[go: up one dir, main page]

WO2025059947A1 - Système d'éclairage à lumière structurée, procédé d'éclairage à lumière structurée et système optique à super-résolution - Google Patents

Système d'éclairage à lumière structurée, procédé d'éclairage à lumière structurée et système optique à super-résolution Download PDF

Info

Publication number
WO2025059947A1
WO2025059947A1 PCT/CN2023/120198 CN2023120198W WO2025059947A1 WO 2025059947 A1 WO2025059947 A1 WO 2025059947A1 CN 2023120198 W CN2023120198 W CN 2023120198W WO 2025059947 A1 WO2025059947 A1 WO 2025059947A1
Authority
WO
WIPO (PCT)
Prior art keywords
light
structured
structured light
light beam
illumination system
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2023/120198
Other languages
English (en)
Chinese (zh)
Inventor
苏泽宇
沈梦哲
黄雁樵
周师岳
徐讯
章文蔚
黎宇翔
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BGI Shenzhen Co Ltd
Original Assignee
BGI Shenzhen Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BGI Shenzhen Co Ltd filed Critical BGI Shenzhen Co Ltd
Priority to PCT/CN2023/120198 priority Critical patent/WO2025059947A1/fr
Publication of WO2025059947A1 publication Critical patent/WO2025059947A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens

