A fluorescence loss principle sensitized by stimulated emission loss and a super-resolution imaging method and device thereof

Abstract

The present invention discloses a fluorescence loss principle of stimulated radiation loss sensitization and a super-resolution imaging method and device thereof, wherein, on the one hand, the fluorescence loss method uses near-infrared continuous laser to induce the stimulated radiation process of sensitized ions in rare-earth doped fluorescent nanoparticles, blocks the energy transfer process between the sensitized ions and one or more activated ions from the excitation source, quenches the luminescence of all energy levels of all activated ions, thereby realizing the simultaneous loss of fluorescence of different energy levels of different activated ions by a single beam loss laser. On the other hand, based on the loss method, a method and device for full-spectrum multicolor super-resolution imaging is realized, thereby performing full-spectrum multicolor super-resolution microscopic imaging of multiple luminescent ions or molecules based on the same pair of near-infrared wavelength excitation light / loss light, which has the advantages of no photobleaching, low phototoxicity, large depth, high resolution, low complexity, low cost, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical microscopy, and in particular relates to a fluorescence loss principle sensitized by stimulated emission loss and a super-resolution imaging method and device thereof. Technical Background

[0002] In optical microscopy, due to the existence of the diffraction limit, the maximum resolution that an optical microscope can achieve is about 200nm, which is difficult to meet the needs of observing nanoscale structures. In order to break through the optical diffraction limit, researchers have proposed a series of far-field optical super-resolution microscopy methods, the most representative of which is stimulated emission depletion microscopy (STED) based on laser scanning. This method uses a Gaussian focused laser to scan the sample while using another annular beam of light to focus the fluorescence around the depleted fluorescent point to obtain a smaller effective fluorescence point spread function, which is then scanned to obtain a super-resolution fluorescence image. Due to its real-time, fast and ultra-high resolution characteristics, it is widely used in the observation of subcellular structures and other cell biology issues.

[0003] In order to reduce the power requirements of STED, the applicant has developed STED technology based on upconversion nanoparticles in the past, which has achieved near-infrared laser excitation and loss, completely no photobleaching and light blinking, and long-term super-resolution imaging. However, this STED technology still has many limitations, such as the loss mechanism is not universal, and the fluorescence loss is limited to a single fluorescence band of a single rare earth ion, namely Tm 3+ Therefore, it is of great significance to develop a universal up-conversion stimulated emission depletion mechanism to realize full-spectrum multicolor STED imaging of multiple luminescent ions or molecules with a pair of near-infrared wavelength excitation light / depletion light. Summary of the invention

[0004] In view of the defects of the prior art, the present invention aims to provide a fluorescence loss principle sensitized by stimulated emission loss, with the aim of overcoming the limitations of the existing up-conversion stimulated emission loss mechanism. Furthermore, the present invention is based on a beam of near-infrared loss light with a wavelength matching the energy level gap of the sensitized ion, which causes a stimulated emission loss process, quenches the energy of a specific energy level, and blocks the energy transfer process between the sensitized ion and one or more activated ions from the excitation source, thereby quenching the luminescence of all energy levels of all activated ions.

[0005] Secondly, another object of the present invention is to utilize the above-mentioned fluorescence depletion method to provide a method and device for full-spectrum multi-color super-resolution imaging of multiple luminescent ions or molecules through the same pair of near-infrared wavelength excitation light / depletion light.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A fluorescence loss principle sensitized by stimulated radiative depletion, the method comprising the following steps:

[0008] S1 constructs rare earth ion-doped fluorescent nanomaterials of different components; wherein the rare earth ions include sensitizing ions and one or more activating ions. After the action of excitation light, the sensitizing ions can absorb energy and transfer it to the one or more activating ions, so that the one or more activating ions emit fluorescence of different bands;

[0009] S2 adds a single-beam loss laser for loss; wherein, the wavelength of the single-beam loss laser matches the energy gap between two energy levels in the sensitized ion, quenches the energy of a specific energy level by inducing a stimulated radiation loss process, blocks the energy transfer process between the sensitized ion and the one or more activated ions from the excitation source, and quenches the luminescence of all energy levels of all one or more activated ions, thereby achieving the simultaneous loss of fluorescence of different energy levels of the one or more activated ions by the single-beam loss light.

[0010] It should be noted that the sensitizing ion is Nd 3+ , the activated ion is Nd 3+ , Er 3+ 、Ho 3+ , Pr 3+ 、Tm 3+ 、Eu 3+ or Tb 3+ One or more of .

