WO2020211018A1 - 一种受激辐射损耗超分辨成像系统及方法 - Google Patents

一种受激辐射损耗超分辨成像系统及方法 Download PDF

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Publication number
WO2020211018A1
WO2020211018A1 PCT/CN2019/083072 CN2019083072W WO2020211018A1 WO 2020211018 A1 WO2020211018 A1 WO 2020211018A1 CN 2019083072 W CN2019083072 W CN 2019083072W WO 2020211018 A1 WO2020211018 A1 WO 2020211018A1
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light
excitation light
excitation
unit
erasing
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PCT/CN2019/083072
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English (en)
French (fr)
Inventor
严伟
王佳林
张佳
王璐玮
郭勇
屈军乐
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深圳大学
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Priority to PCT/CN2019/083072 priority Critical patent/WO2020211018A1/zh
Publication of WO2020211018A1 publication Critical patent/WO2020211018A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes

Definitions

  • the invention relates to the field of optical imaging microscopy, in particular to a super-resolution imaging system and method for stimulated radiation loss.
  • Stimulated radiation loss Emission Depletion (STED) microscopy is a super-resolution imaging technology, which can break through the limitation of diffraction limit on the resolution of optical microscopes and realize super-resolution microscopy imaging.
  • STED technology is a spatial processing technology for scanning imaging systems. Its principle is to use two laser beams for microscopic imaging, one of which is excitation light for exciting fluorescent materials; and the other is The spot shape is circular, and the erasure light (also called STED light) that is coaxial with the excitation light and whose wavelength matches the emission wavelength of the fluorescent molecule is used to generate stimulated radiation in the overlapping area of the two beams, and in the central area
  • the fluorescent molecules are not affected by erasing light, only spontaneous emission occurs. Since the wavelength of the spontaneous radiation and the wavelength of the stimulated radiation are different, the light of the stimulated radiation can be filtered out through the filter to achieve the goal of reducing the imaging spot, thereby improving the resolution of the system.
  • the main purpose of the embodiments of the present invention is to provide a stimulated radiation loss super-resolution imaging system and method, which can at least solve the problem of generating excitation light and rubbing light by setting two separate lasers when performing STED super-resolution imaging in related technologies.
  • the problems of high system hardware redundancy, limited application range and high imaging cost are caused.
  • the first aspect of the embodiments of the present invention provides a stimulated radiation loss super-resolution imaging system, including: a laser, a beam splitting unit, a first optical path adjustment unit, a scanning unit, and an imaging unit;
  • the laser is used to generate incident light
  • the light splitting unit is used to separate the incident light into excitation light and erasure light with different wavelengths
  • the first optical path adjustment unit is used to adjust the directions of the excitation light and the erasing light, and overlap the excitation light and the erasing light;
  • the scanning unit is used to synchronously scan the overlapping excitation light and erasing light to the sample to be imaged;
  • the imaging unit is used to perform STED super-resolution imaging on the fluorescent signal generated by the excitation of the sample to be imaged.
  • the second aspect of the embodiments of the present invention provides a stimulated radiation loss super-resolution imaging method, which is applied to the above-mentioned stimulated radiation loss super-resolution imaging system, and the stimulated radiation loss super-resolution imaging method includes:
  • the light splitting unit separates the incident light generated by the laser into excitation light and erasure light with different wavelengths
  • the first light path adjustment unit adjusts the directions of the excitation light and the erasing light, and overlaps the excitation light and the erasing light;
  • the scanning unit synchronously scans the overlapping excitation light and erasing light to the sample to be imaged
  • the imaging unit performs STED super-resolution imaging on the fluorescent signal generated by the excitation of the sample to be imaged.
  • the incident light generated by the laser is separated into excitation light and erasing light with different wavelengths by a light splitting unit; then the excitation light is adjusted by the first optical path adjustment unit And the direction of the erasing light, overlap the excitation light and erasing light; then the scanning unit scans the overlapped excitation light and erasing light to the sample to be imaged; finally, the fluorescence generated by the excitation of the sample to be imaged by the imaging unit
  • the signal undergoes STED super-resolution imaging.
  • the light splitting unit separates the two types of lasers required for STED super-resolution imaging from a single light source through light splitting, which can effectively reduce system hardware redundancy, expand the application range of the system, and save Imaging costs.
  • FIG. 1 is a structural block diagram of a super-resolution imaging system for stimulated radiation loss according to the first embodiment of the present invention
  • FIG. 2 is a schematic diagram of STED super-resolution imaging provided by the first embodiment of the present invention.
  • FIG. 3 is a schematic structural diagram of a two-color stimulated radiation loss super-resolution imaging system provided by a second embodiment of the present invention.
  • 4a to 4d are respectively the transmittance graphs of DM1, DM2, DMD1, and DMD2 provided by the second embodiment of the present invention.
  • FIG. 5 is a diagram showing the overlapping effect of excitation light and erasure light provided by the second embodiment of the present invention.
  • FIG. 6 is a schematic flow chart of super-resolution imaging of stimulated radiation loss provided by the third embodiment of the present invention.
  • the stimulated radiation loss STED super-resolution imaging system proposed in this embodiment includes: a laser 101, a beam splitting unit 102, a first Optical path adjustment unit 103, scanning unit 104, and imaging unit 105;
  • the laser 101 is used to generate incident light
  • the light splitting unit 102 is used to separate incident light into excitation light and erasure light with different wavelengths
  • the first light path adjustment unit 103 is used to adjust the directions of the excitation light and the erasing light, and overlap the excitation light and the erasing light;
  • the scanning unit 104 is configured to synchronously scan the overlapped excitation light and erasing light to the sample to be imaged;
  • the imaging unit 105 is used to perform STED super-resolution imaging on the fluorescent signal generated by the excitation of the sample to be imaged.
  • the excitation light and the erasing light source required for STED super-resolution imaging come from the same light source, and two types of light can be obtained from a single light source in the form of light splitting by using a light splitting unit.
  • the laser can be a white light laser, and the incident light generated by the laser supports at least two different wavelengths of light.
  • the white light laser can be a supercontinuum femtosecond pulsed white light laser.
  • the two separated lights are superimposed and then irradiated on the sample, so that the sample is stimulated by radiation, and the fluorescent material on the sample is excited to generate a fluorescent signal, and then the fluorescent signal is transmitted Perform STED super-resolution imaging to the imaging unit for observation processing.
  • FIG. 2 shows the principle diagram of STED super-resolution imaging provided by this embodiment.
  • the stimulated radiation loss super-resolution imaging system further includes: a second optical path adjustment unit; the second optical path adjustment unit includes a low-pass filter and a polarizer.
  • the low-pass filter is used to filter out the light of the target wavelength from the incident light generated by the laser, and the polarizer is used to shape the light of the target wavelength emitted by the low-pass filter into linearly polarized light; correspondingly, the light splitting unit It is specifically used to separate the linearly polarized light after shaping into excitation light and erasure light with different wavelengths.
  • the incident light generated by the laser can be transmitted to the low-pass filter through the single-mode fiber, and the laser light emitted from the femtosecond laser can be modulated through the single-mode fiber.