Definitions

  • the present invention relates to the field of optical imaging technology, and in particular to a structured light illumination system, a structured light illumination method and a super-resolution optical system.
  • Gene sequencing technology is widely used in many research fields of life science and medicine, including various genomics, etiology of complex diseases, prenatal diagnosis, personalized drug treatment, etc.
  • the commonly used gene sequencing method is to use a high-resolution fluorescence microscopy imaging system, where fluorescent groups emit fluorescence of different wavelengths after being excited by lasers of different wavelengths; sequencing is achieved by taking pictures of the sequencing chip.
  • the resolution of current super-resolution optical imaging systems can be reduced to less than 100 nanometers.
  • the stimulated emission depletion method requires high excitation light intensity and is not suitable for long sequencing.
  • its point scanning characteristics make it more suitable for rapid imaging of small area samples.
  • Photoactivated localization microscopy and random optical reconstruction microscopy can achieve very high resolution, but due to their single-molecule localization characteristics, the acquisition time of each super-resolution image is basically in the order of minutes, and the sequencing speed is slow.
  • structured illumination microscopy and spinning disk confocal microscopy based on pixel redistribution can meet the requirements of large-scale, high-speed, and high-resolution imaging, and have no specific requirements for fluorescent dyes.
  • the required light intensity is also relatively low, and it has been combined with existing sequencing technology.
  • the projection method which uses a spatial light modulator (SLM) or a digital microlens array (DMD) to project the interference fringe pattern onto the object surface.
  • SLM spatial light modulator
  • DMD digital microlens array
  • the advantage of this method is that the modulation speed is fast, but the resolution of SLM and DMD is generally below 1920*1200, and the corresponding object field of view is only tens to hundreds of microns.
  • the damage threshold of SLM is low and cannot withstand high-power laser irradiation.
  • the other is the grating interferometry method, which uses a grating to separate two beams of light and then passes through Certain optical designs finally converge on the object side to form interference fringes.
  • the grating interferometry method is not limited by size, and its corresponding object field of view can reach above the millimeter level. At the same time, according to different resolution requirements, interference fringes with a period of several hundred nanometers can be obtained. Based on these characteristics, the grating interferometry method is particularly suitable for high-throughput requirements such as gene sequencing.
  • the cost of gene sequencing is mainly concentrated on sequencing reagents.
  • the amount of sequencing data per unit area can be increased, and the utilization rate of sequencing reagents can be improved.
  • the reagent cost will drop significantly by improving the resolution of the system.
  • the cost of reagent consumables is inversely proportional to the square of the sample density, how to use super-resolution imaging technology to increase the sample density of sequencing chips and further reduce the cost of gene sequencing is an urgent problem to be solved.
  • the field of view of existing super-resolution imaging technology is mostly tens to hundreds of microns, and a rotating stage is required to rotate the grating to switch different structured light directions.
  • the speed of the rotating stage is slow, and it generally takes hundreds of milliseconds to rotate 90°, resulting in slow imaging speed and low information throughput, which makes it difficult to meet the increasingly high high-throughput sequencing requirements on the market.
  • the technical problem to be solved by the present invention is to overcome the defects of small imaging field of view, slow speed and low information flux in the prior art, and to provide a structured light illumination system, a structured light illumination method and a super-resolution optical system.
  • the present invention provides a structured light illumination system, the structured light illumination system comprising:
  • At least two gratings respectively located on the light paths of the at least two light sources, the gratings being used to generate interference fringes;
  • a translation stage the grating is arranged on the translation stage, and the translation stage is used to move the grating to perform phase switching;
  • a beam merging and guiding unit wherein the light paths converge at the beam merging and guiding unit, and the beams
  • the merging and guiding unit is used for guiding the light beams to merge into the object space.
  • the light beam merging and guiding unit comprises:
  • a galvanometer the optical paths intersect at the galvanometer, and the galvanometer is used to reflect the light beams to the object space by swinging at at least two preset angles.
  • the structured light illumination system comprises:
  • a first light source and a second light source wherein the first light source and the second light source are used to generate light beams with different polarization directions;
  • the light beam merging and guiding unit comprises:
  • a polarization beam combiner wherein the optical paths intersect at the polarization beam combiner, and the polarization beam combiner is used to reflect the light beam of the first light source to the object space, and the light beam of the second light source passes through the polarization beam combiner to reach the object space.
  • the at least two gratings are arranged in directions orthogonal to each other so as to generate mutually orthogonal interference fringes; and/or,
  • the at least two gratings include a modulated grating having a high transmittance at a preselected energy level.
  • the light source is specifically used to generate light beams of at least two different preset wavelengths; and/or,
  • the structured light illumination system further comprises:
  • At least two collimating lenses are respectively located on the optical path, and the light beam reaches the grating after being collimated by the collimating lenses.
  • the structured light illumination system further comprises:
  • At least two filtering modules are respectively located on the optical path and are used to filter out light beams outside the preselected energy level so that the light beams with the preselected energy level can reach the light beam merging and guiding unit.
  • the filtering module comprises:
  • the first lens and the second lens are used to form a first 4f system; the grating is located on the front focal plane of the first 4f system, and the light beam merging and guiding unit is located on the back focal plane of the first 4f system;
  • the energy level filter is located on the spectrum of the first 4f system and is used to filter out the pre-selected energy level.
  • the outer light beam is combined and guided by the light beam so that the light beam with the preselected energy level reaches the light beam merging and guiding unit.
  • the object side signal generated by the structured light illumination is received after passing through the dichroic mirror.
  • the present invention further provides a structured light illumination method, which is applied to the structured light illumination system as described above, and comprises:
  • the translation stage is controlled to sequentially move the first grating to other preset positions, and the first light source emits a light beam, which is guided by the light beam merging and guiding unit, and then merged into the object space to generate structured light of other phases in the first direction;
  • the translation stage is controlled to sequentially move the other gratings of the at least two gratings to preset positions, and the other light source of the at least two light sources emits a light beam, which is guided by the light beam merging and guiding unit and merged into the object space to generate structured light in other directions.
  • the present invention also provides a super-resolution optical system, which comprises a substrate, an imaging module, an auto-focus module and an illumination module; the imaging module, the auto-focus module and the illumination module are fixed on the substrate; the illumination module adopts the structured light illumination system as described above;
  • the auto-focus module is coupled to the lighting module via a galvanometer, and the auto-focus module is used to drive the objective lens to achieve auto-focus;
  • the lighting module is used to emit a lighting structure light beam which is projected onto the surface of the sample to be photographed through the galvanometer and the objective lens;
