WO2019119458A1 - 超分辨成像系统 - Google Patents

超分辨成像系统 Download PDF

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Publication number
WO2019119458A1
WO2019119458A1 PCT/CN2017/118137 CN2017118137W WO2019119458A1 WO 2019119458 A1 WO2019119458 A1 WO 2019119458A1 CN 2017118137 W CN2017118137 W CN 2017118137W WO 2019119458 A1 WO2019119458 A1 WO 2019119458A1
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Prior art keywords
light
dissipated
fluorescent signal
disposed
excitation light
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PCT/CN2017/118137
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English (en)
French (fr)
Inventor
严伟
屈军乐
王璐玮
叶彤
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Shenzhen University
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Shenzhen University
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Priority to PCT/CN2017/118137 priority Critical patent/WO2019119458A1/zh
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Anticipated expiration legal-status Critical
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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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B26/00—Optical devices or arrangements for the control of light using movable or deformable optical elements
    • G02B26/06—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the phase of light
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06N—COMPUTING ARRANGEMENTS BASED ON SPECIFIC COMPUTATIONAL MODELS
    • G06N3/00—Computing arrangements based on biological models
    • G06N3/12—Computing arrangements based on biological models using genetic models

Definitions

  • the present invention relates to the field of optical microscopy, and more particularly to a super-resolution imaging system.
  • STED synchronous transition equivalent decomposition
  • This STED super-resolution imaging system is an ideal STED super-resolution imaging system.
  • STED super-resolution imaging systems often contain many optical components, such as optical components including many lenses, slides, dichroic mirrors, and mirrors, and the artificially built STED super-resolution imaging system does not allow precise control of light.
  • biological samples have surface irregularities and non-uniformity of internal refractive index distribution. These factors will bring aberrations to the STED super-resolution imaging system, reduce the imaging depth and spatial resolution of the STED super-resolution imaging system, and affect imaging. Quality limits its use in biomedical applications.
  • the existing STED super-resolution imaging system has aberrations caused by many optical components, surface irregularities of the biological sample and non-uniformity of the internal refractive index distribution, which will bring aberrations to the STED super-resolution imaging system.
  • the imaging depth and spatial resolution of the STED super-resolution imaging system are reduced, which affects the imaging quality and limits its technical problems in biomedical applications.
  • the main object of the present invention is to provide a super-resolution imaging system, which aims to solve the existing STED super-resolution imaging system, which has non-uniformity due to surface irregularities and internal refractive index distribution of many optical components and biological samples. Factors such as characteristics will bring aberrations to the STED super-resolution imaging system, reduce the imaging depth and spatial resolution of the STED super-resolution imaging system, affect imaging quality, and limit its technical problems in biomedical applications.
  • the present invention provides a super-resolution imaging system, the system comprising: a femtosecond laser for generating dissipative light and incident on a first light modulation device;
  • the first light modulating device is disposed on an outgoing light path of the femtosecond laser for modulating polarization characteristics and intensity of the dissipated light;
  • a glass rod disposed on the outgoing light path of the first light modulating device for performing pulse width broadening processing on the modulated dissipated light such that the pulse width of the dissipated light is 1 picosecond;
  • a first lens group disposed on an outgoing light path of the glass rod for expanding a spot diameter of the dissipated light having a pulse width of 1 picosecond;
  • a first lens disposed on the outgoing optical path of the first lens group for focusing and coupling the dissipated light whose spot diameter is enlarged to a 100 m single mode polarization maintaining fiber;
  • the 100 meter single mode polarization maintaining fiber is disposed on the outgoing optical path of the first lens for widening the pulse width of the dissipated light having a pulse width of 1 picosecond to 200 picoseconds;
  • a second lens disposed on the outgoing optical path of the 100-meter single-mode polarization-maintaining fiber for amplifying the spot diameter of the dissipated light having a pulse width of 200 picoseconds, and incident on the second light modulation device;
  • the second light modulating device is disposed on an outgoing light path of the second lens for modulating polarization characteristics and intensity of the dissipated light;
  • the spatial light modulator is disposed on an outgoing light path of the second light modulating device, and reflects the dissipated light emitted from the second light modulating device to the second lens group;
  • the second lens group is configured to reduce the spot diameter of the incident dissipated light, and the dissipated light that reduces the spot diameter is injected into the galvanometer scanning system;
  • a picosecond laser for generating excitation light and incident on the first single mode fiber
  • the first single mode fiber is disposed on an exiting optical path of the picosecond laser for mode control of the incident excitation light
  • a third lens disposed on the outgoing optical path of the first single-mode optical fiber, for expanding the spot diameter of the mode-controlled excitation light, and incident on the third light modulation device;
  • the third light modulating device is configured to modulate polarization characteristics and intensity of the excitation light
  • a corner reflector disposed on an outgoing light path of the third light modulating device for changing an optical path of the optical path where the excitation light is located, and controlling a pulse between the excitation light and the dissipative light in time Intervaling and injecting the excitation light into the galvanometer scanning system;
  • the galvanometer scanning system is configured to perform synchronous area array scanning on the overlapping excitation light and the dissipated light;
  • a quarter slide for polarizing the excitation light and the dissipated light scanned by the galvanometer scanning system, and modulating the excitation light and the dissipated light into linearly polarized light by linearly polarized light ;
  • a high numerical aperture objective for focusing overlapping excitation light and dissipating light, projecting the focused overlapping excitation light and dissipated light onto the sample, and collecting the fluorescent signal reflected by the sample;
  • a filter for filtering the fluorescent signal, injecting a fluorescent signal of a preset wavelength band into the second single mode fiber, and filtering out the fluorescent signal outside the preset wavelength band;
  • a second single mode fiber for transmitting a fluorescent signal obtained by filtering through the filter to a photomultiplier tube
  • the photomultiplier tube is configured to amplify a fluorescence signal obtained by filtering through the filter;
  • a first data acquisition card for collecting and analyzing the fluorescent signal collected by the photomultiplier tube
  • a second data acquisition card configured to convert the fluorescent signal collected by the photomultiplier tube into voltage information, and the voltage information is used as a fitness value in a genetic algorithm, and the voltage information is calculated according to a genetic algorithm to obtain a The absolute value of the maximum voltage, and the phase map corresponding to the absolute value of the maximum voltage is used as the phase compensation map;
  • the spatial light modulator is configured to superimpose the phase compensation map on the dissipated light, perform aberration correction on the dissipated light, and further to load the liquid crystal surface on the spatial light modulator Dissipating the spiral phase information of the light, converting the dissipated light into Gaussian light into ring light.
  • the picosecond laser is connected to the femtosecond laser by an external wiring, and the picosecond laser is triggered by the femtosecond laser to output the excitation light.
  • the first light modulation device includes a first half wave plate and a first Glan laser prism
  • the second light modulation device includes a second half wave plate and a second Glan laser prism
  • the third The light modulation device includes a third half wave plate and a third Glan laser prism
  • the first half wave plate is disposed on an outgoing light path of the femtosecond laser for modulating a polarization characteristic of the dissipated light emitted from the femtosecond laser;
  • the first Glan laser prism is disposed on an outgoing light path of the first half wave plate for adjusting the intensity of the modulated dissipated light
  • the second half wave plate is configured to modulate a polarization characteristic of the dissipated light emitted from the second lens
  • the second Glan laser prism is disposed on an outgoing light path of the second half wave plate for adjusting an intensity of the modulated dissipated light that is incident;
  • the third half wave plate is configured to modulate a polarization characteristic of the excitation light emitted from the first single mode fiber
  • the third Glan laser prism is disposed on an outgoing light path of the third wave plate for adjusting the intensity of the modulated excitation light that is incident.
  • the high numerical aperture objective lens has a magnification of 100 times and a numerical aperture of 1.4.
