CN113567410A - Low-light-intensity single-beam large-field-of-view laser point scanning super-resolution microscopic imaging device and method - Google Patents
Low-light-intensity single-beam large-field-of-view laser point scanning super-resolution microscopic imaging device and method Download PDFInfo
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Abstract
The invention discloses a low-light-intensity single-beam large-visual-field laser point scanning super-resolution microscopic imaging device and a method, and particularly relates to a single-beam near-infrared continuous laser excitation rare earth doped nanoparticle, which is used for triggering photon avalanche up-conversion fluorescence radiation of a plurality of different wave bands under the excitation of low-power laser, wherein the photon avalanche fluorescence has high-order nonlinear response to the excitation light intensity, and is excited by using an excitation light spot focused to a diffraction limit, so that a fluorescence point diffusion function which is simultaneously compressed in three dimensions can be obtained, the limitation of the optical diffraction limit is broken through, and a large-visual-field super-resolution image can be obtained in a short time through rapid point-by-point scanning and recording of the laser. Based on the method, a microscopic imaging device consisting of an excitation light generation module, a laser scanning module and a photoelectric detection module is built, and a three-dimensional super-resolution microscopic technology which has the advantages of no light bleaching, low phototoxicity, high resolution, real-time imaging, low complexity, low cost and easiness in use is realized under the excitation of low-power single-beam laser.
Description
Technical Field
The invention belongs to the technical field of optical microscopy, and particularly relates to a low-light-intensity single-beam large-field laser point scanning super-resolution microscopic imaging method and device.
Technical Field
In optical microscopy imaging, due to the existence of diffraction limit, the limit resolution which can be achieved by an optical microscope is about 200nm, and the observation of a nano-scale structure is difficult to meet. In order to break through the optical diffraction limit, researchers have proposed a series of far-field optical super-resolution microscopic imaging methods, the most representative of which is Stimulated emission depletion microscopy (STED) based on laser scanning, in which while scanning a sample with gaussian focused laser, another beam of ring-shaped light is used to focus fluorescence around a depletion fluorescence spot, so as to obtain a smaller effective fluorescence point spread function, and then scanning to obtain a super-resolution fluorescence image. Due to the characteristics of real time, rapidness, ultrahigh resolution and the like, the method is widely applied to the research of observing subcellular structures and other cell biological problems.
However, the STED technology needs to use high-power-loss light, inevitably brings about the problems of photobleaching, photodamage and the like, and meanwhile, two beams of light need to be spatially coupled and time-sequence modulated, so that the system complexity is high, the operation is difficult, and an expensive pulse light source is often used in the STED technology, so that the cost is high. If the defects can be overcome, a more simple real-time scanning imaging super-resolution technology which breaks through the optical diffraction limit is developed, and the super-resolution technology has the advantages of ultralow light intensity, no photobleaching, extremely low phototoxicity, simple and easy system, suitability for ultra-long time-range living cell imaging and the like, and has important significance for enhancing the international influence of China on the basic research of super-resolution microscopic imaging and instrument development.
Disclosure of Invention
The present invention aims to overcome the inherent inefficiencies of stimulated emission depletion microscopy and provide a method for breaking the optical diffraction limit by the photon avalanche fluorescence effect. The inventor provides a novel rare earth ion doping system by taking an ultrahigh-order nonlinear response modulation compression imaging point diffusion function as a breakthrough, and realizes a photon avalanche luminescence physical effect with ultrahigh-order nonlinear optical response in a nano material. Through the realization of the photon avalanche fluorescence nanoprobe, the fluorescence point diffusion function detected in the imaging process can be greatly compressed in three dimensions of XYZ, so that a smaller area can be resolved in the point-by-point scanning process, and the imaging resolution breaking through the diffraction limit is obtained.
The invention also aims to solve the problem that the traditional photon avalanche effect has too long response time and cannot be applied to large-field-of-view point scanning imaging. The invention provides a novel photon avalanche fluorescence nanoprobe which has high-order nonlinear optical response and short fluorescence rise time, and can realize super-resolution imaging of large-field point scanning under the excitation of low-power single-beam laser.