  • the imaging module is used to receive the optical signal generated by the sample to be photographed after being irradiated by the illumination structure light beam; the optical signal is divided into a plurality of optical signals with different wavelengths after passing through the objective lens and a plurality of dichroic mirrors.
  • the present invention also provides an electronic device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the structured light illumination method as described above when executing the computer program.
  • the present invention also provides a computer-readable medium on which computer instructions are stored.
  • the computer instructions are executed by a processor, the structured light illumination method as described above is implemented.
  • the structured light illumination system utilizes a translation stage to move a grating to quickly switch the phase of structured light stripes, and a beam merging and guiding unit guides light beams of different angles to merge into an object space to quickly switch the angles of structured light stripes, thereby improving the imaging speed, thereby improving the flux of the optical system, improving the resolution of the optical system, and significantly reducing the imaging cost.
  • FIG. 1 is a first structural schematic diagram of a structured light illumination system in Embodiment 1 of the present invention.
  • Figure 2 shows the original OTF spectrum of the structured light super-resolution imaging principle.
  • Figure 3 shows the extended OTF spectrum of the structured light super-resolution imaging principle.
  • FIG. 4 is a second schematic diagram of the structure of the structured light illumination system in Embodiment 2 of the present invention.
  • FIG. 5 is a front view of the structured light illumination system in Embodiment 2 of the present invention.
  • FIG. 6 is a perspective view of the structured light illumination system in Embodiment 2 of the present invention.
  • FIG. 7 is a schematic diagram of the structure of the energy level filter in Embodiment 2 of the present invention.
  • FIG. 8 is a third schematic diagram of the structure of the structured light illumination system in Embodiment 2 of the present invention.
  • FIG. 9 is a flow chart of the structured light illumination method in Embodiment 3 of the present invention.
  • FIG. 10 is a timing flow chart of a structured light illumination system using a high-speed galvanometer in Embodiment 3 of the present invention.
  • FIG. 11 is a front view of the structure of the super-resolution optical system in Example 4 of the present invention.
  • FIG12 is a perspective view of the first structure of the super-resolution optical system in Example 4 of the present invention.
  • FIG. 14 is a schematic diagram showing the optical path structure of the super-resolution optical system in Embodiment 4 of the present invention.
  • FIG. 15 is a first structural schematic diagram of an imaging module of a super-resolution optical system in Embodiment 4 of the present invention.
  • FIG. 16 is a second structural schematic diagram of an imaging module of a super-resolution optical system in Embodiment 4 of the present invention.
  • FIG17 is a schematic diagram of the structure of an electronic device in Embodiment 5 of the present invention.
  • first and second in this document and the descriptions of first, second, etc. that appear in this document are only used for illustration and distinction of the objects of description, and have no order, nor do they represent a special limitation on the number of devices in this document, and cannot constitute any limitation on this document.
  • a first element may be referred to as a second element without departing from the scope of this disclosure, and similarly, a second element may be referred to as a first element.
  • FIG1 is a first structural diagram of a structured light illumination system in this embodiment.
  • the structured light illumination system includes:
  • At least two light sources 1, the light sources are used to generate light beams of preset wavelengths;
  • the light beam merging and guiding unit 4 where the light paths converge, is used to guide the light beams to merge into the object space.
  • the structured light illumination system uses a translation stage to move the grating to quickly switch the phase of the structured light stripes, and the beam merging and guiding unit guides light beams of different angles to merge into the object space to quickly switch the angle of the structured light stripes, thereby improving the imaging speed and thus improving the optical
  • the system throughput improves the resolution of the optical system while significantly reducing the imaging cost.
  • the structured light illumination system of this embodiment is a further improvement on the embodiment 1, specifically:
  • the light beam merging and guiding unit comprises:
  • the galvanometer where the optical path intersects, is used to reflect the light beam to the object space by swinging at at least two preset angles.
  • the high-speed galvanometer can swing at a small angle at a high speed to achieve rapid switching of the structured light direction.
  • the structured light illumination system includes:
  • a first light source and a second light source, the first light source and the second light source are used to generate light beams with different polarization directions;
  • the beam combining and guiding unit includes:
  • Polarization beam combiner where the optical paths intersect, is used to reflect the light beam from the first light source to the object space, and the light beam from the second light source passes through the polarization beam combiner to reach the object space.
  • the illumination structured lights in two different directions use light sources with different polarization states respectively, and then are combined using a polarization beam combiner (PBC) to improve the light efficiency of the system.
  • PBC polarization beam combiner
  • the light source is specifically used to generate at least two light beams of different preset wavelengths; the setting directions of the at least two gratings are orthogonal to each other to generate mutually orthogonal interference fringes; the at least two gratings include modulated gratings, and the modulated gratings have high transmittance at a preselected energy level;
  • the structured light illumination system also includes:
  • At least two collimating lenses are respectively located on the optical path, and the light beam reaches the grating after being collimated by the collimating lenses.
  • the structured light illumination system further includes:
  • At least two filter modules are respectively located on the optical path and are used to filter out the light beams outside the preselected energy level so that the light beams of the preselected energy level can reach the light beam merging and guiding unit.
  • the filtering module includes:
  • the first lens and the second lens are used to form a first 4f system;
  • the grating is located on the front focal plane of the first 4f system, and the galvanometer is located on the back focal plane of the first 4f system;
  • the energy level filter is located on the spectrum plane of the first 4f system and is used to filter out the light beam outside the pre-selected energy level so that the light beam of the pre-selected energy level reaches the galvanometer.
  • the structured light illumination system further includes:
  • the third lens and the fourth lens are used to form a second 4f system; the light beam merging and guiding unit is located on the front focal plane of the second 4f system, and the object plane is located on the back focal plane of the second 4f system.
  • the structured light illumination system also includes:
  • the dichroic mirror is located on the optical path between the third lens and the fourth lens, and is used to reflect the structured light emitted by the third lens toward the object; the object signal generated by the structured light irradiation is received after passing through the dichroic mirror.
  • the point spread function can be characterized by a Bessel function, and the PSF is represented by an optical transfer function (OTF) in the frequency domain.
  • OTF optical transfer function
  • the OTF in the frequency domain, the OTF is equivalent to a spectrum filter.
  • the size of the OTF determines the resolution of the optical system.
  • the low-frequency information within the circle can be collected by the optical system, and the high-frequency information outside the circle will be filtered.
  • the image information collected by the system will contain information of three different frequencies: k+k 0 , k, kk 0.
  • the information of the three frequencies can be calculated through a super-resolution reconstruction algorithm to obtain an expanded frequency domain space, thereby improving the resolution of the system.
  • Figure 4 is a second structural diagram of the structured light illumination system in this embodiment.
  • Figure 4 shows a V-shaped structured light illumination system, in which two structured lights in different directions are generated by two sides of the V-shaped illumination system, and a high-speed galvanometer is installed at the vertex of the V-shaped illumination system.