  • the light splitting unit 102 includes: a first beam splitting sub-unit, a second beam splitting sub-unit, and a band-pass filtering sub-unit; the first beam splitting sub-unit is used to separate incident light into units of different wavelengths. Channel excitation light and erasing light; the band-pass filter subunit is used to separate the single channel excitation light into multiple channels of excitation light; the second beam splitting subunit is used to separate the single channel erasing light into multiple channels corresponding to the multiple channels of excitation light Road erase light.
  • the laser of a single wavelength can only achieve monochromatic STED super-resolution imaging.
  • the single-channel excitation can be achieved through the band-pass filter subunit. The light is separated into multiple paths, and the single-path erasing light is separated into multiple erasing lights corresponding to the excitation light by the second beam splitting subunit.
  • the first light path adjustment unit is specifically configured to adjust the directions of the multiple excitation light and the multiple erasing light, overlap each excitation light with the corresponding erasing light, and combine the formed multiple overlapping light Adjusted to be coaxial
  • the scanning unit is specifically used to synchronously scan multiple overlapping lights to the sample to be imaged
  • the imaging unit is specifically used to perform multi-color STED super-resolution imaging of all fluorescent signals generated by the sample to be imaged.
  • the band-pass filter sub-unit is realized by two band-pass filters, that is, one band-pass filter is used to filter one excitation light, and the second beam splitting sub-unit Then a single beam splitter is realized.
  • a corner reflector can also be provided on the transmission light path of the multiple excitation light to change the light path of the excitation light, which can control the time between the excitation light and the erasing light. Pulse interval.
  • the light splitting unit 102 further includes: a spiral phase plate; the spiral phase plate is used to convert the wavefront of the multipath erased light from a Gaussian distribution to a ring distribution.
  • a spiral phase plate is used to convert the erasing light into a vortex beam, that is, the axis in the propagation direction of the wave front of the beam spirals forward. This rotation causes the beams to cancel each other out at the optical axis, and the projection On a flat surface, a halo with a dark hole in the center is formed.
  • the first light path adjustment unit 103 includes : The first dichroic mirror, the second dichroic mirror, the third dichroic mirror and the fourth dichroic mirror; the first dichroic mirror is used to reflect the first excitation light and transmit corresponding to the first The first erasing light of the excitation light is used to overlap the first excitation light and the first erasing light; the second dichroic mirror is used to reflect the second excitation light and transmit it corresponding to the second excitation The second path of light is used for erasing light to overlap the second path of excitation light and the second path of erasing light; the third dichroic mirror is used to reflect the overlapped second path of excitation light and second path of erasing light; The fourth dichroic mirror is used to reflect the overlapping first path of excitation light and first path of erasing light.
  • the separated excitation light and erasing light are respectively two paths.
  • the first excitation light and the second excitation light The wavelengths of the light, the first erasing light, and the second erasing light may be 488 nm, 635 nm, 592 nm, and 775 nm, respectively.
  • the excitation light and erasing light separated in this embodiment need to be adjusted in the optical path, that is, through the first dichroic mirror and the second dichroic mirror respectively to form an overlapped light, and then pass the third and second The dichroic mirror and the fourth dichroic mirror adjust the formed two overlapping lights to be coaxial.
  • the third dichroic mirror and the fourth dichroic mirror can be arranged on the optical path between the scanning unit and the imaging unit, so that the third dichroic mirror The color mirror and the fourth dichroic mirror are also used to transmit the fluorescent signal during the transmission of the fluorescent signal to the imaging unit.
  • the scanning unit 104 includes: a scanning galvanometer, a quarter glass slide, and a high numerical aperture objective lens set at a time in the propagation direction of the optical path.
  • the scanning galvanometer is used to synchronously scan the overlapping excitation light and erased light;
  • the quarter glass slide is used to convert the light emitted by the scanning galvanometer from linear polarization to circular polarization; for high numerical aperture objective lenses To focus the light emitted from a quarter glass slide to the sample to be imaged.
  • the imaging unit 105 includes a narrow band pass filter, a photomultiplier tube, and a display and processing subunit.
  • the narrow-band band-pass filter is used to filter the stray light outside the target band in the fluorescent signal generated by the excitation of the sample to be imaged;
  • the photomultiplier tube is used to amplify the fluorescent signal emitted by the narrow-band band-pass filter; display and processing
  • the sub-unit is used to perform STED super-resolution imaging processing on the fluorescence signal emitted by the photomultiplier tube.
  • a multimode optical fiber can be used for transmission.
  • the fiber core can be used as a small hole to receive the fluorescent signal focused by the lens and eliminate impurities. The effect of astigmatism.
  • the incident light generated by the laser is separated into excitation light and erasing light with different wavelengths through a light splitting unit; then the first optical path adjustment unit adjusts the excitation light and erasing light Direction, the excitation light and the erasing light are overlapped; then the scanning unit scans the overlapped excitation light and erasing light to the sample to be imaged; finally the imaging unit performs STED super-resolution on the fluorescence signal generated by the excitation of the sample to be imaged Imaging.
  • the light splitting unit separates the two types of lasers required for STED super-resolution imaging from a single light source through light splitting, which can effectively reduce system hardware redundancy, expand the application range of the system, and save Imaging costs.
  • FIG. 3 shows a schematic structural diagram of the dual-color stimulated radiation loss super-resolution imaging system provided by this embodiment, including:
  • Supercontinuum femtosecond pulsed white light laser used to generate white light source that can be divided into four wavelengths;
  • Single-mode fiber is used for mode control of the laser emitted from the femtosecond laser
  • Low-pass filter (F1) used to filter out the light wavelength within the required range
  • Polarizer (H) used to ensure that the laser in the optical path is linearly polarized
  • the beam splitter (PBS1, PBS2) can split the beam and adjust the splitting ratio;
  • VPP Spiral phase plate
  • Dichroic mirror (DM1) used to reflect the first excitation light (488nm), transmit the first erasing light (592nm) and the second excitation light (635nm), and it can also fine-tune the first excitation light Transmission direction, so that excitation light and loss light can overlap well;
  • the dichroic mirror (DM2) is used to reflect the second excitation light (635nm) and transmit the second erasing light (775nm). At the same time, it can also fine-tune the transmission direction of the second excitation light to make the excitation light and the loss light Can overlap well;
  • Dichroic mirror used to reflect the second excitation light and erasure light (635nm and 775nm), while transmitting the light of the fluorescent signal generated by the sample after being excited;
  • Dichroic mirror used to reflect the first excitation light and erasure light (488nm and 592nm), transmit the second excitation light and erasure light (635nm and 775nm), and transmit the sample after being excited The fluorescent signal of light;
  • the corner reflector (RR) is used to change the light path of the excitation light, which can control the pulse interval between the excitation light and the erasing light in time;
  • Lens used to focus or diverge the incident laser light, the combined use can enlarge or reduce the spot diameter
  • the galvanometer scanning system (Scanner) is used to scan the overlapped excitation light and erasure light to realize the area array imaging of the sample;
  • Quarter glass used to convert the erasing laser from linear polarization to circular polarization
  • Bandpass filters F2, F3, F4, F5
  • Narrow band pass filter used to transmit the fluorescence signal of the band to be collected, and filter the stray light outside this band;
  • Multimode fiber is used to transmit the collected fluorescent signal to the photomultiplier tube PMT;
  • the fiber core can be used as a small hole to receive the fluorescent signal focused by the lens L3 and eliminate the influence of stray light;
  • Photomultiplier tube used to receive the fluorescent signal of the sample and amplify the signal
  • a high numerical aperture objective lens with a magnification of 100 times and a numerical aperture of 1.4, is used to focus the overlapping excitation light and loss light, while collecting the fluorescence signal reflected by the sample.
