A dual-excitation light excitation-based photothermal modulation differential microscopic imaging method

Abstract

The application discloses a kind of photothermal modulation differential microscopic imaging methods based on double excitation light excitation, it relates to a kind of photothermal modulation differential microscopic imaging methods.The present application is to solve the problem that current photothermal microscopic imaging technology is limited to diffraction limit, cannot break through minimum resolution limit.The steps of the present application include step 1, clear to be detected sample, places sample in photothermal modulation differential microscopic imaging system;Step 2, start photothermal modulation differential microscopic imaging system;Step 3, start excitation light, probe light laser power supply and refrigerator;Step 4, set detection parameters in the photothermal modulation differential microscopic imaging system software in computer;Step 5, select gold nanoparticles as imaging object, carry out multiple detection;Step 6, adopt multi-arm carbon nanotube as sample, carry out multiple detection;Step 7, close equipment after 5 minutes of experiment end.The present application belongs to photothermal science and detection signal processing technical field.

Description

Technical Field

[0001] This invention relates to a photothermal modulation differential microscopy imaging method, belonging to the field of photothermal science and detection signal processing technology. Background Technology

[0002] Optical microscopy plays a crucial role in the development of microscopy and has become an indispensable tool in other fields such as materials science, medicine, optics, and energy. However, optical microscopy has long been limited by the diffraction effect of light, preventing the direct observation of structures below 200 nm. Traditional microscopy methods also suffer from problems such as complex sample preparation, weak fluorescence signals, and significant background interference. In recent decades, with the further improvement of related optical technologies and materials research, more and more super-resolution microscopy techniques that break 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. Therefore, for the microscopic imaging and detection of nanoscale particles, it is crucial to find a microscopy technique that can achieve stable imaging and reduce background interference. Photothermal heterodyne microscopy is a novel far-field microscopy technique that utilizes the photothermal properties of objects. When a probe beam passes through a sample material, optical parameters change. By detecting these changes, the characteristic information of the material under test can be obtained. This technique has advantages such as being less affected by background scattering, less affected by changes in the size of the object under test, and being able to perform microscopic imaging of non-fluorescent objects.

[0003] In existing research, Li Yiming et al. (patent number: CN202110390429.6) disclosed a super-resolution microscopy imaging system. This system includes: an excitation light source generation module, an imaging module, a focal plane locking module, and a control and data acquisition module. The system controls and adjusts the optical elements in the excitation light source generation module, imaging module, and focal plane locking module through the control and data acquisition module, and acquires fluorescence data obtained by the imaging module for data analysis. This system realizes a compact, highly automated, adjustable and controllable single-molecule localization microscopy technology, and improves precision and accuracy. Yang Qing et al. (Patent No.: CN202411133674.9) disclosed a fast frequency-shifting super-resolution microscopy imaging method based on ring illumination. It acquires a single wide-field image when the sample is illuminated by normal incidence, and acquires multiple low-resolution images with corresponding single frequency-shifting under tilted illumination. The single wide-field image and multiple low-resolution images are used to achieve fast frequency-shifting super-resolution microscopy imaging through a super-resolution imaging model built based on deep learning, thereby obtaining a super-resolution image. This improves the imaging speed and is expected to be applied to video-level super-resolution microscopy imaging. Wang Shufeng et al. (Patent No.: CN202110540951.8) disclosed a super-resolution microscopy imaging system and imaging method based on two-photon nonlinear effects. This invention utilizes the threshold of the two-photon nonlinear effect to naturally form an excitation probe exceeding the diffraction limit in space; it uses a spatial light phase modulator to control the pulse width of the laser, thereby dynamically adjusting the scale of the excitation probe at the center of the spot. This invention overcomes some common shortcomings in existing super-resolution imaging technologies. The peak power of the excitation light is greatly reduced after passing through the shaping system, thus avoiding photodamage or photobleaching to the sample, making it suitable for long-exposure imaging and live-cell imaging. Yang Shuming et al. (Patent No.: CN201510292588.7) disclosed a super-resolution confocal microscopy imaging device and method. In this device, the movement of the object under test and the reflective microscope objective is accomplished through three-dimensional PZT and one-dimensional PZT, respectively, to achieve tomographic imaging of the object. This device comprehensively uses a super-oscillating zone plate and a reflective microscope objective to achieve the convergence of the scanning beam, avoiding the assembly and adjustment errors caused by the use of multiple lenses in existing confocal imaging devices, thereby significantly improving the imaging effect of the confocal imaging device. This invention eliminates the need for lens assemblies, resulting in a simple and stable device that can be used for super-resolution confocal imaging of micro-devices. To fully utilize the advantages of differential microscopy in improving lateral resolution and the thermal lensing effect of materials to achieve precise three-dimensional detection of defect size / morphology, this invention relates to a photothermal modulation differential microscopy imaging method based on dual-excitation light. This method, based on vector diffraction theory, heat transfer, and electromagnetic field theory, can effectively improve system resolution (the FWHM of the modulated light source gradually decreases from the initial 654nm to 192nm), enabling super-resolution photothermal microscopy imaging detection of shallow surface defects in metallic materials and polymers. Summary of the Invention

[0004] To address the problem that current photothermal microscopy imaging technology is limited by the diffraction limit and cannot break through the minimum resolution limitation, this invention proposes a photothermal modulation differential microscopy imaging method based on dual-excitation light excitation.