  • the high-speed and small-angle swing of the galvanometer can realize the rapid switching of the structured light direction.
  • the scheme is described as follows:
  • elements 101 to 106 constitute a first illumination optical path
  • elements 111 to 116 constitute a second illumination optical path.
  • the two illumination optical paths have similar structures, except that the gratings are installed in different directions, forming a 90-degree angle with each other.
  • the two optical paths have similar structures.
  • descriptions such as “light source 101/111” are used below, it means that the two light sources have the same characteristics.
  • descriptions such as “first light source 101, second light source 111” are used, it means that the two light sources have different functions or properties.
  • Light source 101/111 is a laser with optical fiber output as an illumination excitation light source.
  • Light source 101/111 generally includes two laser wavelengths, 532nm (nanometers) and 660nm. The two wavelengths share one optical fiber output, and the power of each wavelength is 4W (watts).
  • the parallel light emitted by the light source 101/111 is collimated by the lens 102/112 and reaches the grating 103/113.
  • the grating 103/113 generates a +-1 level light beam which is projected onto the object plane 124 after passing through several lenses to generate structured light fringes.
  • the installation directions of grating 103 and grating 113 are mutually orthogonal to produce interference fringes in two mutually orthogonal directions; the direction angles of the two gratings can be 0° and 90°, 15° and 105°, etc.
  • the solid line represents the light beam of the first illumination light path
  • the dotted line represents the light beam of the second illumination light path.
  • Grating 103/113 is a modulated grating with a higher transmittance at level +-1.
  • the preferred total transmittance of grating 103/113 at level +-1 is greater than 70%, which improves the energy utilization of the optical system compared to ordinary gratings.
  • the grating 103/113 is mounted on a one-dimensional translation stage (not shown), and the grating is driven to move back and forth by the one-dimensional translation stage to achieve different grating phase switching.
  • the three different phases are
  • the one-dimensional displacement stage is driven by piezoelectric ceramics and can achieve fast movement.
  • the piezoelectric displacement stage load grating moves in 2um (micrometer) steps, its setting time should be less than 10ms (milliseconds) to meet the fast imaging requirements of the system.
  • Lenses 104/114 and lenses 106/116 form a Fourier 4f system, in which gratings 103/113
  • the energy level filter 105/115 is placed on the front focal plane of the 4f system, and the galvanometer 107/117 is placed on the back focal plane of the 4f system.
  • the energy level filter 104/114 only allows the +-1 level light beam emitted from the grating 103/113 to pass through, and the light beams of other energy levels will be filtered out to avoid interference with the structured light.
  • the energy level filter can select a spatial filter.
  • 107 indicates that the galvanometer is at a first angle
  • 117 indicates that the galvanometer is at a second angle.
  • the angle between the two illumination light paths is designed according to the actual mechanical layout requirements. Generally speaking, the smaller the angle, the faster the galvanometer switching speed. Preferably, the angle between the two light paths can be set to 10°.
  • the galvanometer 107/117 reflects the structured light of two different illumination light paths onto the object surface by quickly switching the angle.
  • the structured light of the two light paths has the same phase and the angles are orthogonal to each other.
  • the setting time should be less than 20ms, and the preferred galvanometer system can reach less than 10ms.
  • the galvanometer is at the first angle 107, the solid line light beam of the first illumination light path is reflected into the object space; when the galvanometer rotates to the second angle 117, the dotted line light beam of the second illumination light path will be reflected into the object space.
  • Lens 121 and objective lens 123 form a second 4f system
  • galvanometer 107/117 is on the front focal plane of the 4f system
  • object plane 124 is on the back focal plane of the 4f system.
  • the +-1 level light beams generated by grating 103/113 are overlapped on the object plane through the front and rear 4f systems, thereby generating the required structured light stripes.
  • lens 104/114, lens 106/116, and lens 121 should be a group of achromatic lenses; preferably, through appropriate optical design, lens 104/114, lens 106/116, and lens 121 can be the same lens to reduce hardware costs.
  • the illumination light beam emitted from lens 121 toward the object is reflected by the dichroic mirror and reaches the object plane 124.
  • the dichroic mirror 122 has a high reflectivity near wavelengths of 532nm and 660nm, and can reflect the illumination structured light onto the object plane 124. At the same time, it has a high transmittance for other visible light bands, allowing the fluorescence emitted from the object plane 124 to be transmitted to the camera 126.
  • the sample 124 is generally based on a silicon wafer or glass substrate, on which a biological tissue slice with a fluorescent dye or a regularly arranged DNA (deoxyribonucleic acid) nanosphere is attached.
  • the DNA nanospheres are spaced 360 nm apart. When irradiated by laser, they emit a fluorescent signal.
  • the fluorescent signal passes through the objective lens 123 and the tube lens 125 and is collected by the camera 126.
  • the tube lens 125 and the camera 126 can be one or more. For example, in a common 4-color imaging gene sequencing system, the tube lens 125 and the camera 126 will have four channels.
  • Figure 5 is a front view of the structured light illumination system in this embodiment
  • Figure 6 is an oblique view of the structured light illumination system in this embodiment.
  • the structured light illumination system is used to generate structured light required for illumination, and includes a laser light source, a grating, a piezoelectric displacement stage, a galvanometer, and a plurality of lenses.
  • the left and right optical paths in Figure 5 are arranged symmetrically, and the angle between the grating installation directions of the two optical paths is 90°, thereby generating two mutually orthogonal structured light stripes, and at the same time, the direction of the structured light stripes can be switched at a high speed by quickly swinging the galvanometer 221.
  • all components are installed with the substrate 200 as a reference plane, so as to ensure that the optical axes are on the same plane.
  • the light source 201/211 is a laser light source, using an optical fiber output of NA0.22 (numerical aperture), and the optical fiber port type is FC/APC (a type of optical fiber port) or SMA (a type of optical fiber port).
  • the light source 201/211 is connected to the lighting system through the corresponding optical fiber interface.
  • the light source 201/211 includes two wavelengths, 532nm and 660nm, respectively, and the power of each wavelength is 4W.
  • the light beam emitted by the light source 201/211 is projected onto the grating 203/213 after passing through the collimating lens 202/212.
  • the collimating lens 202/212 is an achromatic lens with a focal length of 50 mm (millimeter).
  • the installation directions of the first grating 203 and the second grating 213 are orthogonal to each other.
  • the first grating 203 is installed at 15° and the second grating 213 is installed at 105°.
  • the grating 203/213 is a modulated grating, and its ⁇ 1st order diffraction efficiency is close to 80%, and its line pair number is 117lp/mm.
  • the first grating 203 and the second grating 213 are mounted on the first piezoelectric displacement stage 204 and the second piezoelectric displacement stage 214 respectively.
  • the piezoelectric displacement stage 204/214 loads the grating 203/213 to move back and forth to achieve switching of three different phases.
  • lens 205/215, lens 207/217, and lens 222 are the same lens. It is an achromatic doublet lens with an effective focal length of 91mm.
  • Lens 205/215 and lens 207/217 form the first set of 4f optical system
  • grating 203/213 is placed on the front focal plane of the 4f optical system
  • galvanometer 221 is placed on the back focal plane of the 4f optical system
  • energy level filter 206/216 is placed on the relay plane of the 4f optical system to achieve energy level filtering function.
  • Lens 205/215, lens 207/217, and energy level filter 206/216 have a mechanical structure of fine adjustment up and down to ensure the accuracy of the focal plane of the 4f system.
  • the energy level filter 206/216 only allows the +-1 order light beams emitted by the grating 203/213 to pass through, and the light beams of other energy levels will be blocked. There is a marking point in the center of the energy level filter 206/216 for aligning the zero order light beam emitted by the grating 203/213.