[0011] It should be noted that the additional sensitizing ion Yb 3+ , which is used to assist energy from Nd 3+ Further transferred to one or more activated ions.

[0012] It should be noted that when the one or more activated ions undergo up-conversion luminescence under the action of the sensitizing ions, the higher the multi-photon order, the higher the fluorescence loss efficiency and the lower the required loss laser intensity.

[0013] It should be noted that the sensitizing ion Nd 3+ It has a four-level system that is easy to realize stimulated radiation loss. It can be excited by a laser with a wavelength of 740, 800 or 860nm and lost by a laser with a wavelength of 1064nm. 3+ Under the action of excitation light, the energy level 4 I 9 / 2 Direct transition to excited state4 F 3 / 2 , or excited to a higher energy level and then assisted by phonons to relax to 4 F 3 / 2 , in 4 F 3 / 2 Nd 3+ It can transfer energy to activated ions to emit up-conversion fluorescence, and under the action of loss light whose wavelength matches the energy level gap, 4 F 3 / 2 and 4 I 11 / 2 Stimulated radiation occurs between the two energy levels. 4 F 3 / 2 Nd used to sensitize the activated ions 3+ The number is greatly reduced and the luminescence process is blocked.

[0014] The present invention also provides a multicolor super-resolution imaging method based on the principle of fluorescence loss sensitized by stimulated emission loss, the method comprising the following steps:

[0015] S1 has two optical paths; in one optical path, the first near-infrared fiber-coupled laser emits a single-mode laser with a wavelength of 740nm, 800nm ​​or 860nm, which is processed by a collimating beam expander and a pinhole aperture filter to obtain a Gaussian excitation beam;

[0016] S2 In another optical path, the second near-infrared fiber-coupled laser generates a single-mode laser with a wavelength of 1064nm. After being filtered by a collimating beam expander and a pinhole aperture, the single-mode laser is modulated by a spatial phase modulator to form a hollow beam with a central light intensity of zero, thereby obtaining a hollow-type loss beam. The wavelength of the hollow-type loss beam needs to match the energy gap between the two energy levels of the stimulated radiation process in the sensitized ions.

[0017] S3 performs spatial coaxial coupling of the focused Gaussian excitation beam and the hollow loss beam, and focuses and irradiates the rare earth ion-doped nanomaterial. The Gaussian excitation beam excites the nanomaterial to produce fluorescence, while the hollow loss beam triggers the stimulated radiation process of the sensitized ions, making it difficult for the sensitized ions to transfer energy to one or more activated ions, thereby suppressing the emission of fluorescence in the loss light irradiation area from the source of the excitation process;

[0018] S4 uses biochemical methods to mark nanoparticles doped with one or more activated ions on different parts or structures of the sample, and uses the same pair of near-infrared excitation light and near-infrared loss light to focus on the sample through an objective lens and perform scanning in the XYZ direction. Different up-conversion fluorescence signals are separated by dichroic mirrors and filters, and the super-resolution fluorescence signals are detected by a photodetector to obtain a super-resolution image.

[0019] It should be noted that for multi-color imaging, multiple photodetectors are used to synchronously detect signals of different wavelengths to obtain fluorescence images of different bands, and then the images of different channels are dyed and superimposed to obtain a multi-color super-resolution image.

[0020] The present invention further provides a microscopic imaging device for realizing a multi-color super-resolution imaging method, the device comprising an excitation light generating module, a loss light generating module, a polarization beam splitter, a multi-photon microscopic scanning module and a photoelectric detection module, the excitation light generating module is used to generate a near-infrared Gaussian beam used as excitation light, the loss light generating module is used to generate a near-infrared hollow beam used as loss light; the polarization beam splitter spatially conjugate couples the near-infrared Gaussian excitation beam and the near-infrared hollow loss beam that are perpendicular to each other on a horizontal plane into a coupled laser beam, and the coupled laser beam is focused on a sample on a stage through the multi-photon microscopic scanning module; the photoelectric detection module is used to detect the super-resolution fluorescence signal generated after the above-mentioned sample is excited and lost.

[0021] It should be noted that the excitation light generating module includes a first near-infrared continuous laser, and a first filter, a first collimating beam expander, a first half-wave plate, and a first polarizer which are placed in sequence along the direction of the laser beam emitted by the laser. The first near-infrared continuous laser generates a continuous near-infrared steady-state laser beam output, and the wavelength of the beam matches the excitation wavelength of the sensitized ions of the rare-earth-doped nanomaterial. The laser beam is filtered and collimated by the first filter and the first collimating beam expander, and its power is adjusted by the cooperation of the first half-wave plate and the first polarizer.