[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 tested and place it 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 for the excitation and probe lasers and the cooler;

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

[0010] Step 5: Select gold nanoparticles as the imaging object, use glass slide samples, perform multiple detections, and the computer reads the signals from each point from the data acquisition card, processes them, and obtains the detection result images. Summarize the detection results to obtain photothermal microscopic images of nanoparticle clusters.

[0011] Step 6: Using multi-arm carbon nanotubes as samples, and in the form of glass slide samples, multiple tests are performed. The computer reads the signals from each point from the data acquisition card, processes them, and obtains the detection result images. The detection results are summarized to obtain photothermal microscopic images of carbon nanotube clusters.

[0012] Step 7: After the experiment is completed, wait 5 minutes before turning off the laser power supply, function generator, lock-in amplifier, data acquisition card, galvanometer controller, and computer.

[0013] Furthermore, the samples to be tested in step 1 include nanoparticle clusters and carbon nanotube clusters.

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

[0015] Furthermore, in step 3, the operating temperature of the laser power supply is maintained at 20°C to ensure the normal operation of the laser equipment.

[0016] Furthermore, step 4 involves setting detection parameters, including setting laser power / current parameters, setting different differential coefficients γ, and scanning the sample for detection.

[0017] Furthermore, in step 5, gold nanoparticles with a diameter of 60 nm are selected as the imaging target.

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

[0019] The beneficial effects of this invention are:

[0020] 1. This invention uses two beams of Gaussian excitation light with the same frequency and opposite phase modulation and hollow excitation light to simultaneously act on the sample for imaging. Compared with the traditional photothermal microscopy imaging detection method, this invention can effectively improve the system resolution (the FWHM of the modulated differential light source gradually decreases from the initial 654nm to 192nm), enabling super-resolution photothermal microscopy imaging detection of shallow surface defects in metallic materials and polymers.

[0021] 2. This invention is applicable to the field of precise non-destructive testing and evaluation of material defects / damage in aerospace, microelectronics, and micro / nano structures. Attached Figure Description

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

[0023] Figure 2 These are experimental images 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 profile of the cut-off signal in a photothermal modulated differential microscopy image;

[0026] Figure 3 These are experimental images 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 profile of the cut-off signal in a photothermal modulation differential microscopy image. Detailed Implementation

[0029] Specific implementation method one: as follows Figure 1 As shown, a photothermal modulation differential microscopy imaging method based on dual-excitation light excitation includes the following specific steps:

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

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

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

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

[0034] Step 5: Select gold nanoparticles with a diameter of 60nm as the imaging object, use glass slide sample form, perform multiple detections, the computer reads the signals from each point from the data acquisition card, processes the signals to obtain the detection result image, summarize the detection results, and obtain the photothermal microscopic image of the nanoparticle cluster.

[0035] Step 6: Using multi-arm carbon nanotubes with a diameter of 10-30nm as samples, and in the form of glass slide samples, multiple tests are performed. The computer reads the signals from each point from the data acquisition card, processes them, and obtains the detection result images. The detection results are summarized to obtain photothermal microscopic images of carbon nanotube clusters.

[0036] Step 7: After the experiment is completed, wait 5 minutes before turning off the laser power supply, function generator, lock-in amplifier, data acquisition card, galvanometer controller, and computer.

[0037] Among them, such as Figure 2 As shown, the FWHM of the signal profile gradually decreased from the initial 654nm to 192nm, which is close to the FWHM value of the time-spread function corresponding to the differential coefficient. This indicates that the modulation differential technique significantly improved the resolution of photothermal microscopy and achieved high-resolution imaging.

[0038] Among them, such as Figure 3 As shown, the resolution of photothermal imaging has been greatly improved. At the same time, the background noise of the image has also been effectively reduced due to the use of this method. This indicates that high-resolution microscopy can also be achieved for nanotube samples using photothermal modulation differential microscopy.

[0039] Working principle

[0040] like Figure 1 As shown, this invention employs two beams of Gaussian excitation light modulated with the same frequency but opposite phase, along with a hollow excitation light, to simultaneously act on the sample for imaging. To meet the above requirements, the two beams need to be modulated separately and then combined in front of the incident objective lens.