  • the galvanometer 221 is a high-speed galvanometer that can achieve rapid deflection at a small angle. By changing the angle of the galvanometer 221, the left illumination beam or the right illumination beam can be selected to enter the object space.
  • the left illumination beam and the right illumination beam have different structured light direction angles, thereby achieving the purpose of switching the stripe direction.
  • the time taken for the galvanometer 221 to swing at an angle of 10° is less than 10ms.
  • the lens 222 and the objective lens form a second 4f optical system, thereby imaging the structured light onto the object surface.
  • the lens 222 has the function of front and back adjustment, which can adjust the focal plane of the structured light to ensure that it is imaged onto the sample surface.
  • This embodiment also proposes another structured light illumination system, which can replace the above structured light illumination system.
  • the technical key point of the structured light illumination system is that two illumination structured lights in different directions use light sources with different polarization states, and then combine them using a polarization beam combiner (PBC) to improve the system light efficiency.
  • PBC polarization beam combiner
  • the vertical illumination light path and the horizontal illumination light path have different structured light directions and polarization directions.
  • the structured light direction of the vertical illumination light path is 0°, and the polarization state is s polarization; the structured light direction of the horizontal direction is 90°, and the polarization state is p polarization.
  • the polarization beam combiner 610 both have a transmission efficiency of more than 90%.
  • the two light beams pass through the focusing lens 621 and enter the object side.
  • the subsequent optical path structure is the same as that of the first embodiment.
  • the light source 601/611 is a laser light source, and the light source 601/611 has two output wavelengths, 532nm and 660nm, and the output power of each wavelength is 4W.
  • a spatial light output is used, wherein the polarization state of the first light source 601 is p-polarization, and the polarization state of the second light source 611 is s-polarization.
  • the light beams emitted by the light sources 601/611 directly illuminate the gratings 602/612 to generate structured light in different directions, wherein the polarization state of the first light source 601 is p polarization, and the corresponding stripe direction is 90°; the polarization state of the second light source 611 is s polarization, and the corresponding stripe direction is 0°.
  • Lenses 603/613 and lenses 605/615 form a 4f system
  • grating 602/612 is placed on the front focal plane of the 4f system
  • energy level filter 604/614 is placed on the spectrum plane of the 4f system
  • polarization beam combiner 610 is placed on the back focal plane of the 4f system.
  • Energy level filter 604/614 filters the light beam emitted by grating 602/612 and only allows ⁇ 1 level to pass.
  • the polarization beam combiner 610 is a common polarization optical component, which has a transmittance of more than 90% for p-polarized light incident in the horizontal optical path, and a reflectivity of more than 90% for s-polarized light in the vertical direction. Using the polarization beam combiner 610 instead of the traditional semi-transparent and semi-reflective mirror can greatly improve the light efficiency of the system.
  • the lens 621 and the objective lens 623 form a second 4f system
  • the polarization beam combiner 610 is located on the front focal plane of the 4f system
  • the object plane 624 is located on the back focal plane of the 4f system.
  • the +-1 level light beams generated by the grating 602/612 are overlapped on the object plane through the front and rear 4f systems, thereby generating the required structured light stripes.
  • the illumination light beam emitted from the lens 621 to the object side is reflected by the dichroic mirror 622 to reach the object plane 624.
  • the dichroic mirror 622 has a high reflectivity near the wavelengths of 532nm and 660nm, and can reflect the illumination structured light to the object plane 624. At the same time, it has a high transmittance for other visible light bands, allowing the fluorescence emitted from the object plane 624 to be transmitted to the camera 626.
  • the sample 624 is generally based on a silicon wafer or glass substrate, on which a biological tissue slice with fluorescent dye or regularly arranged DNA nanospheres is attached.
  • the DNA nanospheres are spaced 360nm apart.
  • the tube lens 625 and the camera 626 can also be one channel or multiple channels.
  • the tube lens 625 and the camera 626 may have four paths.
  • FIG9 is a flow chart of the structured light illumination method in this embodiment.
  • the structured light illumination method is applied to the structured light illumination system in Embodiment 1 or Embodiment 2, and the structured light illumination method includes:
  • the structured light illumination method of this embodiment is further described below by taking examples.
  • this embodiment proposes a set of fast-response lower computer control system. As shown in Figure 10, the lower computer system is in the dotted box, and its logic sequence is as follows:
  • the host computer issues a photo-taking command, the galvanometer rotates to a first angle 107, and at the same time, the first translation stage (not shown) carries the first grating 103 and moves it to a first position, corresponding to a first direction and a first phase of the structured light;
  • the first laser 101 After completing action (1), the first laser 101 is turned on, and the camera 126 collects a first set of images. After the collection is completed, the first laser 101 is turned off;
  • the first grating 103 moves to the second and third positions respectively, corresponding to the structured light
  • the second phase in the first direction, the third phase in the first direction, and then the camera 126 collects the second and third sets of pictures respectively. So far, three sets of phase pictures in the first direction of the structured light are collected;
  • the galvanometer mirror rotates to a second angle 117; the second translation stage (not shown) loads the second grating 113 and moves it to the first position;
  • the second laser 111 is turned on, and the camera 126 simultaneously collects the fourth set of images. After the collection is completed, the second laser 111 is turned off;
  • the second grating 113 moves to the second and third positions respectively, corresponding to the second direction phase 2 and the second direction phase 3 of the structured light, and then the camera 126 collects the fifth and sixth groups of pictures respectively, and the laser 2 111 is turned off; at this point, all six groups of pictures are collected for the current FOV;
  • the current FOV image acquisition process ends, the lower computer sends a feedback signal to the upper computer, moves to the next FOV to take a picture, and repeats the above actions.
  • the photographing timing of the structured light illumination method of this embodiment is as follows:
  • the first grating 602 moves to a first phase position, the first laser 601 is turned on, and the camera 626 collects a first set of pictures;
  • the first grating 602 moves to the second and third phase positions respectively, and the camera 626 collects the second and third groups of pictures. After the collection is completed, the first laser 601 is turned off;
  • the second grating 612 moves to the second and third phase positions respectively, and the camera 626 collects the fifth and sixth groups of pictures. After the collection is completed, the second laser 611 is turned off.
  • the lower computer sends a feedback signal to the upper computer, moves to the next FOV to take a picture, and repeats the above actions.
  • This embodiment provides a super-resolution optical system, which includes a substrate, an imaging module, an auto-focus module, and an illumination module; the imaging module, the auto-focus module, and the illumination module are fixed on the substrate; the illumination module adopts the structured light illumination system in Embodiment 1 or Embodiment 2;
  • the autofocus module is coupled to the illumination module via a galvanometer, and the autofocus module is used to drive the objective lens to achieve autofocus;
  • the lighting module is used to emit a lighting structure light beam which is projected onto the surface of the sample to be photographed through the galvanometer and the objective lens;
  • the imaging module is used to receive the optical signal generated by the sample to be photographed after being irradiated by the illumination structure light beam; the optical signal is divided into several optical signals with different wavelengths after passing through the objective lens and several dichroic mirrors.