  • Figures 4a to 4d respectively show the transmittance curves of DM1, DM2, DMD1 and DMD2 provided in this embodiment.
  • Table 1 shows the model table of each filter and dichroic mirror provided in this embodiment.
  • the white-light laser emits broadband laser. After passing through the low-pass filter F1, only the laser with wavelength below 800nm is allowed to pass, and then the laser is linearly polarized by the polarizer H, and then passes through the beam splitter PBS1, and the divided two lights can be used as excitations respectively. Light and erase light.
  • the excitation light passes through the wheel, and F2 and F3 are arranged on the wheel, which can pass the excitation light of 488nm and 635nm respectively, and the specific excitation wavelength can be selected by rotating the wheel; the 488nm excitation light and the 635nm excitation light enter through the same path Corner reflector (RR), the corner reflector can adjust the optical path of the excitation light, and can adjust the excitation light of both wavelengths, provided that the position of the corner reflector must be strictly perpendicular to the optical path, otherwise the light When the path is adjusted, the propagation of the optical path will be biased. After the delay, the excitation light reaches the position of the first dichroic mirror DM1. The best position is when the dichroic mirror in the system forms an angle of 45° with the incident light.
  • the erased light passes through two lenses L1 and L2 (the focal length of L2 is twice that of L1) to magnify the spot twice, then passes through the spiral phase plate VPP to form a ring light, and then is divided into two beams by the beam splitter PBS2, one of which The light passes through the band-pass filter F4 to become a 592nm narrow-band laser, which is used as the first erasing light to reach the first dichroic mirror DM1; the other beam passes through the band-pass filter F5 to become a 775nm narrow-band laser as the second The erasing light reaches the second dichroic mirror DM2.
  • the wheel filter When the wheel filter is selected as F2, there is only 488nm excitation light in the excitation light path. At this time, the 488nm excitation light is reflected at DM1, the first 592nm erasing light is transmitted here, and the 488nm excitation light is The 592nm erasing light overlaps at DM1, and DM1 can adjust the overlap of the 488nm excitation light and 592nm erasing light to form the effect shown in FIG. 5. As shown in FIG. 5, the excitation light and erasing provided by this embodiment The effect of light overlap. After the 488nm excitation light and 592nm erasing light are overlapped, they are reflected by DMD2.
  • the overlapped light passes through the mirror M and enters the scanning galvanometer Scanner for synchronous scanning, and then passes through the quarter glass QWP to convert the erasing light from linear polarization to linear polarization. Circular polarization, and finally through a high numerical aperture objective lens (OL), the sample is scanned to form an area array imaging. After the sample emits fluorescence, the fluorescence returns along the incident light path to reach DMD2 and transmits. It also transmits at DMD1, and then passes through lens L3 focusing and narrow band pass filter F6 to pass through the fluorescence signal of the wavelength to be collected, and filter out the band. The stray light enters the multimode fiber (MMF) to reach the photomultiplier tube PMT. Finally, the PMT performs signal amplification and transmits the signal data to the display and processing sub-unit (such as a computer) for imaging for observation and processing.
  • MMF multimode fiber
  • the wheel filter When the wheel filter is selected as F3, there is only 635nm wavelength excitation light in the excitation light path. At this time, the 635nm excitation light is transmitted at DM1 and reflected at DM2, and the second 775nm erasure light occurs here. In transmission, 635nm excitation light and 775nm erasing light overlap at DM2, and DM2 can adjust the overlap of 635nm excitation light and 775nm erasing light. After the 635nm excitation light and 775nm erasure light overlap, they are reflected by DMD1 and transmitted at DMD2.
  • the coincident light then enters the scanning galvanometer for synchronous scanning, and then passes through a quarter glass slide to convert the erased light from linear polarization to circular polarization, and finally passes through the objective lens to scan the sample to form an area array Imaging.
  • the fluorescence returns along the incident light path to reach DMD2 and then transmits. It also transmits at DMD1, and then passes through L3 focusing and F6 to transmit the fluorescence signal of the wavelength band to be collected, and filter the stray light outside this band to enter the multimode Optical fiber (MMF) reaches the PMT.
  • the PMT performs signal amplification and transmits the signal data to the display and processing sub-unit (such as a computer) for imaging for observation and processing.
  • the system design can achieve dual-color STED imaging using a single white light source, which also expands the application range of the STED system while saving material costs.
  • This embodiment also provides a stimulated radiation loss super-resolution imaging method, which is applied to the stimulated radiation including the laser, the beam splitting unit, the first optical path adjustment unit, the scanning unit, and the imaging unit described in the previous embodiment.
  • Loss super-resolution imaging system as shown in FIG. 6, the stimulated radiation loss super-resolution imaging method proposed in this embodiment includes the following steps:
  • Step 601 The light splitting unit separates the incident light generated by the laser into excitation light and erasure light with different wavelengths;
  • Step 602 The first light path adjustment unit adjusts the directions of the excitation light and the erasing light, and overlaps the excitation light and the erasing light.
  • Step 603 The scanning unit synchronously scans the overlapped excitation light and erasing light to the sample to be imaged;
  • Step 604 The imaging unit performs STED super-resolution imaging on the fluorescent signal generated by the excitation of the sample to be imaged.
  • the excitation light and the erasing light source required for STED super-resolution imaging come from the same light source, and two types of light can be obtained from a single light source in the form of light splitting.
  • the sample is stimulated by radiation, so that the fluorescent material on the sample is excited to generate a fluorescent signal, and then the fluorescent signal is transmitted to the imaging unit for STED ultrasound.
  • Resolution imaging for observation processing
  • the stimulated radiation loss super-resolution imaging system further includes: a second optical path adjustment unit; the second optical path adjustment unit includes a low-pass filter and a polarizer; the beam splitting unit combines the incident light generated by the laser Before separating into excitation light and erasing light with different wavelengths, it also includes: a low-pass filter filters out the light of the target wavelength from the incident light generated by the laser; the polarizer will obtain the light of the target wavelength emitted by the low-pass filter into Linearly polarized light.
  • the splitting unit separating the incident light generated by the laser into excitation light and erasing light with different wavelengths includes: the splitting unit separates the linearly polarized light after shaping into excitation light and erasing light with different wavelengths.
  • the light splitting unit includes: a first beam splitting subunit, a second beam splitting subunit, and a bandpass filter subunit; the light splitting unit separates the incident light generated by the laser into excitation light with different wavelengths and erases
  • the light includes: the first beam splitting sub-unit separates the incident light generated by the laser into single-channel excitation light and erasing light with different wavelengths; the band-pass filter sub-unit separates the single-channel excitation light into multiple excitation lights, and the second beam-splitting sub-unit
  • the unit separates the single-path erasing light into multiple-path erasing light corresponding to the multiple-path excitation light.
