Photo-thermal modulation differential microscopic imaging method based on double excitation light excitation

Abstract

The invention discloses a photo-thermal modulation differential microscopic imaging method based on double excitation light excitation, and relates to a photo-thermal modulation differential microscopic imaging method. The invention aims to solve the problem that the existing photo-thermal microscopic imaging technology is limited by the diffraction limit and cannot break through the minimum resolution limit. The method comprises the following steps: step 1, determining a sample piece to be detected, and placing the sample piece in a photo-thermal modulation differential microscopic imaging system; step 2, starting a photo-thermal modulation differential microscopic imaging system; 3, starting an exciting light laser power supply, a probe light laser power supply and a refrigerator; step 4, setting detection parameters in photo-thermal modulation differential microscopic imaging system software in the computer; 5, gold nanoparticles are selected as imaging objects, and multiple times of detection are carried out; step 6, adopting a multi-arm carbon nanotube as a sample, and carrying out multiple times of detection; and 7, closing the equipment after the experiment is finished for 5 minutes. The invention belongs to the technical field of photo-thermal science and detection signal processing.

Description

Technical Field

[0001] The present invention relates to a photothermal modulation differential microscopy imaging method, belonging to the technical field of photothermal science and detection signal processing. Background Art

[0002] Optical microscopy imaging technology plays an important role in the development of microscopy and has become an indispensable important tool in other fields such as materials science, medicine, optics, and energy. However, optical microscopy imaging technology has always been limited by the diffraction effect of light, resulting in the inability to directly observe structures below 200 nm by optical microscopy methods; traditional microscopy imaging methods have problems such as complex sample preparation, weak fluorescence signals, or large background signal interference. In recent decades, with the further improvement of related optical technologies and material research, more and more super-resolution microscopy imaging technologies that break through the diffraction limit have been proposed. The 2014 Nobel Prize in Chemistry was awarded to this field, further promoting the development of super-resolution microscopy technology. Therefore, for the microscopic imaging detection of nanoscale particles, it is very important to find a microscopy imaging technology that can achieve stable imaging and reduce background signal interference. Photothermal heterodyne microscopy imaging technology is a new type of far-field microscopy imaging technology. This technology utilizes the photothermal properties of objects. When a probe beam passes through the sample material, optical parameter changes occur. By detecting the optical parameter changes of the probe beam, the characteristic information of the material to be measured can be obtained. It has the advantages of being less affected by background scattering, less affected by the size change of the object to be measured, and can realize non-fluorescent object microscopy imaging.

[0003] In existing research, Li Yiming et al. (Patent No.: CN202110390429.6) disclosed a super-resolution microscopy system. The system includes an excitation light source generation module, an imaging module, a focal plane locking module, and a control and data acquisition module. By controlling and adjusting the optical elements in the excitation light source generation module, the imaging module, and the focal plane locking module through the control and data acquisition module, and collecting the fluorescence data obtained by the imaging module for data analysis, the system realizes a single-molecule localization microscopy technology with high compactness, high automation, adjustable and controllable, and improves the precision and accuracy; Yang Qing et al. (Patent No.: CN202411133674.9) disclosed a fast frequency-shifting super-resolution microscopy method based on annular illumination, collecting a single wide-field image when the sample is illuminated normally, illuminating obliquely and collecting multiple low-resolution images corresponding to the mixed frequency-shifted single images, and realizing fast frequency-shifting super-resolution microscopy imaging by passing the single wide-field image and multiple low-resolution images through a super-resolution imaging model constructed based on deep learning to obtain a super-resolution image, improving the imaging speed and promising to be applied to video-level super-resolution microscopy imaging; Wang Shufeng et al. (Patent No.: CN202110540951.8) disclosed a super-resolution microscopy system and imaging method based on two-photon nonlinear effects. The present invention utilizes the threshold of two-photon nonlinear effects to naturally form an excitation probe exceeding the diffraction limit in space; using a spatial light phase modulator to control the pulse width of the laser to dynamically adjust the scale of the excitation probe at the center of the light spot; the present invention overcomes some common disadvantages in existing super-resolution imaging technologies, and the peak power of the excitation light is greatly reduced after passing through the shaping system, so it will not cause optical damage or photobleaching to the sample, and is suitable for long-time exposure imaging and live cell imaging; Yang Shuming et al. (Patent No.: CN201510292588.7) disclosed a super-resolution confocal microscopy device and method. The movement of the object to be measured and the reflective microscope objective lens in the device are respectively completed by a three-dimensional PZT and a one-dimensional PZT to realize tomographic imaging of the object. The device comprehensively uses a superoscillatory zone plate and a reflective microscope objective lens to realize the convergence of the scanning beam, avoiding the alignment error caused by using multiple lenses in the existing confocal imaging device, thus significantly improving the imaging effect of the confocal imaging device. The present invention does not require a lens assembly, the structure of the device is simple, and the performance is stable, and can be used for super-resolution confocal imaging of micro-devices; in order to make full use of the advantages of differential microscopy in improving the lateral resolution and the characteristics of the material thermal lens effect to realize the three-dimensional precise detection of the defect size / morphology, the present invention relates to a photo-thermal modulation differential microscopy imaging method based on dual-excitation light excitation. This method is based on the vector diffraction theory, heat transfer theory, and electromagnetic field theory, and can effectively improve the system resolution (the FWHM of the modulated differential light source gradually decreases from the initial 654 nm to 192 nm), and perform super-resolution photo-thermal microscopy imaging detection on the defects in the shallow surface layer of metal materials and polymer polymers. Summary of the Invention