  • the system further includes: a beam splitter and a charge coupled component;
  • the beam splitter is configured to divide the fluorescent signal collected and emitted by the high numerical aperture objective into two parts, one part is reflected into the charge coupling element, and the other part is transmitted into the filter;
  • the charge coupled device is configured to monitor in real time the overlap of the spot of the dissipated light and the spot of the excitation light in the incident fluorescent signal.
  • the system further includes: a first dichroic mirror and a second dichroic mirror;
  • the first dichroic mirror is configured to transmit the dissipated light emitted from the second lens group and reflect the fluorescent signal
  • the second dichroic mirror is configured to reflect excitation light emitted from the corner reflector and transmit the fluorescent signal.
  • the second dichroic mirror is further configured to adjust a transmission direction of the dissipated light and a transmission direction of the excitation light in the fluorescent signal, so that the dissipated light and the The excitation light overlaps.
  • the beam splitter splits the fluorescent signal into two parts according to 9:1.
  • the interval between the dissipated light and the pulse peak of the excitation light is 160 to 200 ps.
  • the system further includes an electronically controlled baffle that blocks the dissipated light emitted from the second lens group when the electronically controlled baffle is closed.
  • the invention provides a super-resolution imaging system, including a femtosecond laser, a picosecond laser, a first data acquisition card, a second data acquisition card, a spatial light modulator, etc., a dispersive light generated by a femtosecond laser and a picosecond laser
  • a fluorescent signal is generated
  • the second data acquisition card converts the fluorescent signal into voltage information
  • the voltage information is used as a fitness value in the genetic algorithm
  • the voltage information is calculated according to the genetic algorithm to obtain a maximum
  • the absolute value of the voltage, and the phase map corresponding to the absolute value of the maximum voltage is used as a phase compensation map, and the phase compensation map is superimposed on the dissipated light to perform aberration correction on the dissipated light.
  • the embodiment of the present invention introduces an aberration correction system of a genetic algorithm in a STED super-resolution imaging system, and obtains a phase compensation map according to a genetic algorithm, and superimposes the phase compensation map on the dissipated light to dissipate
  • the optical aberration correction overcomes the aberration introduced in the STED imaging process and improves the imaging depth and spatial resolution of the STED super-resolution imaging system, so that it can be widely used in the field of medicine and the like.
  • FIG. 1 is a schematic structural diagram of a super-resolution imaging system according to a first embodiment of the present invention
  • FIG. 2 is a phase compensation diagram to be loaded on a spatial light modulator SLM for aberration correction of Gaussian-type dissipative light
  • FIG. 3 is a schematic diagram of spiral phase information of dissipated light loaded on a spatial light modulator SLM;
  • FIG. 4 is a schematic diagram of an annular light having an aberration correction effect after superimposing a phase compensation map and spiral phase information
  • Figure 5 is a schematic view of the diffraction limit after the depletion of light and the excitation light overlap
  • Figure 6 (a) is a schematic view of confocal imaging of fluorescent beads without aberration correction
  • Fig. 6(b) is a schematic diagram of STED super-resolution imaging of stimulated emission loss of fluorescent beads.
  • FIG. 1 is a schematic structural diagram of a super-resolution imaging system according to a first embodiment of the present invention, including: a femtosecond laser Laser1 for generating dissipative light and incident on a first light modulation device;
  • a first light modulating device disposed on an outgoing light path of the femtosecond laser Laser1 for modulating polarization characteristics and intensity of the dissipated light;
  • a glass rod GR disposed on the outgoing light path of the first light modulating device for performing pulse width broadening on the modulated dissipated light such that the pulse width of the dissipated light is 1 picosecond;
  • a first lens group disposed on an outgoing light path of the glass rod GR for expanding a spot diameter of the dissipated light having a pulse width of 1 picosecond;
  • the first lens L3 is disposed on the outgoing light path of the first lens group for focusing and dissipating the dissipated light with the enlarged spot diameter to the 100 m single mode polarization maintaining fiber Fiber0;
  • the second lens L4 is disposed on the outgoing light path of the 100-meter single-mode polarization-maintaining fiber Fiber0, and is used for expanding the spot diameter of the dissipated light having a pulse width of 200 picoseconds, and is incident on the second light modulation device;
  • a second light modulating device disposed on the outgoing light path of the second lens L4 for modulating polarization characteristics and intensity of the dissipated light
  • a spatial light modulator SLM disposed on the exiting optical path of the second light modulating device to reflect the dissipated light emitted from the second light modulating device to the second lens group;
  • a second lens group for reducing the spot diameter of the incident dissipated light, and dissipating the scattered light of the spot diameter into the galvanometer scanning system Scanner;
  • a picosecond laser Laser2 for generating excitation light and incident on the first single mode fiber Fiber1;
  • the first single-mode fiber Fiber1 is disposed on the outgoing light path of the picosecond laser Laser2 for mode control of the incident excitation light;
  • a third lens disposed on the outgoing optical path of the first single-mode fiber Fiber1 for expanding the spot diameter of the mode-controlled excitation light and incident on the third light modulation device;
  • a third light modulating device for modulating polarization characteristics and intensity of the excitation light
  • the corner reflector R1 is disposed on the outgoing light path of the third light modulating device for changing the optical path of the optical path where the excitation light is located, controlling the pulse interval between the excitation light and the dissipative light in time, and emitting the excitation light Scanner scanning system Scanner;
  • the galvanometer scanning system Scanner is used for synchronous area scanning of overlapping excitation light and dissipated light
  • the quarter glass slide Q1 is used for polarizing the excitation light and the dissipated light after scanning by the Scanner scanning system Scanner, and modulating the excitation light and the dissipated light into linearly polarized light by linearly polarized light;
  • a high numerical aperture objective Obj for focusing overlapping excitation light and dissipating light, projecting the focused overlapping excitation light and dissipated light onto the sample, and collecting the fluorescent signal reflected by the sample;
  • the filter F1 is configured to filter the fluorescent signal, and the fluorescent signal of the preset wavelength band is injected into the second single-mode fiber Fiber2, and the fluorescent signal outside the preset wavelength band is filtered out;
  • a second single-mode fiber Fiber2 for transmitting the fluorescent signal obtained by filtering through the filter F1 to the photomultiplier tube PMT;
  • a photomultiplier tube PMT for amplifying a fluorescent signal obtained by filtering through the filter F1;
  • the first data acquisition card NI1 is used for collecting and analyzing the fluorescent signal collected by the photomultiplier tube PMT;
  • the second data acquisition card NI2 is used to convert the fluorescent signal collected by the photomultiplier tube PMT into voltage information, and the voltage information is used as the fitness value in the genetic algorithm, and the voltage information is calculated according to the genetic algorithm to obtain a maximum absolute value of the voltage. And using a phase map corresponding to the absolute value of the maximum voltage as a phase compensation map;
  • the spatial light modulator SLM is used to superimpose the phase compensation map on the dissipated light and perform aberration correction on the dissipated light; and is also used to load the spiral phase information of the dissipated light on the liquid crystal surface of the spatial light modulator SLM, Dissipative light is converted into circular light by Gaussian light.
  • the picosecond laser Laser2 is connected to the femtosecond laser Laser1 through an external connection, and the picosecond laser Laser2 is triggered by the femtosecond laser Laser1 to output excitation light.
  • the first light modulating device includes a first half wave plate H1 and a first Glan laser prism G1
  • the second light modulating device includes a second half wave plate H2 and a second Glan laser prism G2
  • the third light modulating device The third half wave plate H3 and the third Glan laser prism G3 are included;
  • the first half wave plate H1 is disposed on the outgoing light path of the femtosecond laser Laser1 for modulating the polarization characteristics of the dissipated light emitted from the femtosecond laser Laser1;
  • the first Glan laser prism G1 is disposed on the outgoing light path of the first half-wave plate H1 for adjusting the intensity of the modulated dissipated light;
  • a second half-wave plate H2 for modulating a polarization characteristic of the dissipated light emitted from the second lens
  • the second Glan laser prism G2 is disposed on the outgoing light path of the second half-wave plate H2 for adjusting the intensity of the modulated dissipated light that is incident;
  • a third half-wave plate H3 for modulating a polarization characteristic of the excitation light emitted from the first single-mode fiber Fiber1;
  • the third Glan laser prism G3 is disposed on the outgoing light path of the third wave plate for adjusting the intensity of the modulated excitation light that is incident.