The purpose of the invention is realized by the following technical scheme: the realization of the novel photon avalanche fluorescence nanoprobe comprises the following steps:
(1) synthesizing rare earth ion doped photon avalanche nano particles with a core-shell structure, and realizing the emission of photon avalanche fluorescence under the nanoscale through the construction of a novel multi-ion doped system. The core of the nano-particle is formed by water-storing ions Yb with a dual-energy level structure3+With three-level structure avalanche ion Pr3+Co-doped in fluoride nanocrystals, Yb3+Higher doping concentration, Pr3+The doping concentration is low, and the shell layer is an inert fluoride nanocrystal;
(2) exciting photon avalanche fluorescence nano probe by using a beam of continuous near-infrared exciting light, wherein the photon energy of the wavelength is not completely matched with Pr3+From3H4To1G4Is absorbed but perfectly matched to Pr3+From1G4To3P0Is absorbed by the excited state of (1). Under the excitation of a certain power of near infrared laser, is in3H4Is first at least partly excited to1G4Is then rapidly excited to3P0，Pr3+And Yb3+There is aAn efficient energy transfer process, Pr3+In the middle of3P0The particles of (a) transfer energy to Yb3+Middle ground state energy level2F7/2Post-particle relaxation of1G4Yb of3+The particles of ground state energy level are excited to2F5/2Then, the energy is transferred back to Pr3+To make Pr3+Excitation of particles in the ground state to1G4Thereby realizing1G4Doubling the number of particles, after several cycles,1G4the number of particles shows avalanche growth, thereby1G4Particles excited to other energy levels also undergo avalanche-like growth phenomena, e.g.3P0、3P0And1D2the fluorescence emitted from the energy levels has ultrahigh-order nonlinear response to the exciting light, a response curve (a logarithmic relation curve of fluorescence intensity and exciting light intensity) presents an S shape, and after the power reaches a certain threshold value, the nonlinear effect in a certain power range starts to be sharply enhanced;
(3) when the probe is in a high-order nonlinear response stage, a fluorescence point diffusion function generated by excitation of a Gaussian light spot is greatly compressed, the half-height width of the point diffusion function in the transverse direction and the axial direction is reduced to 1/V N along with the increase of a nonlinear order N, and when the nonlinear order is high enough, the imaging resolution can break through the optical diffraction limit in three dimensions.
Based on the novel photon avalanche fluorescence nanoprobe, the invention provides a super-resolution imaging method for scanning a low-power single-beam large-field-of-view point, which comprises the following steps:
(1) the near-infrared laser generates a stable continuous near-infrared exciting light beam, and the laser is collimated and focused by the high-power objective lens to generate a Gaussian light spot with the diffraction limit size;
(2) marking the photon avalanche fluorescence nanoprobes on different parts or structures of a sample by a biochemical method, fixing the sample on an objective table, and generating different nonlinear effects by the photon avalanche fluorescence nanoprobes under the irradiation of Gaussian solid light spots with different powers, wherein when the power of exciting light reaches a photon avalanche threshold value, the nonlinear effect is the largest, and the imaging resolution ratio is the highest;
(3) photon avalanche fluorescence signals emitted by the photon avalanche fluorescence nanoprobe under the action of the Gaussian solid light spots are collected and detected by a photoelectric detector, meanwhile, the Gaussian light spots are moved by a scanning device to carry out XYZ three-dimensional point-by-point scanning on the sample, the distribution of the fluorescence signals of the sample in a three-dimensional space is obtained, and a three-dimensional super-resolution fluorescence image is obtained through reconstruction.
In order to achieve the technical purpose, the invention also provides a low-power single-beam large-visual-field-point scanning super-resolution imaging device, which comprises an excitation light generation module, a multi-photon micro-scanning module and a photoelectric detection module, wherein the excitation light generation module is used for generating a continuous near-infrared steady-state laser beam serving as excitation light, the laser beam is focused to a sample fixed on an objective table and marked by a photon avalanche fluorescence nano probe through the multi-photon micro-scanning module and scans different areas, and the photoelectric detection module is used for detecting a super-resolution fluorescence signal of the sample which is scanned and excited.
