Gold nanoparticle fluorescence flicker characteristic regulation and control method and super-resolution imaging system

Abstract

The invention belongs to the technical field of fluorescence imaging, and discloses a gold nanoparticle fluorescence flicker characteristic regulation and control method and a super-resolution imaging system. Comprising a femtosecond laser, a spatial light modulator, a microscopic system, gold nanoparticles and a detection system for receiving fluorescence signals emitted after the gold nanoparticles are excited by femtosecond light emitted by the femtosecond laser. According to the method for regulating and controlling the fluorescence flicker characteristics of the gold nanoparticles and the super-resolution imaging system, the fluorescence flicker characteristics of the gold nanoparticles are regulated and controlled by changing parameters such as the frequency and the amplitude of phase change on the spatial light modulator; the problem that high-order super-resolution optical fluctuation imaging technology processing depends on fluorescent samples with different inherent flicker characteristics is fundamentally solved, and a super-resolution image can be obtained under low-power excitation.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence imaging, and in particular, to a method for regulating the fluorescence blinking characteristics of gold nanoparticles and a super-resolution imaging system. Background Art

[0002] Optical fluorescence microscopes have been widely used in exploring various biological studies. However, due to the limitation of optical diffraction, the spatial resolution of traditional optical microscopes is limited. Therefore, the fine structures within cells cannot be distinguished. To overcome the diffraction barrier, various super-resolution techniques have been introduced. These super-resolution techniques can be classified into two categories: (1) light source modulation, such as stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM); (2) random blinking / fluctuation modulation, such as photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), Bayesian analysis of blinking and bleaching (3B), and super-resolution optical fluctuation imaging (SOFI). Although PALM, STORM, and 3B can achieve higher resolution compared to SOFI, they have certain sacrifices in terms of data processing, temporal resolution, system complexity, and phototoxicity.

[0003] Compared with other super-resolution techniques, SOFI improves spatial resolution based on the analysis of temporal random intensity fluctuations. Therefore, SOFI does not require the use of a complex optical system and can be compatible with different imaging platforms. The signals of these random intensity fluctuations originate from blinking fluorescent markers, such as fluorescent nanoparticles, organic dyes, and some customized fluorescent proteins, which is mainly based on the intrinsic blinking characteristics of the fluorescent signals. However, compared with the specially designed fluorescent markers, the vast majority of common fluorescent markers that can be conjugated to biological samples have relatively stable fluorescent characteristics during image acquisition, that is, they have weak blinking characteristics. This means that it is not sufficient to support the calculation of higher-order cumulants in SOFI processing, resulting in the appearance of artifacts and the loss of signals in the final imaging results. To improve the blinking characteristics of fluorescent markers, several special methods have been used in imaging, such as Förster resonance energy transfer (FRET), increasing the use of imaging buffers, and random speckle illumination light through a disordered medium. However, these methods increase the complexity of sample processing and the system, limiting the widespread use of SOFI. Therefore, it is very crucial to fundamentally solve the dependence of higher-order SOFI processing on fluorescent samples with different intrinsic blinking characteristics. Summary of the Invention

[0004] The object of the present invention is to provide a method for regulating the fluorescence blinking characteristics of gold nanoparticles and a super-resolution imaging system, which can artificially control the fluorescence blinking characteristics of gold nanoparticles, alleviate the dependence of high-order super-resolution optical fluctuation imaging on fluorescent samples with different inherent blinking characteristics; in addition, by utilizing its coherent enhancement advantage, fluorescence imaging can be obtained at low power, reducing phototoxicity; using an 800-nm femtosecond laser with strong penetrability, biological samples with a certain thickness can be measured.

[0005] To achieve the above object, the present invention provides a super-resolution imaging system for gold nanoparticles, comprising a femtosecond laser, a spatial light modulator, a microscopy system, gold nanoparticles, and a detection system for receiving the fluorescence signal emitted by the gold nanoparticles after being excited by the femtosecond light emitted by the femtosecond laser.

[0006] Preferably, the microscopy system includes a dichroic mirror and an objective lens; the detection system includes a fluorescence filter, a lens, an electron-multiplying charge-coupled device, a single-photon detector, and a time-correlated single-photon counting system;

[0007] The single-photon detector in the detection system, in combination with a data acquisition card and a time-correlated single-photon counting system, records the arrival time of fluorescence photons; the electron-multiplying charge-coupled device is used to collect wide-field fluorescence imaging images for super-resolution optical fluctuation imaging.