  • the high numerical aperture objective Obj has a magnification of 100 times and a numerical aperture of 1.4.
  • system further includes: a beam splitter B1 and a charge coupled device CCD;
  • the beam splitter B1 is configured to divide the fluorescent signal collected and emitted by the high numerical aperture objective Obj into two parts, one part is reflected and then enters the charge coupled element CCD, and the other part is transmitted into the filter F1;
  • the charge coupled device CCD is used for real-time monitoring of the overlap of the spot of the dissipated light and the spot of the excitation light in the incident fluorescent signal.
  • system further includes: a first dichroic mirror DM1 and a second dichroic mirror DM2;
  • a first dichroic mirror DM1 for transmitting the dissipated light emitted from the second lens group and reflecting the fluorescent signal
  • the second dichroic mirror DM2 is for reflecting the excitation light emitted from the corner reflector R1 and transmitting the fluorescent signal.
  • the second dichroic mirror DM2 is further configured to adjust a transmission direction of the dissipated light and a transmission direction of the excitation light in the fluorescence signal, so that the dissipated light and the excitation light in the fluorescence signal overlap.
  • the beam splitter B1 divides the fluorescent signal into two parts in accordance with 9:1.
  • the interval between the peaks of the scattered light and the excitation light is 160 to 200 ps.
  • system further includes an electrically controlled baffle E1 that blocks the dissipated light emitted from the second lens group when the electrically controlled baffle E1 is closed.
  • the system has two laser beams, which are the 780 nm-dissipated light generated by the femtosecond laser Laser1 and the excitation light of 635 nm generated by the picosecond laser Laser2.
  • the picosecond laser Laser2 When working, first open the picosecond laser Laser2 to generate excitation light, then turn on the femtosecond laser Laser1 to generate dissipated light.
  • the common method is to use the control system of the femtosecond laser Laser1 to synchronously trigger the Trigger femtosecond laser Laser1 to generate dissipative light and picoseconds.
  • the laser Laser2 generates excitation light.
  • the light path of the dissipated light and the excitation light is described in detail below. Specifically:
  • the optical path of the dissipated light is: the dissipated light first passes through the first light modulating device, and the first light modulating device includes a first half wave plate H1 and a first Glan laser prism G1, and the first half wave plate H1 is disposed in the femtosecond laser
  • the outgoing light path of Laser1 is used to modulate the polarization characteristics of the dissipated light emitted from the femtosecond laser Laser1, ensuring that the dissipated light entering the optical path system is linearly polarized light, and the first Glan laser prism G1 is disposed in the first half-wave plate.
  • the intensity of the dissipated light that becomes linearly polarized light after modulation is adjusted, and then the dissipative light is incident on the glass rod GR, and the glass rod GR is disposed on the outgoing light path of the first light modulation device, and the radiation is emitted.
  • the modulated dissipated light is subjected to pulse width broadening processing so that the pulse width of the dissipated light is 1 picosecond, and then the first lens group is composed of two lenses, which are respectively lens L1 and In the lens L2, the first lens group is disposed on the outgoing light path of the glass rod GR, and expands the spot diameter of the dissipated light having a pulse width of 1 picosecond to expand the spot diameter to the aperture size of the first lens L3.
  • Lens L3 for injection The astigmatism is focused, and the focused dissipated light is coupled into the 100-meter single-mode polarization-maintaining fiber Fiber0.
  • the 100-meter single-mode polarization-maintaining fiber Fiber0 is placed on the exit path of the first lens, and the pulse width is 1 picosecond.
  • the pulse width of the astigmatism is broadened to 200 picoseconds and is incident on the second lens L4.
  • the second lens L4 is disposed on the outgoing light path of the 100 m single mode polarization-maintaining fiber Fiber0, and the second lens L4 has a pulse width of 200 picoseconds.
  • the spatial light modulator SLM has two functions here. First, the compensation phase is generated by the genetic algorithm, and the dissipated light is corrected for aberration. Second, the spiral phase information of the dissipated light is loaded on the liquid crystal surface of the spatial light modulator SLM, and the dissipated light is converted into the ring light by the Gaussian light, and the dissipated light reflected by the spatial light modulator SLM passes through the second lens.
  • the second lens group is composed of two lenses, which are a lens L5 and a lens L6, respectively, and reduces the spot diameter of the incident dissipated light, and enters the electronic control baffle E1 because the electronically controlled baffle E1 is opened.
  • the dissipated light emitted by the second lens group is incident on the first dichroic mirror DM1 through the electronically controlled baffle E1, and the first dichroic mirror DM1 transmits the dissipated light emitted from the second lens group and is incident.
  • the second dichroic mirror DM2, the second dichroic mirror DM2 mirror transmits the dissipated light incident from the first dichroic mirror DM1, and dissipates the light into the galvanometer scanning system Scanner.
  • the excitation light path is: the excitation light is generated by the picosecond laser Laser2, and is injected into the first single-mode fiber Fiber1.
  • the first single-mode fiber Fiber1 mode-modulates the incident excitation light, and couples the mode-controlled excitation light to
  • the third lens L0 and the third lens L0 enlarge the spot diameter of the mode-controlled excitation light and enter the third light modulating device, and the third light modulating device includes the third half-wave plate H3 and the third granule
  • the laser prism G3, the third half-wave plate H3 is for modulating the polarization characteristics of the excitation light emitted from the third lens L0, ensuring that the excitation light entering the optical path system is linearly polarized light, and the third Glan laser prism G3 is disposed in the third half.
  • the intensity of the excitation light that becomes linearly polarized light after modulation is adjusted, and then incident on the corner reflector R1, and the corner reflector R1 is disposed on the outgoing light path of the third light modulation device, and the excitation is changed.
  • the optical path of the optical path in which the light is located controls the pulse interval between the excitation light and the dissipative light in time so that the excitation light is in front and the light is dissipated, and the pulse interval between the excitation light and the dissipative light should be kept at 160 ⁇ 200p Between s, this ensures that the dissipated light more thoroughly returns the excited state electrons generated by the excitation light to the ground state in the form of stimulated radiation, wherein the specific pulse interval value can be determined by the actual super-resolution imaging effect.
  • the excitation light After the excitation light is emitted from the corner reflector R1, it is incident on the second dichroic mirror DM2, and the second dichroic mirror DM2 reflects the excitation light emitted from the corner reflector R1, and the excitation light is incident on the galvanometer scanning system Scanner.
  • the diameter of the excitation light and the scattered light scattered by the Scanner scanning system is amplified, amplified to the aperture of the high numerical aperture objective Obj, and then transmitted through the beam splitter B1 to the quarter glass Q1, quarter glass
  • the film Q1 polarizes the incident excitation light and the dissipated light, modulates the excitation light and the dissipated light into linearly polarized light, and then emits it to the high numerical aperture objective lens Obj, and the high numerical aperture objective lens Obj focuses and overlaps.
  • the excitation light and the dissipated light are projected onto the sample after the focused overlapping excitation light and the dissipated light, and the fluorescent signal reflected by the sample is collected.
  • the fluorescent signal passes through the beam splitter B1, and the beam splitter B1 divides the fluorescent signal collected and emitted by the high numerical aperture objective Obj into two parts, one part is reflected and enters the CCD of the charge coupled element, and the other part is transmitted into the scanning mirror scanning system Scanner, specifically,
  • the beam splitter B1 divides the fluorescent signal into two parts according to 9:1, one tenth enters the charge coupled element CCD through the lens L8, and the charge coupled element CCD monitors the spot of the dissipated light and the excitation light in the injected fluorescent signal in real time.