  • Figure 11 is a front view of the optical system structure
  • Figure 12 is a first oblique view of the optical system structure
  • Figure 13 is a second oblique view of the optical system structure.
  • some structural support parts are hidden in the figure, but it does not affect the function of the entire optical system.
  • the optical system structure in Figures 11-13 includes a substrate 300, an imaging module 301, an illumination module 302, an autofocus module 303, and a sample 304.
  • the entire optical system is based on the substrate 300, and the imaging module 301, the illumination module 302, and the autofocus module 303 are all installed on the substrate 300.
  • the illumination module 302 has been described in detail in the first embodiment.
  • the structure and function of the imaging module 301 and the autofocus module 303 will be specifically introduced by taking the four-color gene sequencing system as an example.
  • FIG14 shows a schematic diagram of the optical path structure of the imaging module of the super-resolution optical system.
  • the illumination module 406 is coupled with the autofocus module 405 through the galvanometer 404, it enters the object side through the first dichroic mirror 403.
  • the galvanometer 404 is also a dichroic mirror in this embodiment, and the specific specification is a 750nm long-pass dichroic mirror.
  • the operating wavelength of the autofocus module 405 is 820nm.
  • the detection light emitted by the autofocus module 405 passes through the galvanometer 404, the first dichroic mirror 403, the objective lens 402 in sequence, and then reaches the sample 401, and then returns to the autofocus module 405 along the original path.
  • the autofocus module 405 calculates the defocus amount of the sample 401 according to the returned detection signal, it drives the objective lens 402 to perform a focusing action to realize the autofocus function.
  • the illumination structure light beam emitted by the illumination module 406 is reflected by the galvanometer 404 and the first dichroic mirror 403 , and then projected onto the surface of the sample 401 through the objective lens 402 .
  • the fluorescence signal emitted by the sample 401 is collected by the objective lens 402, it is divided into four paths according to the fluorescence wavelength from short to long through a number of dichroic mirrors;
  • the first dichroic mirror 403 has a reflectivity of more than 90% near 532nm, 660nm and 820nm, and a transmittance of more than 90% for other visible light bands; the spectral parameter of the second dichroic mirror 407 is 660nm long pass; the spectral parameter of the third dichroic mirror 408 is 585nm long pass; the spectral parameter of the fourth dichroic mirror 409 is 705nm long pass;
  • the wavelength of the first fluorescence signal is 550nm-580nm, which is reflected by the second dichroic mirror 407, then passes through the third dichroic mirror 408, and finally is focused to the camera 1 412 through the tube lens 1 411;
  • the second fluorescent signal has a wavelength of 590nm-640nm, is reflected by the second dichroic mirror 407, then reflected by the third dichroic mirror 408, and finally focused to the second camera 422 via the second tube lens 421;
  • the third fluorescent signal has a wavelength of 680nm-695nm, passes through the second dichroic mirror 407, is then reflected by the fourth dichroic mirror 409, and is finally focused to the third camera 432 via the third tube lens 431;
  • the fourth fluorescent signal has a wavelength of 710nm-770nm, passes through the second dichroic mirror 407, then passes through the fourth dichroic mirror 409, and finally is focused to the fourth camera 442 via the fourth tube lens 441;
  • FIG15 and FIG16 show the physical structure diagram of the super-resolution optical system imaging module.
  • the imaging module is installed on the imaging substrate 500 , and a plurality of dichroic mirrors, reflective mirrors, tube mirrors and cameras are all installed on the substrate 500 .
  • the illumination module and the autofocus module are connected to the imaging module through the port where the first dichroic mirror 503 is located. After integration, it is shown in Figures 11-13.
  • the fluorescence signal emitted by the sample 501 is collected by the objective lens 502, it is divided into four paths according to the fluorescence wavelength from short to long through a number of dichroic mirrors;
  • the dichroic mirrors 503/507/508/509 all have a structure for fine-tuning the angle to ensure the accuracy of the optical axis angle.
  • the tube lenses 511/521/531/541 collect the fluorescence signals emitted from the object side and focus them into the cameras 512/522/532/542 respectively.
  • the focal length of the tube lenses 511/521/531/541 is 246 mm, and they are all achromatic lenses.
  • the camera 512/522/532/542 is a CMOS camera with a resolution of 7000*7000 pixels and a high quantum conversion efficiency (QE).
  • a reflector 513/523/533/543 is added between the tube lens 511/521/531/541 and the camera 512/522/532/542 to fold the light path and reduce the size of the optical module.
  • FIG17 is a schematic diagram of the structure of an electronic device provided in Example 5 of the present invention.
  • the electronic device includes a memory, a processor, and a computer program stored in the memory and executable on the processor, and the processor implements the structured light illumination method of Example 3 when executing the program.
  • the electronic device 30 shown in FIG17 is only an example and should not bring any limitation to the functions and scope of use of the embodiments of the present invention.
  • the electronic device 30 may be in the form of a general-purpose computing device, for example, it may be a server device.
  • the components of the electronic device 30 may include, but are not limited to: at least one processor 31, at least one memory 32, and a bus 33 connecting different system components (including the memory 32 and the processor 31).
  • the bus 33 includes a data bus, an address bus, and a control bus.
  • the memory 32 may include volatile memory, such as random access memory (RAM) 321 and/or Or cache memory 322 , and may further include read-only memory (ROM) 323 .
  • RAM random access memory
  • ROM read-only memory
  • the memory 32 may also include a program/utility 325 having a set (at least one) of program modules 324, such program modules 324 including but not limited to: an operating system, one or more application programs, other program modules, and program data, each of which or some combination may include an implementation of a network environment.
  • program modules 324 including but not limited to: an operating system, one or more application programs, other program modules, and program data, each of which or some combination may include an implementation of a network environment.
  • the processor 31 executes various functional applications and data processing by running the computer program stored in the memory 32, such as the structured light illumination method of embodiment 3 of the present invention.
  • the electronic device 30 may also communicate with one or more external devices 34 (e.g., keyboards, pointing devices, etc.). Such communication may be performed via an input/output (I/O) interface 35.
  • the model-generated device 30 may also communicate with one or more networks (e.g., a local area network (LAN), a wide area network (WAN), and/or a public network, such as the Internet) via a network adapter 36.
  • the network adapter 36 communicates with other modules of the model-generated device 30 via a bus 33.
  • model-generated device 30 may be used in conjunction with the model-generated device 30, including but not limited to: microcode, device drivers, redundant processors, external disk drive arrays, RAID (RAID) systems, tape drives, and data backup storage systems, etc.
  • This embodiment provides a computer-readable storage medium on which a computer program is stored.
  • the program is executed by a processor, the structured light illumination method of embodiment 3 is implemented.
  • the readable storage medium may include but is not limited to: a portable disk, A hard disk, a random access memory, a read-only memory, an erasable programmable read-only memory, an optical storage device, a magnetic storage device, or any suitable combination of the above.
  • the present invention may also be implemented in the form of a program product, which includes a program code.
  • the program product When the program product is run on a terminal device, the program code is used to enable the terminal device to execute the structured light illumination method of embodiment 3.
  • the program code for executing the present invention can be written in any combination of one or more programming languages, and the program code can be executed completely on the user device, partially on the user device, as an independent software package, partially on the user device and partially on a remote device, or completely on the remote device.