[0008] Preferably, the femtosecond light emitted by the femtosecond laser passes through a first mirror located on the outgoing light path; after being reflected by the first mirror and passing through a first half-wave plate and a first beam splitter prism on its transmitted light path; after being reflected by the first beam splitter prism, it passes through the spatial light modulator for phase modulation of the femtosecond light and then is reflected; the femtosecond light reflected back by the spatial light modulator passes through the first beam splitter prism and is reflected by the dichroic mirror into the objective lens as pump light; the pump light is focused by the objective lens and irradiates the gold nanoparticles prepared on a transparent glass slide.

[0009] Preferably, the femtosecond light transmitted by the first beam splitter prism sequentially passes through a quarter-wave plate and a movable second mirror; the femtosecond light reflected back by the second mirror passes through the first beam splitter prism and is reflected by the dichroic mirror into the objective lens as detection light; the detection light is focused by the objective lens and irradiates the gold nanoparticles prepared on the transparent glass slide again.

[0010] Preferably, the first mirror, the first beam splitter prism, and the movable second mirror set the entire super-resolution imaging system for gold nanoparticles as an equi-arm Michelson interferometer.

[0011] Preferably, the spatial light modulator regulates the magnitude of the phase difference and its changing frequency between the pump light and the detection light by changing the phase change range and frequency parameters of the femtosecond light after being reflected by the first beam splitter prism, thereby changing the time difference between the pump light and the detection light when irradiating the gold nanoparticles.

[0012] Preferably, based on the energy level structure of gold nanoparticles, when the time difference between the pump light and the probe light reaching the gold nanoparticles is near zero delay, the fluorescence signal of the gold nanoparticles shows interference fringes of coherent enhancement and cancellation to the noise level;

[0013] Therefore, by only changing the time difference between the pump light and the probe light reaching the gold nanoparticles, the fluorescence blinking characteristics of the gold nanoparticles can be regulated.

[0014] Preferably, the center wavelength of the femtosecond light emitted by the femtosecond laser is 800 nm, the pulse width is 15 fs, the second mirror is located on a one-dimensional nano-displacement stage with a movement accuracy of 1 nm; the objective lens is an oil immersion objective lens with a magnification of 100 times and a numerical aperture of 1.49; the transmittance of the fluorescence filter in the wavelength range of 300 nm to 750 nm is 99%, and the transmittance above 750 nm is less than 0.001%;

[0015] The spatial light modulator is applicable to the wavelength range of 420 nm to 1100 nm; among them, the phase modulation depth of the spatial light modulator > 2π, the pixel size is 8 μm, the resolution is 1920×1200, the fill factor > 95%, and the maximum refresh rate is 60 Hz.

[0016] Preferably, the method for fabricating gold nanoparticles includes the following steps:

[0017] First, dilute the gold nanoparticle solution grown by the seed-mediated method with deionized water to obtain a gold nanoparticle dilution with an optical density of 1×10 -2 ;

[0018] Then, ultrasonically oscillate the gold nanoparticle dilution to uniformly disperse the gold nanoparticles;

[0019] Finally, spin-coat on a clean transparent glass slide using a spin coater to form gold nanoparticles attached to the transparent glass slide.

[0020] A method for regulating the fluorescence blinking characteristics of gold nanoparticles, applied to the gold nanoparticle super-resolution imaging system described above, includes the following steps:

[0021] Step S1: Focus the femtosecond light emitted by the femtosecond laser on the gold nanoparticles, change the time when the pump light and the probe light reach the gold nanoparticles by moving the displacement stage, find the zero-delay point, and lock the displacement stage at the zero-delay point;

[0022] Step S2: Change the phase change range and frequency parameters of the laser after reflection by the first beam splitter prism through the spatial light modulator, regulate the magnitude and changing frequency of the phase difference between the pump light and the probe light, so as to change the time difference between the pump light and the probe light irradiating on the gold nanoparticles;

[0023] Step S3: Use a single-photon detector in combination with a data acquisition card and a time-correlated single-photon counting system to record the arrival time of fluorescent photons, and analyze the fluorescence blinking characteristics of gold nanoparticles at different phase difference magnitudes and their changing frequencies between the pump light and the probe light.

