A ground state correlation-based super-linear infrared confocal stimulated emission depletion super-resolution system and method

Abstract

The application discloses a kind of based on ground state association's superlinear infrared confocal stimulated radiation super-resolution system and method.The application builds the up-conversion superlinear stimulated radiation loss super-resolution imaging system under the guidance of confocal, by constructing ground state association's dynamic cross relaxation energy transfer model, to accurately obtain the light loss process of the rare earth particles under the action of superlinearity, and can accurately predict the ideal excitation power under high loss efficiency.Based on the super-resolution imaging method under the guidance of the theory, by infrared confocal module, two-photon scanning module, photoelectric detection module Microscopic imaging device, simultaneously utilize the double infrared excitation response of rare earth nanoparticles, the light path precision calibration of stimulated radiation super-resolution imaging process is realized.Using rare earth doped up-conversion nanoparticles and rate equation model accurately simulates the particle number distribution of each energy state of up-conversion nanoparticles, explores the influence of cross relaxation energy transfer process in rare earth nanometer material on the formation process of high-efficiency particle number inversion.Finally, the application can realize the up-conversion stimulated radiation super-resolution imaging system of low loss, high resolution, high signal-to-noise ratio and low system calibration complexity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of super-resolution fluorescence microscopy, and particularly relates to a super-linear infrared confocal stimulated emission depletion super-resolution system and method based on ground state association. BACKGROUND

[0002] Confocal laser scanning microscope (CLSM) is a fluorescence microscope with a laser scanning device. By point-by-point scanning imaging, the fluorescence image of the fine structure inside the living tissue of cells can be obtained. This imaging technology has the advantages of high resolution, optical sectioning, three-dimensional reconstruction, dynamic analysis, etc., and has become an essential tool for biological imaging. Although its resolution is higher than that of ordinary optical microscopes, it still does not break through the optical diffraction limit.

[0003] Stimulated emission depletion (STED) microscopy is a fluorescence microscopy technology that can break through the optical diffraction limit. In the STED microscopy system, two illumination lights are usually needed, one as an excitation light beam and the other as a depletion light beam. The excitation light irradiates the sample to excite the fluorescent molecules within the light spot to the excited state. The depletion light beam makes the electrons outside the center of the doughnut excited to return to the ground state, and the fluorescence in the center spontaneously radiates back to the ground state. The imaging resolution of STED is related to the area of the spontaneous emission fluorescence region. The smaller the area, the higher the imaging resolution. Therefore, a high-power depletion light is needed to reduce the area of the effective region. However, the use of high-power depletion light increases the photo-bleaching and photo-toxicity of the sample, which is not conducive to in vivo imaging. In addition, the perfect alignment of the excitation light beam and the depletion light beam also increases the difficulty of system calibration.

[0004] Upconverting stimulated emission depletion (U-STED) microscopy is a stimulated emission depletion microscopy technology that uses upconversion nanoparticles (UCNPs) as the imaging sample. The advantage of this technology is that the upconversion fluorescent nanoparticles are excited by low-energy light to emit high-energy fluorescence. Therefore, for high-resolution imaging, upconversion nanoparticles can be used to reduce the requirement for high power. For example, under the irradiation of the doughnut depletion light, the intermediate level experiences a photonic avalanche effect, which can partially block the upward upconversion path, thereby promoting the depletion of the high excited state and achieving higher depletion efficiency.

[0005] Although upconversion stimulated emission loss microscopy has the advantages of low loss power and high resolution, there is still no perfect mechanism to balance the spatial resolution and imaging quality. Therefore, finding the appropriate excitation power is of great significance for upconversion stimulated emission microscopy to achieve high resolution and high imaging quality. SUMMARY

[0006] The present application aims to provide a ground state correlation based super-linear infrared confocal stimulated emission super-resolution system.

[0007] The technical solution for achieving the purpose of the present application is: a ground state correlation based super-linear infrared confocal stimulated emission super-resolution system, comprising: an infrared confocal module, a two-photon-like scanning module, a photoelectric detection module, and a stimulated emission loss module; wherein:

[0008] The infrared confocal module includes two infrared excitation lights, one of which includes: a single-mode fiber coupled 980nm laser, a collimating mirror 1, a mirror 1, a polarization beam splitter 1, a mirror 2, a first 4f system, a dichroic mirror 1, and a dichroic mirror 2. The single-mode fiber coupled 980nm laser is used as the excitation source, and the emitted 980nm laser is collimated by the collimating mirror 1, then reflected by the mirror 1 for the first time, and then enters the 1 / 2 wave plate 1 and the polarization beam splitter 1 combination. After the second reflection by the mirror 2, it enters the first 4f system composed of lens 1 and lens 2. The light beam passing through the first 4f system is reflected by the dichroic mirror 1 and the dichroic mirror 2, and then incident on the 1 / 4 wave plate, and then focused on the sample by the oil immersion objective lens.

[0009] The other infrared excitation light includes a single-mode fiber coupled 808nm laser, a collimating mirror 2, a mirror 3, a 1 / 2 wave plate 2, a polarization beam splitter 2, a mirror 4, and a second 4f system. Similarly, the single-mode fiber coupled 808nm laser is used as the excitation source, and the emitted 808nm laser is collimated by the collimating mirror 2, then reflected by the mirror 3 for the first time, and then enters the 1 / 2 wave plate 2 and the polarization beam splitter 2 combination. After the second reflection by the mirror 4, it enters the second 4f system composed of lens 3 and lens 4. The light beam passing through the second 4f system is reflected by the mirror 5 to the dichroic mirror 1. The reflected light is reflected by the dichroic mirror 1, and the dichroic mirror 1 couples the 980nm laser beam and the 808nm laser beam. Then, after being reflected by the dichroic mirror 2, it is focused on the sample by the oil immersion objective lens.

