WO2019119458A1 - Super resolution imaging system - Google Patents

Abstract

A super resolution imaging system, comprising a femtosecond laser (Laser1), a picosecond laser (Laser2), a first data collection card (NI1), a second data collection card (NI2), a spatial light modulator (SLM), etc. After the dissipation light generated by the femtosecond laser (Laser1) and the excitation light generated by the picosecond laser (Laser2) are emitted into a sample, a fluorescent signal is generated; the second data collection card (NI2) converts the fluorescence signal into voltage information; the voltage information acts as a fitness value of a genetic algorithm, and the voltage information is calculated according to the genetic algorithm so as to obtain the maximum voltage absolute value; and a phase diagram corresponding to the maximum voltage absolute value acts as a phase compensation diagram, and the phase compensation diagram is superposed on the dissipation light to perform aberration correction on the dissipation light. By means of the present invention, the aberration introduced in an STED imaging process can be overcome, the imaging depth and the spatial resolution of the STED super resolution imaging system can be improved, and thus the super resolution imaging system can be widely applied in biomedicine and other fields.

Description

Super resolution imaging system

The present invention relates to the field of optical microscopy, and more particularly to a super-resolution imaging system.

In an optical system, if all the light emitted from the object point is focused on a point by a synchronous transition equivalent decomposition (STED) super-resolution imaging system, this point is called a perfect image of the object point, if the object point is When moving in a vertical plane, its perfect image also moves linearly on a vertical plane. This STED super-resolution imaging system is an ideal STED super-resolution imaging system. However, 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. At the same time, 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.

Therefore, 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.

Summary of the invention

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.

To achieve the above object, 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.

Optionally, 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.

Optionally, the first light modulation device includes a first half wave plate and a first Glan laser prism, and 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.

Optionally, the high numerical aperture objective lens has a magnification of 100 times and a numerical aperture of 1.4.

Optionally, 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.

Optionally, 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.

Optionally, 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.

Optionally, the beam splitter splits the fluorescent signal into two parts according to 9:1.

Optionally, the interval between the dissipated light and the pulse peak of the excitation light is 160 to 200 ps.

Optionally, 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 After the excitation light is injected into the sample, a fluorescent signal is generated, and the second data acquisition card converts the fluorescent signal 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 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. Compared with the prior art, 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.

DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below. Obviously, the drawings in the following description are only It is a certain embodiment of the present invention, and those skilled in the art can obtain other drawings according to these drawings without any creative work.

1 is a schematic structural diagram of a super-resolution imaging system according to a first embodiment of the present invention;

2 is a phase compensation diagram to be loaded on a spatial light modulator SLM for aberration correction of Gaussian-type dissipative light;

3 is a schematic diagram of spiral phase information of dissipated light loaded on a spatial light modulator SLM;

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.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. The embodiments are merely a part of the embodiments of the invention, and not all of the embodiments. All other embodiments obtained by a person skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.

In order to explain the technical solution of the present invention, the following description will be made by way of specific embodiments.

Please refer to FIG. 1. 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;

100m single-mode polarization-maintaining fiber Fiber0, disposed on the outgoing light path of the first lens L3, for widening the pulse width of the scattered light with a pulse width of 1 picosecond to 200 picoseconds;

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.

Further, 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.

Further, the first light modulating device includes a first half wave plate H1 and a first Glan laser prism G1, and the second light modulating device includes a second half wave plate H2 and a second Glan laser prism G2, and 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.

Further, the high numerical aperture objective Obj has a magnification of 100 times and a numerical aperture of 1.4.

Further, the 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.

Further, the 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.

Further, 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.

Further, the beam splitter B1 divides the fluorescent signal into two parts in accordance with 9:1.

Further, the interval between the peaks of the scattered light and the excitation light is 160 to 200 ps.

Further, the 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.

[Correct according to Rule 26 08.02.2018]
In the embodiment of the present invention, as shown in FIG. 1, 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. 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. On the outgoing light path of H1, 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. Expanding the spot diameter of the scattered light, expanding the spot diameter to be slightly smaller than the longitudinal width of the liquid crystal surface of the spatial light modulator SLM, and then entering the second light modulating device, the second light modulating device including the second half wave plate H2 and The second Glan laser prism G2 uses the second light modulating device to adjust the polarization characteristics and intensity of the dissipated light again, so that the polarization direction of the dissipated light is parallel to the long axis of the liquid crystal surface of the spatial light modulator SLM to ensure space. The sensitivity of the light modulator SLM to dissipative light modulation. After that, the dissipated light is incident on the spatial light modulator SLM at an incident angle of about 6°. 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. On the outgoing light path of the wave plate H3, 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. 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.

[Correct according to Rule 26 08.02.2018]
After the excitation light and the dissipated light are injected into the galvanometer scanning system Scanner, the galvanometer scanning system scans the overlapping excitation light and the dissipated light for synchronous area array scanning, and then enters the lens L7 and the lens barrel lens T1. 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 overlap of the spots, nine out of ten into the galvanometer scanning system Scanner, and from the galvanometer scanning system Scanner into the second dichroic mirror DM2, 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. To the photomultiplier tube PMT, 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. Wherein, 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. It should be noted that, during the experiment, 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. As a phase compensation diagram (as shown in Fig. 2), 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. By superimposing the phase compensation map and the spiral phase information, 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.

In the embodiment of the present invention, as shown in FIG. 1, 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.

Among them, the high numerical aperture objective Obj has a magnification of 100 times and a numerical aperture of 1.4.

In the experiment, 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. 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. When the electronically controlled baffle E1 is opened, 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.

In the embodiment of the present invention, a super-resolution imaging system is provided, 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, and 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. Compared with the prior art, 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.

In the above embodiments, the descriptions of the various embodiments are all focused, and the parts that are not detailed in a certain embodiment can be referred to the related descriptions of other embodiments.

The above is a description of a super-resolution imaging system provided by the present invention. For those skilled in the art, according to the idea of the embodiment of the present invention, there will be changes in specific implementation modes and application scopes. The description should not be construed as limiting the invention.

Claims (10)

1. A super-resolution imaging system, characterized in that the system comprises:
   a femtosecond laser for generating dissipative light and incident on the 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.
2. The system of claim 1 wherein said picosecond laser is coupled to said femtosecond laser by an external wiring, said picosecond laser being triggered by said femtosecond laser to output said excitation light.
3. The system of claim 1 wherein said first light modulating means comprises a first half wave plate and a first Glan laser prism, said second light modulating means comprising a second half wave plate and said second a Glan laser prism, the third light modulating device comprising 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.
4. The system of claim 1 wherein said high numerical aperture objective has a magnification of 100 and a numerical aperture of 1.4.
5. The system of claim 1 further comprising: 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.
6. The system of claim 1 further comprising: 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.
7. The system according to claim 1, wherein said second dichroic mirror is further configured to adjust a transmission direction of the dissipated light and a transmission direction of the excitation light in said fluorescent signal to cause said fluorescent signal The dissipated light and the excitation light overlap.
8. The system of claim 1 wherein said beam splitter splits said fluorescent signal into two portions in a 9:1 ratio.
9. The system of claim 1 wherein the interval between the dissipative light and the pulse peak of the excitation light is between 160 and 200 ps.
10. The system of claim 1 wherein said system further comprises an electrically controlled baffle that blocks dissipative light emitted from said second lens group when said electronically controlled baffle is closed.