[0022] It should be noted that the loss light generation module includes a second near-infrared continuous laser, and a second filter, a second collimating beam expander, a second half-wave plate, a second polarizer, and a spatial phase modulator placed in sequence along the direction of the laser beam emitted by the laser. The second near-infrared continuous laser generates a continuous near-infrared steady-state laser beam output, and the photon energy of the beam matches the energy gap between the upper and lower energy levels of the stimulated radiation process in the sensitized ions of the rare earth-doped nanomaterials. The laser beam is filtered and collimated by the second filter and the second collimating beam expander, and its power is adjusted by the second half-wave plate and the second polarizer, and then modulated into a hollow beam by the spatial phase modulator.

[0023] It should be noted that the multi-photon microscopy scanning module includes a scanning galvanometer, a high-reflection and low-transmittance dichroic mirror and an objective lens which are placed in sequence along the advancing direction of the coupled laser beam. The coupled laser beam is focused by the objective lens after passing through the scanning galvanometer and the high-reflection and low-transmittance dichroic mirror. The sample on the stage is placed on the focal plane of the objective lens. A motor for driving the stage to move along the Z-axis direction is provided on one side of the stage. Through this motor, combined with a rotating device, a three-dimensional laser scanning fluorescence image can be obtained.

[0024] It should be noted that the photoelectric detection module includes a focusing lens and a photoelectric detector coaxially placed in sequence, the focusing lens and the photoelectric detector are arranged in the opposite direction of the forward direction of the coupled laser beam, the photoelectric detector is connected to an external computer chain, and the sample marked with rare earth-doped nanomaterials emits super-resolution fluorescence signals in all directions, a part of which is collected by the objective lens, and is received by the photoelectric detector after passing through a high-reflection and low-transmittance dichroic mirror and a focusing lens. The scanning galvanometer is arranged on a rotating device, and the rotating device is controlled by a computer. The computer is connected to the photoelectric detector. After the photoelectric detector receives a detection signal, it sends a signal to the computer, and then the computer controls the rotation of the scanning galvanometer through the rotating device, and obtains a two-dimensional laser scanning fluorescence image by scanning the sample with a focused light spot.

[0025] The beneficial effects of the present invention are:

[0026] 1. Compared with traditional STED technology, the present invention greatly reduces the power requirement of loss light, and can achieve high resolution under the action of low-power loss light. In traditional STED technology, loss light often requires higher power to achieve efficient fluorescence loss and high resolution, which can easily irreversibly destroy fluorescent molecules and cause serious photobleaching, and at the same time cause certain photodamage to biological tissues. The stimulated radiation loss method based on the excitation process in the present invention performs loss from the source, with high loss efficiency. It further reduces the loss light power on the basis of the previous STED technology combined with rare earth nanoparticles. Therefore, only lower-power loss laser is required for super-resolution imaging, avoiding the problems of photobleaching and phototoxicity, which is conducive to realizing ultra-long-term real-time super-resolution imaging in cells;

[0027] 2. Compared with the traditional STED technology, the fluorescence loss method of the present invention has the loss efficiency of cascade amplification. Specifically, when the activated ions are up-converted under the action of the sensitized ions, the higher the multi-photon order of the fluorescence, the higher the loss efficiency, and the lower the required loss laser intensity;

[0028] 3. In the traditional STED technology, a single pair of excitation / depletion lasers can only optically control the fluorescence of a single ion in a single band. Therefore, multiple pairs of excitation / depletion lasers are required to achieve multi-color super-resolution imaging. The present invention proposes a fluorescence depletion method that is sensitized by stimulated emission depletion, which is universal for activated ions. Based on the same pair of near-infrared wavelength excitation light / depletion light, full-spectrum multi-color super-resolution imaging of multiple luminescent ions or molecules can be performed, which greatly simplifies the optical path system and greatly reduces the system cost.