Specifically, the excitation light generation module comprises a near-infrared continuous laser, a light filter, a collimation beam expander (including a pinhole filter), a half wave plate and a polaroid, wherein the light filter, the collimation beam expander (including the pinhole filter), the half wave plate and the polaroid are sequentially arranged along the direction of a laser beam emitted by the laser. The near-infrared laser generates continuous near-infrared steady-state laser beams and outputs the continuous near-infrared steady-state laser beams, the photon energy of the laser beams is matched with the excited state absorption of avalanche ions, the laser beams are subjected to filtering treatment and collimated beam expansion by the optical filter and the collimated beam expander, and the power of the laser beams is adjusted by the half wave plate and the polaroid;
the multi-photon microscopic scanning module comprises a scanning vibration mirror, a scanning lens, a tube mirror, a high-reflection low-transmission dichroic mirror and an objective lens which are sequentially arranged along the advancing direction of a laser beam, wherein the scanning vibration mirror controls the light path deflection of the laser beam to perform two-dimensional scanning on a sample, the scanning lens and a field lens focus and collimate the laser beam emitted by the scanning vibration mirror, the high-reflection low-transmission dichroic mirror is used for reflecting near-infrared exciting light and transmitting fluorescence of the sample to separate exciting light and fluorescence, and the laser beam is focused to the sample which is fixed on an objective table and marked by a photon avalanche fluorescence nanoprobe through the objective lens;
specifically, the photoelectric detection module comprises a focusing lens and a photoelectric detector which are coaxially arranged in sequence, the focusing lens and the photoelectric detector are arranged in the advancing direction of the fluorescence collected along the objective lens, the sample emits super-resolution fluorescence in all directions under the excitation of near-infrared laser, the objective lens collects a part of super-resolution fluorescence signals, the signals pass through the high-reflection low-transmission dichroic mirror and the focusing lens and are received by the photoelectric detector, after the photoelectric detector receives the signals detected each time, the signal is sent to a computer, the computer controls the scanning galvanometer to rotate through a rotating device, the focusing light spot is moved to scan the next area so as to obtain a two-dimensional laser scanning super-resolution fluorescence image, a motor for driving the objective table to move along the Z-axis direction is arranged on one side of the objective table and is controlled by the computer, and Z-axis scanning can be performed on the basis, and finally, a three-dimensional laser scanning super-resolution fluorescence image is obtained.
The invention has the beneficial effects that:
1. compared with the traditional STED technology, the invention only needs low-power single-beam continuous near infrared light for excitation, and realizes ultrahigh resolution under low power. Traditional STED technique needs two bunches of laser work, a bunch of exciting light and a bunch of loss light, and wherein the loss light often needs higher power to realize high-efficient fluorescence loss and high resolution, destroys the fluorescence molecule irreversibly easily, causes serious photobleaching phenomenon, and certain light damage can be caused to biological tissue to high power laser simultaneously. Compared with the prior art, the invention only needs low-power laser to excite, avoids the problems of photobleaching and phototoxicity, and is beneficial to realizing real-time super-resolution imaging in cells for a very long time;
2. compared with the traditional STED technology, the invention only needs a single beam of near infrared light to directly excite the sample, and can achieve the resolution ratio of breaking through the diffraction limit. Two bundles of laser in traditional STED technique need carry out strict space coupling and time sequence modulation, greatly increased the complexity of system, complex operation, and the pulse laser ware that uses simultaneously is expensive. Compared with the prior art, the laser scanning microscope system is based on the conventional laser scanning microscope system, single-beam laser is used for excitation, the operation is simple and convenient, the popularization is easy, and the used continuous laser is low in price and easy to industrialize;
3. compared with the traditional multi-photon fluorescent probe, the novel photon avalanche fluorescent probe has high order of nonlinear response, and can realize super-resolution microscopic imaging which breaks through the optical diffraction limit;
4. compared with the traditional photon avalanche fluorescence probe, the novel photon avalanche fluorescence probe only needs millisecond-level rise time for light emission, the rise time of the previous research mostly reaches more than seconds, and the rapid and short fluorescence rise time of the probe provides an important basis for rapid point scanning super-resolution imaging under a large visual field.
Drawings
FIG. 1 shows NaYF as the material of example 14:Yb/Pr(15/0.5％)@NaYF4Transmission electron microscopy images of;
FIG. 2 is a schematic diagram of the multi-ion doped photon avalanche system in example 2;
FIG. 3 is the luminescence spectrum of the photon avalanche fluorescence nanoprobe in example 2;
FIG. 4 is a fluorescence response curve of the photon avalanche fluorescence nanoprobe in example 2;
FIG. 5 is the full width at half maximum of the spread function of the luminous point in different non-linear orders in example 2;
FIG. 6 is a schematic view of the structure of a microscopic imaging apparatus in example 4.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be further described with reference to the accompanying drawings, and it should be noted that the present embodiment is based on the technical solution, and the detailed implementation and the specific operation process are provided, but the protection scope of the present invention is not limited to the present embodiment.