  • the first light path adjustment unit adjusts the directions of the excitation light and the erasing light, and superimposing the excitation light and the erasing light includes: the first light path adjustment unit adjusts the directions of the multiple excitation light and the multiple erasing light, The excitation light is overlapped with the corresponding erasing light, and the formed multiple overlapping lights are adjusted to be coaxial; the scanning unit synchronously scans the multiple overlapping lights to the sample to be imaged, including: the scanning unit uniforms the multiple overlapping lights Synchronous scanning to the sample to be imaged; the imaging unit performs STED super-resolution imaging of the fluorescent signal generated by the excitation of the sample to be imaged, including: the imaging unit performs multi-color STED super-resolution imaging of all the fluorescent signals generated by the excitation of the sample to be imaged.
  • the light splitting unit further includes: a spiral phase plate; before the second beam splitting subunit separates the single-path erasing light into multiple-path erasing light, it further includes: The wavefront of the multipath erased light is converted from a Gaussian distribution to a circular distribution.
  • the second beam splitting subunit separating the single-path erasing light into multiple erasing lights includes: the second beam splitting subunit separates the single-path erasing light emitted through the spiral phase plate into multiple erasing lights.
  • the first light path adjustment unit when the erasing light separated by the second beam splitting subunit and the excitation light separated by the bandpass filter subunit are both two paths, the first light path adjustment unit includes: The color mirror, the second dichroic mirror, the third dichroic mirror and the fourth dichroic mirror; the first light path adjustment unit adjusts the direction of the multi-channel excitation light and the multi-channel erasing light, and respectively and The corresponding erasing light is overlapped, and the formed multi-path overlapped light is adjusted to be coaxial including: the first dichroic mirror reflects the first path of excitation light and transmits the first path corresponding to the first path of excitation light In addition to light, overlap the first excitation light with the first erasing light; the second dichroic mirror reflects the second excitation light and transmits the second erasing light corresponding to the second excitation light, The second excitation light and the second erasing light overlap; the second excitation light and the second erasing light after the overlap of the third dichroic
  • the wavelengths of the first excitation light, the second excitation light, the first erasing light, and the second erasing light are: 488nm, 635nm, 592nm, and 775nm, respectively .
  • the scanning unit includes: a scanning galvanometer, a quarter glass slide, and a high numerical aperture objective lens; the scanning unit synchronously scanning the overlapped excitation light and erasing light to the sample to be imaged includes: Scanning galvanometer scans the overlapped excitation light and erasing light simultaneously; quarter glass converts the light emitted by scanning galvanometer from linear polarization to circular polarization; high numerical aperture objective lens emits quarter glass The light is focused on the sample to be imaged.