[0010] The photoelectric detection module is used to collect the fluorescence signal emitted by the upconversion sample;

[0011] The two-photon-like scanning module uses a three-axis closed-loop nanometer positioner, and a sample holder is arranged on the positioner to fix the sample.

[0012] The stimulated radiation loss module adopts a vortex phase plate, which is arranged at one end of the second 4f system close to the mirror 4 when the super-linear effect under the confocal stimulated radiation super-resolution imaging is performed.

[0013] Preferably, the 4f system is composed of two lenses with a focal length of 300 mm.

[0014] Preferably, the dichroic mirror 1 is a long-pass dichroic mirror, and the dichroic mirror 2 is a short-pass dichroic mirror.

[0015] Preferably, the photoelectric detection module comprises a flip mirror, a tube lens 1, a camera, a mirror 6, a filter, a tube lens 2, a single-mode optical fiber and a single-photon counter. When the flip mirror is arranged as a mirror into the light path, the fluorescence signal is focused on the camera through the flip mirror and the tube lens 1, and the camera is adjusted to keep the image plane conjugate with the entrance of the multi-mode optical fiber.

[0016] When the flip mirror is rotated to be out of the light path, the up-conversion sample is scanned by the confocal method, the fluorescence signal emitted by the up-conversion sample is collected by the oil immersion objective, the reflected laser and the fluorescence signal pass through the 1 / 4 wave plate, the dichroic mirror 2 is used to separate the excitation and emission beams, the fluorescence signal is filtered by the filter, and is converged to the entrance of the multi-mode optical fiber through the imaging lens. The other end of the multi-mode optical fiber is connected to the single-photon counter, and the single-photon counter records the number of photons emitted by the sample.

[0017] The application also provides an ultra-linear infrared confocal stimulated radiation super-resolution method based on ground state association, and the specific steps are as follows.

[0018] Step 1: build an ultra-linear infrared confocal stimulated radiation super-resolution system.

[0019] Step 2: construct a dynamic cross-relaxation energy transfer model based on ground state association to predict the ideal excitation power under high loss efficiency.

[0020] Step 3: adjust the ultra-linear infrared confocal stimulated radiation super-resolution system according to the predicted ideal excitation power under high loss efficiency, and realize the confocal multi-photon super-resolution imaging under the super-linear effect by using the adjusted ultra-linear infrared confocal stimulated radiation super-resolution system.

[0021] Step 4: calibrate the stimulated radiation light path by using the double infrared response of the up-conversion material.

[0022] Step 5: realize the confocal stimulated radiation super-resolution imaging under the super-linear effect by using the ultra-linear infrared confocal stimulated radiation super-resolution system.

[0023] Preferably, the specific steps of constructing the dynamic cross-relaxation energy transfer model based on ground state association to predict the ideal excitation power under high loss efficiency are as follows:

[0024] Step 2.1: Constructing rate equation modeling energy level diagram;

[0025] Step 2.2: Constructing rate equation of each energy state in the energy transfer process according to the energy level diagram constructed in step 2.1;

[0026] Step 2.3: Extracting the rate characteristics of dynamic power-dependent cross-relaxation CR3 under dual laser excitation;

[0027] Step 2.4: Inputting the extracted rate characteristic parameters into the rate equation model of step 2.2 to predict the ideal excitation power under high loss efficiency.

[0028] Preferably, the constructed rate equation of each energy state in the energy transfer process is specifically:

[0029]

[0030]

[0031]

[0032]

[0033]

[0034] m T1 +m T2 =1

[0035] m1+m2+m3+m4+m5=1

[0036] Wherein, s i is the upconversion coefficient between Yb 3+ and Tm 3+ in the i layer, i = 1, 2, 3, 4, m j represents the particle number density, j = 1-5, bij is the branching ratio of Tm 3+ radiation transition from initial state i to final state j, k ij is the cross-relaxation coefficient; P i is the intrinsic decay rate of Tm 3 + in the i layer; P 980 is the absorption rate of Yb 3+ , P 808 is the loss rate of Tm 3+ (P = σλI / hc, λ is the excitation wavelength, I is the excitation intensity at 976 / 808 nm, σ is the absorption cross section of Yb 3+ at 976 / 808 nm, h is the Planck constant, and c is the speed of light.

[0037] Preferably, the specific method for realizing the confocal multi-photon super-resolution imaging under the super-linear effect by adjusting the super-linear infrared confocal stimulated radiation super-resolution system is as follows:

[0038] The 980nm laser emitted by the single-mode fiber-coupled 980nm laser is collimated by the collimating mirror 1, is reflected by the mirror 1 for the first time, enters the 1 / 2 wave plate 1 and the polarization beam splitter 1 combination, is reflected by the mirror 2 for the second time, enters the first 4f system, the first 4f system is composed of the lens 1 and the lens 2, the light beam passing through the first 4f system is reflected by the first dichroic mirror 1 and the second dichroic mirror 2, is incident on the 1 / 4 wave plate, and is focused on the sample by the oil immersion objective, so that the excitation of the 980nm light beam on the sample is completed.

