Multiphoton up-conversion super-resolution microscopic imaging method based on microsphere super-lens enhancement

Abstract

The invention discloses a multiphoton up-conversion super-resolution microscopic imaging method based on microsphere super-lens enhancement, which utilizes the super-resolution advantage of a microsphere super-lens and the nonlinear response advantage of a lanthanide doped rare earth up-conversion nano material to reduce the saturation excitation power of the rare earth nano material. And super-resolution imaging under the condition of a low-magnification and low-numerical-aperture objective lens is realized. An example verifies that under the condition that system complexity is not increased, excitation powers of 455 nm and 808 nm radiation fluorescence of an up-conversion nano material are respectively reduced by 15.40 times and 23.44 times, fluorescence imaging resolutions of 455 nm and 808 nm can be respectively improved by 3.14 times and 3.6 times under a 10x 0.25 NA objective lens, and corresponding transverse resolution limits can respectively reach 358.20 nm and 480.02 nm, so that the fluorescence imaging resolution of the up-conversion nano material can be improved by 3.14 times and 3.6 times under a 10x 0.25 NA objective lens. The method has the advantages of simple imaging system, low cost and good imaging effect, and is of great significance to biological application research.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical super-resolution microscopy, and specifically relates to a multi-photon up-conversion super-resolution microscopy method based on microsphere superlens enhancement. Background Art

[0002] In recent years, super-resolution microscopy techniques that break through the optical diffraction limit have made significant progress, especially the rapid development of single-molecule localization microscopy, stimulated emission depletion microscopy, and structured illumination microscopy. These technologies have successfully broken through the resolution limitations of traditional optical microscopes, allowing researchers to observe biological molecules and internal structures of cells with nanometer-level precision. Although some fluorescence super-resolution imaging technologies have been commercialized, their high prices and complex equipment limit their widespread application. Current technologies still face many challenges, including how to effectively reduce the cost of use, simplify the optical complexity of the system, and how to achieve more general super-resolution imaging of living cells to meet the needs of scientific research and clinical applications.

[0003] On the other hand, as an emerging tool in super-resolution microscopy, upconversion nanomaterials have advantages such as good photostability and low background signals, but their saturation excitation power is high, which limits their application in in vivo imaging to a certain extent. High saturation excitation power may not only cause photodamage to samples, especially living cells, but also increase the demand for laser equipment, thereby increasing the cost of the overall imaging system.

[0004] Therefore, in the field of multiphoton microscopy, there is still an urgent need for an imaging method that can achieve both high resolution and low saturation excitation power to meet the needs of practical applications. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide a multi-photon up-conversion super-resolution microscopic imaging method and device based on microsphere super-lens enhancement.

[0006] In order to achieve the above object, the present invention provides the following technical solution: a multi-photon up-conversion super-resolution microscopic imaging method based on microsphere superlens enhancement, the specific steps are as follows:

[0007] Step 1: Prepare microsphere superlens upconversion sample;

[0008] Step 2: Construct a multimodal multiphoton super-resolution optical microscopy system;

[0009] Step 3: Use a multimodal multiphoton super-resolution optical microscopy system to achieve super-resolution imaging under bright field illumination;

[0010] Step 4: Use a multimodal multiphoton super-resolution optical microscopy system to achieve two-color fluorescence super-resolution imaging under wide-field illumination.

[0011] Preferably, the specific steps of step 1 for preparing a microsphere superlens upconversion sample are as follows:

[0012] Step 1: preparing lanthanide-doped rare earth upconversion nanomaterials;

[0013] Step 2: Evenly disperse and fill the lanthanide-doped rare earth upconversion nanomaterials in the stripes of the DVD disc from which the surface covering has been removed;

[0014] Step 3: drop the microspheres dispersed in anhydrous ethanol onto the surface of the optical disc stripes, and fix the position of the microspheres by the surface tension of anhydrous ethanol;

[0015] Step 4: After the ethanol has completely evaporated, an oil medium with a refractive index of 1.5 is added to the surface of the optical disc stripes and sealed to complete the production of the microsphere superlens upconversion sample.