  • the imaging unit includes: a narrowband bandpass filter, a photomultiplier tube, and a display and processing subunit; the imaging unit performs STED super-resolution imaging on the fluorescent signal generated by the excitation of the sample to be imaged, including: narrowband The band-pass filter filters the stray light outside the target band from the fluorescence signal generated by the excitation of the sample to be imaged; the photomultiplier tube amplifies the fluorescent signal emitted by the narrow band pass filter; the display and processing sub-unit emits the photomultiplier tube The fluorescence signal is processed by STED super-resolution imaging.
  • the stimulated radiation loss super-resolution imaging method in this embodiment can be implemented based on the stimulated radiation loss super-resolution imaging device provided in the foregoing embodiments, and those of ordinary skill in the art can clearly understand that for description It is convenient and concise.
  • the execution process of the stimulated radiation loss super-resolution imaging method described in this embodiment please refer to the corresponding working process in the foregoing device embodiment, which will not be repeated here.
  • the incident light generated by the laser is separated into excitation light and erasing light with different wavelengths by a spectroscopic unit; then the excitation light and erasing light are adjusted by the first optical path adjustment unit The excitation light and the erasing light are overlapped; the scanning unit scans the overlapped excitation light and erasing light to the sample to be imaged; finally, the imaging unit performs STED ultrasound on the fluorescence signal generated by the excitation of the sample to be imaged. Resolution imaging.
  • the light splitting unit separates the two types of lasers required for STED super-resolution imaging from a single light source through light splitting, which can effectively reduce system hardware redundancy, expand the application range of the system, and save Imaging costs.

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Abstract

本发明实施例公开了一种受激辐射损耗超分辨成像系统及方法，通过分光单元将激光器产生的入射光分离为波长不同的激发光和擦除光；然后由第一光路调整单元调整激发光和擦除光的方向，将激发光与擦除光进行重叠；再由扫描单元将重叠后的激发光和擦除光同步扫描至待成像样品；最后成像单元对待成像样品受激发所产生的荧光信号进行STED超分辨成像。通过本发明的实施，由分光单元通过分光的方式从单一光源中分离出STED超分辨成像所需的两种类型的激光，可以有效降低系统硬件冗杂度，扩展了系统的应用范围，并节省了成像成本。

Description

一种受激辐射损耗超分辨成像系统及方法 技术领域
本发明涉及光学成像显微领域，尤其涉及一种受激辐射损耗超分辨成像系统及方法。
 背景技术
受激辐射损耗(Stimulated Emission Depletion, STED)显微技术是一种超分辨成像技术，可以突破衍射极限对光学显微镜的分辨率的限制，实现超分辨显微成像。
具体地说，STED技术是针对扫描成像系统的一种空域处理的技术，其原理为利用两束激光来进行显微成像，其中一束为激发光，用于激发荧光材料；而另一束是光斑形状为圆环状，与激发光共轴且波长与荧光分子发射波长匹配的擦除光（也称为STED光），用来使两束光重叠区域产生受激辐射作用，而在中心区域的荧光分子则不受擦除光的影响，只会发生自发辐射。由于自发辐射的波长和受激辐射的波长不同，因此可以通过滤光片滤掉受激辐射的光，达到减小成像光斑的目标，从而提升系统的分辨率。
由于该显微技术需要依赖于两种类型的光来实现，目前通常是通过设置两个单独的激光器，来分别产生激发光与擦除光，因此目前的STED超分辨显微系统的硬件冗杂度相对较高，应用范围较为局限，成像成本较高。
 技术问题
本发明实施例的主要目的在于提供一种受激辐射损耗超分辨成像系统及方法，至少能够解决相关技术中在进行STED超分辨成像时，通过设置两个单独的激光器来分别产生激发光与擦除光，所导致的系统硬件冗杂度较高、应用范围较为局限以及成像成本较高的问题。
 技术解决方案
为实现上述目的，本发明实施例第一方面提供了一种受激辐射损耗超分辨成像系统，包括：激光器、分光单元、第一光路调整单元、扫描单元以及成像单元；
所述激光器用于产生入射光；
所述分光单元用于将所述入射光分离为波长不同的激发光和擦除光；
所述第一光路调整单元用于调整所述激发光和擦除光的方向，将所述激发光与擦除光进行重叠；
所述扫描单元用于将重叠后的所述激发光和擦除光同步扫描至待成像样品；
所述成像单元用于对所述待成像样品受激发所产生的荧光信号进行STED超分辨成像。
为实现上述目的，本发明实施例第二方面提供了一种受激辐射损耗超分辨成像方法，应用于上述受激辐射损耗超分辨成像系统，该受激辐射损耗超分辨成像方法包括：
所述分光单元将所述激光器产生的入射光分离为波长不同的激发光和擦除光；
所述第一光路调整单元调整所述激发光和擦除光的方向，将所述激发光与擦除光进行重叠；
所述扫描单元将重叠后的所述激发光和擦除光同步扫描至待成像样品；
所述成像单元对所述待成像样品受激发所产生的荧光信号进行STED超分辨成像。
 有益效果
根据本发明实施例所提供的受激辐射损耗超分辨成像系统及方法，通过分光单元将激光器产生的入射光分离为波长不同的激发光和擦除光；然后由第一光路调整单元调整激发光和擦除光的方向，将激发光与擦除光进行重叠；再由扫描单元将重叠后的激发光和擦除光同步扫描至待成像样品；最后成像单元对待成像样品受激发所产生的荧光信号进行STED超分辨成像。通过本发明的实施，由分光单元通过分光的方式从单一光源中分离出STED超分辨成像所需的两种类型的激光，可以有效降低系统硬件冗杂度，扩展了系统的应用范围，并节省了成像成本。
本发明其他特征和相应的效果在说明书的后面部分进行阐述说明，且应当理解，至少部分效果从本发明说明书中的记载变得显而易见。
 附图说明
为了更清楚地说明本发明实施例或相关技术中的技术方案，下面将对实施例或相关技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明第一实施例提供的一种受激辐射损耗超分辨成像系统的结构框图；
图2为本发明第一实施例提供的STED超分辨成像的原理图；
图3为本发明第二实施例提供的双色受激辐射损耗超分辨成像系统的结构示意图；
图4a至4d分别为本发明第二实施例提供的DM1、DM2、DMD1以及DMD2的透过率曲线图；
图5为本发明第二实施例提供的激发光与擦除光的重合效果图；
图6为本发明第三实施例提供的受激辐射损耗超分辨成像的流程示意图。