  • the second dichroic mirror DM2 transmits the fluorescent signal, into the first two directions
  • the color mirror DM1 the first dichroic mirror DM1 reflects the incident fluorescent signal, and is directed to the lens L9, and is focused by the lens L9 to focus the fluorescent signal to the filter F1, and the filter F1 filters the fluorescent signal, which will pre-
  • the fluorescent signal of the band is injected into the second single-mode fiber Fiber2Fiber2, and the fluorescent signal outside the preset band is filtered out, and the fluorescent signal transmitted by the second single-mode fiber Fiber2 after filtering through the filter F1 is transmitted.
  • the photomultiplier tube PMT amplifies the fluorescence signal obtained by filtering through the filter F1, and transmits the amplified fluorescence signal to the first data acquisition card NI1 and the second data acquisition card NI2, the first data The acquisition card NI1 and the second data acquisition card NI2 are installed on the computer Computer.
  • the first data acquisition card NI1 collects and analyzes the fluorescence signal collected by the photomultiplier tube PMT, and uses the fluorescence signal collected by the photomultiplier tube PMT for imaging.
  • the second data acquisition card NI2 converts the fluorescent signal collected by the photomultiplier tube PMT into voltage information, and the voltage information is used as a fitness value in the genetic algorithm, and the voltage information is calculated according to the genetic algorithm to obtain a plurality of absolute values of the voltage.
  • each absolute value of the voltage corresponds to a phase map for judging the effect of the aberration correction and feeding back to the spatial light modulator SLM.
  • the spatial light modulator SLM does not add the spiral phase information, only loads the phase map generated by the genetic algorithm, and compensates the Gaussian-type dissipated light, and generates new ones with the iteration of the genetic algorithm.
  • the phase map because the voltage information is calculated according to the genetic algorithm, the absolute values of the plurality of voltages are gradually increased. After a certain number of iterations, a maximum absolute value of the voltage is obtained, and the phase corresponding to the absolute value of the maximum voltage is obtained.
  • the spatial light modulator SLM loads the spiral phase information of the dissipated light on the liquid crystal surface (as shown in Fig. 3), and the dissipated light is converted into Gaussian light into ring light.
  • a ring-shaped light having an aberration correction effect is formed (as shown in FIG. 4).
  • the annular light having the aberration correction effect is transmitted through the first dichroic mirror DM1 and the second dichroic mirror DM2, and coincides with the Gaussian excitation light after being reflected by the second dichroic mirror DM2, and the galvanometer scanning system Scanner After scanning, the sample is irradiated, and the reflected fluorescent signal is collected and analyzed by the photomultiplier tube PMT and the first data acquisition card NI1 to form a super-resolution image.
  • the mirror M1, the mirror M2, the mirror M3, the mirror M4, the mirror M5, and the mirror M6 are used to change the beam transmission direction.
  • the second dichroic mirror DM2 is further configured to adjust a transmission direction of the dissipated light and a transmission direction of the excitation light in the fluorescence signal, so that the dissipated light and the excitation light in the fluorescence signal overlap, the mirror M5 and the second two
  • the color mirror DM2 can fine tune the excitation light to ensure a spatially high overlap with the dissipated light, as shown in FIG.
  • the high numerical aperture objective Obj has a magnification of 100 times and a numerical aperture of 1.4.
  • a fluorescent bead with a diameter of 170 nm was used as a sample, and an electrically controlled baffle E1 was set on the optical path of the dissipative light (between the lens L6 and the first dichroic mirror DM1), and the electronically controlled baffle E1 was used.
  • the dissipative light emitted from the second lens group is blocked, and only the excitation light is incident on the high numerical aperture objective Obj, which can be regarded as confocal imaging, and the aberration-free correction as shown in FIG. 6(a) is obtained.
  • Fluorescent bead image When closed, the dissipative light emitted from the second lens group is blocked, and only the excitation light is incident on the high numerical aperture objective Obj, which can be regarded as confocal imaging, and the aberration-free correction as shown in FIG. 6(a) is obtained. Fluorescent bead image.
  • both the dissipative light and the picosecond excitation enter the high numerical aperture objective Obj, and the dissipative light and picosecond excitation overlap on the sample to form the stimulated emission loss as shown in Fig. 6(b).
  • STED super-resolution imaging Comparing Fig. 6(a) with Fig. 6(b), it can be observed that the effective point spread function (PSF) of the excitation light is significantly reduced after the dissipative light is applied.
  • PSF point spread function
  • a super-resolution imaging system including a femtosecond laser Laser1, a picosecond laser Laser2, a first data acquisition card NI1, a second data acquisition card NI2, a spatial light modulator SLM, etc., femtosecond
  • the dissipative light generated by the laser Laser1 and the excitation light generated by the picosecond laser Laser2 are injected into the sample to generate a fluorescent signal
  • the second data acquisition card NI2 converts the fluorescent signal into voltage information, which is used as a fitness in the genetic algorithm.
  • the value is calculated according to the genetic algorithm to obtain a maximum absolute value of the maximum voltage, and the phase map corresponding to the absolute value of the maximum voltage is used as the phase compensation map, and the phase compensation map is superimposed on the dissipated light to dissipate the light. Perform aberration correction.
  • the embodiment of the present invention introduces an aberration correction system of a genetic algorithm in a STED super-resolution imaging system, and obtains a phase compensation map according to a genetic algorithm, and superimposes the phase compensation map on the dissipated light to dissipate
  • the optical aberration correction overcomes the aberration introduced in the STED imaging process and improves the imaging depth and spatial resolution of the STED super-resolution imaging system, so that it can be widely used in the field of medicine and the like.