[0016] Preferably, the multimodal multiphoton super-resolution optical microscopy system comprises:

[0017] White light LED light source 1, switchable 50 / 50 beam splitter 2, near-infrared laser light source 3, half wave plate 4, polarization beam splitter cube 5, narrow bandpass filter 6, excitation light path beam expansion system 7, long reflection and short transmission dichroic mirror 8, focusing lens 9, objective lens 10, microsphere superlens upconversion sample 11, reflector 12, emission light path beam expansion system 13, filter wheel 14, imaging lens 15, low-pass filter 16, photodetector 17;

[0018] The multimodal multiphoton super-resolution optical microscopy system includes a bright field imaging mode and a fluorescence imaging mode;

[0019] Bright field imaging mode: white light LED light source 1 is coupled to objective lens 10 through switchable 50 / 50 beam splitter 2, and irradiated to microsphere superlens up-conversion sample 11 through objective lens 10. The scattered light after white light passes through sample is collected by objective lens 10, passes through switchable 50 / 50 beam splitter 2, dichroic mirror 8, reflector 12, and then focuses to the image plane of photodetector 17 through emission light path beam expansion system 13, imaging lens 15, low-pass filter 16;

[0020] In the fluorescence imaging mode, a 980nm near-infrared laser 3 is used as a fluorescence excitation light source. The fluorescence excitation light source passes through a rotatable half-wave plate 4, the light intensity distribution after the polarization beam splitter cube 5, a narrow bandpass filter 6, and a 4f beam expansion system 7. It is reflected by a long reflection short transmission dichroic mirror 8, and the light beam is evenly expanded to fill the incident light hole of the objective lens 10 through a focusing lens 9, and irradiates the microsphere superlens up-conversion sample 11. The fluorescence emitted by the sample passes through a focusing lens 9, the long reflection short transmission dichroic mirror 8, a reflector 12, and then passes through a group of 4f optical systems 13 and a filter wheel 14. After that, the fluorescence is focused onto the image plane of the photodetector through an imaging lens 15 to ensure that the imaging area fills the entire sensing surface of the photodetector 17. At the same time, a low-pass filter 16 is placed in front of the photodetector to filter out light with a wavelength greater than 875nm. The filter wheel 14 can select different bands for multi-band acquisition of fluorescence signals.

[0021] Preferably, in step 3, the specific steps of realizing super-resolution imaging under bright field illumination using a multimodal multiphoton super-resolution optical microscopy system are as follows:

[0022] Step 3.1: Switch the working mode of the system to the bright field imaging mode by switching the 50 / 50 beam splitter in the multimodal multiphoton super-resolution optical microscopy system, and switch the filter wheel position to empty;

[0023] Step 3.2: Place the microsphere metalens upconversion sample upside down just above the objective lens;

[0024] Step 3.3: Adjust the position of the microsphere superlens upconversion sample to find the microsphere distribution area;

[0025] Step 3.4: Adjust the position of the microsphere upconversion sample until clear fringes appear on the image plane of the photodetector.

[0026] Preferably, the specific steps of step 4 using a multimodal multiphoton super-resolution optical microscopy system to achieve dual-color fluorescence super-resolution imaging under wide-field illumination are as follows:

[0027] Step 4.1: Keep the microsphere upconversion sample in place and switch the system working mode to the fluorescence imaging mode by switching the 50 / 50 beam splitter in the multimodal multiphoton super-resolution optical microscopy system;

[0028] Step 4.2: Switch the filter wheel to the bandpass filters with a central wavelength of 455nm and 808nm and a bandwidth of 50nm, respectively, and a clear fringe image is formed on the image plane of the photodetector after each switch;

[0029] Step 4.3: Collect fluorescence images at different powers at the same position by adjusting the rotating half wave plate and controlling the laser power conditions;

[0030] Step 4.4: Summarize the series of images collected in step 3 to obtain the power dependence curves of fluorescence in different bands, and find the threshold point as the optimal fluorescence imaging power.

[0031] Compared with the prior art, the present invention has the following significant advantages: the present invention effectively reduces the saturation excitation power of rare earth up-conversion nanomaterials, and it has been verified that the excitation power can be reduced by 23.44 times. Under the conditions of low-magnification and low numerical aperture objective lenses, the present invention can break through the diffraction limit and significantly improve the imaging resolution by 3.6 times, achieving a lateral resolution of 358.20nm. The present invention reduces the use cost of objective lenses and lasers without increasing the complexity of the imaging system, and at the same time has excellent imaging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0033] Figure 1 Schematic diagram of the microsphere superlens upconversion sample structure.

[0034] Figure 2 Schematic diagram of the structure of the multimodal multiphoton super-resolution optical microscopy device.