[0041] The point spread function of Gaussian excitation light with intensity modulation and the point spread function of hollow excitation light are respectively... PSF G-heating and PSF D-heating The specific definitions are shown in equations (1) and (2); the diffusion function of the modulated excitation spot after beam combining. PSF combined It can be seen as PSF G-heating and PSF D-heating Adding them together, because PSF G-heating and PSF D-heating The difference in light intensity distribution on the transverse section of the focal point divides the beam convergence point spread function into two regions, i and ii, as shown below. Figure 1 As shown. From Figure 1 As can be seen, the change in light intensity after beam combining over time can be decomposed into a modulated part and an unmodulated part. Since photothermal microscopy uses a lock-in amplifier for signal demodulation, only the intensity difference generated by the modulated part can be detected in relation to the temperature field and refractive index modulation of the sample, forming the final photothermal amplitude signal. In region i, the light intensity of the Gaussian point spread function is greater than that of the hollow point spread function, so the phase of the modulated part after beam combining is consistent with the Gaussian light; while in region ii, the light intensity of the hollow point spread function is stronger, and the phase of the modulated part after beam combining is consistent with the hollow light. The amplitude and phase of the point spread function after beam combining are as follows: Figure 1 As shown in (c) and (d).

[0042] The power ratio of the hollow beam to the Gaussian beam is expressed by the difference coefficient γ, from which the modulation excitation spot spread function (PSF) of the combined beam can be obtained. combined Formula (3) is used; taking the amplitude of photothermal microscopy as the photothermal signal, and considering the non-negativity of the amplitude, the point spread function (PSF) of photothermal modulation differential microscopy is obtained. MDPT Formula (4) is used.

[0043]

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

[0045] In formulas (1), (2), (3) and (4), PSF Gaussian The Gaussian beam focal point spread function, PSF Doughnut ω represents the diffusion function at the focal point of the hollow beam, and ω represents the modulation angular frequency (rad / s). Indicates the initial phase (rad).

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent substitutions, and improvements made to the above embodiments without departing from the scope of the present invention, based on the technical essence of the present invention and within the spirit and principles of the present invention, shall still fall within the protection scope 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 tested 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 for the excitation and probe lasers and the cooler; Step 4: Set the detection parameters in the photothermal modulation differential microscopy system software on the computer; Imaging is achieved by simultaneously applying two beams of Gaussian excitation light with the same frequency but opposite phase and a hollow excitation light to the sample. The point spread function of Gaussian excitation light with intensity modulation and the point spread function of hollow excitation light are respectively... PSF G-heating and PSF D-heating The specific definitions are shown in formulas (1) and (2); the diffusion function of the modulated excitation spot after beam combining. PSF combined See as PSF G-heating and PSF D-heating Adding them together, because PSF G-heating and PSF D-heating The difference in light intensity distribution on the cross section of the focal point divides the beam combining point spread function into two regions, i and ii. In region i, the light intensity of the Gaussian point spread function is greater than that of the hollow point spread function, so the phase of the modulated part after beam combining is consistent with that of the Gaussian light. In region ii, the light intensity of the hollow point spread function is stronger, and the phase of the modulated part after beam combining is consistent with that of the hollow light. The power ratio of the hollow beam to the Gaussian beam is expressed using a difference factor. γ This indicates that the diffusion function of the modulated excitation spot after beam combining is obtained. PSF combined Formula (3) is used; taking the amplitude of photothermal microscopy as the photothermal signal, and considering the non-negativity of the amplitude, the point spread function of photothermal modulation differential microscopy is obtained. PSF MDPT Formula (4); （1） （2） （3） （4） In formulas (1), (2), (3) and (4), PSF Gaussian This represents the spread function at the focal point of a Gaussian beam. PSF Doughnut This represents the diffusion function at the focal point of a hollow beam. ω Indicates the modulation angular frequency; φ Indicates the initial phase; Step 5: Select gold nanoparticles as the imaging object, use glass slide samples, perform multiple detections, and the computer reads the signals from each point from the data acquisition card, processes them, and obtains the detection result images. Summarize the detection results to obtain photothermal microscopic images of nanoparticle clusters. Step 6: Using multi-arm carbon nanotubes as samples, and in the form of glass slide samples, multiple tests are performed. The computer reads the signals from each point from the data acquisition card, processes them, and obtains the detection result images. The detection results are summarized to obtain photothermal microscopic images of carbon nanotube clusters. Step 7: After the experiment is completed, wait 5 minutes before turning off the laser power supply, function generator, lock-in amplifier, data acquisition card, galvanometer controller, and computer.

2. The photothermal modulation differential microscopy imaging method based on dual-excitation light excitation according to claim 1, characterized in that, The samples to be tested in step 1 include nanoparticle clusters and carbon nanotube clusters.

3. The photothermal 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, data acquisition card, lock-in amplifier, galvanometer controller, and function generator.

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

5. The photothermal modulation differential microscopy imaging method based on dual-excitation light excitation according to claim 1, characterized in that, Step 4 involves setting the detection parameters, including setting the laser power / current parameters and different differential coefficients. γ The sample is scanned and tested.

6. The photothermal modulation differential microscopy imaging method based on dual-excitation light excitation according to claim 1, characterized in that, In step 5, gold nanoparticles with a diameter of 60 nm were selected as the imaging target.

7. The photothermal modulation differential microscopy imaging method based on dual-excitation light excitation according to claim 1, characterized in that, In step 6, multi-arm carbon nanotubes with a diameter of 10-30 nm were used as samples.

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