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Abstract

一种超分辨成像系统，包括飞秒激光器（Laser1）、皮秒激光器（Laser2）、第一数据采集卡（NI1）、第二数据采集卡（NI2）、空间光调制器（SLM）等，飞秒激光器（Laser1）产生的耗散光与皮秒激光器（Laser2）产生的激发光射入样本后，会产生荧光信号，第二数据采集卡（NI2）将荧光信号转换为电压信息，电压信息作为遗传算法中的适应度值，根据遗传算法对电压信息进行计算，得到一个最大电压绝对值，并将与最大电压绝对值对应的相位图作为相位补偿图，通过将相位补偿图叠加到耗散光上，对耗散光进行像差校正，克服STED成像过程中引入的像差，提高STED超分辨成像系统的成像深度和空间分辨率，使得可以在生物医学等方面得到广泛应用。

Description

超分辨成像系统
本发明涉及光学显微成像领域，尤其涉及一种超分辨成像系统。
在光学系统中，如果从物点发出的所有光线经过受激发射损耗（synchronous transition equivalent decomposition，STED）超分辨成像系统都聚焦于一点，则称此点为物点的完善像，如果物点在垂直平面上移动时，其完善像也在垂直平面上作线性运动，此STED超分辨成像系统是理想的STED超分辨成像系统。但是，STED超分辨成像系统中通常包含许多光学原件，如包含许多透镜、玻片、二向色镜和反射镜等光学原件，而且人工搭建的STED超分辨成像系统无法做到对光线的精准控制，同时生物样品存在表面不平整性和内部折射率分布的非均匀性特点，这些因素都会给STED超分辨成像系统带来像差，降低STED超分辨成像系统的成像深度和空间分辨率，影响成像质量，限制了其在生物医学等方面的应用。
因此，现有的STED超分辨成像系统，存在着由于包含许多光学原件、生物样品存在表面不平整性及内部折射率分布的非均匀性特点等因素，会给STED超分辨成像系统带来像差，降低STED超分辨成像系统的成像深度和空间分辨率，影响成像质量，限制了其在生物医学等方面的应用的技术问题。
发明内容
本发明的主要目的在于提供一种超分辨成像系统 ，旨在解决现有的STED超分辨成像系统，存在的由于包含许多光学原件、生物样品存在表面不平整性及内部折射率分布的非均匀性特点等因素，会给STED超分辨成像系统带来像差，降低STED超分辨成像系统的成像深度和空间分辨率，影响成像质量，限制了其在生物医学等方面的应用的技术问题。
为实现上述目的，本发明提供一种超分辨成像系统，所述系统包括：飞秒激光器，用于产生耗散光，并入射至第一光调制装置；
所述第一光调制装置，设置在所述飞秒激光器的出射光路上，用于调制所述耗散光的偏振特性及强度；
玻璃棒，设置在所述第一光调制装置的出射光路上，用于对经过调制后的耗散光进行脉冲宽度展宽处理，使得所述耗散光的脉冲宽度为1皮秒；
第一透镜组，设置在所述玻璃棒的出射光路上，用于对脉冲宽度为1皮秒的耗散光的光斑直径进行扩大处理；
第一透镜，设置在所述第一透镜组的出射光路上，用于将光斑直径扩大的耗散光聚焦耦合至100米单模保偏光纤；
所述100米单模保偏光纤，设置在所述第一透镜的出射光路上，用于将脉冲宽度为1皮秒的耗散光的脉冲宽度展宽至200皮秒；
第二透镜，设置在所述100米单模保偏光纤的出射光路上，用于对脉冲宽度为200皮秒的耗散光的光斑直径进行扩大处理，并入射至第二光调制装置；
所述第二光调制装置，设置在所述第二透镜的出射光路上，用于调制所述耗散光的偏振特性及强度；
所述空间光调制器，设置在所述第二光调制装置的出射光路上，将从所述第二光调制装置射出的耗散光反射至第二透镜组；
所述第二透镜组，用于对射入的耗散光的光斑直径进行缩小处理，并将光斑直径进行缩小处理的耗散光射入振镜扫描系统；
皮秒激光器，用于产生激发光，并入射至第一单模光纤；
所述第一单模光纤，设置在所述皮秒激光器的出射光路上，用于对射入的激发光进行模式调控；
第三透镜，设置在所述第一单模光纤的出射光路上，用于对进行模式调控后的激发光的光斑直径进行扩大处理，并入射至第三光调制装置；
所述第三光调制装置，用于调制所述激发光的偏振特性及强度；
角反射器，设置在所述第三光调制装置的出射光路上，用于改变所述激发光所在的光路的光程，在时间上控制所述激发光及所述耗散光之间的脉冲间隔，并将所述激发光射入所述振镜扫描系统；
所述振镜扫描系统，用于对重叠的激发光及耗散光进行同步面阵扫描；
四分之一玻片，用于对经过所述振镜扫描系统扫描后的激发光及耗散光进行偏振处理，将所述激发光及所述耗散光由线偏振光调制成圆偏振光；
高数值孔径物镜，用于聚焦重叠的激发光及耗散光，将聚焦后的重叠的激发光及耗散光投射到样品上，同时收集所述样品反射回来的荧光信号；
滤镜，用于对所述荧光信号进行过滤，将预设波段的荧光信号射入第二单模光纤，并过滤掉所述预设波段以外的荧光信号；
第二单模光纤，用于将经过所述滤镜过滤后得到的荧光信号传输给光电倍增管；
所述光电倍增管，用于将经过所述滤镜过滤后得到的荧光信号进行放大；
第一数据采集卡，用于采集和分析所述光电倍增管收集到的荧光信号；
第二数据采集卡，用于将所述光电倍增管收集到的荧光信号转换为电压信息，所述电压信息作为遗传算法中的适应度值，根据遗传算法对所述电压信息进行计算，得到一个最大电压绝对值，并将与最大电压绝对值对应的相位图作为相位补偿图；
所述空间光调制器用于将所述相位补偿图叠加到所述耗散光上，对所述耗散光进行像差校正；还用于在所述空间光调制器的液晶面上加载所述耗散光的螺旋相位信息，将所述耗散光由高斯型光转化成环形光。
可选的，所述皮秒激光器通过外接线与所述飞秒激光器相连接，所述皮秒激光器由所述飞秒激光器触发输出所述激发光。
可选的，所述第一光调制装置包括第一半波片及第一格兰激光棱镜，所述第二光调制装置包括第二半波片及第二格兰激光棱镜，所述第三光调制装置包括第三半波片及第三格兰激光棱镜；
所述第一半波片，设置在所述飞秒激光器的出射光路上，用于调制从所述飞秒激光器射出的耗散光的偏振特性；
所述第一格兰激光棱镜，设置在所述第一半波片的出射光路上，用于对调制后的耗散光的强度进行调整；
所述第二半波片，用于调制从所述第二透镜射出的耗散光的偏振特性；
所述第二格兰激光棱镜，设置在所述第二半波片的出射光路上，用于对射入的调制后的耗散光的强度进行调整；
所述第三半波片，用于调制从所述第一单模光纤射出的激发光的偏振特性；
所述第三格兰激光棱镜，设置在所述第三波片的出射光路上，用于对射入的调制后的激发光的强度进行调整。
可选的，所述高数值孔径物镜的放大倍率为100倍，数值孔径为1.4。
可选的，所述系统还包括：分束器及电荷耦合元件；
所述分束器，用于将所述高数值孔径物镜收集并射出的荧光信号分成两部分，一部分反射后进入所述电荷耦合元件，另一部分透射进入所述滤镜；
所述电荷耦合元件，用于实时监测射入的荧光信号中的耗散光的光斑和激发光的光斑的重叠情况。
可选的，所述系统还包括：第一二向色镜及第二二向色镜；
所述第一二向色镜，用于透射从所述第二透镜组射出的耗散光，并并反射所述荧光信号；
所述第二二向色镜，用于反射从所述角反射器射出的激发光，并透射所述荧光信号。
可选的，所述第二二向色镜，还用于调整所述荧光信号中的耗散光的传输方向及激发光的传输方向，使所述荧光信号中的所述耗散光及所述激发光重叠。
可选的，所述分束器将所述荧光信号按照 9:1 分成两部分。
可选的，所述耗散光和所述激发光的脉冲峰值之间的间隔为160~200ps。
可选的，所述系统还包括电控挡板，当所述电控挡板闭合时，挡住从所述第二透镜组射出的耗散光。