[0029] 4. The present invention uses near-infrared laser for excitation and near-infrared zone II laser for depletion, and combines the advantages of the multi-photon excitation process, which has the potential for low phototoxicity and deep tissue super-resolution imaging in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The transmission electron microscope images of the NaYF4:Nd / Yb / Er and NaYF4:Nd / Yb / Ho nanomaterials in Example 1 are shown;

[0031] Figure 2 A schematic diagram showing that when a beam of continuous near-infrared loss light with a wavelength of 1064 nm is added to Example 1, the higher the multi-photon order, the higher the fluorescence loss efficiency;

[0032] Figure 3 Schematic diagram of the principle of stimulated emission fluorescence loss of NaYF4:Nd / Yb / Er and NaYF4:Nd / Yb / Ho in Example 2;

[0033] Figure 4 The loss spectra of NaYF4:Nd / Yb / Er and NaYF4:Nd / Yb / Ho in Example 2;

[0034] Figure 5 Schematic diagram of the structure of the microscopic imaging device in Example 4. Specific embodiments

[0035] The present invention will be further described below in conjunction with the accompanying drawings. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and a specific operation process, but the protection scope of the present invention is not limited to this embodiment.

[0036] A fluorescence loss principle sensitized by stimulated radiative depletion, the method comprising the following steps:

[0037] S1 constructs rare earth ion-doped fluorescent nanomaterials of different components; wherein the rare earth ions include sensitizing ions and one or more activating ions. After the action of excitation light, the sensitizing ions can absorb energy and transfer it to the one or more activating ions, so that the one or more activating ions emit fluorescence of different bands;

[0038] S2 adds a single-beam loss laser for loss; wherein, the wavelength of the single-beam loss laser matches the energy gap between two energy levels in the sensitized ion, quenches the energy of a specific energy level by inducing a stimulated radiation loss process, blocks the energy transfer process between the sensitized ion and the one or more activated ions from the excitation source, and quenches the luminescence of all energy levels of all one or more activated ions, thereby achieving the simultaneous loss of fluorescence of different energy levels of the one or more activated ions by the single-beam loss light.

[0039] It should be noted that the sensitizing ion is Nd 3+ , the activated ion is Nd 3+ , Er 3+ 、Ho 3+ , Pr 3+ 、Tm 3+ 、Eu 3+ or Tb 3+ One or more of .

[0040] It should be noted that the additional sensitizing ion Yb 3+ , which is used to assist energy from Nd 3+ Further transferred to one or more activated ions.

[0041] It should be noted that when the one or more activated ions perform up-conversion luminescence under the action of the sensitizing ions, the higher the multi-photon order, the higher the fluorescence loss efficiency.

[0042] It should be noted that the sensitizing ion Nd 3+ It has a four-level system that is easy to realize stimulated radiation loss. It can be excited by a laser with a wavelength of 740, 800 or 860nm and lost by a laser with a wavelength of 1064nm. 3+ Under the action of excitation light, the energy level 4 I 9 / 2 Direct transition to excited state 4 F 3 / 2 , or excited to a higher energy level and then assisted by phonons to relax to 4 F 3 / 2 , in 4 F 3 / 2 Nd 3+ It can transfer energy to activated ions to emit up-conversion fluorescence, and under the action of loss light whose wavelength matches the energy level gap, 4 F 3 / 2 and 4 I 11 / 2 Stimulated radiation occurs between the two energy levels. 4 F3 / 2 Nd used to sensitize the activated ions 3+ The number is greatly reduced and the luminescence process is blocked.

[0043] The present invention also provides a multicolor super-resolution imaging method based on the principle of fluorescence loss sensitized by stimulated emission loss, the method comprising the following steps:

[0044] S1 has two optical paths; in one optical path, the first near-infrared fiber-coupled laser emits a single-mode laser with a wavelength of 740nm, 800nm ​​or 860nm, which is processed by a collimating beam expander and a pinhole aperture filter to obtain a Gaussian excitation beam;

[0045] S2 In another optical path, the second near-infrared fiber-coupled laser generates a single-mode laser with a wavelength of 1064nm. After being filtered by a collimating beam expander and a pinhole aperture, the single-mode laser is modulated by a spatial phase modulator to form a hollow beam with a central light intensity of zero, thereby obtaining a hollow-type loss beam. The wavelength of the hollow-type loss beam needs to match the energy gap between the two energy levels of the stimulated radiation process in the sensitized ions.