[0004] In order to solve the problem that the current photothermal microscopy technology is limited by the diffraction limit and cannot break through the minimum resolution limit, a photothermal modulation differential microscopy method based on dual-excitation light excitation is proposed.

[0005] The technical solution adopted by the present invention to solve the above problems is as follows: The steps of the present invention include:

[0006] Step 1: Identify the sample to be detected and place the sample in the photothermal modulation differential microscopy system;

[0007] Step 2: Turn on the photothermal modulation differential microscopy system;

[0008] Step 3: Turn on the power supplies of the excitation light, detection light lasers and the cooler;

[0009] Step 4: Set the detection parameters in the software of the photothermal modulation differential microscopy system in the computer;

[0010] Step 5: Select gold nanoparticles as the imaging object, adopt the form of a glass slide sample, conduct multiple detections, the computer reads the signals at each point from the data acquisition card, obtains the detection result image after processing, summarizes the detection results, and obtains the photothermal microscopy image of the nanoparticle cluster;

[0011] Step 6: Use multi-walled carbon nanotubes as the sample, adopt the form of a glass slide sample, conduct multiple detections, the computer reads the signals at each point from the data acquisition card, obtains the detection result image after processing, summarizes the detection results, and obtains the photothermal microscopy image of the carbon nanotube cluster;

[0012] Step 7: After the experiment, wait for 5 minutes and then turn off the power supplies of the lasers, function generator, lock-in amplifier, data acquisition card, galvanometer controller and computer.

[0013] Further, the sample to be detected in Step 1 includes nanoparticle clusters and carbon nanotube clusters.

[0014] Further, Step 2 specifically includes turning on the computer, data acquisition card, lock-in amplifier, galvanometer controller and function generator.

[0015] Further, in Step 3, maintain the operating temperature of the laser power supply at 20 °C to ensure the normal operation of the laser equipment.

[0016] Further, the detection parameters set in Step 4 include setting the laser power / current parameters, setting different differential coefficients γ, and scanning and detecting the sample to be detected.

[0017] Further, in Step 5, gold nanoparticles with a diameter of 60 nm are selected as the imaging object.

[0018] Further, in step 6, multi-walled carbon nanotubes with a diameter of 10-30 nm are used as samples.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1. In the present invention, two Gaussian excitation lights with the same frequency and opposite phases and a hollow excitation light act on the sample simultaneously for imaging. Compared with the traditional photothermal microscopy detection method, the present invention can effectively improve the system resolution (the FWHM of the modulated differential light source gradually decreases from the initial 654 nm to 192 nm), and perform super-resolution photothermal microscopy detection on the shallow surface defects of metal materials and polymer polymers;

[0021] 2. The present invention is applicable to the fields of precise non-destructive detection and evaluation of material defects / damages in aerospace, microelectronics, micro-nano structures, etc. Description of the Drawings