[0039] Meanwhile, the single-mode fiber-coupled 808nm laser is used as the excitation source, the emitted 808nm laser is collimated by the collimating mirror 2, is reflected by the mirror 3 for the first time, enters the 1 / 2 wave plate 2 and the polarization beam splitter 2 combination, is reflected by the mirror 4 for the second time, enters the second 4f system, the second 4f system is composed of the lens 3 and the lens 4, the light beam passing through the second 4f system is reflected by the mirror 5 to the dichroic mirror 1, the reflected light is reflected by the dichroic mirror 1, the dichroic mirror 1 couples the 980nm laser beam and the 808nm laser beam, and then the light beam is reflected by the dichroic mirror 2 and is focused on the sample by the oil immersion objective.

[0040] The flip mirror is rotated to enter the light path as a mirror, the fluorescence signal is focused on the camera by the flip mirror and the tube lens 1, the camera is adjusted to keep the conjugate with the image plane at the entrance of the multimode optical fiber, and the flip mirror is rotated to not enter the light path.

[0041] The upconversion sample is scanned by the confocal scanning, the fluorescence signal emitted by the upconversion sample is collected by the oil immersion objective, the reflected laser and the fluorescence signal pass through the 1 / 4 wave plate, the dichroic mirror 2 is used to separate the excitation and emission beams, the fluorescence signal is filtered by the filter, is converged to the entrance of the multimode optical fiber by the imaging lens, and the other end of the multimode optical fiber is connected to the single-photon counter, the single-photon counter records the number of photons emitted by the sample to obtain a high-resolution confocal multi-photon super-resolution image.

[0042] Preferably, the specific method for calibrating the stimulated radiation light path by using the dual infrared response of the upconversion material is as follows:

[0043] The angle of the dichroic mirror 1 is adjusted to couple the 980nm light beam and the 808nm light beam.

[0044] Rotate the flip mirror to make it as a mirror into the light path, observe the imaging of the reference camera, take the same detection site as the conjugate calibration point of the excitation of the double light path, adjust the angle of the mirror and the dichroic mirror, so that the photon intensity of the detection site reaches the maximum, and the alignment of the stimulated radiation light path is completed.

[0045] Preferably, the specific method for realizing super-linear infrared confocal stimulated radiation super-resolution imaging under super-linear effect is realized by using a super-linear infrared confocal stimulated radiation super-resolution system.

[0046] A vortex phase plate is inserted in front of the 4f system of the 808nm excitation light path, and a donut-shaped beam is generated as the loss beam of the stimulated radiation loss system by using the vortex phase plate;

[0047] The angle of the dichroic mirror 1 is adjusted to couple the 980nm light beam and the 808nm light beam;

[0048] Rotate the flip mirror to make it as a mirror into the light path, observe the imaging of the reference camera, and ensure that the point spread functions of the two laser beams coincide in the X-Y and Z axes;

[0049] Rotate the flip mirror to make it not enter the light path, filter the fluorescence signal by the filter, converge the fluorescence signal to the entrance of the multimode optical fiber through the tube lens 2, and connect the other end of the multimode optical fiber to the single photon counter, so that the single photon counter records the number of photons emitted by the sample to obtain a high-resolution stimulated radiation loss super-resolution image.

[0050] Compared with the prior art, the present application has the following advantages: the present application can break through the diffraction limit and realize super-resolution imaging under confocal multi-photon imaging; the present application realizes accurate light path calibration of the super-resolution imaging process of the stimulated radiation by simultaneously utilizing the double infrared excitation response of the rare earth nanoparticles; and the present application finally realizes a low-loss, high-resolution, high signal-to-noise ratio, and low system calibration complexity upconversion confocal stimulated radiation imaging system. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 It is a super-linear infrared confocal stimulated radiation super-resolution imaging system diagram.

[0052] Figure 2 It is a super-linear infrared confocal stimulated radiation super-resolution imaging method flowchart based on ground state association.

[0053] Figure 3 It is an energy transfer process diagram under different power 980nm excitation light beams. 3+ Cross relaxation under 980nm excitation. 3+The electronic distribution of the.

[0054] Figure 4 The numerical simulation results of dynamic CR3 under dual laser.

[0055] Figure 5 The multi-photon-like super-resolution imaging and the super-linear effect under the stimulated radiation-like super-resolution imaging under different excitation powers. DETAILED DESCRIPTION

[0056] In order to more clearly illustrate the embodiments of the present application or related technical schemes, the present application will be further described below with reference to the accompanying drawings, but the protection scope of the present application is not limited to the present application.

[0057] The present application builds a super-linear stimulated radiation loss super-resolution imaging system under confocal guidance, and by constructing a dynamic cross-relaxation energy transfer model associated with the ground state, the light loss process of the rare earth particles under super-linear effect is accurately obtained, and the ideal excitation power under high loss efficiency can be accurately predicted.

[0058] The technical scheme adopted by the present application is: a super-linear infrared confocal stimulated radiation super-resolution imaging method based on ground state correlation, the flow of the method is as shown in Figure 2 The specific steps are as follows:

[0059] Step 1: Build a super-linear infrared confocal stimulated radiation super-resolution system. Figure 1 As shown in the figure.