[0046] S3 performs spatial coaxial coupling of the focused Gaussian excitation beam and the hollow loss beam, and focuses and irradiates the rare earth ion-doped nanomaterial. The Gaussian excitation beam excites the nanomaterial to produce fluorescence, while the hollow loss beam triggers the stimulated radiation process of the sensitized ions, making it difficult for the sensitized ions to transfer energy to one or more activated ions, thereby suppressing the emission of fluorescence in the loss light irradiation area from the source of the excitation process;

[0047] S4 uses biochemical methods to mark nanoparticles doped with one or more activated ions on different parts or structures of the sample, and uses the same pair of near-infrared excitation light and near-infrared loss light to focus on the sample through an objective lens and perform scanning in the XYZ direction. Different up-conversion fluorescence signals are separated by dichroic mirrors and filters, and the super-resolution fluorescence signals are detected by a photodetector to obtain a super-resolution image.

[0048] It should be noted that for multi-color imaging, multiple photodetectors are used to synchronously detect signals of different wavelengths to obtain fluorescence images of different bands, and then the images of different channels are dyed and superimposed to obtain a multi-color super-resolution image.

[0049] The present invention further provides a microscopic imaging device for realizing a multi-color super-resolution imaging method, the device comprising an excitation light generating module, a loss light generating module, a polarization beam splitter, a multi-photon microscopic scanning module and a photoelectric detection module, the excitation light generating module is used to generate a near-infrared Gaussian beam used as excitation light, the loss light generating module is used to generate a near-infrared hollow beam used as loss light; the polarization beam splitter spatially conjugate couples the near-infrared Gaussian excitation beam and the near-infrared hollow loss beam that are perpendicular to each other on a horizontal plane into a coupled laser beam, and the coupled laser beam is focused on a sample on a stage through the multi-photon microscopic scanning module; the photoelectric detection module is used to detect the super-resolution fluorescence signal generated after the above-mentioned sample is excited and lost.

[0050] It should be noted that the excitation light generating module includes a first near-infrared continuous laser, and a first filter, a first collimating beam expander, a first half-wave plate, and a first polarizer which are placed in sequence along the direction of the laser beam emitted by the laser. The first near-infrared continuous laser generates a continuous near-infrared steady-state laser beam output, and the wavelength of the beam matches the excitation wavelength of the sensitized ions of the rare-earth-doped nanomaterial. The laser beam is filtered and collimated by the first filter and the first collimating beam expander, and its power is adjusted by the cooperation of the first half-wave plate and the first polarizer.

[0051] It should be noted that the loss light generation module includes a second near-infrared continuous laser, and a second filter, a second collimating beam expander, a second half-wave plate, a second polarizer, and a spatial phase modulator placed in sequence along the direction of the laser beam emitted by the laser. The second near-infrared continuous laser generates a continuous near-infrared steady-state laser beam output, and the photon energy of the beam matches the energy gap between the upper and lower energy levels of the stimulated radiation process in the sensitized ions of the rare earth-doped nanomaterials. The laser beam is filtered and collimated by the second filter and the second collimating beam expander, and its power is adjusted by the second half-wave plate and the second polarizer, and then modulated into a hollow beam by the spatial phase modulator.

[0052] It should be noted that the multi-photon microscopy scanning module includes a scanning galvanometer, a high-reflection and low-transmittance dichroic mirror and an objective lens which are placed in sequence along the advancing direction of the coupled laser beam. The coupled laser beam is focused by the objective lens after passing through the scanning galvanometer and the high-reflection and low-transmittance dichroic mirror. The sample on the stage is placed on the focal plane of the objective lens. A motor for driving the stage to move along the Z-axis direction is provided on one side of the stage. Through this motor, combined with a rotating device, a three-dimensional laser scanning fluorescence image can be obtained.

[0053] It should be noted that the photoelectric detection module includes a focusing lens and a photoelectric detector coaxially placed in sequence, the focusing lens and the photoelectric detector are arranged in the opposite direction of the forward direction of the coupled laser beam, the photoelectric detector is connected to an external computer chain, and the sample marked with rare earth-doped nanomaterials emits super-resolution fluorescence signals in all directions, a part of which is collected by the objective lens, and is received by the photoelectric detector after passing through a high-reflection and low-transmittance dichroic mirror and a focusing lens. The scanning galvanometer is arranged on a rotating device, and the rotating device is controlled by a computer. The computer is connected to the photoelectric detector. After the photoelectric detector receives a detection signal, it sends a signal to the computer, and then the computer controls the rotation of the scanning galvanometer through the rotating device, and obtains a two-dimensional laser scanning fluorescence image by scanning the sample with a focused light spot.