The invention relates to a realization method of a super-resolution imaging device utilizing low-power single-beam large-field-of-view point scanning, which comprises the following steps:
the method comprises the following steps that S1 a laser emits a stable near-infrared laser beam, photon energy of the laser beam is matched with excited state absorption energy of avalanche ions, the laser beam is filtered by a filter to remove laser beams with other wavelengths, collimated and expanded by a double-convex lens and filtered and shaped by a pinhole filter, and then enters a microscope scanning system to be focused by an objective lens to obtain a Gaussian spot with a diffraction limit size;
s2 the focused Gaussian light spot excites the photon avalanche fluorescence nanoprobe to generate photon avalanche up-conversion fluorescence, the laser power is adjusted, when the power slightly exceeds the photon avalanche threshold, the photon avalanche fluorescence emitted by the nanoprobe has an ultra-high order nonlinear response relation to the excitation light intensity, the fluorescence point spread function is greatly compressed, namely the resolution ratio can be greatly improved, so that the diffraction limit is broken through;
s3, marking the photon avalanche fluorescence nanoprobes on different parts or structures of the sample by a biochemical method, focusing the same near infrared laser with the power slightly exceeding the photon avalanche threshold value on the sample, scanning point by point in the XYZ direction, and detecting photon avalanche fluorescence signals corresponding to each point by a photoelectric detector to obtain a three-dimensional super-resolution fluorescence image.
The nanoprobe is obtained by doping rare earth ions to synthesize a specific core-shell structure up-conversion nanomaterial, and the core of the nanoprobe is formed by water storage ions Yb with a dual-energy level structure3+With three-level structure avalanche ion Pr3+Co-doped in fluoride nanocrystals, Yb3+Higher doping concentration, Pr3+The doping concentration is low and the shell is an inert fluoride nanocrystal.
It should be noted that, the nanoprobe performs scanning imaging in a multi-photon microscopic imaging system using a single low-power laser, the fluorescence point spread function is greatly compressed in three dimensions of XYZ during imaging, the resolution of breaking through the diffraction limit is reached, and based on a short response time, rapid point scanning imaging can be performed in a large field of view.
The energy of photons with the wavelength is matched with the excited state absorption of avalanche ions, the avalanche ions undergo energy level transition through weak ground state absorption and strong excited state absorption, energy is conducted between the two ions circularly under the assistance of water storage ions, the laser power is continuously increased after reaching an avalanche threshold, the number of particles at multiple energy levels in the avalanche ions and the water storage ions is increased in an avalanche manner, and the fluorescence radiation corresponding to the energy levels has an ultrahigh-order nonlinear response relation to the excitation light intensity. .
The invention also provides a device for realizing the super-resolution imaging of the low-power single-beam large-field-of-view point scanning, which comprises an excitation light generation module, a multi-photon micro-scanning module and a photoelectric detection module; the excitation light generation module is used for generating continuous near-infrared steady-state laser beams serving as excitation light, the laser beams are focused on a sample which is fixed on an object stage and marked by a photon avalanche fluorescence nano probe through the multi-photon micro scanning module, different areas of the sample are scanned, and the photoelectric detection module is used for detecting fluorescence signals of the sample which are scanned and excited.
It should be noted that the excitation light generation module includes a near-infrared continuous laser, and an optical filter, a collimating beam expander, a half-wave plate and a polarizer which are sequentially disposed along a laser beam direction emitted by the laser; the near-infrared laser generates continuous near-infrared steady-state laser beams and outputs the continuous near-infrared steady-state laser beams, photon energy of the laser beams is matched with excited state absorption of avalanche ions, the laser beams are filtered and collimated and expanded by the optical filter and the collimating and expanding lens, and power of the laser beams is adjusted by matching of the half wave plate and the polaroid.