[0060] The super-linear infrared confocal stimulated radiation super-resolution system comprises: an infrared confocal module, a two-photon-like scanning module, a photoelectric detection module, and a stimulated radiation loss module.

[0061] In a further embodiment, the infrared confocal module comprises two infrared excitation lights, one of which comprises: a single-mode fiber-coupled 980 nm laser, a collimating mirror 1, a mirror 1, a polarization beam splitter 1, a mirror 2, a first 4f system, a dichroic mirror 1, and a dichroic mirror 2. The single-mode fiber-coupled 980 nm laser is used as an excitation source. The emitted 980 nm laser light is collimated by the collimating mirror 1, reflected by the mirror 1 for the first time, and then enters the 1 / 2 wave plate 1 and the polarization beam splitter 1 combination. After being reflected by the mirror 2 for the second time, the light enters the first 4f system, which is composed of a lens 1 and a lens 2. The light beam passing through the first 4f system is reflected by the first dichroic mirror 1, reflected by the second dichroic mirror 2, and then enters the 1 / 4 wave plate. The light is focused onto the sample by the oil immersion objective lens. The other infrared excitation light comprises: a single-mode fiber-coupled 808 nm laser, a collimating mirror 2, a mirror 3, a 1 / 2 wave plate 2, a polarization beam splitter 2, a mirror 4, a second 4f system, and a mirror 5. The single-mode fiber-coupled 808 nm laser is used as an excitation source. The emitted 808 nm laser light is collimated by the collimating mirror 2, reflected by the mirror 3 for the first time, and then enters the 1 / 2 wave plate 2 and the polarization beam splitter 2 combination. After being reflected by the mirror 4 for the second time, the light enters the second 4f system, which is composed of a lens 3 and a lens 4. The light beam passing through the second 4f system is reflected by the mirror 5 to the dichroic mirror 1. The reflected light is reflected by the dichroic mirror 1. The dichroic mirror 1 couples the 980 nm laser beam and the 808 nm laser beam. The light is then reflected by the dichroic mirror 2 and focused onto the sample by the oil immersion objective lens.

[0062] In a further embodiment, the photoelectric detection module is used to collect the upconversion sample emitted fluorescence signal. The two reflected excitation lights (980 nm excitation light and 808 nm excitation light) and the fluorescence signal emitted by the sample pass through the 1 / 4 wave plate. The dichroic mirror 2 separates the reflected excitation light beam and the emitted fluorescence light beam. The detection of the fluorescence signal is divided into two paths. First, the first detection light path is used as the adjustment light path of the system. The flip mirror is rotated to enter the light path as a mirror. The fluorescence signal is reflected by the flip mirror and focused by the tube lens 1 to the reference camera. The camera is used as an adjustment device of the system. The position of the camera is adjusted to keep the image plane at the entrance of the multi-mode optical fiber. After the fluorescence signal enters the camera, the second detection light path is used for confocal stimulated emission depletion imaging. The flip mirror is rotated to not enter the light path. The fluorescence signal is reflected by the mirror 6, filtered by the filter, and finally focused by the tube lens 2 to the entrance of the multi-mode optical fiber. After the adjustment of the first detection light path, the fluorescence perfectly enters the multi-mode optical fiber. The other end of the multi-mode optical fiber is connected to a single photon counter.

[0063] In further embodiments, the two-photon-like scanning module adopts a high-precision three-axis closed-loop nanometer positioner, a sample holder is fixed above the positioner, when scanning the up-conversion nanoparticle sample, the sample is fixed on the holder, the X, Y and Z axes of the positioner are adjusted, the intensity of the light spot is observed by the reference camera, and when the intensity of the light spot in the X, Y and Z directions is the maximum, the flip mirror is switched to scan the up-conversion nanoparticle sample in the XY and YZ planes. The sample is scanned point by point by the nanometer positioner according to the set scanning route, and the nanometer positioner stops working after the scanning task is completed.

[0064] The stimulated radiation loss module is a vortex phase plate inserted in front of the 4f system of the 808nm excitation light path. By using the vortex phase plate, a donut-shaped light beam can be generated as the loss light beam of the super-linear infrared stimulated radiation loss system.

[0065] When performing super-linear infrared confocal multi-photon super-resolution imaging, two lasers of 980nm and 880nm are used as excitation light to image the up-conversion sample. The super-linear infrared stimulated radiation super-resolution imaging is similar to the confocal multi-photon super-resolution imaging, and a 980nm laser is used as the excitation light beam and an 808nm laser is used as the loss light beam.

[0066] Step 2: Construct a dynamic cross-relaxation energy transfer model associated with the ground state. Using this energy transfer model, the particle number distribution of each relevant energy state of lanthanide elements is simulated, and the ideal excitation power under high loss efficiency is predicted. The specific steps are as follows:

[0067] Step 2.1: Construct a rate equation modeling energy level diagram. As shown in Figure 3 The simplified energy level diagram is used in this example to simulate the energy transfer process between the high-concentration (>2mol.%) sensitizer Yb3+ and the emitter Tm3+. Throughout the example, up-conversion nanoparticles β-NaYF4 are used to study the energy transfer involving 8mol.% Yb3+ and Tm3+ dopants. Figures a and b describe the main cross-relaxation phenomena under low and high excitation power of 980nm, respectively. Figures c and d show the energy transfer process and Tm3+ ion distribution under low and high excitation power of 980nm, respectively, with a fixed power of 808nm loss light beam.