Landscapes

  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un système d'éclairage à lumière structurée, un procédé d'éclairage à lumière structurée et un système optique à super-résolution. Le système d'éclairage à lumière structurée comprend : au moins deux sources de lumière (1), les sources de lumière (1) étant utilisées pour générer des faisceaux lumineux d'une longueur d'onde prédéfinie ; au moins deux réseaux (2) situés respectivement sur les trajets optiques desdites au moins deux sources de lumière (1), les réseaux (2) étant utilisés pour générer des franges d'interférence ; des plateformes de déplacement (3), les réseaux (2) étant disposés sur les plateformes de déplacement (3), et les plateformes de déplacement (3) étant utilisées pour déplacer les réseaux (2) de façon à effectuer une commutation de phase ; et une unité de convergence et de guidage de faisceau lumineux (4), les trajets optiques se croisant au niveau de l'unité de convergence et de guidage de faisceau lumineux (4), et l'unité de convergence et de guidage de faisceau lumineux (4) étant utilisée pour guider les faisceaux lumineux pour converger vers un espace d'objet. Le système d'éclairage à lumière structurée peut effectuer une commutation d'angle et de phase rapide sur des franges de lumière structurée et augmente la vitesse d'imagerie, augmentant ainsi le débit du système optique, et réduisant considérablement les coûts d'imagerie tout en améliorant la résolution du système optique.
PCT/CN2023/120198 2023-09-20 2023-09-20 Système d'éclairage à lumière structurée, procédé d'éclairage à lumière structurée et système optique à super-résolution Pending WO2025059947A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/120198 WO2025059947A1 (fr) 2023-09-20 2023-09-20 Système d'éclairage à lumière structurée, procédé d'éclairage à lumière structurée et système optique à super-résolution