The multi-photon micro-scanning module comprises a scanning galvanometer, a scanning lens, a tube lens, a high-reflection low-transmission dichroic mirror and an objective lens which are sequentially arranged along the advancing direction of a laser beam; the scanning galvanometer controls the light path deflection of the laser beam to perform two-dimensional scanning on a sample, the scanning lens and the field lens focus and collimate the laser beam emitted by the scanning galvanometer, the high-reflection low-transmission dichroic mirror can reflect near-infrared exciting light and transmit sample fluorescence and is used for separating the exciting light and the fluorescence, and the laser beam is focused to the sample which is fixed on the objective table and marked by the photon avalanche fluorescence nano probe through the objective lens.
It should be noted that the photoelectric detection module includes a focusing lens and a photoelectric detector coaxially disposed in sequence, the focusing lens and the photoelectric detector are disposed in the forward direction of the fluorescence collected by the objective lens, the sample emits photon avalanche fluorescence in all directions under the excitation of the near-infrared laser, the objective lens collects a part of fluorescence signals, the signals pass through the high-reflection low-transmission dichroic mirror and the focusing lens and are received by the photoelectric detector, the photoelectric detector receives the detected signals each time, the signals are sent to the computer, the computer controls the scanning galvanometer to rotate through the rotating device, the focusing light spot is moved to scan the next pixel, so as to obtain a two-dimensional laser scanning super-resolution fluorescence image, a motor for driving the objective table to move in the Z-axis direction is disposed on one side of the objective table, and the Z-axis scanning can be performed on the basis through the control of the computer, finally, a three-dimensional laser scanning super-resolution fluorescence image is obtained.
Example 1
The embodiment provides a realization method of a novel photon avalanche fluorescence nanoprobe. In the embodiment, the photon avalanche fluorescence nanoprobe is constructed by the core-shell structure up-conversion nanoparticles, and the core of the nanoparticles is formed by water storage ions Yb3+And avalanche ion Pr3 +Co-doped in NaYF4Nanocrystal of Yb3+The doping concentration is about 15 percent, and Pr3+The doping concentration is about 0.5%, and the shell layer is a fluoride nanocrystal.
The method for synthesizing the photon avalanche fluorescence nano material with the core-shell structure by adopting a solvothermal method comprises the following steps: the core structure was synthesized by first, adding 5mL of 0.2M Ln (CH) to a 100mL round-bottomed flask at room temperature (23-25 deg.C)3COO)3To the solution (Ln ═ Y/Yb/Pr), 7mL of oleic acid and 17.5mL of 1-octadecene were added in this order, and the mixture was reacted at 150 ℃ for 40 minutes to obtain a precursor. The heating mantle was removed and the reaction mixture was allowed to cool to 40 ℃ with stirring, and 10mL NH was added quickly4A mixture of F-methanol solution (0.4M) and 2.5mL of NaOH-methanol solution (1M) was reacted at 40 ℃ for 4 hours, followed by reaction at 110 ℃ for 30 minutes under vacuum to remove methanol. After evaporation of the methanol, the temperature was raised to 300 ℃ under an argon atmosphere and reacted at the same temperature for 1.5 hours. The reaction was allowed to cool to room temperature while stirring with the heating mantle removed, 10mL of absolute ethanol was added, the mixture was centrifuged at 7500r.p.m. for 5 minutes, and the supernatant was removed and collectedCollecting the product, cleaning with mixed solution of ethanol and cyclohexane, and oven drying to obtain core NaYF of upconversion nanoparticles4Yb/Pr dispersed in 9mL of cyclohexane. By adjusting Y3+、Pr3+And Yb3+The NaYF with determined components can be obtained4Yb/Pr (15/0.5%) nanoparticles.