[0068] Step 2.2: According to the energy level diagram constructed in step 2.1, the rate equations of each energy state in the energy transfer process are derived as follows:

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] m T1 +m T2 = 1 (6)

[0075] m1+m2+m3+m4+m5= 1 (7)

[0076] CR1, CR2 and CR3 refer to the cross-relaxation processes between the ground and the upper excited levels of Yb 1 G4, 3 H4),( 1 G4, 3 H4), and( 1 D2, 3 H6) states, resulting in the population distributions of( 1 D2, 3 F4),( 3 F4, 1 D2) and( 3 F2, 3 H4), respectively. Tm 3+ of 3 H6, 3 H5 / 3 F4 and 3 F2,3 / 3 H4, 1 G4, 1 D4 levels are denoted as 1, 2, 3, 4 and 5, respectively. Yb 3+ of 2 F 7 / 2 and 2 F 5 / 2 levels are denoted as T1 and T2, respectively,

[0077] where s i (i = 1, 2, 3, 4) is the upconversion coefficient between the i-th excited Yb 3+ and Tm 3+ , m i (i = 1-5) represents the population density, bij is the branching ratio of the transition from the initial state i to the final state j, k 3+ is the cross-relaxation coefficient. P ij is the intrinsic decay rate of the i-th layer of Tm i ; P 3+ is the absorption rate of Yb 980 , P 3+ is the emission rate of Tm 808 . 3+loss rate (P = σλI / hc, λ is the excitation wavelength, I is the excitation intensity at 976 / 808 nm, σ is the Yb 3+ absorption cross section at 976 / 808 nm, h is the Planck constant, and c is the speed of light);

[0078] Step 2.3: Extract the rate characteristics of the dynamic power-dependent cross-relaxation CR3 under dual-laser excitation.

[0079] By ignoring the small cross-relaxation and non-radiative relaxation, this example simulates the transient response of 8 mol.% Tm3+-doped upconversion nanoparticles (UCNPs) under dual-laser excitation by using a 980 nm laser as the excitation beam before 500 us and coupling an 808 nm laser as the coupling beam after 500 us. This example measures the 1 power-dependent transition response of 455 nm at the D2 level and 800 nm at the 3H4 level.

[0080] Step 2.4: Input the extracted rate characteristics into the rate equation model of Step 2.2 to predict the ideal excitation power under high loss efficiency.

[0081] As shown in Figure 4 (a), when the excitation power is higher than a certain value, the intensity of the 800 nm (3H4-3H6) emission shows a downward trend, indicating that the emission intensity of the power-dependent cross-relaxation CR3 decreases, while the 455 nm (1D2-3H6) emission always shows an upward trend with the increase of power at high excitation power.

[0082] As shown in Figure 4 (b), based on the constructed ground-state-dependent dynamic cross-relaxation energy transfer model, according to the simulated population distribution of the emitter under low excitation power, it is found that the use of a 980 nm low excitation power laser produces a higher loss efficiency; Figure 4 (c) is the population distribution of the emitter under high excitation power. It can be seen that, when the 808 nm loss beam is fixed, a low excitation power can obtain a higher loss efficiency. On the contrary, a high excitation power obtains a lower loss efficiency. Therefore, the cross-relaxation can control the critical population distribution of the intermediate energy level. When the cross-relaxation coefficient exceeds 1 the intrinsic decay rate of the D2 level, a higher loss efficiency is produced. The simulation shows that, when performing dual-infrared confocal multi-photon super-resolution imaging, the ideal excitation power is fixed at 1.1 mW; when realizing confocal stimulated emission super-resolution imaging, the ideal excitation power and loss power are fixed at 1.1 mW and 27 mW, respectively.

[0083] Step 3: Adjust the super-linear infrared confocal stimulated radiation super-resolution system according to the predicted ideal excitation power under high loss efficiency, and realize confocal multi-photon super-resolution imaging under super-linear effect using the adjusted super-linear infrared confocal stimulated radiation super-resolution system.

[0084] The sample for high-resolution imaging in the present application uses highly doped upconversion nanoparticles (beta-NaYF4: 20 mol. % Yb 3+ , 8 mol. % Tm 3+ After the sample is synthesized, an appropriate amount of the sample is fixed on a sample holder, and the sample holder is fixed on a three-axis closed-loop nanometer positioner.

[0085] First, a single-mode optical fiber coupled 980 nm laser is used as an excitation source. An objective lens is used as a collimator, and after collimation, the laser is reflected by a mirror for the first time, enters a 1 / 2 wave plate and a polarization beam splitter prism combination, which can maintain the polarization purity of the laser. After the second reflection, it enters a 4f system composed of two lenses with a focal length of 300 mm. The 980 nm excitation beam passes through the first long-pass dichroic mirror and is reflected by the second short-pass dichroic mirror to a 1 / 4 glass plate, and then focused on the sample slide by an oil immersion objective lens. This process completes the excitation of the sample by the 980 nm beam.

[0086] At the same time, another single-mode optical fiber coupled 808 nm laser is used as an excitation source, and the setup is similar to that of the 980 nm laser. The 980 nm laser beam and the 808 nm laser beam are coupled by the first long-pass dichroic mirror, and then focused on the sample slide by the oil immersion objective lens. This process completes the excitation of the sample by the 808 nm beam. The excitation power of the dual infrared excitation light used in this example is fixed at 1.1 mW.

