluorescence detection system; 11. Ring-shaped phase modulation element; 111. First phase modulation region; 112. Second phase modulation region; 113. Microstructure; 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

[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 and Figure 2The 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 an annular partitioned phase modulation element 11. The illumination mechanism 12 is used to provide a first beam and a second beam that are coaxially incident. The annular partitioned phase modulation element 11 includes a first phase modulation region 111 and a second phase modulation region 112. The first phase modulation region 111 is circular, and the second phase modulation region 112 is annular. The second phase modulation region 112 is connected to the outer periphery of the first phase modulation region 111. The first beam and the second beam are incident perpendicularly to the annular partitioned phase modulation element 11. The first phase modulation region 111 is configured to perform phase modulation and focusing on the first beam, forming a first spot at the focal plane of the stimulated emission depletion microscope. The second phase modulation region 112 is configured to perform phase modulation and focusing on the second beam, forming 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 that can cover the annular partitioned phase modulation element.

[0025] In some embodiments, see Figure 3 and Figure 4 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.

[0026] 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 and no longer spontaneously emitting fluorescence, so that only the central region of the excitation spot retains spontaneous fluorescence. This allows for the acquisition of super-resolution images after each excitation spot is scanned.

[0027] When the first and second beams provided by the illumination mechanism 12 are coaxial and perpendicularly incident on the annular partitioned phase modulation element, the first phase modulation region 111 of the annular partitioned phase modulation element 11 can diffract the first beam and perform phase modulation and focusing on the first beam to form a first spot at the focal plane of the microscope. The second phase modulation region 112 of the annular partitioned phase modulation element 11 can diffract the second beam and perform phase modulation and focusing on the second beam to form a second spot at the focal plane of the microscope that corresponds to the first spot and coincides with its center. The first spot and the second spot formed at the focal plane of the microscope can be stable and strictly coincident with their centers for a long time. The elimination of the high numerical aperture illumination objective used in traditional stimulated emission depletion microscopy not only improves the problem of large light energy loss caused by high numerical aperture illumination objectives in traditional stimulated emission depletion microscopy, which helps to achieve system miniaturization, but also allows the use of a relatively low numerical aperture objective for the collection objective, reducing energy loss of fluorescence signals and improving image quality. In addition, it also improves the wavelength sensitivity of traditional diffractive optical elements, enabling the microscopic imaging system to be properly adapted to use commercial lasers with large bandwidth and wavelength drift as excitation and depletion beams.

[0028] Please see Figure 2 According to some embodiments of this application, the annular partitioned phase modulation element includes a substrate and microstructures 113 disposed on the substrate. The substrate is provided with a first phase modulation region 111 and a second phase modulation region 112. The first phase modulation region 111 and the second phase modulation region 112 are respectively etched with microstructures 113 distributed 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 and a second spot that correspond one-to-one and coincide in center at the focal plane of the stimulated emission depletion microscope after passing through the annular partitioned phase modulation element 11.

[0029] Specifically, the microstructure 113 is fabricated using micro-nano fabrication technology and photolithographically applied to a substrate. The core function of the microstructure 113 is to precisely control the phase, amplitude, or polarization state of light, causing specific diffraction and interference of the incident light, thereby achieving customized optical functions such as beam shaping, beam splitting, and focusing.

[0030] 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.

[0031] 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. Therefore, by etching microstructures 113 according to a preset phase distribution in the first phase modulation region 111 and the second phase modulation region 112 of the substrate, the preset phase distribution can be obtained through simulation iterative training to accurately control the phase of the incident light. This enables the first and second beams of coaxial incident light to form a one-to-one corresponding and centered first and second light spots at the focal plane of the stimulated emission loss microscope after passing through the annular partitioned phase modulation element. Understandably, each pair of first and second light spots formed at the focal plane of the microscope, which are one-to-one correspondences and whose centers are strictly coincident, are independent super-resolution imaging units.

[0032] In some embodiments, the preset phase distribution of each microstructure 113 on the annular partitioned phase modulation element 11 can be optimized by a deep learning algorithm. In order to construct the target image, the three electric field components of the first light spot can be calculated by the following formula:

[0033] The three electric field components of the second light spot can be calculated using the following formula:

[0034] in, , and These are the three components of the electric field vector at point P in the spatial field. 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.

[0035] The depth of each microstructure 113 on the annular partition phase modulation element 11 is further optimized. The optimization targets for the first wavelength and the second wavelength are the first light spot and the second light spot, respectively. When the algorithm converges, a pair of light spots that meet the requirements and whose centers coincide can be obtained.