[0054] Example 1

[0055] This embodiment provides a method of 3+ The invention is a method for realizing a super-resolution fluorescent nanoprobe sensitized by stimulated radiative depletion of activated ions. In this embodiment, a super-resolution fluorescent nanoprobe is constructed by rare earth doped upconversion nanoparticles. The nanoparticles are composed of Nd as both sensitizing ions and activating ions. 3+ Doped in NaYF4 nanocrystals.

[0056] A continuous near-infrared excitation light with a wavelength of 740, 800 or 860 nm is used to excite the Nd 3+ Doped super-resolution fluorescent nanoprobe, Nd 3+ Under the action of excitation light, the energy level 4 I 9 / 2 Direct transition to excited state 4 F 3 / 2 , or excited to a higher energy level and then assisted by phonons to relax to 4 F 3 / 2 , in 4 F 3 / 2 Nd 3+ Can transfer energy to the activated ion Nd for luminescence 3+ Jump to 2 F 5 / 2 , 2 P 1 / 2 as well as 4 D 5 / 2 , thereby emitting up-conversion fluorescence in the 867nm / 523nm / 450nm band. For the specific process, see Figure 1 .

[0057] Add a continuous near-infrared loss light with a wavelength of 1064nm to sensitize the Nd activated ions. 3+The number is greatly reduced, the luminescence process is blocked, and the upconversion fluorescence is greatly lost. It also shows that the higher the multiphoton order, the higher the fluorescence loss efficiency. Figure 2 .

[0058] Example 2

[0059] This embodiment provides a method of 3+ Or Ho 3+ The invention is a method for realizing a super-resolution fluorescent nanoprobe sensitized by stimulated radiative depletion of activated ions. In this embodiment, a super-resolution fluorescent nanoprobe is constructed by rare earth doped upconversion nanoparticles. The nanoparticles are composed of sensitizing ions Nd 3+ , auxiliary sensitizing ion Yb 3+ and the activated ion Er 3+ Or Ho 3+ Co-doped in NaYF4 nanocrystals.

[0060] A continuous near-infrared excitation light with a wavelength of 740, 800 or 860 nm is used to excite the optical super-resolution fluorescent nanoprobe synthesized in Example 1. 3+ Under the action of excitation light, the energy level 4 I 9 / 2 Direct transition to excited state 4 F 3 / 2 , or excited to a higher energy level and then assisted by phonons to relax to 4 F 3 / 2 , in 4 F 3 / 2 Nd 3+ Can transfer energy to the auxiliary sensitizing ion Yb in the ground state 3+ Jump to 2 F 5 / 2 , Yb 3+ Then transfer the energy to Er in the ground state 3+ Or Ho 3+ , Er 3+ After excitation, it can emit up-conversion fluorescence in the bands of 655nm / 540nm / 525nm, Ho 3+ After excitation, it can emit up-conversion fluorescence in the wavelength bands of 645nm / 540nm / 488nm. For the specific process, see Figure 3 .

[0061] Add a beam of continuous near-infrared loss light with a wavelength of 1064nm, 4 F 3 / 2 Nd 3+ Stimulated radiation transition to 4 I 11 / 2 , used to sensitize the Nd activated ions 3+The number is greatly reduced, the luminescence process is blocked, Er 3+ Or Ho 3+ The emitted upconversion fluorescence is significantly depleted, see Figure 4 .

[0062] Example 3

[0063] This embodiment provides a specific synthesis method of stimulated emission depletion sensitized super-resolution fluorescent nanoprobe. The above rare earth doped upconversion nanoparticles are synthesized by solvothermal method. Taking NaYF4:Nd / Yb / Er as an example, the steps are as follows: First, at room temperature (23-25°C), 5mL of 0.2M Ln(CH3COO)3 solution (Ln=Y / Nd / Yb / Er) is added to a 100mL round-bottom flask, and then 7mL of oleic acid and 17.5mL of 1-octadecene are added in sequence, and reacted at 150°C for 40 minutes to form a precursor. Remove the heating jacket and allow the reaction mixture to cool to 40°C while stirring, add 0.148g of ammonium fluoride solid and 0.761g of sodium oleate solid in sequence, and then react at 40°C for 2 hours, and then react at 100°C in vacuum for 30 minutes to remove moisture. After the water evaporates, heat to 270°C in an argon atmosphere and react at this temperature for 1.5 hours. The heating jacket was removed and the reactants were allowed to cool to room temperature while stirring. Then 10 mL of anhydrous ethanol was added and the mixture was centrifuged at 7500 rpm for 5 minutes. The supernatant was removed to collect the precipitated product, which was washed with a mixture of ethanol and cyclohexane and dried to obtain upconversion nanoparticles NaYF4:Nd / Yb / Er, which were dispersed in 10 mL of cyclohexane.