本发明提供一种超分辨成像系统，包括飞秒激光器、皮秒激光器、第一数据采集卡、第二数据采集卡、空间光调制器等，飞秒激光器产生的耗散光与皮秒激光器产生的激发光射入样本后，会产生荧光信号，第二数据采集卡将荧光信号转换为电压信息，该电压信息作为遗传算法中的适应度值，根据遗传算法对电压信息进行计算，得到一个最大电压绝对值，并将与最大电压绝对值对应的相位图作为相位补偿图，通过将相位补偿图叠加到耗散光上，对耗散光进行像差校正。与现有技术相比，本发明实施例在STED超分辨成像系统中引入遗传算法的像差校正系统，根据遗传算法得到相位补偿图，通过将相位补偿图叠加到耗散光上，对耗散光进行像差校正，克服STED成像过程中引入的像差，提高STED超分辨成像系统的成像深度和空间分辨率，使得可以在物医学等方面得到广泛应用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有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超分辨成像系统的结构示意图；
图2为对高斯型耗散光进行像差校正的待加载在空间光调制器SLM上的相位补偿图；
图3为加载在空间光调制器SLM上的耗散光的螺旋相位信息示意图；
图4为将相位补偿图与螺旋相位信息叠加后的具有像差校正效果的环形光示意图；
图5为耗尽光与激发光重叠后超越衍射极限的示意图；
图6（a）为无像差校正时的荧光珠的共聚焦成像示意图；
图6（b）为荧光珠的受激发射损耗STED超分辨成像示意图。
具体实施方式
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。
为了说明本发明的技术方案，下面通过具体实施例来进行说明。
请参阅图1，图1为本发明第一实施例提供的一种超分辨成像系统的结构示意图，包括：飞秒激光器Laser1，用于产生耗散光，并入射至第一光调制装置；
第一光调制装置，设置在飞秒激光器Laser1的出射光路上，用于调制耗散光的偏振特性及强度；
玻璃棒GR，设置在第一光调制装置的出射光路上，用于对经过调制后的耗散光进行脉冲宽度展宽处理，使得耗散光的脉冲宽度为1皮秒；
第一透镜组，设置在玻璃棒GR的出射光路上，用于对脉冲宽度为1皮秒的耗散光的光斑直径进行扩大处理；
第一透镜L3，设置在第一透镜组的出射光路上，用于将光斑直径扩大的耗散光聚焦耦合至100米单模保偏光纤Fiber0；
100米单模保偏光纤Fiber0，设置在第一透镜L3的出射光路上，用于将脉冲宽度为1皮秒的耗散光的脉冲宽度展宽至200皮秒；
第二透镜L4，设置在100米单模保偏光纤Fiber0的出射光路上，用于对脉冲宽度为200皮秒的耗散光的光斑直径进行扩大处理，并入射至第二光调制装置；
第二光调制装置，设置在第二透镜L4的出射光路上，用于调制耗散光的偏振特性及强度；
空间光调制器SLM，设置在第二光调制装置的出射光路上，将从第二光调制装置射出的耗散光反射至第二透镜组；
第二透镜组，用于对射入的耗散光的光斑直径进行缩小处理，并将光斑直径进行缩小处理的耗散光射入振镜扫描系统Scanner；
皮秒激光器Laser2，用于产生激发光，并入射至第一单模光纤Fiber1；
第一单模光纤Fiber1，设置在皮秒激光器Laser2的出射光路上，用于对射入的激发光进行模式调控；
第三透镜，设置在第一单模光纤Fiber1的出射光路上，用于对进行模式调控后的激发光的光斑直径进行扩大处理，并入射至第三光调制装置；
第三光调制装置，用于调制激发光的偏振特性及强度；
角反射器R1，设置在第三光调制装置的出射光路上，用于改变激发光所在的光路的光程，在时间上控制激发光及耗散光之间的脉冲间隔，并将激发光射入振镜扫描系统Scanner；
振镜扫描系统Scanner，用于对重叠的激发光及耗散光进行同步面阵扫描；
四分之一玻片Q1，用于对经过振镜扫描系统Scanner扫描后的激发光及耗散光进行偏振处理，将激发光及耗散光由线偏振光调制成圆偏振光；
高数值孔径物镜Obj，用于聚焦重叠的激发光及耗散光，将聚焦后的重叠的激发光及耗散光投射到样品上，同时收集样品反射回来的荧光信号；
滤镜F1，用于对荧光信号进行过滤，将预设波段的荧光信号射入第二单模光纤Fiber2，并过滤掉预设波段以外的荧光信号；
第二单模光纤Fiber2，用于将经过滤镜F1过滤后得到的荧光信号传输给光电倍增管PMT；
光电倍增管PMT，用于将经过滤镜F1过滤后得到的荧光信号进行放大；
第一数据采集卡NI1，用于采集和分析光电倍增管PMT收集到的荧光信号；
第二数据采集卡NI2，用于将光电倍增管PMT收集到的荧光信号转换为电压信息，电压信息作为遗传算法中的适应度值，根据遗传算法对电压信息进行计算，得到一个最大电压绝对值，并将与最大电压绝对值对应的相位图作为相位补偿图；
空间光调制器SLM用于将相位补偿图叠加到耗散光上，对耗散光进行像差校正；还用于在空间光调制器SLM的液晶面上加载耗散光的螺旋相位信息，将耗散光由高斯型光转化成环形光。
进一步的，皮秒激光器Laser2通过外接线与飞秒激光器Laser1相连接，皮秒激光器Laser2由飞秒激光器Laser1触发输出激发光。
进一步的，第一光调制装置包括第一半波片H1及第一格兰激光棱镜G1，第二光调制装置包括第二半波片H2及第二格兰激光棱镜G2，第三光调制装置包括第三半波片H3及第三格兰激光棱镜G3；
第一半波片H1，设置在飞秒激光器Laser1的出射光路上，用于调制从飞秒激光器Laser1射出的耗散光的偏振特性；
第一格兰激光棱镜G1，设置在第一半波片H1的出射光路上，用于对调制后的耗散光的强度进行调整；
第二半波片H2，用于调制从第二透镜射出的耗散光的偏振特性；
第二格兰激光棱镜G2，设置在第二半波片H2的出射光路上，用于对射入的调制后的耗散光的强度进行调整；
第三半波片H3，用于调制从第一单模光纤Fiber1射出的激发光的偏振特性；
第三格兰激光棱镜G3，设置在第三波片的出射光路上，用于对射入的调制后的激发光的强度进行调整。
进一步的，高数值孔径物镜Obj的放大倍率为100倍，数值孔径为1.4。
进一步的，系统还包括：分束器B1及电荷耦合元件CCD；
分束器B1，用于将高数值孔径物镜Obj收集并射出的荧光信号分成两部分，一部分反射后进入电荷耦合元件CCD，另一部分透射进入滤镜F1；
电荷耦合元件CCD，用于实时监测射入的荧光信号中的耗散光的光斑和激发光的光斑的重叠情况。
进一步的，系统还包括：第一二向色镜DM1及第二二向色镜DM2；
第一二向色镜DM1，用于透射从第二透镜组射出的耗散光，并并反射荧光信号；
第二二向色镜DM2，用于反射从角反射器R1射出的激发光，并透射荧光信号。
进一步的，第二二向色镜DM2，还用于调整荧光信号中的耗散光的传输方向及激发光的传输方向，使荧光信号中的耗散光及激发光重叠。
进一步的，分束器B1将荧光信号按照 9:1 分成两部分。
进一步的，耗散光和激发光的脉冲峰值之间的间隔为160~200ps。
进一步的，系统还包括电控挡板E1，当电控挡板E1闭合时，挡住从第二透镜组射出的耗散光。
[根据细则26改正08.02.2018]　
在本发明实施例中，如图1所示，本系统有两束激光，分别为飞秒激光器Laser1产生的波长为780nm的耗散光和皮秒激光器Laser2产生的波长为635nm的激发光。工作时，先打开皮秒激光器Laser2产生激发光，后打开飞秒激光器Laser1产生耗散光，常用手段是利用飞秒激光器Laser1的控制系统来同步触发Trigger飞秒激光器Laser1产生耗散光及皮秒激光器Laser2产生激发光，下面详细介绍耗散光及激发光的光路，具体的：