[0036] In some embodiments, the annular partitioned phase modulation element 11 is millimeter-sized. Replacing the illumination objective and a conventional phase plate, it allows the excitation beam and the loss beam to be phase-modulated and converged in a millimeter-scale coaxial manner. Furthermore, it enables the first and second light spots, after phase modulation and convergence by the annular partitioned phase modulation element 11, to remain stable and strictly coincident at their centers for extended periods.

[0037] According to some embodiments of this application, the incident surface of the first phase modulation region 111 is coated with a first bandpass film, which is configured to transmit only a first light beam, and the incident surface of the second phase modulation region 112 is coated with a second bandpass film, which is configured to transmit only a second light beam.

[0038] A bandpass thin film is an optical thin film designed based on the multi-beam interference effect of light. Essentially, it is achieved by depositing multiple layers of dielectric films with different refractive indices and thicknesses on the substrate surface to "filter" the wavelength of incident light, allowing only the target wavelength to pass through while satisfying the constructive interference condition, and reflecting or absorbing other wavelengths due to destructive interference.

[0039] As an example, the first bandpass film is only adapted to the wavelength of the first light beam (excitation beam) (e.g., a bandwidth of 646.94 nm - 647.05 nm). The first bandpass film allows efficient transmission of light within this wavelength range (transmittance ≥ 95%), while the transmittance for the second light beam (i.e., the loss beam, e.g., a bandwidth of 774.94 nm - 775.05 nm) is ≤ 3%, ensuring that the loss beam cannot enter the central region. The second bandpass film is only adapted to the wavelength of the second light beam (e.g., a bandwidth of 774.94 nm - 775.05 nm). This second bandpass film allows efficient transmission of light within this wavelength range (transmittance ≥ 95%), while the transmittance for the first light beam (excitation beam) is ≤ 3%, ensuring that the excitation beam cannot enter the annular region.

[0040] By setting targeted first and second bandpass films for different regions (first phase modulation region 111 and second phase modulation region 112) of the annular partitioned phase modulation element 11, selective transmission of specific wavelength beams can be achieved by utilizing the interference effect of light. Combined with the physical isolation of spatial partitioning, it is ensured that the first beam and the second beam can be independently phase modulated after entering the corresponding regions, reducing the cross interference between the first beam and the second beam, and ultimately forming a target beam pair with strictly overlapping centers.

[0041] According to some embodiments of this application, the diameter of the first phase modulation region 111 is set in the range of 3mm-5mm; the outer diameter of the second phase modulation region 112 is set in the range of 7mm-9mm.

[0042] The diameter of the first phase modulation region 111 is set to be in the range of 3mm-5mm, which matches the aperture of the parallel beam emitted by a conventional commercial laser. This ensures that the first beam (excitation beam) can completely cover the micron-level modulation cell in the central region, reducing phase modulation unevenness caused by beam edge overflow and reducing spot distortion caused by mismatch between region size and beam aperture.

[0043] The outer diameter of the second phase modulation region 112 is designed to be within the range of 7mm-9mm, providing sufficient physical space for the annular phase modulation of the second beam (loss beam) and adapting to the edge modulation requirements of the loss beam forming an annular loss spot. This helps to improve the uniformity of the annular loss spot's width and its edge sharpness, and reduces problems such as insufficient modulation of the loss beam and collapse of the center of the annular loss spot caused by the annular region being too narrow.

[0044] 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.

[0045] 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 align, the second reflector 1225 is used to reflect the first beam and the second beam, and make the first beam and the second beam perpendicularly incident on the quarter-wave plate 1226 and the annular partitioned phase modulation element 11. The quarter-wave plate 1226 is used to convert the first beam and the second beam from linearly polarized light to circularly polarized light, ensuring that the intensity distribution of the second light spot 300 is uniform and symmetrical, so that the annular loss light spot can uniformly and efficiently extinguish the edge fluorescence of the solid excitation light spot, thereby compressing the size of the effective light-emitting area, ensuring super-resolution, and helping to improve the uniformity of imaging.

[0046] 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.

[0047] Please see Figure 1 In some embodiments, a second reflector 1225 is provided between the dichroic element 1224 and the quarter-wave plate 1226. The second reflector 1225 is used to reflect the first beam and the second beam, which are coaxially incident after passing through the dichroic element 1224, to the quarter-wave plate 1226.

[0048] 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.

[0049] 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.

[0050] The dichroic element 1224 is a dichroic sheet that can reflect the first beam and project the second beam, so that the first beam and the second beam are coaxial and perpendicularly incident on the annular partition phase modulation element 11.

