A confocal differential based super-resolution non-contact photoacoustic microscopic device and method

Abstract

The present application is applicable to the field of super-resolution non-contact photoacoustic microscopic imaging, in order to overcome the problem of low imaging resolution of the PARS microscope in the prior art, the present application provides a kind of super-resolution non-contact photoacoustic microscopic device and method based on confocal difference, provides two kinds of detection light path structures to realize the design of PARS system of "confocal difference", under the premise of ensuring that endogenous photoacoustic signal is unchanged, realize the super-resolution PARS imaging.The present application provides an effective new strategy in realizing super-resolution photoacoustic microscopic imaging, which helps to break through the dilemma of the existing photoacoustic microscopic imaging system limited by resolution capability, and promotes the application and development of optical imaging field.

Description

Technical Field

[0001] This invention patent belongs to the field of optical microscopy imaging, and particularly relates to a super-resolution non-contact photoacoustic microscopy device and method based on confocal differential imaging. Background Technology

[0002] Photoacoustic microscopy (PAM), a functional imaging method based on optical absorption and using ultrasound as a carrier, has been extensively studied in recent years. However, traditional PAM relies on contact detection through ultrasonic transducer coupling with a liquid, limiting its application in contact-sensitive scenarios. As an alternative, photoacoustic remote sensing (PARS), based on the photoelastic effect, can detect photoacoustic signals and obtain high-quality images in a non-contact manner, becoming a powerful tool in biomedical research and industrial non-destructive testing. In a PARS microscope, the excitation and probe beams operate in a confocal state. When the excitation beam illuminates the absorber, the absorber converts light energy into heat energy, causing thermal expansion and generating an initial sound pressure level on the order of megapascals (photoacoustic effect). The high initial sound pressure modulates the refractive index at the absorption point of the object, and the higher the initial sound pressure, the more significant the change in refractive index (photoelastic effect). The change in refractive index then affects the reflectivity. By detecting the change in the intensity of the probe beam's reflection, the absorption of the excitation beam by the absorber can be reflected, thereby obtaining image information of the target location. Based on the aforementioned "photoacoustic-elastic-optical" theory, incoherent PARS avoids the phase noise of coherent detection in principle, enabling the acquisition of high-quality images with high sensitivity and high resolution.

[0003] High-resolution photomicrography (PAM) is of great significance for accurately reflecting cellular and tissue structures and monitoring physiological and pathological processes. Improving imaging resolution is an important research direction in PAM microscopy. PARS systems rely on optical focusing of the objective lens to achieve resolution in the micrometer to sub-micrometer range. Both their lateral and axial resolutions are limited by the optical diffraction limit (currently, a lateral resolution of 2.1 μm and an axial resolution of 43 μm have been achieved under 532 nm excitation light, and lateral resolutions of 2.3 μm and 1.6 μm, and an axial resolution of 32.27 μm have been achieved under 1064 nm excitation light). Further research is needed to further improve the imaging resolution of PARS microscopes and even break through the diffraction limit to achieve super-resolution PARS imaging.

[0004] The resolving power of an optical imaging system is related to its point spread function (PSF). The PSF is the energy distribution of an ideal geometric point after passing through an optical system; the smaller the PSF, the higher the resolution. In PARS, the effective PSF is related to the excitation and probe PSFs. Existing optical super-resolution imaging techniques are based on PSF modulation. If the concept of PSF modulation is applied to PARS systems, and appropriate PSF processing methods are used to modulate the PSF of the excitation or probe light, optimizing the PARS imaging resolution, then PARS images with clearer details and higher contrast can be obtained.

[0005] Based on the above background, this patent proposes a super-resolution non-contact photoacoustic microscopy device and method based on confocal differential imaging, which improves the imaging resolution of the PARS microscope and realizes super-resolution non-contact photoacoustic imaging. Summary of the Invention

[0006] To overcome the problem of low imaging resolution in existing PARS microscopes, this invention proposes a super-resolution non-contact photoacoustic microscopy device and method based on confocal differential. It provides two probe optical path structures to realize the PARS system design of "confocal differential", and achieves super-resolution PARS imaging while ensuring that the intrinsic photoacoustic signal remains unchanged.

[0007] This invention provides a confocal differential super-resolution PARS microscopy imaging device using multi-wavelength probe light. The imaging device comprises an excitation light module, a probe light module, a first beam splitting module, a beam combining module, a scanning focusing module, a data acquisition and image processing module, a test object module, and a variable module. The beam combining module includes a first beam combining module and a second beam combining module. The data acquisition and image processing module includes a first photodetector, a second photodetector, a data acquisition card, and a computer.

[0008] The excitation light module is used to generate pulsed excitation light.