[0022] Figure 1 is a schematic diagram of the principle of a photothermal modulation differential microscopy method based on dual-excitation light excitation;

[0023] Figure 2 is an experimental diagram of photothermal modulation differential microscopy of nanoparticle clusters;

[0024] Figure 2 (a) is an experimental diagram of photothermal modulation differential microscopy;

[0025] Figure 2 (b) is a cross-section signal contour diagram in the photothermal modulation differential microscopy image;

[0026] Figure 3 is an experimental diagram of photothermal modulation differential microscopy of carbon nanotube clusters;

[0027] Figure 3 (a) is an experimental diagram of photothermal modulation differential microscopy;

[0028] Figure 3 (b) is a cross-section signal contour diagram in the photothermal modulation differential microscopy image. Detailed Embodiments

[0029] Detailed Embodiment 1: As Figure 1 shown, a photothermal modulation differential microscopy method based on dual-excitation light excitation specifically includes the following steps:

[0030] Step 1. Identify the sample to be detected and place the sample in the photothermal modulation differential microscopy system; the sample to be detected includes nanoparticle clusters and carbon nanotube clusters;

[0031] Step 2: Turn on the photo-thermal modulation differential microscopy imaging system; including turning on the computer, data acquisition card, lock-in amplifier, galvanometer controller, and function generator.

[0032] Step 3: Turn on the power supplies of the excitation light and detection light lasers and the cooler; maintain the operating temperature of the laser power supply at 20 °C to ensure the normal operation of the laser equipment.

[0033] Step 4: Set the detection parameters in the photo-thermal modulation differential microscopy imaging system software in the computer; setting the detection parameters includes setting the laser power / current parameters, setting different differential coefficients γ, and scanning and detecting the test sample.

[0034] Step 5: Select gold nanoparticles with a diameter of 60 nm as the imaging object, adopt the form of a glass slide sample, conduct multiple detections, the computer reads the signals at each point from the data acquisition card, processes them, obtains the detection result image, and summarizes the detection results to obtain the photo-thermal microscopy image of the nanoparticle cluster.

[0035] Step 6: Use multi-walled carbon nanotubes with a diameter of 10 - 30 nm as the sample, adopt the form of a glass slide sample, conduct multiple detections, the computer reads the signals at each point from the data acquisition card, processes them, obtains the detection result image, and summarizes the detection results to obtain the photo-thermal microscopy image of the carbon nanotube cluster.

[0036] Step 7: After the experiment, turn off the laser power supply, function generator, lock-in amplifier, data acquisition card, galvanometer controller, and computer after an interval of 5 minutes.

[0037] Among them, as Figure 2 shown; it can be seen that the FWHM of the signal profile gradually decreases from the initial 654 nm to 192 nm, approaching the FWHM value of the point spread function at the corresponding differential coefficient, indicating that the modulation differential technology significantly improves the resolution of photo-thermal microscopy imaging and realizes high-resolution imaging.

[0038] Among them, as Figure 3 shown; it can be seen that the resolution of photo-thermal imaging has been greatly improved, and at the same time, the background noise of the image is effectively reduced by using this method, which indicates that high-resolution microscopy imaging can also be achieved for nanotube samples using the photo-thermal modulation differential microscopy method.

[0039] Working principle

[0040] As Figure 1 shown, the present invention uses two Gaussian excitation lights and a hollow excitation light with the same frequency and opposite-phase modulation to act on the sample simultaneously for imaging. To meet the above requirements, the two lights need to be modulated separately and combined before entering the objective lens.