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/120198 WO2025059947A1 (fr) 2023-09-20 2023-09-20 Système d'éclairage à lumière structurée, procédé d'éclairage à lumière structurée et système optique à super-résolution

Publications (1)

Publication Number Publication Date
WO2025059947A1 true WO2025059947A1 (fr) 2025-03-27

Family

ID=95073367

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/120198 Pending WO2025059947A1 (fr) 2023-09-20 2023-09-20 Système d'éclairage à lumière structurée, procédé d'éclairage à lumière structurée et système optique à super-résolution

Country Status (1)

Country Link
WO (1) WO2025059947A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110093291A (ko) * 2010-02-12 2011-08-18 한국과학기술원 십자형 구조 조명 선형 패턴을 이용한 구조 조명 공초점 현미경
US20150211986A1 (en) * 2012-06-29 2015-07-30 Zhejiang University Super-resolution microscopy method and device
WO2019143564A1 (fr) * 2018-01-16 2019-07-25 Illumina, Inc. Imagerie à éclairage structuré par sélection spatiale d'angle de motif
US20200142171A1 (en) * 2017-05-12 2020-05-07 Suzhou Institute Of Biomedical Engineering And Technology, Chinese Academy Of Sciences Structured illumination microscopic imaging system
US20200218052A1 (en) * 2018-01-16 2020-07-09 Illumina, Inc. Multi-Arm Structured Illumination Imaging
CN112798564A (zh) * 2020-12-22 2021-05-14 中国科学院苏州生物医学工程技术研究所 随机光学重建与结构光照明复合超分辨成像系统

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110093291A (ko) * 2010-02-12 2011-08-18 한국과학기술원 십자형 구조 조명 선형 패턴을 이용한 구조 조명 공초점 현미경
US20150211986A1 (en) * 2012-06-29 2015-07-30 Zhejiang University Super-resolution microscopy method and device
US20200142171A1 (en) * 2017-05-12 2020-05-07 Suzhou Institute Of Biomedical Engineering And Technology, Chinese Academy Of Sciences Structured illumination microscopic imaging system
WO2019143564A1 (fr) * 2018-01-16 2019-07-25 Illumina, Inc. Imagerie à éclairage structuré par sélection spatiale d'angle de motif
US20200218052A1 (en) * 2018-01-16 2020-07-09 Illumina, Inc. Multi-Arm Structured Illumination Imaging
CN112798564A (zh) * 2020-12-22 2021-05-14 中国科学院苏州生物医学工程技术研究所 随机光学重建与结构光照明复合超分辨成像系统

Similar Documents

Publication Publication Date Title
CN107167929B (zh) 基于dmd的双模式光学超分辨显微成像装置及方法
CN102928970B (zh) 一种大样本快速三维显微成像的方法和系统
CN105929560B (zh) 一种宽带远场超分辨成像装置
CN103713390B (zh) 一种多波长激光合束选通调试系统及方法
CN104568872B (zh) 具有光学层析能力的荧光显微光谱成像系统
US9753265B2 (en) Variable orientation illumination-pattern rotator
CN109682819B (zh) 一种平铺光片选择性平面照明显微镜
US9766442B2 (en) Confocal scanner and confocal microscope
US10520713B2 (en) System for confocal illumination of a sample
JP2025515943A (ja) 超解像度単対物レンズ光学シート顕微結像光学システム及びその結像システム
JP4854880B2 (ja) レーザー顕微鏡
CN110794562A (zh) 一种显微镜阵列
WO2024217203A1 (fr) Système d'imagerie par fluorescence tridimensionnelle à feuille de lumière à objectif unique
US20220397751A1 (en) Re-scan microscope system and method
KR20000073448A (ko) 반사형 칼라 프로젝터
CN111504968A (zh) 四色激光照明荧光显微镜
CN111650739B (zh) 基于dmd的单帧曝光快速三维荧光成像系统及方法
WO2025059947A1 (fr) Système d'éclairage à lumière structurée, procédé d'éclairage à lumière structurée et système optique à super-résolution
WO2012177206A1 (fr) Systèmes de commandes de polarisation
CN106979460A (zh) 一种荧光光源及荧光显微成像系统
CN207018828U (zh) 一种荧光光源及荧光显微成像系统
CN203303034U (zh) 一种生物神经环路活体成像系统
CN104083146B (zh) 一种生物神经环路活体成像系统
WO2025129599A1 (fr) Système et procédé d'imagerie, appareil et procédé d'identification, séquenceur et procédé de séquençage
CN107941777B (zh) 一种抗漂白单分子定位三维超分辨显微系统

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23952607

Country of ref document: EP

Kind code of ref document: A1