[0024] Step S4: An electron-multiplying charge-coupled device collects wide-field fluorescence imaging images of gold nanoparticles under different fluorescence blinking characteristics, and uses super-resolution optical fluctuation imaging technology to process the acquired images to obtain super-resolution imaging results.

[0025] Therefore, the present invention adopts the above-mentioned method for regulating the fluorescence blinking characteristics of gold nanoparticles and a super-resolution imaging system. By changing parameters such as the frequency and amplitude of phase changes on the spatial light modulator, the fluorescence blinking characteristics of gold nanoparticles are regulated, fundamentally solving the problem of the dependence of high-order super-resolution optical fluctuation imaging technology on fluorescent samples with different inherent blinking characteristics, and super-resolution images can be obtained under low-power excitation.

[0026] Next, through the drawings and embodiments, the technical solutions of the present invention will be further described in detail. Brief Description of the Drawings

[0027] Figure 1 It is an experimental device diagram of a gold nanoparticle super-resolution imaging system of the present invention;

[0028] Figure 2 It is the interference envelope near zero delay of the gold nanoparticles used in the present invention;

[0029] Figure 3 It is a schematic diagram of different fluorescence blinking characteristics provided by an example of the present invention; among them, (a) is a schematic diagram of fluorescence blinking characteristics when the phase change frequency of the spatial light modulator is set to 10 Hz; (b) is a schematic diagram of fluorescence blinking characteristics when the phase change frequency of the spatial light modulator is set to 1 Hz.

[0030] Figure 4 is Figure 3 the analysis result diagram of the power-law distribution of the fluorescence trajectory in Figure 3 among them, (a) is the analysis result diagram of the power-law distribution of the fluorescence trajectory in (a) of Figure 3 ; (b) is the analysis result diagram of the power-law distribution of the fluorescence trajectory in (b) of

[0031] Figure 5 It is a confocal imaging schematic diagram provided by an application example of the present invention;

[0032] Figure 6 It is a wide-field imaging schematic diagram provided by an application example of the present invention;

[0033] Figure 7For Figure 5 Schematic diagram of imaging for super-resolution fluorescence fluctuation microscopy method;

[0034] Figure 8 For Figure 6 Schematic diagram of imaging for super-resolution fluorescence fluctuation microscopy method;

[0035] Figure 9 For Figure 5 Normalized intensity distribution curves of wide-field imaging and 2nd - 6th order super-resolution fluorescence fluctuation microscopy along line;

[0036] Figure 10 For Figure 6 Normalized intensity distribution curves of wide-field imaging and 2nd - 6th order super-resolution fluorescence fluctuation microscopy along line.

[0037] Reference numerals

[0038] 1. Femtosecond laser; 2. First reflector; 3. First half-wave plate; 4. First beam splitter prism; 5. Spatial light modulator; 6. Quarter-wave plate; 7. Second reflector; 8. One-dimensional nano-displacement stage; 9. Dichroic mirror; 10. Objective lens; 11. Transparent glass slide; 12. Gold nanoparticles; 13. Fluorescence filter; 14. Lens; 15. Electron-multiplying charge-coupled device; 16. Single-photon detector; 17. Data acquisition system. Detailed implementation manners

[0039] The technical solutions of the present invention will be further described below with reference to the drawings and embodiments.

[0040] Embodiment

[0041] As Figure 1 shown, the present invention provides a gold nanoparticle super-resolution imaging system, including a femtosecond laser 1, a spatial light modulator 5, a microscopy system, gold nanoparticles 12, and a detection system for receiving the fluorescence signal emitted by the gold nanoparticles after being excited by the femtosecond light emitted by the femtosecond laser.

[0042] Among them, the microscopy system includes a dichroic mirror 9 and an objective lens 10. The detection system includes a fluorescence filter 13, a lens 14, an electron-multiplying charge-coupled device 15, a single-photon detector 16, and a time-correlated single-photon counting system 17. The single-photon detector 16 in the detection system, in combination with a data acquisition card and the time-correlated single-photon counting system 17, records the arrival time of fluorescence photons; the electron-multiplying charge-coupled device 15 is used to collect wide-field fluorescence imaging images for super-resolution optical fluctuation imaging (SOFI).