[0087] Secondly, the flip mirror is rotated to enter the light path as a mirror, and the fluorescence signal is focused by a tube lens to a camera. The camera is used as an adjustment device to maintain conjugation with the image plane at the entrance of the multimode optical fiber by adjusting the camera. The upconversion sample is scanned by the confocal scanning module. This module is similar to the scanning module of a conventional confocal system, which uses a high-precision three-axis closed-loop nanometer positioner to scan the upconversion nanoparticle sample in the XY and YZ planes.

[0088] Finally, the fluorescence signal emitted by the upconversion sample is collected by an oil immersion objective. The reflected laser and fluorescence signals pass through a 1 / 4 wave plate, and the excitation and emission beams are separated by a dichroic mirror 2 (750 nm short-pass filter). The fluorescence signal is filtered by a filter and then focused by an imaging lens onto the entrance of a multimode fiber, the other end of which is connected to a single photon counter. The single photon counter records the number of photons emitted by the sample to obtain a high-resolution confocal-like multi-photon super-resolution image.

[0089] For the control of each hardware, including two lasers, a three-axis closed-loop nanometer positioner, a camera, a single photon counter, and a flip mirror, an external triggering mode is used. The external triggering ports of each device are connected to the input and output ports of the NI acquisition card, the USB end of the acquisition card is connected to the computer, and the LabVIEW program is used to control the switching of each hardware and the scanning of the three-axis nanometer positioner. Whenever the single photon counter receives a fluorescence signal, the counter sends the signal to the computer, and the computer sends a pulse signal to the three-axis nanometer positioner after receiving the signal. By controlling the positioner to move a specific distance, the focused spot scans the sample and a two-dimensional image is obtained.

[0090] In the non-saturated excitation process, multi-level energy transfer through nonlinear interaction is used to obtain confocal-like multi-photon super-resolution imaging results.

[0091] This example verifies the ground state correlation-based super-linear infrared confocal stimulated emission super-resolution imaging system. As shown in Figure 5 (a), the power-dependent response of the system under 455 nm emission is measured, and it is found that the emission of the upconversion nanoparticles has a strong super-linear relationship with the excitation. The critical value in the curve is the trigger point of the 455 nm emission excitation state population process, and the particle number in this state reaches a dynamic balance between the intermediate state and the excited state, and a saturation point appears. However, the cross-relaxation effect of the upconversion nanoparticles changes this balance, indicating that changing the excitation power can control the distribution of particles in each energy level.

[0092] As shown in Figure 5 (c), confocal super-resolution under super-linear use, the full width at half maximum (FWHM) under different excitation powers is measured, and it is found that confocal scanning imaging under super-linear use can break through the diffraction limit and achieve super-resolution imaging when the excitation power is small.

[0093] Step 4: High-precision calibration of the stimulated emission light path using the dual infrared response of the upconversion material.

[0094] By using the dual excitation characteristics of upconversion nanoparticles, a nanoparticle or heterogeneous nanoprobe under multiple excitation is constructed, and rare earth upconversion nanoparticles with multi-channel compatible response are used as calibration particles. The biggest feature is to monitor the position of the conjugate point of the two beams in real time by exciting fluorescence, and to realize accurate alignment. The specific steps are: adjust the angle of dichroic mirror 1 to couple the 980nm light beam and the 808nm light beam. Rotate the flip mirror to make it enter the light path as a mirror, observe the imaging of the reference camera, and use the same detection site as the conjugate calibration point of the dual light path excitation. The photon intensity of the detection site reflects the coincidence degree of the excitation of the conjugate point. At this time, the angles of the mirror and the dichroic mirror are adjusted to make the photon intensity of the detection site reach the maximum, and the stimulated radiation light path realizes accurate alignment. The dual excitation rare earth nanoparticles with high optical stability can complete long-time dynamic calibration without optical quenching, further reduce the system deviation problem caused by replacing sample particles, and increase the convenience and usability.

[0095] Step 5: Use the super-linear infrared confocal stimulated radiation super-resolution system to realize super-linear confocal stimulated radiation super-resolution imaging.

[0096] The upconversion super-linear stimulated radiation loss super-resolution imaging is similar to the confocal multi-photon super-resolution, and 980nm laser is used as the excitation light beam, and 808nm laser is used as the loss light beam. A vortex phase plate is inserted in front of the 4f system of the 808nm excitation light path, and the vortex phase plate is used to generate a donut-shaped beam as the loss light beam of the stimulated radiation loss system.

[0097] Adjust the angle of dichroic mirror 1 to couple the 980nm light beam and the 808nm light beam. Rotate the flip mirror to make it enter the light path as a mirror, observe the imaging of the reference camera, and ensure that the point spread functions (PSF) of the two laser beams perfectly coincide in the X-Y and Z axes. Rotate the flip mirror to make it not enter the light path, filter the fluorescence signal by the filter, and finally converge the fluorescence signal to the multi-mode fiber entrance through the tube lens 2. The other end of the multi-mode fiber is connected to a single photon counter, which records the number of photons emitted by the sample to obtain a high-resolution stimulated radiation loss super-resolution image.