After obtaining the core structure of the nanoparticle, the shell structure of the upconversion nanoparticle was synthesized in the same way. To a 100mL round bottom flask was added 5mL of 0.2M Y (CH3COO)3And adding 7mL of oleic acid and 17.5mL of 1-octadecene into the solution in sequence, heating to 120 ℃ to react for 10 minutes to remove water, and then reacting at 150 ℃ for 40 minutes to form a precursor. The heating mantle was removed and the reaction mixture was allowed to cool to 80 ℃ while stirring, then 3mL of the previously synthesized core structure of the upconverting nanoparticle was added and held for 20 minutes to remove cyclohexane. Cooled to 40 ℃ for 4 hours, followed by reaction at 110 ℃ under vacuum for 30 minutes to remove methanol. After evaporation of the methanol, the temperature was raised to 300 ℃ under an argon atmosphere and the reaction was carried out at this temperature for 1.5 hours at constant temperature. Then, the same operation as the previous operation is carried out, the temperature is reduced to the room temperature, 10mL of absolute ethyl alcohol is added, the centrifugal operation is carried out, the supernatant is removed, the product is collected, the ethanol and cyclohexane mixed solution is used for cleaning and drying, and finally, cyclohexane is added for dissolving, so that the prepared nuclear shell structure photon avalanche nanoparticle NaYF is obtained4:Yb/Pr(15/0.5％)@NaYF4。
The transmission electron microscope image of the successfully synthesized core-shell structure photon avalanche nanoparticle is shown in fig. 1.
Example 2
Based on the implementation method of the novel photon avalanche fluorescence nanoprobe in the embodiment 1, the embodiment illustrates the implementation principle of the ultra-high order nonlinear response of the novel photon avalanche fluorescence nanoprobe.
The photon avalanche fluorescence nanoprobe synthesized in example 1 is excited by a beam of continuous near infrared excitation light, and the photon energy of the wavelength is not completely matched with Pr3+From3H4To1G4Is absorbed but perfectly matched to Pr3+From1G4To3P0Excited state ofThe absorption, in this example, was chosen to be 852 nm. Under the excitation of 852nm near infrared laser with certain power, the specific energy level transition process is as shown in figure 2 and is at3H4Is first at least partly excited to1G4Is then rapidly pumped to3P0，Pr3+And Yb3+With an efficient energy transfer process in between, Pr3+In the middle of3P0The particles of (a) transfer energy to Yb3+Middle ground state energy level2F7/2Post-particle relaxation of1G4Yb of3+The particles of ground state energy level are excited to2F5/2Then, the energy is transferred back to Pr3+To make Pr3+Excitation of particles in the ground state to1G4Thereby realizing1G4Doubling the number of particles, after several cycles,1G4the number of particles shows avalanche growth, thereby1G4Particles excited to higher energy levels also undergo avalanche-like growth phenomena, e.g.3P1、3P0And1D2etc. the fluorescence corresponding to these energy levels has ultrahigh-order nonlinearity, and the fluorescence emission peaks in this embodiment include 484nm, 642nm, 609nm, 525nm, etc., and the fluorescence spectrum is shown in fig. 3.
Under the action of near-infrared excitation light with different powers, photon avalanche fluorescence has different nonlinear effects, the nonlinear effects are firstly enhanced and then saturated along with the increase of the power of the excitation light, the overall nonlinear response curve of the fluorescence is S-shaped, and when the power of the excitation light is near the photon avalanche threshold, the nonlinear effect is the maximum, as shown in FIG. 4. When the probe is in a high-order nonlinear effect stage, a fluorescence point diffusion function generated by excitation of a Gaussian light spot is greatly compressed, the half-height width of the point diffusion function in the transverse direction and the axial direction is reduced to 1/V N along with the increase of a nonlinear order N, when the nonlinear order is high enough, the imaging resolution can break through the optical diffraction limit in three dimensions, and the increase condition of the resolution along with the increase of the nonlinear order is shown in FIG. 5.
Example 3
The embodiment provides a super-resolution imaging method based on photon avalanche fluorescence for low-power single-beam large-field-of-view point scanning, which comprises the following steps:
(1) a laser emits a continuous near-infrared laser beam with the wavelength of 852nm, and after the laser beam is subjected to filtering processing such as an optical filter, a collimation beam expander, a pinhole filter and the like and collimation beam expansion, a focused Gaussian spot is obtained;
(2) the focused Gaussian spot excitation component is NaYF4:Yb/Pr(15/0.5％)@NaYF4The photon avalanche fluorescence nanometer probe generates photon avalanche fluorescence, the laser power is adjusted, when the power is near the photon avalanche threshold, the photon avalanche fluorescence emitted by the nanometer probe has super high-order nonlinear response, the fluorescence point diffusion function is greatly compressed, and the resolution ratio of breaking through the diffraction limit is achieved;
(3) the method comprises the steps of marking photon avalanche fluorescence nanoprobes on different parts or structures of a sample by a biochemical method, fixing the sample on an objective table, adjusting laser power, focusing the same near infrared laser with the power slightly exceeding a photon avalanche threshold value on the sample, scanning point by point in the XYZ direction, detecting photon avalanche fluorescence signals by a photoelectric detector, and obtaining a three-dimensional super-resolution fluorescence image.