 本发明的实施方式
为使得本发明的发明目的、特征、优点能够更加的明显和易懂，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而非全部实施例。基于本发明中的实施例，本领域技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
第一实施例：
为了解决相关技术中在进行STED超分辨成像时，通过设置两个单独的激光器来分别产生激发光与擦除光，所导致的系统硬件冗杂度较高、应用范围较为局限以及成像成本较高的技术问题，本实施例提出了一种受激辐射损耗超分辨成像系统，具体请参见图1，本实施例提出的受激辐射损耗STED超分辨成像系统包括：激光器101、分光单元102、第一光路调整单元103、扫描单元104以及成像单元105；
激光器101用于产生入射光；
分光单元102用于将入射光分离为波长不同的激发光和擦除光；
第一光路调整单元103用于调整激发光和擦除光的方向，将激发光与擦除光进行重叠；
扫描单元104用于将重叠后的激发光和擦除光同步扫描至待成像样品；
成像单元105用于对待成像样品受激发所产生的荧光信号进行STED超分辨成像。
具体的，在本实施例中，STED超分辨成像所需的激发光和擦除光源自于同一光源，通过采用分光单元以分光的形式来实现从单一光源中得到两种类型的光，在实际应用中，激光器可以采用白光激光器，该激光器所产生的入射光至少支持被分为两种不同波长的光，更进一步地，该白光激光器可以为超连续谱飞秒脉冲白光激光器。并且，本实施例中通过将所分出的两束光进行重叠，然后照射至样品上，以使样品受激辐射，而使得样品上的荧光材料被激发产生荧光信号，然后该荧光信号被传输至成像单元进行STED超分辨成像以进行观察处理。如图2所示为本实施例提供的STED超分辨成像的原理图。
在本实施例的一种实施方式中，受激辐射损耗超分辨成像系统还包括：第二光路调整单元；第二光路调整单元包括低通滤波片和偏光镜。
具体的，低通滤波片用于从激光器产生的入射光中筛选出目标波长的光，偏光镜用于将得到低通滤波片出射的目标波长的光整形为线偏振光；对应的，分光单元则具体用于将整形后的线偏振光分离为波长不同的激发光和擦除光。此外，还应当说明的是，本实施例中可以将激光器产生的入射光通过单模光纤传输至低通滤波片，通过单模光纤对飞秒激光器中出射的激光进行模式调控。
在本实施例的一种实施方式中，分光单元102包括：第一分束子单元、第二分束子单元以及带通滤波子单元；第一分束子单元用于将入射光分离为波长不同的单路激发光和擦除光；带通滤波子单元用于将单路激发光分离为多路激发光；第二分束子单元用于将单路擦除光分离为对应于多路激发光的多路擦除光。
具体的，由于单一波长的激发光只能对一部分的荧光材料具有激发效应，从而单一波长的激光只能实现单色STED超分辨成像，本实施例中可以通过带通滤波子单元将单路激发光分离为多路，并由第二分束子单元将单路擦除光分离为与激发光分别对应的多路擦除光。并且对应的，第一光路调整单元具体用于调整多路激发光和多路擦除光的方向，将各路激发光分别与对应的擦除光进行重叠，并将所形成的多路重叠光调整为共轴，扫描单元具体用于将多路重叠光均同步扫描至待成像样品，成像单元具体用于对待成像样品受激发所产生的所有荧光信号进行多色STED超分辨成像。应当理解的是，在需要实现双色STED超分辨成像时，带通滤波子单元采用两个带通滤波器实现，也即分别通过一个带通滤波器滤过一路激发光，而第二分束子单元则单个分束器实现。此外，还应当说明的是，本实施例中多路激发光的传输光路上还可以设置有角反射器，用于改变激发光的光路，可在时间上控制激发光和擦除光之间的脉冲间隔。
进一步地，在本实施例的一种实施方式中，分光单元102还包括：螺旋相位板；螺旋相位板用于将多路擦除光的波前由高斯型分布转换为环形分布。
具体的，本实施例中通过螺旋相位板来将擦除光转换为涡旋光束，也即该光束的波前沿传播方向上的轴螺旋前进，这种旋转导致光束在光轴处相互抵消，投影到一个平面上形成中心具有暗孔的光环。
进一步地，在本实施例的一种实施方式中，在第二分束子单元所分离的擦除光以及带通滤波子单元所分离的激发光均为两路时，第一光路调整单元103包括：第一二向色镜、第二二向色镜、第三二向色镜以及第四二向色镜；第一二向色镜用于反射第一路激发光，并透射对应于第一路激发光的第一路擦除光，将第一路激发光与第一路擦除光进行重叠；第二二向色镜用于反射第二路激发光，并透射对应于第二路激发光的第二路擦除光，将第二路激发光与第二路擦除光进行重叠；第三二向色镜用于反射重叠后的第二路激发光与第二路擦除光；第四二向色镜用于反射重叠后的第一路激发光与第一路擦除光。
具体的，本实施例中在进行双色STED超分辨成像时，所分离出的激发光和擦除光分别为两路，在一种优选的实施方式中，第一路激发光、第二路激发光、第一路擦除光以及第二路擦除光的波长可以分别为：488nm、635nm、592nm以及775nm。本实施例中所分离出的激发光和擦除光需要进行光路调整，也即分别通过第一二向色镜和第二二向色镜分别形成一路重叠后的光，然后再通过第三二向色镜和第四二向色镜将所形成的两路重叠光调整为共轴。应当说明的是，在本实施例一种优选的实施方式中，可以将第三二向色镜以及第四二向色镜设置于扫描单元与成像单元之间的光路上，从而第三二向色镜以及第四二向色镜还用于在荧光信号传输至成像单元的过程中，透射荧光信号。
在本实施例的一种实施方式中，扫描单元104包括：在光路的传播方向上一次设置的扫描振镜、四分之一玻片以及高数值孔径物镜。
具体的，扫描振镜用于对重叠后的激发光和擦除光进行同步扫描；四分之一玻片用于将扫描振镜出射的光由线偏振转化为圆偏振；高数值孔径物镜用于将四分之一玻片出射的光聚焦至待成像样品。
在本实施例的一种实施方式中，成像单元105包括：窄带带通滤波片、光电倍增管以及显示与处理子单元。
具体的，窄带带通滤波片用于对待成像样品受激发所产生的荧光信号中，目标波段之外的杂散光进行过滤；光电倍增管用于放大窄带带通滤波片出射的荧光信号；显示与处理子单元用于将光电倍增管出射的荧光信号进行STED超分辨成像处理。还应当说明的是，在将窄带带通滤波片出射的荧光信号传输至光电倍增管时，可以采用多模光纤进行传输，光纤纤芯可作为小孔，接收透镜聚焦的荧光信号，并排除杂散光的影响。
本实施例提供的受激辐射损耗超分辨成像系统，通过分光单元将激光器产生的入射光分离为波长不同的激发光和擦除光；然后由第一光路调整单元调整激发光和擦除光的方向，将激发光与擦除光进行重叠；再由扫描单元将重叠后的激发光和擦除光同步扫描至待成像样品；最后成像单元对待成像样品受激发所产生的荧光信号进行STED超分辨成像。通过本发明的实施，由分光单元通过分光的方式从单一光源中分离出STED超分辨成像所需的两种类型的激光，可以有效降低系统硬件冗杂度，扩展了系统的应用范围，并节省了成像成本。
第二实施例：
为了更加直观的理解本发明实施例中的受激辐射损耗超分辨成像系统，本发明实施例二以一个具体的示例对受激辐射损耗超分辨成像系统进行详细说明。如图3所示为本实施例提供的双色受激辐射损耗超分辨成像系统的结构示意图，包括：
超连续谱飞秒脉冲白光激光器（Laser），用于产生可分为四种波长的白光光源；
单模光纤(SMF)，用于对飞秒激光器中出射的激光进行模式调控；
低通滤波片（F1），用于筛选出所需要范围内的光波长；
反射镜（M），用于反射入射光，改变光束传输方向；
偏光镜（H），用于保证所在光路的激光为线偏振光；
分束器（PBS1，PBS2），可对光束进行分光，并可调节分光比例；
螺旋相位板(VPP)，用于将两路擦除光的波前由高斯型转换成环形分布；
二向色镜(DM1)，用于反射第一路激发光(488nm)，透射第一路擦除光(592nm)和第二路激发光（635nm），同时还可以微调第一路激发光的传输方向，使激发光和损耗光能够很好的重叠；
二向色镜(DM2)，用于反射第二路激发光（635nm），透射第二路擦除光（775nm），同时还可以微调第二路激发光的传输方向，使激发光和损耗光能够很好的重叠；
二向色镜（DMD1），用于反射第二路激发光与擦除光（635nm和775nm），同时透射样品（sample）被激发后所产生的荧光信号的光；
二向色镜（DMD2），用于反射第一路激发光与擦除光（488nm和592nm），透射第二路激发光与擦除光（635nm和775nm），同时透射样品被激发后所产生的荧光信号的光；
角反射器（RR），用于改变激发光的光路，可在时间上控制激发光和擦除光之间的脉冲间隔；
透镜(L1，L2，L3)，用于对入射激光进行聚焦或发散，组合使用可以扩大或缩小光斑直径；
振镜扫描系统(Scanner)，用于对重合后的激发光和擦除光进行扫描，实现对样品的面阵成像；
四分之一玻片(QWP)，用于将擦除激光由线偏振转化为圆偏振；
带通滤波片(F2，F3，F4，F5)，用于筛选出单一波长的激发光及其对应的擦除光；