[0098] The present application designs a super-linear upconversion confocal super-resolution, emission intensity power dependence measurement function and a multi-wavelength overlapping confocal system, which are respectively used to obtain analog parameter data of the super-linear process, single nanoparticle optical characteristic value representation, light intensity power dependence measurement of each fluorescence band, system quenching efficiency measurement, etc. At the same time, the upconversion multi-photon-like infrared imaging mode and the upconversion lifetime imaging are realized.

[0099] Fix the excitation power and the loss power, and measure the loss efficiency of the upconversion emission at 455nm, such asFigure 5 (d) shows. The half width of the super-resolution stimulated emission upconversion decreases with the decrease of the excitation power, as shown in Figure 5 (c). With the change of the excitation power, the pump rate and the intermediate state loss rate change nonlinearly. The reason for this change is that the electron generation rate in the upconversion system depends on the number of electrons. By changing the excitation power, the curve of the loss particle number versus the total particle number is drawn, as shown in Figure 5 (e). The curve shows that, with the decrease of the excitation power, the power-dependent cross-relaxation CR3 rate approaches the optimal point, and the loss rate also approaches the generation rate, at which the loss efficiency is the highest, and the corresponding loss power is the minimum loss power, which is fixed at 27 mW, as shown in Figure 5 (b). This power is the minimum power to achieve the optimal loss efficiency.

Claims

1. A ground state correlation based superlinear infrared confocal stimulated emission depletion super-resolution system, characterized in that, The application relates to an infrared confocal module, a two-photon-like scanning module, a photoelectric detection module and a stimulated radiation loss module. The infrared confocal module comprises two infrared excitation light paths, one of which comprises a single-mode fiber-coupled 980nm laser, a first collimating mirror, a first reflecting mirror, a first polarization beam splitter, a second reflecting mirror, a first 4f system, a first dichroic mirror and a second dichroic mirror; the single-mode fiber-coupled 980nm laser is used as an excitation source, and the emitted 980nm laser is collimated by the first collimating mirror, reflected by the first reflecting mirror for the first time, enters the first 1 / 2 wave plate and the first polarization beam splitter combination, is reflected by the second reflecting mirror for the second time and then enters the first 4f system; the first 4f system is composed of a first lens and a second lens; the light beam passing through the first 4f system is reflected by the first dichroic mirror, the second dichroic mirror, a 1 / 4 wave plate and an oil immersion objective lens and then focused on a sample. The other infrared excitation light path comprises a single-mode fiber-coupled 808nm laser, a second collimating mirror, a third reflecting mirror, a second 1 / 2 wave plate, a second polarization beam splitter, a fourth reflecting mirror and a second 4f system; the single-mode fiber-coupled 808nm laser is also used as an excitation source, and the emitted 808nm laser is collimated by the second collimating mirror, reflected by the third reflecting mirror for the first time, enters the second 1 / 2 wave plate and the second polarization beam splitter combination, is reflected by the fourth reflecting mirror for the second time and then enters the second 4f system; the second 4f system is composed of a third lens and a fourth lens; the light beam passing through the second 4f system is reflected by a fifth reflecting mirror, the first dichroic mirror, the second dichroic mirror, a 1 / 4 wave plate and an oil immersion objective lens and then focused on the sample. The photoelectric detection module is used for collecting the fluorescent signal emitted by the up-conversion sample. The two-photon-like scanning module adopts a three-axis closed-loop nanometer positioner, and a sample holder is arranged on the positioner and used for fixing the sample. The stimulated radiation loss module adopts a vortex phase plate, which is arranged at one end of the second 4f system close to the fourth reflecting mirror when the confocal stimulated radiation super-resolution imaging is performed under the super-linear effect. The photoelectric detection module comprises a flip mirror, a first tube lens, a camera, a sixth reflecting mirror, a filter, a second tube lens, a single-mode optical fiber and a single-photon counter; when the flip mirror is arranged in the light path as a reflecting mirror, the fluorescent signal is focused on the camera through the flip mirror and the first tube lens; the camera is adjusted to keep the image plane at the entrance of the multi-mode optical fiber conjugated; when the flip mirror is rotated and does not enter the light path, the up-conversion sample is scanned by the confocal method, the fluorescent signal emitted by the up-conversion sample is collected by the oil immersion objective lens, the reflected laser and the fluorescent signal pass through the 1 / 4 wave plate, the second dichroic mirror is used to separate the excitation and emission light beams, the fluorescent signal is filtered by the filter, is converged on the entrance of the multi-mode optical fiber through the imaging lens, and the other end of the multi-mode optical fiber is connected to the single-photon counter; the single-photon counter records the number of photons emitted by the sample. ​ 2. The ground-state correlation-based superlinear infrared confocal stimulated- emission depletion super-resolution system of claim 1, wherein, The first 4f system and the second 4f system are both composed of two lenses with a focal length of 300 mm.

3. The ground-state correlation-based superlinear infrared confocal stimulated- emission depletion super-resolution system of claim 1, wherein, The first dichroic mirror is a long-pass dichroic mirror, and the second dichroic mirror is a short-pass dichroic mirror.

4. A ground state correlation based superlinear infrared confocal stimulated emission depletion super-resolution method, characterized in that, The specific steps are: Step 1: build the super-linear infrared confocal stimulated radiation super-resolution system according to any one of claims 1-3; Step 2: construct a dynamic cross-relaxation energy transfer model associated with the ground state to predict the ideal excitation power under high loss efficiency; Step 3: adjust the super-linear infrared confocal stimulated radiation super-resolution system according to the predicted ideal excitation power under high loss efficiency, and realize confocal multi-photon super-resolution imaging under super-linear action using the adjusted super-linear infrared confocal stimulated radiation super-resolution system; Step 4: use the double infrared response of the upconversion material to calibrate the stimulated radiation light path; Step 5: realize confocal stimulated radiation super-resolution imaging under super-linear action using the super-linear infrared confocal stimulated radiation super-resolution system.