Example 4
The structure of the super-resolution imaging device is shown in fig. 6, and the device comprises an excitation light generation module, a multiphoton micro-scanning module and a photoelectric detection module.
The excitation light generation module comprises a near-infrared continuous laser 1, an optical filter 2, a collimation beam expander 3 (including a pinhole filter), a half wave plate 4 and a polaroid 5. The near-infrared laser generates continuous Gaussian laser with the wavelength of 852nm and outputs, the filter filters stray light of other wave bands in the laser, the collimating beam expander enlarges the size of an excitation light spot, the utilization rate of the power of the excitation light is improved, the pinhole filter is arranged at the focus to filter high-frequency stray light, and the half wave plate 4 is arranged on the rotatable mounting seat and matched with the linear polarizer 5 to adjust the power of the laser beam.
The multiphoton micro-scanning module comprises a scanning galvanometer 6, a high-reflection low-transmission dichroic mirror 7, a scanning lens 8, a tube mirror 9, an objective lens 10 and a photon avalanche nano material or a marked sample thereof 11 arranged on an objective table. The scanning galvanometer controls the light path deflection of the laser beam to realize two-dimensional scanning of a sample, the high-reflection low-transmission dichroic mirror reflects the laser beam, the scanning lens and the tube mirror focus and collimate the emergent light beam of the scanning galvanometer, so that the laser beam still matches the entrance pupil size of the microscope objective in the scanning process, and finally the objective focuses the laser on photon avalanche nano material arranged on an objective table or a marked sample thereof.
The photo-detection module comprises a focusing lens 10 and a photo-detector 11. A focusing lens and a photodetector are disposed in the proceeding direction of the fluorescence collected along the objective lens, the photodetector being connected to an external computer. After the photoelectric detector receives a detection signal once, the signal is sent to a computer, then the computer controls the scanning galvanometer to rotate, a two-dimensional laser scanning super-resolution image is obtained by utilizing a mode that a focusing light spot scans a sample point by point, a motor for driving the objective table to move along the Z-axis direction is arranged on one side of the objective table, and the three-dimensional laser scanning super-resolution image can be obtained by combining the motor with a rotating device.
Various modifications may be made by those skilled in the art based on the above teachings and concepts, and all such modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Claims (8)
1. A method for laser spot scanning super-resolution imaging with low light intensity, single light beam and large visual field is characterized by comprising the following steps:
the method comprises the following steps that S1 a laser emits a stable near-infrared laser beam, photon energy of the laser beam is matched with excited state absorption energy of avalanche ions, the laser beam is filtered by a filter to remove laser beams with other wavelengths, collimated and expanded by a double-convex lens and filtered and shaped by a pinhole filter, and then enters a microscope scanning system to be focused by an objective lens to obtain a Gaussian spot with a diffraction limit size;
s2 the focused Gaussian light spot excites the photon avalanche fluorescence nanoprobe to generate photon avalanche up-conversion fluorescence, laser power is adjusted, when the power reaches a photon avalanche threshold, photon avalanche fluorescence of a plurality of different wave bands emitted by the nanoprobe has an ultra-high order nonlinear response relation to the excitation light intensity, a fluorescence point diffusion function is greatly compressed, namely the resolution ratio can be greatly improved, so that the diffraction limit is broken through;
s3, marking the photon avalanche fluorescence nanoprobes on different parts or structures of a sample by an immunofluorescence marking method, focusing the same near infrared laser with power reaching the photon avalanche threshold value on the sample, scanning point by point in the XYZ direction, and detecting photon avalanche fluorescence signals corresponding to each point by a photoelectric detector to obtain a three-dimensional super-resolution fluorescence image.
2. The method for super-resolution imaging with low power, single beam and large visual field point scanning according to claim 1, wherein the nanoprobe is obtained by synthesizing a specific core-shell structure up-conversion nano material by doping rare earth ions, and the core of the nanoprobe is formed by double-energy-level structure water-storage ions Yb3+With three-level structure avalanche ion Pr3+Co-doped in fluoride nanocrystals, Yb3+Higher doping concentration, Pr3+The doping concentration is low and the shell is an inert fluoride nanocrystal.