[0035] Figure 3 To achieve super-resolution imaging results under bright field illumination using a multimodal multiphoton super-resolution optical microscopy system.

[0036] Figure 4 For ordinary samples without microspheres, two-color fluorescence super-resolution imaging under wide-field illumination is achieved in a multimodal multiphoton super-resolution optical microscopy system.

[0037] Figure 5 The microsphere super-lens upconversion sample is used to achieve two-color fluorescence super-resolution imaging under wide-field illumination in a multi-modal multi-photon super-resolution optical microscopy system.

[0038] Figure 6 The power dependence curves of 455nm fluorescence and 808nm fluorescence measured with and without microspheres are shown.

[0039] Attached Figure 1 Marking Description:

[0040] 1- white light LED light source; 2- switchable 50 / 50 beam splitter; 3- near-infrared laser light source; 4- half wave plate; 5- polarization beam splitter cube; 6- narrow bandpass filter; 7- excitation light path beam expansion system; 8- long reflection and short transmission dichroic mirror; 9- focusing lens; 10- objective lens; 11- microsphere superlens upconversion sample; 12- reflector; 13- emission light path beam expansion system; 14- filter wheel; 15- imaging lens; 16- low-pass filter; 17- photodetector. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0042] The present invention provides Figure 1-6 The multi-photon up-conversion super-resolution microscopy imaging method based on microsphere superlens enhancement is shown, and its operation steps are as follows:

[0043] Step 1: Prepare microsphere superlens upconversion samples such as Figure 1 As shown, the specific steps are as follows:

[0044] Step 1.1: Preparation of lanthanide-doped rare earth upconversion nanomaterials. The present invention uses 4% Tm 3+ and 20% Yb 3+ Co-doped NaYF4:Yb,Tm@NaYF4 core-shell nanoparticles.

[0045] Step 1.2: Disperse the nanomaterial in cyclohexane and dilute it to 0.15mg / ml for later use. Prepare a DVD disc with the surface cover removed, about 1cm2 in size, and drop 20ul of the dispersed nanomaterial on the surface of the prepared DVD disc, shake it in the air, and then rinse it vertically with cyclohexane, and drop 20ul of the nanomaterial again. Repeat the above process 3-5 times to ensure that the nanomaterial is evenly filled in the stripe grooves of the DVD.

[0046] Step 1.3: Add the microspheres dispersed in anhydrous ethanol to the surface of the optical disc stripes prepared in step 1.2 where the cyclohexane is completely volatilized, and fix the position of the microspheres by the surface tension of anhydrous ethanol. The microsphere lenses used in this step are usually high refractive index dielectric materials, such as polystyrene n~1.59, fused quartz n~1.46, titanium dioxide n~2.1, barium titanate n~1.9-2.2, etc. The diameter of the microsphere lenses is mostly between a few microns and tens of microns, and a few microsphere lenses can reach hundreds of microns in diameter. In order to take into account both imaging resolution and imaging field of view, microsphere lenses of different materials and sizes need to be selected accordingly. The microspheres used in the invention example are barium titanate n~1.9, with a diameter of about 20um.

[0047] Step 1.4: After the anhydrous ethanol on the surface of the sample in step 1.3 has completely evaporated, add a high refractive index oil medium of n to 1.5, cover the cover glass, and use a transparent sealing medium to fix the position of the cover glass around to ensure complete sealing.

[0048] Step 2: Construct a multimodal multiphoton super-resolution optical microscopy system such as Figure 2 As shown, the specific steps are as follows:

[0049] The multimodal multiphoton super-resolution optical microscopy system includes a white light LED light source 1, a switchable 50 / 50 beam splitter 2, a near-infrared laser light source 3, a half-wave plate 4, a polarization beam splitter cube 5, a narrow bandpass filter 6, an excitation light path beam expansion system 7, a long reflection and short transmission dichroic mirror 8, a focusing lens 9, an objective lens 10, a microsphere superlens up-conversion sample 11, a reflector 12, an emission light path beam expansion system 13, a filter wheel 14, an imaging lens 15, a low-pass filter 16, and a photodetector 17.