[0064] Example 4

[0065] This embodiment provides a method for performing full-spectrum multicolor super-resolution imaging of multiple luminescent ions or molecules by using the same pair of near-infrared wavelength excitation light / depletion light, the method comprising:

[0066] (1) The optical path is divided into two. In the first optical path, the first near-infrared fiber-coupled laser emits a single-mode laser beam with a wavelength of 740, 800 or 860 nm. After the laser is processed by a collimating beam expander and a pinhole aperture filter, a Gaussian excitation beam is obtained.

[0067] (2) In another optical path, a second near-infrared fiber-coupled laser generates a single-mode laser with a wavelength of 1064 nm. After being filtered by a collimating beam expander and a pinhole aperture, the laser is modulated by a spatial phase modulator to form a hollow beam with a central light intensity of zero, thereby obtaining a hollow-type loss beam. The wavelength of the hollow-type loss beam needs to match the energy gap between the two energy levels where the stimulated radiation process occurs in the sensitized ions.

[0068] (3) The Gaussian excitation beam and the hollow-type loss beam are spatially coaxially coupled and focused to irradiate the super-resolution fluorescent nanoprobe. The Gaussian excitation beam excites the nanoprobe to produce fluorescence, while the hollow-type loss beam triggers the stimulated radiation process of the sensitized ions, making it difficult for the sensitized ions to transfer energy to the auxiliary sensitized ions and then to the activated ions, thereby suppressing the emission of fluorescence in the irradiated area of ​​the loss light from the source of the excitation process.

[0069] (4) The super-resolution fluorescent nanoprobe is marked on different parts or structures of the sample by biochemical methods, and the same pair of near-infrared excitation light and near-infrared loss light is focused by an objective lens and applied to the sample, and scanned in the XYZ direction, and different up-conversion fluorescent signals are separated by a dichroic mirror and a filter, and the super-resolution fluorescent signal is detected by a photodetector to obtain a super-resolution image; for multi-color imaging, multiple photodetectors are used to synchronously detect signals of different wavelengths to obtain fluorescent images of different bands, and then the images of different channels are dyed and superimposed to obtain a multi-color super-resolution image;

[0070] Example 5

[0071] This embodiment provides a microscopic imaging device based on the above-mentioned super-resolution imaging method. The structure of the device is shown in Figure 4 , including an excitation light generation module, a loss light generation module, a polarization beam splitter prism, a multi-photon microscopy scanning module and a photoelectric detection module.

[0072] The excitation light generation module includes a first near-infrared continuous laser 1, and a first filter 2, a first collimating beam expander 3 (including a first pinhole diaphragm), a first half-wave plate 4, and a first polarizer 5, which are sequentially placed along the direction of the laser beam emitted by the laser. The loss light generation module includes a second near-infrared continuous laser 7, and a second filter 8, a second collimating beam expander 9, a second half-wave plate 10, a second polarizer 11, and a spatial phase modulation plate 12, which are sequentially placed along the direction of the laser beam emitted by the laser. The output wavelength of the first near-infrared laser is 740 nm. A continuous Gaussian laser is used as the excitation light, and a second near-infrared laser outputs a continuous Gaussian laser with a wavelength of 1064nm as the loss light. The filter filters out stray light in other bands of the laser, and the collimating beam expander expands the size of the excitation spot to improve the utilization rate of the excitation light power. At the same time, a pinhole filter is placed at the focus to filter out high-frequency stray light. A half-wave plate is installed on a rotatable mount and is matched with a polarizer to adjust the power of the laser beam. The spatial phase modulator modulates the loss light into a hollow beam. The excitation light and the loss light are spatially coupled through a high-reflection and low-transmittance dichroic mirror 13.

[0073] The multiphoton microscopic scanning module includes a scanning galvanometer 14, a high-reflection and low-transmittance dichroic mirror 15, a scanning lens 16, a tube lens 17, an objective lens 18, and a photon avalanche nanomaterial or a labeled sample 19 placed on the stage. The scanning galvanometer controls the optical path deflection of the laser beam to achieve two-dimensional scanning of the sample, the high-reflection and low-transmittance dichroic mirror reflects the laser beam, and the scanning lens and the tube lens focus and collimate the outgoing light beam of the scanning galvanometer so that the laser beam still matches the entrance pupil size of the microscope objective lens during the scanning process. Finally, the objective lens focuses the laser onto the super-resolution fluorescent nanomaterial or the labeled sample placed on the stage.