5. The ground-state correlation-based superlinear infrared confocal stimulated- emission depletion super-resolution method according to claim 4, wherein, The specific steps for constructing a dynamic cross-relaxation energy transfer model associated with the ground state to predict the ideal excitation power under high loss efficiency are: Step 2.1: construct a rate equation modeling energy level diagram; Step 2.2: construct the rate equation of each energy state in the energy transfer process according to the energy level diagram constructed in step 2.1; Step 2.3: extract the rate characteristics of dynamic power-dependent cross-relaxation CR3 under double laser excitation; Step 2.4: input the extracted rate characteristic parameters into the rate equation model of step 2.2 to predict the ideal excitation power under high loss efficiency.

6. The ground-state correlation-based superlinear infrared confocal stimulated- emission depletion super-resolution method of claim 4, wherein, The specific method for adjusting the super-linear infrared confocal stimulated radiation super-resolution system according to the predicted ideal excitation power under high loss efficiency, and realizing confocal multi-photon super-resolution imaging under super-linear action using the adjusted super-linear infrared confocal stimulated radiation super-resolution system is: A single-mode fiber-coupled 980nm laser is used as an excitation source to emit 980nm laser light, which is collimated by a first collimating mirror, reflected by a first reflecting mirror for the first time, and enters a first 1 / 2 wave plate and a first polarization beam splitter combination. After the second reflection by a second reflecting mirror, it enters a first 4f system composed of a first lens and a second lens. The light beam passing through the first 4f system is reflected by a first dichroic mirror, a second dichroic mirror, and a 1 / 4 wave plate, and then focused onto the sample by an oil immersion objective, completing the excitation of the sample by 980nm light beam; Meanwhile, a single-mode fiber-coupled 808nm laser is used as an excitation source to emit 808nm laser light, which is collimated by a second collimating mirror, reflected by a third reflecting mirror for the first time, and enters a second 1 / 2 wave plate and a second polarization beam splitter combination. After the second reflection by a fourth reflecting mirror, it enters a second 4f system composed of a third lens and a fourth lens. The light beam passing through the second 4f system is reflected by a fifth reflecting mirror to the first dichroic mirror, and the reflected light is reflected by the first dichroic mirror. The first dichroic mirror couples the 980nm laser beam and the 808nm laser beam, and then the light is reflected by the second dichroic mirror and focused onto the sample by the oil immersion objective; Rotating the flip mirror to enter the light path as a mirror, the fluorescent signal passes through the flip mirror and the first tube lens to focus the spot on the camera, and the camera is adjusted to keep the image plane conjugate with the multimode fiber entrance, and the flip mirror is rotated to not enter the light path; The upconversion sample is subjected to confocal scanning, the fluorescent signal emitted by the upconversion sample is collected through an oil immersion objective, the reflected laser and fluorescent signal pass through a 1 / 4 wave plate, a second dichroic mirror is used to separate the excitation and emission beams, the fluorescent signal is filtered by a filter, and is converged to the multimode fiber entrance through an imaging lens, and the other end of the multimode fiber is connected to a single photon counter, and the single photon counter records the number of photons emitted by the sample to obtain a high-resolution confocal-like multi-photon super-resolution image.

7. The ground-state correlation-based superlinear infrared confocal stimulated- emission depletion super-resolution method of claim 4, wherein, The specific method for calibrating the stimulated radiation light path by using the dual infrared response of the upconversion material is as follows: The angle of the first dichroic mirror is adjusted to couple the 980nm light beam and the 808nm light beam; The flip mirror is rotated to enter the light path as a mirror, the imaging of the reference camera is observed, the same detection site is used as the conjugate calibration point of excitation of the dual light path, the angles of the mirror and the dichroic mirror are adjusted to maximize the photon intensity of the detection site, and the alignment of the stimulated radiation light path is completed.

8. The ground-state correlation-based superlinear infrared confocal stimulated- emission depletion super-resolution method of claim 4, wherein, The specific method for realizing confocal stimulated radiation super-resolution imaging under super-linear effect by using the super-linear infrared confocal stimulated radiation super-resolution system is as follows: 980nm laser is used as the excitation light beam, and 808nm laser is used as the loss light beam, a vortex phase plate is inserted before the second 4f system of the 808nm excitation light path, and the vortex phase plate is used to generate a donut-shaped light beam as the loss light beam of the stimulated radiation loss system; The angle of the first dichroic mirror is adjusted to couple the 980nm light beam and the 808nm light beam; The flip mirror is rotated to enter the light path as a mirror, the imaging of the reference camera is observed, and the point spread functions of the two laser beams are ensured to coincide in the X-Y and Z axes; The flip mirror is rotated to not enter the light path, the fluorescent signal is filtered by a filter, and is converged to the multimode fiber entrance through a second tube lens, the other end of the multimode fiber is connected to a single photon counter, and the single photon counter records the number of photons emitted by the sample to obtain a high-resolution stimulated radiation loss super-resolution image.

Images