[0050] The white light LED light source 1 is used as a bright field illumination light source, and is coupled to the optical path through a switchable 50 / 50 beam splitter 2, and can flexibly switch the imaging mode to a bright field imaging mode or a fluorescence imaging mode to meet different imaging requirements. For the bright field imaging mode, the white light LED light source 1 is coupled to the optical path through a switchable 50 / 50 beam splitter 2, and then irradiated to the microsphere superlens conversion sample 11 through an objective lens 10. The scattered light after the white light passes through the sample is collected by the objective lens 10, passes through the switchable 50 / 50 beam splitter 2 and the dichroic mirror 8, and then passes through the emission optical path beam expansion system 13, and is focused on the image plane of the photodetector 17 through the imaging lens 15 (the filter wheel 14 should be guaranteed to be "empty" at this time), ensuring that the imaging area occupies the entire photodetector sensing surface.

[0051] For the fluorescence imaging mode, a 980nm near-infrared laser 3 is used as the fluorescence excitation light source. The laser first passes through a rotatable half-wave plate 4 to change the polarization state of the laser, thereby adjusting the light intensity distribution after passing through the polarization beam splitter cube 5. After that, the laser passes through a narrow bandpass filter 6 to ensure its monochromaticity, and then passes through a 4f beam expansion system 7 to evenly expand the light beam to fill the incident light hole of the objective lens 10. The laser is reflected by the long reflection short transmission dichroic mirror 8 and irradiated to the microsphere superlens conversion sample 11, while the fluorescence emitted by the sample passes through the long reflection short transmission dichroic mirror 8. In order to focus the laser on the sample, the light passes through a focusing lens 9 before entering the objective lens 10 to ensure that divergent light is output after entering the objective lens 10, thereby realizing wide-field laser illumination. After the fluorescence emitted by the sample passes through the long reflection short transmission dichroic mirror 8, it passes through a reflector 12 and then passes through a group of 4f optical systems 13 to further amplify the fluorescence signal. The filter wheel 14 can select different bands for multi-band collection of fluorescence signals, and then the fluorescence is focused onto the image plane of the photodetector through the imaging lens 15, ensuring that the imaging area occupies the entire sensing surface of the photodetector 17. At the same time, a low-pass filter 16 is placed in front of the photodetector to filter out light with a wavelength greater than 875nm to prevent the photodetector from being damaged by strong lasers and ensure the stable operation of the system.

[0052] In the process of image acquisition, the external control part is involved. The control elements of this part include laser controller and photodetector. The control program is written through LabVIEW. The function is to record the image detected on the photodetector every time the laser power increases by a specific step length. The laser start and end power and step length can be set. The exposure time, gain and other related parameters of the photodetector are all set on the corresponding control software of the detector. The external control program only controls whether to collect images.

[0053] Step 3: Use a multimodal multiphoton super-resolution optical microscopy system to achieve super-resolution imaging under bright field illumination. The specific method is as follows:

[0054] Step 3.1: Switch the system working mode to bright field imaging mode by switching the 50 / 50 beam splitter in the multimodal multiphoton super-resolution optical microscopy system, and switch the filter wheel position to "empty".

[0055] Step 3.2: Place the microsphere metalens upconversion sample upside down just above the objective lens.

[0056] Step 3.3: Adjust the position of the microsphere superlens upconversion sample to find the microsphere distribution area.

[0057] Step 3.4: Adjust the position of the microsphere upconversion sample until clear stripes appear on the image plane of the photodetector. It is worth noting that the image plane position should be above the microsphere of the inverted sample.

[0058] Step 4: Use a multimodal multiphoton super-resolution optical microscopy system to achieve dual-color fluorescence super-resolution imaging under wide-field illumination. The specific steps are as follows:

[0059] Step 4.1: Keep the microsphere upconversion sample in place and switch the system working mode to the fluorescence imaging mode by switching the 50 / 50 beam splitter in the multimodal multiphoton super-resolution optical microscopy system.

[0060] Step 4.2: Switch the filter wheel to the bandpass filters with a central wavelength of 455 nm and 808 nm and a bandwidth of 50 nm, respectively. After each switch, a clear fringe image is formed on the image plane of the photodetector.

[0061] Step 4.3: Taking 455nm fluorescence image acquisition as an example, use an external hardware control program to acquire fluorescence images at different powers at the same position. Different laser powers can be finely adjusted by adjusting the rotating half wave plate and controlling the laser power. The 808nm fluorescence image acquisition method is the same as the 455nm fluorescence image acquisition method.

[0062] Step 4.4: Summarize the series of images collected in step 3 to obtain the power dependence curves of fluorescence in different bands, and find the threshold point as the optimal fluorescence imaging power.