3. The method of super-resolution imaging of low-power single-beam large-field-of-view point scanning according to claim 1 or 2, characterized in that the nanoprobe is scanned and imaged in a multi-photon microscopic imaging system using a single-beam low-power laser, the fluorescence point spread function is greatly compressed in three dimensions of XYZ during imaging, the resolution of breaking through the diffraction limit is reached, and based on the shorter response time, rapid point scanning imaging can be performed in a large field of view.
4. The method of super-resolution imaging with low power, single beam and large field of view point scanning according to claim 3, characterized in that a beam of near-infrared laser is used for excitation, the photon energy of the wavelength matches the excited state absorption of the avalanche ion, the avalanche ion undergoes energy level transition through the weaker ground state absorption and the stronger excited state absorption, the energy is conducted cyclically between the two ions under the assistance of the water-stored ion, when the laser power reaches the avalanche threshold, the number of particles in the avalanche ion and the water-stored ion increases in an avalanche manner, and the fluorescence radiation corresponding to the energy level has a super-high order nonlinear response relation to the excitation light intensity.
5. A device for realizing super-resolution imaging of low-power single-beam large-field-of-view point scanning is characterized by comprising an excitation light generation module, a multi-photon micro-scanning module and a photoelectric detection module; the excitation light generation module is used for generating continuous near-infrared steady-state laser beams serving as excitation light, the laser beams are focused on a sample which is fixed on an object stage and marked by a photon avalanche fluorescence nano probe through the multi-photon micro scanning module, different areas of the sample are scanned, and the photoelectric detection module is used for detecting fluorescence signals of the sample which are scanned and excited.
6. The device for super-resolution imaging with large field-of-view point scanning and low power single beam as claimed in claim 5, wherein the excitation light generation module comprises a near-infrared continuous laser, and a filter, a collimating beam expander, a half-wave plate and a polarizer which are sequentially arranged along the direction of the laser beam emitted by the laser; the near-infrared laser generates continuous near-infrared steady-state laser beams and outputs the continuous near-infrared steady-state laser beams, photon energy of the laser beams is matched with excited state absorption of avalanche ions, the laser beams are filtered and collimated and expanded by the optical filter and the collimating and expanding lens, and power of the laser beams is adjusted by matching of the half wave plate and the polaroid.
7. The device for super-resolution imaging by scanning the large-view-point with the low-power single beam as claimed in claim 7, wherein the multi-photon micro-scanning module comprises a scanning galvanometer, a scanning lens, a tube lens, a high-reflection low-transmission dichroic mirror and an objective lens which are sequentially arranged along the advancing direction of the laser beam; the scanning galvanometer controls the light path deflection of the laser beam to perform two-dimensional scanning on a sample, the scanning lens and the field lens focus and collimate the laser beam emitted by the scanning galvanometer, the high-reflection low-transmission dichroic mirror can reflect near-infrared exciting light and transmit sample fluorescence and is used for separating the exciting light and the fluorescence, and the laser beam is focused to the sample which is fixed on the objective table and marked by the photon avalanche fluorescence nano probe through the objective lens.
8. The device for super-resolution imaging with low power and single beam wide field of view spot scanning as claimed in claim 7, wherein the photo-detection module comprises a focusing lens and a photo-detector coaxially disposed in sequence, the focusing lens and the photo-detector are disposed in the forward direction of the fluorescence collected by the objective lens, the sample emits photon avalanche fluorescence in all directions under the excitation of the near infrared laser, the objective lens collects a portion of the fluorescence signal, the signal passes through the high-reflective low-transmissive dichroic mirror and the focusing lens and is received by the photo-detector, the photo-detector sends the signal to the computer after receiving the signal detected each time, the computer controls the scanning galvanometer to rotate through the rotating device, the focusing spot scans the next pixel to obtain a two-dimensional laser scanning super-resolution fluorescence image, and a motor for driving the stage to move along the Z-axis is disposed on one side of the stage, and through computer control, Z-axis scanning can be carried out on the basis, and finally a three-dimensional laser scanning super-resolution fluorescence image is obtained.
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