[0043] The femtosecond light emitted by the femtosecond laser 1 passes through the first reflector 2 located on the outgoing light path; after being reflected by the first reflector 2, it passes through the first 1 / 2 wave plate 3 and the first beam splitter prism 4 on its transmission light path; after being reflected by the first beam splitter prism 4, it passes through the spatial light modulator 5 for phase modulation of the femtosecond light and then is reflected; the femtosecond light reflected back by the spatial light modulator 5 is transmitted by the first beam splitter prism 4 and then reflected by the dichroic mirror 9 into the objective lens 10 as the pump light; the pump light is focused by the objective lens 10 and then irradiates the gold nanoparticles 12 prepared on the transparent glass slide 11.

[0044] The femtosecond light transmitted by the first beam splitter prism 4 successively passes through the 1 / 4 wave plate 6 and the movable second reflector 7; the femtosecond light reflected back by the second reflector 7 is reflected by the first beam splitter prism 4 and then reflected by the dichroic mirror 9 into the objective lens 10 as the probe light; the probe light is focused by the objective lens 10 and then irradiates the gold nanoparticles 12 prepared on the transparent glass slide 11 again.

[0045] The first reflector 2, the first beam splitter prism 4 and the movable second reflector 7 set the entire gold nanoparticle super-resolution imaging system as an equi-arm Michelson interferometer; the spatial light modulator 5 regulates the magnitude and the changing frequency of the phase difference between the pump light and the probe light by changing parameters such as the phase change range and frequency of the femtosecond light after being reflected by the first beam splitter prism 4, thereby changing the time difference between the pump light and the probe light when they irradiate the gold nanoparticles 12. Based on the above process, not only is a large-time-scale delay adjustment ensured, but also the time delay between the pump light and the probe light can be finely controlled.

[0046] Due to its unique energy level structure, when the time difference between the pump light and the probe light reaching the gold nanoparticles 12 is near zero delay, the fluorescence signal of the gold nanoparticles 12 shows an almost hundred-fold coherent enhancement and interference fringes that cancel out to the noise level. Thus, it can be seen that the fluorescence blinking characteristics of the gold nanoparticles 12 can be regulated only by changing the time difference between the pump light and the probe light when they reach the gold nanoparticles 12.

[0047] The central wavelength of the femtosecond light emitted by the femtosecond laser 1 is 800 nm, the pulse width is 15 fs, the second reflector 7 is located on the one-dimensional nano-displacement stage 8, and the moving accuracy can reach 1 nm; the objective lens 10 is an oil-immersion objective lens with a magnification of 100 times and a numerical aperture of 1.49; the transmittance of the fluorescence filter 13 between 300 nm and 750 nm is 99%, and the transmittance above 750 nm is less than 0.001%. The spatial light modulator 5 is applicable to the 420 nm - 1100 nm band; among them, the phase modulation depth of the spatial light modulator 5 > 2π, the pixel size is 8 μm, the resolution is 1920×1200, the fill factor > 95%, and the maximum refresh rate is 60 Hz.

[0048] The method for fabricating gold nanoparticles is as follows: First, dilute the gold nanoparticle solution grown by the seed-mediated method with deionized water to obtain a gold nanoparticle diluent with an optical density of 1×10 -2 ; then, perform ultrasonic oscillation on the gold nanoparticle diluent to uniformly disperse the gold nanoparticles; finally, spin-coat them on a cleaned transparent glass slide to form gold nanoparticles attached to the transparent glass slide.

[0049] Based on the above content, the present invention also proposes a method for regulating the fluorescence blinking characteristics of gold nanoparticles, which is applied to the above-mentioned gold nanoparticle super-resolution imaging system, and includes the following steps:

[0050] Step S1: Focus the femtosecond light emitted by the femtosecond laser on the gold nanoparticles, change the arrival time of the pump light and the probe light at the gold nanoparticles by moving the displacement stage, find the zero-delay point, and lock the displacement stage at the zero-delay point.

[0051] Step S2: Change parameters such as the phase change range and frequency of the laser after reflection by the first beam splitter prism through the spatial light modulator, regulate the magnitude of the phase difference between the pump light and the probe light and its changing frequency, so as to change the time difference between the pump light and the probe light irradiating on the gold nanoparticles.