[0063] Example 1

[0064] In order to verify the effectiveness of the present invention, a multimodal multiphoton super-resolution optical microscopy device was built. The super-resolution imaging results under bright field illumination mode are shown in the figure below. Figure 3 As shown. Figure 3 (a) is the SEM result of DVD stripes. Figure 3 (b) Microsphere and super-resolution fringe imaging results under bright field illumination mode. Figure 3 (c) is a distance analysis diagram of the super-resolution stripes shown in (b) in bright field mode. In bright field imaging mode, when using a 10x 0.25NA objective lens for imaging, the imaging resolution under white light is about 1342nm according to the diffraction limit calculation. The DVD disc stripes with a spacing of 740nm cannot be distinguished. After adding a microsphere super lens to the sample, the DVD disc stripes with a spacing of 740nm can be clearly distinguished.

[0065] Example 2

[0066] The results of dual-color fluorescence imaging without microspheres in fluorescence imaging mode are as follows Figure 4 As shown. Among them, Figure 4 (a) is the overall fluorescence imaging results of dispersed upconversion nanomaterials at 455nm and 808nm at different powers. Figure 4(bd) are the imaging results of dispersed upconversion nanomaterials in the 455nm fluorescence band at different powers. Figure 4 (eg) is the imaging result of the 808nm fluorescence band of dispersed upconversion nanomaterials at different powers. Figure 4 (h) is a curve analysis of the change of the point spread function of the 455nm fluorescence band with power in wide-field illumination mode. (i) is a curve analysis of the change of the point spread function of the 808nm fluorescence band with power in wide-field illumination mode. When using a 10x 0.25NA objective lens for imaging, the highest lateral imaging resolution that can be achieved for 455nm and 808nm fluorescence imaging is 1110.2nm and 1971.52nm respectively according to the diffraction limit calculation, and the actual measured results are 1125.75nm and 1731.27nm respectively.

[0067] Example 3

[0068] The results of super-resolution dual-color fluorescence imaging of microspheres in fluorescence imaging mode are as follows Figure 5 As shown. Among them, Figure 5 (a) Overall fluorescence imaging results of the microsphere upconversion sample at 455nm and 808nm at different powers. Figure 5 (bd) are the imaging results of the microsphere upconversion sample in the 455nm fluorescence band at different powers. Figure 5 (eg) is the imaging result of the microsphere upconversion sample in the 808nm fluorescence band at different powers. Figure 5 (h) is a curve analysis of the change of the point spread function of the 455nm fluorescence band of the microsphere upconversion sample under wide-field illumination mode with the power. (i) is a curve analysis of the change of the point spread function of the 808nm fluorescence band of the microsphere upconversion sample under wide-field illumination mode with the power. At this time, according to the calculation that the spacing between two adjacent stripes of the DVD disc is 740nm, it can be concluded that the imaging resolution of 455nm and 808nm fluorescence can be increased to 358.20nm and 480.02nm, respectively, which is increased by 3.14 times and 3.6 times respectively.

[0069] Example 4

[0070] The power dependence curves of 455nm fluorescence and 808nm fluorescence with and without microspheres are shown in the figure. Figure 6 According to the measured curve, it can be seen that the excitation power requirement of 455nm fluorescence is reduced by 15.4 times under the action of microspheres, and the excitation power requirement of 808nm fluorescence is reduced by 23.44 times under the action of microspheres.

Claims

1. A multi-photon up-conversion super-resolution microscopy imaging method based on microsphere superlens enhancement, characterized in that: The specific steps are as follows: Step 1: Prepare microsphere superlens upconversion sample; Step 2: Construct a multimodal multiphoton super-resolution optical microscopy system; Step 3: Use a multimodal multiphoton super-resolution optical microscopy system to achieve super-resolution imaging under bright field illumination; Step 4: Use a multimodal multiphoton super-resolution optical microscopy system to achieve two-color fluorescence super-resolution imaging under wide-field illumination.

2. The multi-photon up-conversion super-resolution microscopy imaging method based on microsphere superlens enhancement as claimed in claim 1, characterized in that: Step 1 The specific steps for preparing the microsphere superlens upconversion sample are as follows: Step 1: preparing lanthanide-doped rare earth upconversion nanomaterials; Step 2: Evenly disperse and fill the lanthanide-doped rare earth upconversion nanomaterials in the stripes of the DVD disc from which the surface covering has been removed; Step 3: drop the microspheres dispersed in anhydrous ethanol onto the surface of the optical disc stripes, and fix the position of the microspheres by the surface tension of anhydrous ethanol; Step 4: After the ethanol has completely evaporated, an oil medium with a refractive index of 1.5 is added to the surface of the optical disc stripes and sealed to complete the production of the microsphere superlens upconversion sample.