[0074] The photoelectric detection module includes a focusing lens 20 and a photoelectric detector 21. The focusing lens and the photoelectric detector are arranged along the forward direction of the fluorescence collected by the objective lens, and the photoelectric detector is connected to an external computer. After the photoelectric detector receives a detection signal, it sends a signal to the computer, and then the computer controls the scanning galvanometer to rotate, and obtains a two-dimensional laser scanning super-resolution image by scanning the sample point by point using the focused light spot.

[0075] Those skilled in the art can make various corresponding changes according to the above technical solutions and concepts, and all of these changes should be included in the protection scope of the claims of the present invention.

Claims

1. A fluorescence depletion method sensitized by stimulated emission depletion, characterized in that: The method comprises the following steps: S1 constructs rare earth ion-doped fluorescent nanomaterials of different components; wherein the rare earth ions include sensitizing ions and one or more activating ions. After the action of excitation light, the sensitizing ions can absorb energy and transfer it to the one or more activating ions, so that the one or more activating ions emit fluorescence of different bands; S2 adds a single-beam loss laser for loss; wherein the wavelength of the single-beam loss laser matches the energy gap between the two energy levels in the sensitized ion, and by inducing a stimulated radiation loss process, quenches the energy of a specific energy level, blocks the energy transfer process between the sensitized ion and the one or more activated ions from the excitation source, quenches the luminescence of all energy levels of all the one or more activated ions, thereby achieving the simultaneous loss of fluorescence of different energy levels of the one or more activated ions by the single-beam loss light; the sensitized ion is Nd 3+ , the activated ion is Nd 3+ , Er 3+ 、Ho 3+ , Pr 3+ 、Tm 3 + 、Eu 3+ or Tb 3+ One or more of; Among them, the additional sensitizing ion Yb 3+ , which is used to assist energy from Nd 3+ Further transferred to one or more activated ions.

2. The stimulated emission depletion sensitized fluorescence depletion method according to claim 1, characterized in that: When the one or more activated ions perform up-conversion luminescence under the action of the sensitizing ions, the higher the multi-photon order, the higher the fluorescence loss efficiency and the lower the required loss laser light intensity.

3. A multicolor super-resolution imaging method based on the fluorescence loss method based on stimulated emission loss sensitization according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: S1 has two optical paths; in one optical path, the first near-infrared fiber-coupled laser emits a single-mode laser with a wavelength of 740 nm, 800 nm or 860 nm, which is processed by a collimating beam expander and a pinhole aperture filter to obtain a Gaussian excitation beam; S2 In another optical path, the second near-infrared fiber-coupled laser generates a single-mode laser with a wavelength of 1064 nm. After being filtered by a collimating beam expander and a pinhole aperture, the single-mode laser is modulated by a spatial phase modulator to form a hollow beam with a central light intensity of zero, thereby obtaining a hollow-type loss beam. The wavelength of the hollow-type loss beam needs to match the energy gap between the two energy levels where the stimulated radiation process occurs in the sensitized ions. S3 The focused Gaussian excitation beam and the hollow loss beam are spatially coaxially coupled and focused to irradiate the rare earth ion-doped nanomaterials. The Gaussian excitation beam excites the nanomaterials to produce fluorescence, while the hollow loss beam triggers the stimulated radiation process of the sensitized ions, making it difficult for the sensitized ions to transfer energy to one or more activated ions, thereby suppressing the emission of fluorescence in the irradiated area of ​​the loss beam from the source of the excitation process. S4 Nanoparticles doped with one or more activated ions are marked on different parts or structures of the sample through biochemical methods. The same pair of near-infrared excitation light and near-infrared loss light are focused by the objective lens and applied to the sample. The sample is scanned in the XYZ direction, and different super-resolution fluorescence signals are separated by dichroic mirrors and filters. The super-resolution fluorescence signals are detected by photodetectors to obtain super-resolution images.

4. The multi-color super-resolution imaging method based on stimulated emission depletion sensitized fluorescence depletion method according to claim 3, characterized in that: For multi-color imaging, multiple photodetectors are used to synchronously detect signals of different wavelengths to obtain fluorescence images of different bands. The images of different channels are then dyed and superimposed to obtain a multi-color super-resolution image.

Images