[0051] Please see Figure 1 and Figure 6 The 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 being phase-modulated and converged by an annular partitioned phase modulation element to excite the fluorescent material in the sample to generate a fluorescence signal; and a fluorescence detection system 30 for collecting and detecting the fluorescence signal and converting the fluorescence signal into an electrical signal.

[0052] 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.

[0053] See Figure 5 and Figure 6 By imaging extracellular vesicle samples using both a conventional confocal microscope and the stimulated emission depletion microscope provided in this application, it is evident that... Figure 5 Confocal images generated using traditional confocal microscopy cannot distinguish nanoparticles within the dashed frame. Figure 5 (The area within the dashed box), while the images generated using the stimulated emission depletion microscope provided in this application clearly identify the fluorescent particles ( Figure 5 (The area within the dashed box) has a resolution that is significantly better than that of a traditional confocal microscope.

[0054] 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.

[0055] 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.

[0056] The signal collection assembly may include a collection objective lens 31 and an optical filter. The signal conversion element 36 may be a photodetector.

[0057] 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.

[0058] As an example, signal conversion element 36 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.

[0059] 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.

[0060] 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.

[0061] Please see Figure 1 According to some embodiments of this application, the signal collection component includes a collection objective lens 31, a third reflector 32, a third reflector 33, 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 reflector 32 is used to reflect the fluorescence signal collected and converged by the collection objective lens 31 to the fourth reflector 33, the fourth reflector 33 is used to reflect the fluorescence signal to the third filter 34, the third filter 34 is a fluorescence filter, and the third lens 35 is used to converge the fluorescence signal.

[0062] 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.

[0063] Please see Figure 1According to some embodiments of this application, the signal collection component further includes a fourth reflector 33, which is used to reflect the fluorescence signal reflected by the third reflector 33 back to the third filter 34.

[0064] For example, the mirror surfaces of the third mirror 32 and the fourth mirror 33 are arranged opposite each other and are both set at an angle 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, so that other stray light other than fluorescence can be filtered out by the third filter 34.

[0065] 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; An annular partitioned phase modulation element includes a first phase modulation region and a second phase modulation region. The first phase modulation region is circular, and the second phase modulation region is annular. The second phase modulation region is connected to the outer periphery of the first phase modulation region. The first beam and the second beam are incident perpendicularly to the annular partitioned phase modulation element. The first phase modulation region is configured to perform phase modulation and focusing on the first beam, and to form a first spot on the focal plane of the stimulated emission depletion microscope. The second phase modulation region is configured to perform phase modulation and focusing on the second beam, and to form a second spot on 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 annular partition phase modulation element.

2. The microscopic imaging system according to claim 1, characterized in that, The annular partitioned phase modulation element includes a substrate and microstructures disposed on the substrate. The substrate has a first phase modulation region and a second phase modulation region. The first phase modulation region and the second phase modulation region are respectively etched with the microstructures distributed according to a preset phase. The preset phase distribution is configured to enable the first beam and the second beam to form a first spot and a second spot that correspond to each other and coincide in center at the focal plane of the stimulated emission depletion microscope after passing through the annular partitioned phase modulation element.

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 incident surface of the first phase modulation region is coated with a first bandpass film, which is configured to transmit only the first light beam. The incident surface of the second phase modulation region is coated with a second bandpass film, which is configured to transmit only the second light beam.

5. The microscopic imaging system according to claim 1 or 2, characterized in that, The diameter of the first phase modulation region is set in the range of 3mm-5mm; And / or, the outer diameter of the second phase modulation region is set in the range of 7mm-9mm.

6. 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, the second reflector is used to reflect the first beam and the second beam and make the first beam and the second beam perpendicularly incident on the quarter-wave plate and the annular partitioned phase modulation element, and the quarter-wave plate is used to convert the first beam and the second beam from linearly polarized light to circularly polarized light.

7. The microscopic imaging system according to claim 6, characterized in that, The first filter and the second filter are neutral density filters; And / or, the dichroic element is a dichroic sheet.

8. 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 1-7, 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 annular partition phase modulation element; 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 assembly includes a collection objective, a third reflector, a third filter, and a third lens arranged sequentially along the propagation direction of the fluorescence signal. The collection objective is used to collect and converge the fluorescence signal excited by the sample. The third reflector is used to reflect the fluorescence signal collected and converged by the collection objective to the third filter. The third filter is a fluorescence filter. The third lens is used to converge the fluorescence signal passing through the fluorescence filter.

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