窄带带通滤波片（F6），用于透过所要收集波段的荧光信号，并过滤此波段以外的杂散光；
多模光纤(MMF)，用于将收集到的荧光信号传输给光电倍增管PMT；光纤纤芯可作为小孔，接收透镜L3聚焦的荧光信号，并排除杂散光的影响；
光电倍增管(PMT)，用于接收样品的荧光信号并放大该信号；
高数值孔径物镜(OL)，放大倍率为100倍，数值孔径为1.4，用于聚焦重叠的激发光和损耗光，同时收集样品反射回来的荧光信号。
如图4a至4d分别为本实施例提供的DM1、DM2、DMD1以及DMD2的透过率曲线图。如表1所示为本实施例提供的各滤波片与二向色镜的型号表。
表1
代号 型号
F1 FF01-890/SP-25
F2 FF01-488/10-25
F3 ET632/28m
F4 FF01-590/20-25
F5 ET775/50X
DM1 ZT561rdc
DM2 ZT640rdc
DMD1 ZT488/647/780
DMD2 FF395/495/610
白光激光器发出宽带激光，经低通滤波片F1后仅允许800nm以下波光的激光通过，然后经偏光镜H使激光为线偏振光，随后经过分束器PBS1，分成的两路光可分别作为激发光和擦除光。激发光经过转轮，转轮上安置了F2和F3，分别可通过488nm与635nm波长的激发光，旋转转轮即可选择使用特定的激发波长；488nm激发光与635nm激发光经过相同的路径进入角反射器（RR），角反射器可以对激发光的光程进行调节，且对两个波长的激发光都具有调节作用，前提是角反射器的位置必须与光路保持严格垂直，否则进行光程调节时会对光路的传播产生偏差。经过延时后激发光到达第一个二向色镜DM1的位置，系统中的二向色镜与入射光线成45°夹角时为最佳位置。
擦除光经过L1和L2两个透镜（L2焦距为L1的两倍）将光斑放大两倍，然后经过螺旋相位板VPP形成环形光，随后再由分束器PBS2分成两束光，其中一束光经过带通滤波片F4，成为592nm的窄带激光，作为第一路擦除光到达第一个二向色镜DM1处；另一束经过带通滤波片F5成为775nm的窄带激光，作为第二路擦除光到达第二个二向色镜DM2处。
在转轮滤波片选择为F2时，则激发光光路仅存在488nm波长的激发光，此时在DM1处488nm激发光发生反射，第一路592nm擦除光在此处发生透射，488nm激发光与592nm擦除光在DM1处产生重合，DM1可调节488nm激发光与592nm擦除光的重合度，形成如图5所示的效果，如图5所示为本实施例提供的激发光与擦除光的重合效果图。488nm激发光与592nm擦除光重合之后经过DMD2均发生反射，重合光经过反射镜M进入扫描振镜Scanner进行同步扫描，再经过四分之一玻片QWP，将擦除光由线偏振转变为圆偏振，最后经过高数值孔径物镜(OL)后对样品（sample）进行扫描形成面阵成像。样品发出荧光后，荧光沿着入射光路返回到达DMD2处发生透射，在DMD1处同样发生透射，再经过透镜L3聚焦和窄带带通滤波片F6透过所要收集波段的荧光信号，并过滤此波段以外的杂散光进入多模光纤（MMF）到达光电倍增管PMT。最后PMT进行信号放大并将信号数据传到显示与处理子单元（如电脑）成像以进行观察和处理。
在转轮滤波片选择为F3时，则激发光光路仅存在635nm波长的激发光，此时在DM1处635nm激发光发生透射，在DM2处发生反射，第二路775nm擦除光在此处发生透射，635nm激发光与775nm擦除光在DM2处产生重合，DM2可调节635nm激发光与775nm擦除光的重合度。635nm激发光与775nm擦除光重合之后经过DMD1发生反射，在DMD2处均发生透射。与第一路光相同，重合光随后进入扫描振镜进行同步扫描，再经过四分之一玻片，将擦除光由线偏振转变为圆偏振，最后经过物镜后对样品进行扫描形成面阵成像。样品发出荧光后，荧光沿着入射光路返回到达DMD2处发生透射，在DMD1处同样发生透射，再经过L3聚焦和F6透过所要收集波段的荧光信号，并过滤此波段以外的杂散光进入多模光纤（MMF）到达PMT。最后PMT进行信号放大并将信号数据传到显示与处理子单元（如电脑）成像以进行观察和处理。
通过本发明实施例提供的受激辐射损耗超分辨成像系统，该系统设计可以达到利用单一白光光源实现双色STED成像，在节省材料成本的情况下，也拓展了STED系统的应用范围。
第三实施例：
为了解决相关技术中在进行STED超分辨成像时，通过设置两个单独的激光器来分别产生激发光与擦除光，所导致的系统硬件冗杂度较高、应用范围较为局限以及成像成本较高的技术问题，本实施例还提供了一种受激辐射损耗超分辨成像方法，应用于前述实施例中所述的包括激光器、分光单元、第一光路调整单元、扫描单元以及成像单元的受激辐射损耗超分辨成像系统，如图6所示，本实施例提出的受激辐射损耗超分辨成像方法包括以下的步骤：
步骤601、分光单元将激光器产生的入射光分离为波长不同的激发光和擦除光；
步骤602、第一光路调整单元调整激发光和擦除光的方向，将激发光与擦除光进行重叠；
步骤603、扫描单元将重叠后的激发光和擦除光同步扫描至待成像样品；
步骤604、成像单元对待成像样品受激发所产生的荧光信号进行STED超分辨成像。
其中，在本实施例中，STED超分辨成像所需的激发光和擦除光源自于同一光源，通过分光的形式来实现从单一光源中得到两种类型的光。通过将所分出的两束光进行重叠，然后照射至样品上，以使样品受激辐射，而使得样品上的荧光材料被激发产生荧光信号，然后该荧光信号被传输至成像单元进行STED超分辨成像以进行观察处理。
在本实施例的一些实施方式中，受激辐射损耗超分辨成像系统还包括：第二光路调整单元；第二光路调整单元包括低通滤波片和偏光镜；在分光单元将激光器产生的入射光分离为波长不同的激发光和擦除光之前，还包括：低通滤波片从激光器产生的入射光中筛选出目标波长的光；偏光镜将得到低通滤波片出射的目标波长的光整形为线偏振光。对应的，分光单元将激光器产生的入射光分离为波长不同的激发光和擦除光包括：分光单元将将整形后的线偏振光分离为波长不同的激发光和擦除光。
在本实施例的一些实施方式中，分光单元包括：第一分束子单元、第二分束子单元以及带通滤波子单元；分光单元将激光器产生的入射光分离为波长不同的激发光和擦除光包括：第一分束子单元将激光器产生的入射光分离为波长不同的单路激发光和擦除光；带通滤波子单元将单路激发光分离为多路激发光，以及第二分束子单元将单路擦除光分离为分别对应于多路激发光的多路擦除光。对应的，第一光路调整单元调整激发光和擦除光的方向，将激发光与擦除光进行重叠包括：第一光路调整单元调整多路激发光和多路擦除光的方向，将各路激发光分别与对应的擦除光进行重叠，并将所形成的多路重叠光调整为共轴；扫描单元将多路重叠光同步扫描至待成像样品包括：扫描单元将多路重叠光均同步扫描至待成像样品；成像单元对待成像样品受激发所产生的荧光信号进行STED超分辨成像包括：成像单元对待成像样品受激发所产生的所有荧光信号进行多色STED超分辨成像。
进一步地，在本实施例的一些实施方式中，分光单元还包括：螺旋相位板；在第二分束子单元将单路擦除光分离为多路擦除光之前，还包括：螺旋相位板将多路擦除光的波前由高斯型分布转换为环形分布。对应的，第二分束子单元将单路擦除光分离为多路擦除光包括：第二分束子单元将经过螺旋相位板出射的单路擦除光分离为多路擦除光。
在本实施例的一些实施方式中，在第二分束子单元所分离的擦除光以及带通滤波子单元所分离的激发光均为两路时，第一光路调整单元包括：第一二向色镜、第二二向色镜、第三二向色镜以及第四二向色镜；第一光路调整单元调整多路激发光和多路擦除光的方向，将各路激发光分别与对应的擦除光进行重叠，并将所形成的多路重叠光调整为共轴包括：第一二向色镜反射第一路激发光，并透射对应于第一路激发光的第一路擦除光，将第一路激发光与第一路擦除光进行重叠；第二二向色镜反射第二路激发光，并透射对应于第二路激发光的第二路擦除光，将第二路激发光与第二路擦除光进行重叠；第三二向色镜反射重叠后的第二路激发光与第二路擦除光，第四二向色镜反射重叠后的第一路激发光与第一路擦除光，将两路重叠光调整为共轴。
进一步地，在本实施例的一些实施方式中，第一路激发光、第二路激发光、第一路擦除光以及第二路擦除光的波长分别为：488nm、635nm、592nm以及775nm。
在本实施例的一些实施方式中，扫描单元包括：扫描振镜、四分之一玻片以及高数值孔径物镜；扫描单元将重叠后的激发光和擦除光同步扫描至待成像样品包括：扫描振镜对重叠后的激发光和擦除光进行同步扫描；四分之一玻片将扫描振镜出射的光由线偏振转化为圆偏振；高数值孔径物镜将四分之一玻片出射的光聚焦至待成像样品。
在本实施例的一些实施方式中，成像单元包括：窄带带通滤波片、光电倍增管以及显示与处理子单元；成像单元对待成像样品受激发所产生的荧光信号进行STED超分辨成像包括：窄带带通滤波片对待成像样品受激发所产生的荧光信号中，目标波段之外的杂散光进行过滤；光电倍增管放大窄带带通滤波片出射的荧光信号；显示与处理子单元将光电倍增管出射的荧光信号进行STED超分辨成像处理。