[0052] Step S3: Use a single-photon detector combined with a data acquisition card and a time-correlated single-photon counting system to record the arrival time of fluorescence photons, and analyze the fluorescence blinking characteristics of the gold nanoparticles under different phase difference magnitudes and their changing frequencies between the pump light and the probe light.

[0053] Step S4: The electron-multiplying charge-coupled device collects the wide-field fluorescence imaging images of the gold nanoparticles under different fluorescence blinking characteristics, and uses the super-resolution optical fluctuation imaging (SOFI) technology to process the obtained images to obtain the super-resolution imaging result.

[0054] By moving within a range of 0.06 mm near the zero-delay point at a speed of 0.005 mm / s, the fluorescence interference envelope of the gold nanoparticles can be obtained, as Figure 2 shown. As can be seen from Figure 2 , near the zero-delay point, the fluorescence signal of the gold nanoparticles shows interference fringes with nearly a hundred-fold coherent enhancement and cancellation to the noise level.

[0055] Change parameters such as the phase change range and frequency of the laser after reflection by the first beam splitter prism through the spatial light modulator, regulate the magnitude of the phase difference between the pump light and the probe light and its changing frequency, so as to change the time difference between the pump light and the probe light irradiating on the gold nanoparticles. When the phase change frequencies of the spatial light modulator are set to 10 Hz and 1 Hz respectively, the fluorescence trajectories of the gold nanoparticles are obtained as Figure 3As shown, the analysis results of the power-law distribution of the fluorescence blinking characteristics are as Figure 4 shown, and it can be seen that the fluorescence blinking characteristics have changed.

[0056] When the phase change frequency of the spatial light modulator is set to 10 Hz, confocal imaging is obtained by collecting with an electron multiplying charge coupled device as Figure 5 shown and wide-field imaging images as Figure 6 shown. Processed with the SOFI algorithm, the super-resolution imaging results are obtained, as Figure 7 and Figure 8 shown. Among them, Figure 5 and Figure 7 The size of the whole image is about 1.49 μm; Figure 6 and Figure 8 The size of the whole image is about 4 μm.

[0057] Figure 5 The cross-sectional normalized intensity values of the fluorescence microscopy images along the line and the cross-sectional normalized intensity values of the 2nd to 6th order SOFI images are as Figure 9 shown. Figure 6 The cross-sectional normalized intensity values of the fluorescence microscopy images along the line and the cross-sectional normalized intensity values of the 2nd to 6th order SOFI images are as Figure 10 shown.

[0058] Therefore, the present invention adopts the above-mentioned method for regulating the fluorescence blinking characteristics of gold nanoparticles and the super-resolution imaging system. By changing parameters such as the frequency and amplitude of the phase change on the spatial light modulator, the fluorescence blinking characteristics of gold nanoparticles are regulated, fundamentally solving the problem of the dependence of the high-order super-resolution optical fluctuation imaging technology on fluorescence samples with different inherent blinking characteristics, and super-resolution images can be obtained under low-power excitation.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A gold nanoparticle super-resolution imaging system, characterized in that: It includes a femtosecond laser, a spatial light modulator, a microscopic system, gold nanoparticles, and a detection system that receives the fluorescence signal emitted by the gold nanoparticles after being excited by the femtosecond light emitted by the femtosecond laser.

2. The super-resolution imaging system of gold nanoparticles according to claim 1, wherein: The microscopic system includes a dichroic mirror and an objective lens; the detection system includes a fluorescence filter, a lens, an electron-multiplying charge-coupled device, a single-photon detector, and a time-correlated single-photon counting system; In the detection system, the single-photon detector combines with a data acquisition card and the time-correlated single-photon counting system to record the arrival time of fluorescence photons; the electron-multiplying charge-coupled device is used to collect wide-field fluorescence imaging images for super-resolution optical fluctuation imaging.

3. The super-resolution imaging system of gold nanoparticles according to claim 2, characterized in that: The femtosecond light emitted by the femtosecond laser passes through a first reflecting mirror located on the outgoing light path; it is reflected by the first reflecting mirror and passes through a first half-wave plate and a first beam splitter prism on its transmission light path; after being reflected by the first beam splitter prism, it passes through the spatial light modulator for phase modulation of the femtosecond light and then is reflected; The femtosecond light reflected back by the spatial light modulator is transmitted by the first beam splitter prism and then reflected by the dichroic mirror into the objective lens as pump light; the pump light is focused by the objective lens and irradiates the gold nanoparticles prepared on a transparent glass slide.