[0041] The point spread function of the Gaussian excitation light with modulated light intensity change and the point spread function of the hollow excitation light are represented by PSF G-heating and PSF D-heating respectively, and the specific definitions are shown in Equations (1) and (2); the point spread function of the modulated excitation light after beam combination PSF combined can be regarded as PSF G-heating and PSF D-heating added together. Due to PSF G-heating and PSF D-heating the difference in the light intensity distribution on the transverse cross-section of the focus, the beam combination point spread function is divided into two regions, i and ii, as shown in Figure 1 . It can be seen from Figure 1 that the change of the light intensity after beam combination with time can be decomposed into a modulated part and an unmodulated part. Since the lock-in amplifier is used for signal demodulation in the photo-thermal microscopy technology, only the light intensity difference generated by the modulated part acting on the sample to produce the medium temperature field and refractive index modulation change can be detected, forming the final photo-thermal amplitude signal. In region i, the light intensity of the Gaussian point spread function is greater than that of the hollow point spread function. Therefore, the phase of the modulated part after beam combination is consistent with that of the Gaussian light; while in region ii, the light intensity of the hollow point spread function is stronger. At this time, the phase of the modulated part after beam combination is consistent with that of the hollow light. The amplitude and phase of the point spread function after beam combination are shown in Figure 1 (c) and (d) respectively.

[0042] Denote the power ratio of the hollow beam to the Gaussian beam by the differential coefficient γ. At this time, the point spread function PSF combined of the modulated excitation light after beam combination can be obtained as Equation (3); take the amplitude of the photo-thermal microscopy as the photo-thermal signal. Considering the non-negativity of the amplitude, the point spread function PSF MDPT of the photo-thermal modulation differential microscopy is obtained as Equation (4);

[0043]

[0044] PSF MDPT =|PSF G-heating -γ·PSF D-heating |·PSF probe (4)

[0045] In Equations (1), (2), (3) and (4), PSF Gaussian represents the Gaussian beam focused point spread function, PSF Doughnut represents the hollow beam focused point spread function, and ω represents the modulation angular frequency (rad / s); Indicates the initial phase (rad).

[0046] The above are only the preferred embodiments of the present invention, and do not impose any formal restrictions on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the above-disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent replacement, and improvement made to the above embodiments within the spirit and principle of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A photothermal modulation differential microscopy imaging method based on dual-excitation light excitation, characterized in that, The specific steps include: Step 1: Identify the sample to be detected and place it in the photothermal modulation differential microscopy system; Step 2: Turn on the photothermal modulation differential microscopy system; Step 3: Turn on the power supplies of the excitation light laser, the detection light laser, and the cooler; Step 4: Set the detection parameters in the software of the photothermal modulation differential microscopy system in the computer; Step 5: Select gold nanoparticles as the imaging object, use the glass slide sample form, conduct multiple detections, the computer reads the signals at each point from the data acquisition card, processes them, obtains the detection result image, summarizes the detection results, and obtains the photothermal microscopy image of the nanoparticle clusters; Step 6: Use multi-walled carbon nanotubes as the sample, use the glass slide sample form, conduct multiple detections, the computer reads the signals at each point from the data acquisition card, processes them, obtains the detection result image, summarizes the detection results, and obtains the photothermal microscopy image of the carbon nanotube clusters; Step 7: After the experiment ends, wait for 5 minutes, and then turn off the power supplies of the laser, the function generator, the lock-in amplifier, the data acquisition card, the galvanometer controller, and the computer.

2. The method for photo-thermal modulation differential microscopy imaging based on dual-excitation light excitation according to claim 1, wherein The sample to be detected in Step 1 includes nanoparticle clusters and carbon nanotube clusters.

3. A photo-thermal modulation differential microscopy imaging method based on dual-excitation light excitation according to claim 1, characterized in that, Step 2 specifically includes turning on the computer, the data acquisition card, the lock-in amplifier, the galvanometer controller, and the function generator.

4. A photothermal modulation differential microscopy imaging method based on dual-excitation light excitation according to claim 1, characterized in that, In Step 3, maintain the operating temperature of the laser power supply at 20 °C to ensure the normal operation of the laser equipment.

5. A photothermal modulation differential microscopy imaging method based on dual-excitation light excitation according to claim 1, characterized in that The detection parameters set in Step 4 include setting the laser power / current parameters, setting different differential coefficients γ, and scanning and detecting the sample to be detected.

6. The photo-thermal modulation differential microscopy imaging method based on dual-excitation light excitation according to claim 1, wherein In Step 5, select gold nanoparticles with a diameter of 60 nm as the imaging object.

7. A photothermal modulation differential microscopy imaging method based on dual-excitation light excitation according to claim 1, characterized in that In Step 6, use multi-walled carbon nanotubes with a diameter of 10 - 30 nm as the sample.

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