应当说明的是，本实施例中的受激辐射损耗超分辨成像方法均可基于前述实施例提供的受激辐射损耗超分辨成像装置实现，所属领域的普通技术人员可以清楚的了解到，为描述的方便和简洁，本实施例中所描述的受激辐射损耗超分辨成像方法的部分执行流程，可以参考前述装置实施例中的对应工作过程，在此不再赘述。
采用本实施例提供的受激辐射损耗超分辨成像方法，通过分光单元将激光器产生的入射光分离为波长不同的激发光和擦除光；然后由第一光路调整单元调整激发光和擦除光的方向，将激发光与擦除光进行重叠；再由扫描单元将重叠后的激发光和擦除光同步扫描至待成像样品；最后成像单元对待成像样品受激发所产生的荧光信号进行STED超分辨成像。通过本发明的实施，由分光单元通过分光的方式从单一光源中分离出STED超分辨成像所需的两种类型的激光，可以有效降低系统硬件冗杂度，扩展了系统的应用范围，并节省了成像成本。
以上内容是结合具体的实施方式对本发明实施例所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (10)

  1. 一种受激辐射损耗超分辨成像系统，其特征在于，包括：激光器、分光单元、第一光路调整单元、扫描单元以及成像单元；
    所述激光器用于产生入射光；
    所述分光单元用于将所述入射光分离为波长不同的激发光和擦除光；
    所述第一光路调整单元用于调整所述激发光和擦除光的方向，将所述激发光与擦除光进行重叠；
    所述扫描单元用于将重叠后的所述激发光和擦除光同步扫描至待成像样品；
    所述成像单元用于对所述待成像样品受激发所产生的荧光信号进行STED超分辨成像。
  2. 如权利要求1所述的受激辐射损耗超分辨成像系统，其特征在于，所述分光单元包括：第一分束子单元、第二分束子单元以及带通滤波子单元；
    所述第一分束子单元用于将所述入射光分离为波长不同的单路激发光和擦除光；所述带通滤波子单元用于将所述单路激发光分离为多路激发光；所述第二分束子单元用于将所述单路擦除光分离为对应于所述多路激发光的多路擦除光；
    所述第一光路调整单元具体用于调整所述多路激发光和多路擦除光的方向，将各路激发光分别与对应的擦除光进行重叠，并将所形成的多路重叠光调整为共轴；
    所述扫描单元具体用于将所述多路重叠光均同步扫描至待成像样品；
    所述成像单元具体用于对所述待成像样品受激发所产生的所有荧光信号进行多色STED超分辨成像。
  3. 如权利要求2所述的受激辐射损耗超分辨成像系统，其特征在于，所述分光单元还包括：螺旋相位板；
    所述螺旋相位板用于将所述多路擦除光的波前由高斯型分布转换为环形分布。
  4. 如权利要求2所述的受激辐射损耗超分辨成像系统，其特征在于，在所述第二分束子单元所分离的擦除光以及所述带通滤波子单元所分离的激发光均为两路时，所述第一光路调整单元包括：第一二向色镜、第二二向色镜、第三二向色镜以及第四二向色镜；
    所述第一二向色镜用于反射第一路激发光，并透射对应于所述第一路激发光的第一路擦除光，将所述第一路激发光与第一路擦除光进行重叠；
    所述第二二向色镜用于反射第二路激发光，并透射对应于所述第二路激发光的第二路擦除光，将所述第二路激发光与第二路擦除光进行重叠；
    所述第三二向色镜用于反射重叠后的所述第二路激发光与第二路擦除光；
    所述第四二向色镜用于反射重叠后的所述第一路激发光与第一路擦除光。
  5. 如权利要求4所述的受激辐射损耗超分辨成像系统，其特征在于，所述第一路激发光、所述第二路激发光、所述第一路擦除光以及所述第二路擦除光 的波长分别为：488nm、635nm、592nm以及775nm。
  6. 如权利要求1所述的受激辐射损耗超分辨成像系统，其特征在于，所述扫描单元包括：扫描振镜、四分之一玻片以及高数值孔径物镜；
    所述扫描振镜用于对重叠后的所述激发光和擦除光进行同步扫描；
    所述四分之一玻片用于将所述扫描振镜出射的光由线偏振转化为圆偏振；
    所述高数值孔径物镜用于将所述四分之一玻片出射的光聚焦至所述待成像样品。
  7. 如权利要求1所述的受激辐射损耗超分辨成像系统，其特征在于，所述成像单元包括：窄带带通滤波片、光电倍增管以及显示与处理子单元；
    所述窄带带通滤波片用于对所述待成像样品受激发所产生的荧光信号中，目标波段之外的杂散光进行过滤；
    所述光电倍增管用于放大所述窄带带通滤波片出射的荧光信号；
    所述显示与处理子单元用于将所述光电倍增管出射的荧光信号进行STED超分辨成像处理。
  8. 如权利要求1所述的受激辐射损耗超分辨成像系统，其特征在于，还包括：第二光路调整单元；所述第二光路调整单元包括低通滤波片和偏光镜；
    所述低通滤波片用于从所述激光器产生的入射光中筛选出目标波长的光；
    所述偏光镜用于将得到低通滤波片出射的目标波长的光整形为线偏振光；
    所述分光单元具体用于将所述整形后的线偏振光分离为波长不同的激发光和擦除光。
  9. 一种受激辐射损耗超分辨成像方法，其特征在于，应用于如权利要求1至8中任意一项所述的受激辐射损耗超分辨成像系统，包括：
    所述分光单元将所述激光器产生的入射光分离为波长不同的激发光和擦除光；
    所述第一光路调整单元调整所述激发光和擦除光的方向，将所述激发光与擦除光进行重叠；
    所述扫描单元将重叠后的所述激发光和擦除光同步扫描至待成像样品；
    所述成像单元对所述待成像样品受激发所产生的荧光信号进行STED超分辨成像。
  10. 如权利要求9所述的受激辐射损耗超分辨成像方法，其特征在于，所述分光单元包括：第一分束子单元、第二分束子单元以及带通滤波子单元；
    所述分光单元将所述激光器产生的入射光分离为波长不同的激发光和擦除光包括：
    所述第一分束子单元将所述激光器产生的入射光分离为波长不同的单路激发光和擦除光；
    所述带通滤波子单元将所述单路激发光分离为多路激发光，以及所述第二分束子单元将所述单路擦除光分离为对应于所述多路激发光的多路擦除光；
    所述第一光路调整单元调整所述激发光和擦除光的方向，将所述激发光与擦除光进行重叠包括：
    所述第一光路调整单元调整所述多路激发光和多路擦除光的方向，将各路激发光分别与对应的擦除光进行重叠，并将所形成的多路重叠光调整为共轴；
    所述扫描单元将重叠后的所述激发光和擦除光同步扫描至待成像样品包括：
    所述扫描单元将所述多路重叠光均同步扫描至待成像样品；
    所述成像单元对所述待成像样品受激发所产生的荧光信号进行STED超分辨成像包括：
    所述成像单元对所述待成像样品受激发所产生的所有荧光信号进行多色STED超分辨成像。
PCT/CN2019/083072 2019-04-17 2019-04-17 一种受激辐射损耗超分辨成像系统及方法 WO2020211018A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103676123A (zh) * 2013-12-18 2014-03-26 中国科学院苏州生物医学工程技术研究所 多模式光学高分辨显微镜
US20150226950A1 (en) * 2012-08-23 2015-08-13 Isis Innovation Limited Stimulated emission depletion microscopy
CN105241857A (zh) * 2015-09-30 2016-01-13 深圳大学 一种超分辨成像系统
CN108957720A (zh) * 2018-09-26 2018-12-07 中国科学院化学研究所 用于sted光学显微镜的照明系统及sted光学显微镜

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150226950A1 (en) * 2012-08-23 2015-08-13 Isis Innovation Limited Stimulated emission depletion microscopy
CN103676123A (zh) * 2013-12-18 2014-03-26 中国科学院苏州生物医学工程技术研究所 多模式光学高分辨显微镜
CN105241857A (zh) * 2015-09-30 2016-01-13 深圳大学 一种超分辨成像系统
CN108957720A (zh) * 2018-09-26 2018-12-07 中国科学院化学研究所 用于sted光学显微镜的照明系统及sted光学显微镜

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