3. The multi-photon up-conversion super-resolution microscopy imaging method based on microsphere superlens enhancement as claimed in claim 1, characterized in that: The multi-modal multi-photon super-resolution optical microscopy system comprises: White light LED light source 1, switchable 50 / 50 beam splitter 2, near-infrared laser light source 3, half wave plate 4, polarization beam splitter cube 5, narrow bandpass filter 6, excitation light path beam expansion system 7, long reflection and short transmission dichroic mirror 8, focusing lens 9, objective lens 10, microsphere superlens upconversion sample 11, reflector 12, emission light path beam expansion system 13, filter wheel 14, imaging lens 15, low-pass filter 16, photodetector 17; The multimodal multiphoton super-resolution optical microscopy system includes a bright field imaging mode and a fluorescence imaging mode; Bright field imaging mode: white light LED light source 1 is coupled to objective lens 10 through switchable 50 / 50 beam splitter 2, and irradiated to microsphere superlens up-conversion sample 11 through objective lens 10. The scattered light after white light passes through sample is collected by objective lens 10, passes through switchable 50 / 50 beam splitter 2, dichroic mirror 8, reflector 12, and then focuses to the image plane of photodetector 17 through emission light path beam expansion system 13, imaging lens 15, low-pass filter 16; In the fluorescence imaging mode, a 980nm near-infrared laser 3 is used as a fluorescence excitation light source. The fluorescence excitation light source passes through a rotatable half-wave plate 4, the light intensity distribution after the polarization beam splitter cube 5, a narrow bandpass filter 6, and a 4f beam expansion system 7. It is reflected by a long reflection short transmission dichroic mirror 8, and the light beam is evenly expanded to fill the incident light hole of the objective lens 10 through a focusing lens 9, and irradiates the microsphere superlens up-conversion sample 11. The fluorescence emitted by the sample passes through a focusing lens 9, the long reflection short transmission dichroic mirror 8, a reflector 12, and then passes through a group of 4f optical systems 13 and a filter wheel 14. After that, the fluorescence is focused onto the image plane of the photodetector through an imaging lens 15 to ensure that the imaging area fills the entire sensing surface of the photodetector 17. At the same time, a low-pass filter 16 is placed in front of the photodetector to filter out light with a wavelength greater than 875nm. The filter wheel 14 can select different bands for multi-band acquisition of fluorescence signals.

4. The multi-photon up-conversion super-resolution microscopy imaging method based on microsphere superlens enhancement as claimed in claim 3, characterized in that: In step 3, the specific steps of realizing super-resolution imaging under bright field illumination using a multimodal multiphoton super-resolution optical microscopy system are as follows: Step 3.1: Switch the working mode of the system to the bright field imaging mode by switching the 50 / 50 beam splitter in the multimodal multiphoton super-resolution optical microscopy system, and switch the filter wheel position to empty; Step 3.2: Place the microsphere metalens upconversion sample upside down just above the objective lens; Step 3.3: Adjust the position of the microsphere superlens upconversion sample to find the microsphere distribution area; Step 3.4: Adjust the position of the microsphere upconversion sample until clear fringes appear on the image plane of the photodetector.

5. The multi-photon up-conversion super-resolution microscopy imaging method based on microsphere superlens enhancement as claimed in claim 4, characterized in that: Step 4 The specific steps of realizing dual-color fluorescence super-resolution imaging under wide-field illumination using a multimodal multiphoton super-resolution optical microscopy system are as follows: Step 4.1: Keep the microsphere upconversion sample in place and switch the system working mode to the fluorescence imaging mode by switching the 50 / 50 beam splitter in the multimodal multiphoton super-resolution optical microscopy system; Step 4.2: Switch the filter wheel to the bandpass filters with a central wavelength of 455nm and 808nm and a bandwidth of 50nm, respectively, and a clear fringe image is formed on the image plane of the photodetector after each switch; Step 4.3: Collect fluorescence images at different powers at the same position by adjusting the rotating half wave plate and controlling the laser power conditions; Step 4.4: Summarize the series of images collected in step 3 to obtain the power dependence curves of fluorescence in different bands, and find the threshold point as the optimal fluorescence imaging power.

Images