耗散光的光路为：耗散光首先通过第一光调制装置，第一光调制装置包括第一半波片H1及第一格兰激光棱镜G1，第一半波片H1设置在飞秒激光器Laser1的出射光路上，用于调制从飞秒激光器Laser1射出的耗散光的偏振特性，确保进入光路系统的耗散光为线偏振光，第一格兰激光棱镜G1设置在第一半波片H1的出射光路上，对调制后变成线偏振光的耗散光的强度进行调整，之后耗散光射入玻璃棒GR，玻璃棒GR设置在第一光调制装置的出射光路上，对射入的经过调制后的耗散光进行脉冲宽度展宽处理，使得耗散光的脉冲宽度为1皮秒后，射入第一透镜组，第一透镜组由两个透镜组成，分别是透镜L1和透镜L2，第一透镜组设置在玻璃棒GR的出射光路上，对脉冲宽度为1皮秒的耗散光的光斑直径进行扩大处理，将光斑直径扩大至第一透镜L3的孔径大小，第一透镜L3对射入的耗散光进行聚焦，并将聚焦后的耗散光耦合进100米单模保偏光纤Fiber0，100米单模保偏光纤Fiber0设置在第一透镜的出射光路上，将脉冲宽度为1皮秒的耗散光的脉冲宽度展宽至200皮秒，并射入第二透镜L4，第二透镜L4设置在100米单模保偏光纤Fiber0的出射光路上，第二透镜L4对脉冲宽度为200皮秒的耗散光的光斑直径进行扩大处理，将光斑直径扩大至略小于空间光调制器SLM的液晶面的纵向宽度，然后进入第二光调制装置，第二光调制装置包括第二半波片H2及第二格兰激光棱镜G2，利用第二光调制装置再一次调节耗散光的偏振特性和强度，使得耗散光的偏振方向平行于空间光调制器SLM的液晶面的长轴，以保证空间光调制器SLM对耗散光调制的灵敏度。之后，耗散光以6°左右的入射角射入空间光调制器SLM，空间光调制器SLM在这里有两个作用，一是由遗传算法控制产生补偿相位，对耗散光进行像差校正，二是在空间光调制器SLM的液晶面上加载耗散光的螺旋相位信息，将耗散光由高斯型光转化成环形光，空间光调制器SLM反射后的耗散光经过第二透镜组，第二透镜组由两个透镜组成，分别是透镜L5和透镜L6，对射入的耗散光的光斑直径进行缩小处理，射入电控挡板E1，因电控挡板E1是打开的，第二透镜组射出的耗散光穿过电控挡板E1射入第一二向色镜DM1，第一二向色镜DM1透射从第二透镜组射出的耗散光，并射入第二二向色镜DM2，第二二向色镜DM2镜透射从第一二向色镜DM1射入的耗散光，耗散光进入振镜扫描系统Scanner。
激发光的光路为：激发光由皮秒激光器Laser2产生，射入第一单模光纤Fiber1，第一单模光纤Fiber1对射入的激发光进行模式调控，并将模式调控后的激发光耦合至第三透镜L0，第三透镜L0对进行模式调控后的激发光的光斑直径进行扩大处理，并入射至第三光调制装置，第三光调制装置包括第三半波片H3及第三格兰激光棱镜G3，第三半波片H3用于调制从第三透镜L0射出的激发光的偏振特性，确保进入光路系统的激发光为线偏振光，第三格兰激光棱镜G3设置在第三半波片H3的出射光路上，对调制后变成线偏振光的激发光的强度进行调整，之后射入角反射器R1，角反射器R1设置在第三光调制装置的出射光路上，改变激发光所在的光路的光程，在时间上控制激发光及耗散光之间的脉冲间隔，使得激发光在前，耗散光在后，激发光和耗散光之间的脉冲间隔应保持在160~200ps之间，这样可以保证耗散光更彻底地将激发光产生的激发态电子以受激辐射的形式返回到基态，其中，具体的脉冲间隔数值可通过实际的超分辨成像效果来确定。激发光从角反射器R1射出后，射入第二二向色镜DM2，第二二向色镜DM2反射从角反射器R1射出的激发光，激发光射入进入振镜扫描系统Scanner。
[根据细则26改正08.02.2018]　
激发光和耗散光都射入振镜扫描系统Scanner后，振镜扫描系统Scanner对重叠的激发光及耗散光进行同步面阵扫描，之后射入透镜L7和镜筒透镜T1，对经过振镜扫描系统Scanner扫描后的激发光及耗散光的光斑直径进行放大，放大至高数值孔径物镜Obj的孔径大小，之后透过分束器B1射向四分之一玻片Q1，四分之一玻片Q1对射入的激发光及耗散光进行偏振处理，将激发光及耗散光由线偏振光调制成圆偏振光后，射向高数值孔径物镜Obj，高数值孔径物镜Obj聚焦重叠的激发光及耗散光，将聚焦后的重叠的激发光及耗散光投射到样品上，同时收集样品反射回来的荧光信号。荧光信号经过分束器B1，分束器B1将高数值孔径物镜Obj收集并射出的荧光信号分成两部分，一部分反射后进入电荷耦合元件CCD，另一部分透射进入振镜扫描系统Scanner，具体的，分束器B1将荧光信号按照 9:1 分成两部分，十分之一通过透镜L8进入电荷耦合元件CCD，电荷耦合元件CCD实时监测射入的荧光信号中的耗散光的光斑和激发光的光斑的重叠情况，十分之九进入振镜扫描系统Scanner，并从振镜扫描系统Scanner射入第二二向色镜DM2，第二二向色镜DM2透射荧光信号，射入第一二向色镜DM1，第一二向色镜DM1反射射入的荧光信号，并射向透镜L9，经过透镜L9的聚焦，将荧光信号聚焦到滤镜F1，滤镜F1对荧光信号进行过滤，将预设波段的荧光信号射入第二单模光纤Fiber2Fiber2，并过滤掉预设波段以外的荧光信号，第二单模光纤Fiber2将经过滤镜F1过滤后得到的荧光信号传输给光电倍增管PMT，光电倍增管PMT将经过滤镜F1过滤后得到的荧光信号进行放大，并将放大后的荧光信号传输至第一数据采集卡NI1及第二数据采集卡NI2，第一数据采集卡NI1及第二数据采集卡NI2是安装在电脑Computer上的，第一数据采集卡NI1采集和分析光电倍增管PMT收集到的荧光信号，将光电倍增管PMT收集到的荧光信号用于成像，第二数据采集卡NI2将光电倍增管PMT收集到的荧光信号转换为电压信息，该电压信息作为遗传算法中的适应度值，根据遗传算法对电压信息进行计算，得到多个电压绝对值，其中，每一个电压绝对值都对应着一个相位图，用以判断像差校正的效果，并反馈给空间光调制器SLM。需要注意的是，实验时，空间光调制器SLM先不加螺旋相位信息，只加载由遗传算法生成的相位图，对高斯型耗散光进行相位补偿，随着遗传算法的迭代不断生成新的相位图，因根据遗传算法对电压信息进行计算，得到的多个电压绝对值以逐渐增加的趋势变化，经过一定次数的迭代，会得到一个最大电压绝对值，将与最大电压绝对值对应的相位图作为相位补偿图（如图2所示），空间光调制器SLM在液晶面上加载耗散光的螺旋相位信息（如图3所示），则耗散光由高斯型光转化成环形光，将相位补偿图与螺旋相位信息叠加，就形成具有像差校正效果的环形光（如图4所示）。具有像差校正效果的环形光经过第一二向色镜DM1和第二二向色镜DM2透射，与经过第二二向色镜DM2反射之后的高斯型激发光重合，经振镜扫描系统Scanner扫描之后照射到样品上，通过光电倍增管PMT和第一数据采集卡NI1收集和分析反射的荧光信号，进而形成超分辨图像。
在本发明实施例中，如图1，反射镜M1、反射镜M2、反射镜M3、反射镜M4、反射镜M5、反射镜M6用来改变光束传输方向。
其中，第二二向色镜DM2还用于调整荧光信号中的耗散光的传输方向及激发光的传输方向，使荧光信号中的耗散光及激发光重叠，反射镜M5和第二二向色镜DM2可以对激发光进行微调，以确保与耗散光在空间上高度重合，如图 5 所示。
其中，高数值孔径物镜Obj的放大倍率为100倍，数值孔径为1.4。