4. A gold nanoparticle super-resolution imaging system according to claim 3, characterized in that: The femtosecond light transmitted by the first beam splitter prism sequentially passes through a quarter-wave plate and a movable second reflecting mirror; the femtosecond light reflected back by the second reflecting mirror is reflected by the first beam splitter prism and then reflected by the dichroic mirror into the objective lens as detection light; the detection light is focused by the objective lens and irradiates the gold nanoparticles prepared on the transparent glass slide again.

5. The super-resolution imaging system of gold nanoparticles according to claim 3, characterized in that: The first reflecting mirror, the first beam splitter prism, and the movable second reflecting mirror set the entire gold nanoparticle super-resolution imaging system as an equal-arm Michelson interferometer.

6. The super-resolution imaging system of gold nanoparticles according to claim 4, wherein: The spatial light modulator regulates the magnitude of the phase difference between the pump light and the detection light and its changing frequency by changing the phase change range and frequency parameters of the femtosecond light after being reflected by the first beam splitter prism, thereby changing the time difference between the pump light and the detection light when they irradiate the gold nanoparticles.

7. The super-resolution imaging system of gold nanoparticles according to claim 6, characterized in that: Based on the energy level structure of the gold nanoparticles, when the time difference between the pump light and the detection light reaching the gold nanoparticles is near zero delay, the fluorescence signal of the gold nanoparticles shows interference fringes of coherent enhancement and cancellation to the noise level; Therefore, by only changing the time difference between the pump light and the detection light reaching the gold nanoparticles, the fluorescence blinking characteristics of the gold nanoparticles can be regulated.

8. A gold nanoparticle super-resolution imaging system according to claim 3, wherein: The central wavelength of the femtosecond light emitted by the femtosecond laser is 800 nm, the pulse width is 15 fs, the second reflecting mirror is located on a one-dimensional nanometer displacement stage with a movement accuracy of 1 nm; the objective lens is an oil immersion objective lens with a magnification of 100 times and a numerical aperture of 1.49; the transmittance of the fluorescence filter in the wavelength range of 300 nm to 750 nm is 99%, and the transmittance above the 750 nm wavelength range is less than 0.001%; The spatial light modulator is applicable to the wavelength range of 420 nm to 1100 nm; Among them, the phase modulation depth of the spatial light modulator > 2π, the pixel size is 8 μm, the resolution is 1920×1200, the fill factor > 95%, and the maximum refresh rate is 60 Hz.

9. A gold nanoparticle super-resolution imaging system according to claim 1, wherein The manufacturing method of the gold nanoparticles includes the following steps: First, the gold nanoparticle solution grown by the seed-mediated method is diluted with deionized water to obtain a gold nanoparticle dilution with an optical density of 1×10 -2 ; Then, the gold nanoparticle dilution is subjected to ultrasonic oscillation to uniformly disperse the gold nanoparticles; Finally, spin coating is used to form gold nanoparticles attached to a cleaned transparent glass slide on the transparent glass slide.

10. A method for regulating the fluorescence blinking characteristics of gold nanoparticles, which is applied to a gold nanoparticle super-resolution imaging system according to any one of claims 1-9, characterized in that, It includes the following steps: Step S1: Focus the femtosecond light emitted by the femtosecond laser on the gold nanoparticles. By moving the displacement stage, change the time when the pump light and the probe light reach the gold nanoparticles. Find the zero-delay point and lock the displacement stage at the zero-delay point. Step S2: Change the phase change range and frequency parameters of the laser after reflection by the first beam splitter prism through the spatial light modulator, and regulate the magnitude of the phase difference between the pump light and the probe light and its changing frequency, so as to change the time difference when the pump light and the probe light irradiate the gold nanoparticles. Step S3: Use a single-photon detector combined with a data acquisition card and a time-correlated single-photon counting system to record the arrival time of fluorescent photons, and analyze the fluorescence scintillation characteristics of the gold nanoparticles under different phase difference magnitudes and their changing frequencies between the pump light and the probe light. Step S4: The electron-multiplying charge-coupled device collects the wide-field fluorescence imaging images of the gold nanoparticles under different fluorescence scintillation characteristics, and uses super-resolution optical fluctuation imaging technology to process the acquired images to obtain the super-resolution imaging results.

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