在实验中，将直径为170nm的荧光珠作为样品，通过在耗散光的光路上（透镜L6与第一二向色镜DM1之间）设置一块电控挡板E1，当电控挡板E1闭合时，挡住从第二透镜组射出的耗散光，只有激发光射入高数值孔径物镜Obj，此时可视为共聚焦成像，得到如图6（a）所示的无像差校正时的荧光珠图像。当电控挡板E1打开时，耗散光及皮秒激发都进入高数值孔径物镜Obj，耗散光及皮秒激发在样品上重叠，形成如图6（b）所示的受激发射损耗STED超分辨成像。对比图6（a）与图6（b），可以观察到耗散光作用后，激发光的有效点扩展函数（point spread function，PSF）明显减小。
在本发明实施例中，提供了一种超分辨成像系统，包括飞秒激光器Laser1、皮秒激光器Laser2、第一数据采集卡NI1、第二数据采集卡NI2、空间光调制器SLM等，飞秒激光器Laser1产生的耗散光与皮秒激光器Laser2产生的激发光射入样本后，会产生荧光信号，第二数据采集卡NI2将荧光信号转换为电压信息，该电压信息作为遗传算法中的适应度值，根据遗传算法对电压信息进行计算，得到一个最大电压绝对值，并将与最大电压绝对值对应的相位图作为相位补偿图，通过将相位补偿图叠加到耗散光上，对耗散光进行像差校正。与现有技术相比，本发明实施例在STED超分辨成像系统中引入遗传算法的像差校正系统，根据遗传算法得到相位补偿图，通过将相位补偿图叠加到耗散光上，对耗散光进行像差校正，克服STED成像过程中引入的像差，提高STED超分辨成像系统的成像深度和空间分辨率，使得可以在物医学等方面得到广泛应用。
在上述实施例中，对各个实施例的描述都各有侧重，某个实施例中没有详述的部分，可以参见其它实施例的相关描述。
以上为对本发明所提供的一种超分辨成像系统的描述，对于本领域的技术人员，依据本发明实施例的思想，在具体实施方式及应用范围上均会有改变之处，综上，本说明书内容不应理解为对本发明的限制。

Claims (10)

  1. 一种超分辨成像系统，其特征在于，所述系统包括：
    飞秒激光器，用于产生耗散光，并入射至第一光调制装置；
    所述第一光调制装置，设置在所述飞秒激光器的出射光路上，用于调制所述耗散光的偏振特性及强度；
    玻璃棒，设置在所述第一光调制装置的出射光路上，用于对经过调制后的耗散光进行脉冲宽度展宽处理，使得所述耗散光的脉冲宽度为1皮秒；
    第一透镜组，设置在所述玻璃棒的出射光路上，用于对脉冲宽度为1皮秒的耗散光的光斑直径进行扩大处理；
    第一透镜，设置在所述第一透镜组的出射光路上，用于将光斑直径扩大的耗散光聚焦耦合至100米单模保偏光纤；
    所述100米单模保偏光纤，设置在所述第一透镜的出射光路上，用于将脉冲宽度为1皮秒的耗散光的脉冲宽度展宽至200皮秒；
    第二透镜，设置在所述100米单模保偏光纤的出射光路上，用于对脉冲宽度为200皮秒的耗散光的光斑直径进行扩大处理，并入射至第二光调制装置；
    所述第二光调制装置，设置在所述第二透镜的出射光路上，用于调制所述耗散光的偏振特性及强度；
    所述空间光调制器，设置在所述第二光调制装置的出射光路上，将从所述第二光调制装置射出的耗散光反射至第二透镜组；
    所述第二透镜组，用于对射入的耗散光的光斑直径进行缩小处理，并将光斑直径进行缩小处理的耗散光射入振镜扫描系统；
    皮秒激光器，用于产生激发光，并入射至第一单模光纤；
    所述第一单模光纤，设置在所述皮秒激光器的出射光路上，用于对射入的激发光进行模式调控；
    第三透镜，设置在所述第一单模光纤的出射光路上，用于对进行模式调控后的激发光的光斑直径进行扩大处理，并入射至第三光调制装置；
    所述第三光调制装置，用于调制所述激发光的偏振特性及强度；
    角反射器，设置在所述第三光调制装置的出射光路上，用于改变所述激发光所在的光路的光程，在时间上控制所述激发光及所述耗散光之间的脉冲间隔，并将所述激发光射入所述振镜扫描系统；
    所述振镜扫描系统，用于对重叠的激发光及耗散光进行同步面阵扫描；
    四分之一玻片，用于对经过所述振镜扫描系统扫描后的激发光及耗散光进行偏振处理，将所述激发光及所述耗散光由线偏振光调制成圆偏振光；
    高数值孔径物镜，用于聚焦重叠的激发光及耗散光，将聚焦后的重叠的激发光及耗散光投射到样品上，同时收集所述样品反射回来的荧光信号；
    滤镜，用于对所述荧光信号进行过滤，将预设波段的荧光信号射入第二单模光纤，并过滤掉所述预设波段以外的荧光信号；
    第二单模光纤，用于将经过所述滤镜过滤后得到的荧光信号传输给光电倍增管；
    所述光电倍增管，用于将经过所述滤镜过滤后得到的荧光信号进行放大；
    第一数据采集卡，用于采集和分析所述光电倍增管收集到的荧光信号；
    第二数据采集卡，用于将所述光电倍增管收集到的荧光信号转换为电压信息，所述电压信息作为遗传算法中的适应度值，根据遗传算法对所述电压信息进行计算，得到一个最大电压绝对值，并将与最大电压绝对值对应的相位图作为相位补偿图；
    所述空间光调制器用于将所述相位补偿图叠加到所述耗散光上，对所述耗散光进行像差校正；还用于在所述空间光调制器的液晶面上加载所述耗散光的螺旋相位信息，将所述耗散光由高斯型光转化成环形光。
  2. 根据权利要求1所述的系统，其特征在于，所述皮秒激光器通过外接线与所述飞秒激光器相连接，所述皮秒激光器由所述飞秒激光器触发输出所述激发光。
  3. 根据权利要求1所述的系统，其特征在于，所述第一光调制装置包括第一半波片及第一格兰激光棱镜，所述第二光调制装置包括第二半波片及第二格兰激光棱镜，所述第三光调制装置包括第三半波片及第三格兰激光棱镜；
    所述第一半波片，设置在所述飞秒激光器的出射光路上，用于调制从所述飞秒激光器射出的耗散光的偏振特性；
    所述第一格兰激光棱镜，设置在所述第一半波片的出射光路上，用于对调制后的耗散光的强度进行调整；
    所述第二半波片，用于调制从所述第二透镜射出的耗散光的偏振特性；
    所述第二格兰激光棱镜，设置在所述第二半波片的出射光路上，用于对射入的调制后的耗散光的强度进行调整；
    所述第三半波片，用于调制从所述第一单模光纤射出的激发光的偏振特性；
    所述第三格兰激光棱镜，设置在所述第三波片的出射光路上，用于对射入的调制后的激发光的强度进行调整。
  4. 根据权利要求1所述的系统，其特征在于，所述高数值孔径物镜的放大倍率为100倍，数值孔径为1.4。
  5. 根据权利要求1所述的系统，其特征在于，所述系统还包括：分束器及电荷耦合元件；
    所述分束器，用于将所述高数值孔径物镜收集并射出的荧光信号分成两部分，一部分反射后进入所述电荷耦合元件，另一部分透射进入所述滤镜；
    所述电荷耦合元件，用于实时监测射入的荧光信号中的耗散光的光斑和激发光的光斑的重叠情况。
  6. 根据权利要求1所述的系统，其特征在于，所述系统还包括：第一二向色镜及第二二向色镜；
    所述第一二向色镜，用于透射从所述第二透镜组射出的耗散光，并并反射所述荧光信号；
    所述第二二向色镜，用于反射从所述角反射器射出的激发光，并透射所述荧光信号。
  7. 根据权利要求1所述的系统，其特征在于，所述第二二向色镜，还用于调整所述荧光信号中的耗散光的传输方向及激发光的传输方向，使所述荧光信号中的所述耗散光及所述激发光重叠。
  8. 根据权利要求1所述的系统，其特征在于，所述分束器将所述荧光信号按照 9:1 分成两部分。
  9. 根据权利要求1所述的系统，其特征在于，所述耗散光和所述激发光的脉冲峰值之间的间隔为160~200ps。
  10. 根据权利要求1所述的系统，其特征在于，所述系统还包括电控挡板，当所述电控挡板闭合时，挡住从所述第二透镜组射出的耗散光。
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