[0009] The detection optical module is used to generate wavelength λ det1 The first probe light and wavelength is λ det2 The second probe light; the first probe light and the second probe light are combined at the second beam combining point after passing through the first beam splitting module; the combined probe light is then transmitted to the first beam combining module to be combined with the pulse excitation light for a second beam combining.

[0010] The scanning focusing module confocals the received secondary beam of combined light onto the test object module.

[0011] The first detection light reflected by the test object module passes through the scanning focusing module, the beam combining module, and the first beam splitting module before being received by the first photodetector in the data acquisition and image processing module, thus realizing the detection of photoacoustic signals.

[0012] The second probe light reflected by the test object module is received by the variable module after passing through the scanning focusing module, the beam combining module, and the first beam splitting module.

[0013] The variable module is used to modulate the point spread function of the received second probe light and input it into the second photodetector to realize the detection of photoacoustic signals.

[0014] The data acquisition card acquires the first photoacoustic image signal obtained by the first photodetector and the second photoacoustic image signal obtained by the second photodetector, and transmits them to the computer for differential processing to obtain a super-resolution image. Specifically:

[0015] I sub (x,y)=I det2 (x,y)-α(I det1 (x,y)-I det2 (x,y)) (1)

[0016] Among them, I det1 (x,y) corresponds to the photoacoustic image obtained by the first photodetector, I det2 (x,y) corresponds to the photoacoustic image obtained by the second photodetector, and α is an adjustment parameter used to adjust the difference size.

[0017] This invention also provides a super-resolution photoacoustic microscopy imaging device employing confocal differential modulation of a single-path probe light with a variable pinhole. This device uses only one photodetector within its data acquisition module, with a variable module inserted before the photodetector. Two scanning images are performed by changing the size of the variable module. To ensure a certain difference in noise and signal between the two photoacoustic images corresponding to the variable module, and to significantly improve the image resolution after differential imaging, the difference between the two images cannot be too small.

[0018] The imaging device comprises an excitation light module, a probe light module, a first beam splitting module, a beam combining module, a scanning focusing module, a data acquisition and image processing module, a test object module, and a variable module; the data acquisition and image processing module includes a first photodetector, a data acquisition card, and a computer.

[0019] The excitation light module is used to generate pulsed excitation light.

[0020] The detection optical module is used to generate wavelength λ det1 The first probe light; after passing through the first beam splitting module, the first probe light is combined with the excitation light generated by the excitation light module at the beam combining module.

[0021] The scanning and focusing module confocals the received combined beam onto the test object module, thereby achieving three-dimensional scanning of the test object with the optical focus on it.

[0022] The probe light reflected by the test object module passes through the scanning focusing module, the beam combining module, and the first beam splitting module before being received by the variable module.

[0023] The variable module modulates the point spread function of the received probe light before inputting it into the first photodetector to detect the photoacoustic signal. The PARS AC voltage signal acquired by the first photodetector is digitally sampled by the data acquisition card and transmitted to the computer for image reconstruction. After the first image acquisition is completed, the size of the variable module is changed, and image acquisition is repeated. A super-resolution image is obtained by differential processing of the photoacoustic images obtained from the two scans, specifically:

[0024] I sub (x,y)=I det-small (x,y)-α(I det-big (x,y)-I det-small (x,y)) (2)

[0025] Among them, I det-big (x,y) corresponds to the photoacoustic image obtained when the variable module size is large, I det-small (x,y) corresponds to the photoacoustic image obtained when the variable module size is small, and α is an adjustment parameter used to adjust the difference size.

[0026] This invention proposes a "confocal differential" technique to improve the resolution of PARS microscopy imaging systems. The core of this technique lies in compressing the PSF size of the probe beam. Its principle is simple and efficient, and theoretically applicable to PARS microscopy imaging systems across all wavelengths. This invention provides an effective new strategy for achieving super-resolution photoacoustic microscopy imaging, helping to overcome the limitations of existing photoacoustic microscopy imaging systems in terms of resolution, and promoting the application and development of optical imaging.

[0027] This invention proposes multiple PARS "confocal differential" system designs based on the "photoacoustic-elastic-optical" theory to achieve all-optical non-contact super-resolution photoacoustic imaging. Furthermore, these systems can be combined with other optical imaging technologies to form multimodal high-resolution optical imaging systems, providing structural and functional information at the cellular or molecular level.

[0028] This invention is expected to realize all-optical non-contact three-dimensional super-resolution photoacoustic microscopy, which can provide strong technical support for the biomedical clinical applications of PARS technology in intraoperative rapid pathology, early cancer diagnosis and monitoring, etc. At the same time, it can promote the realization of ultra-high resolution non-destructive testing and high-precision identification of complex structural materials in industry, and expand its application in aerospace and other industrial fields. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 Schematic diagram of super-resolution implementation using image difference;

[0031] Figure 2 This is a schematic diagram of the super-resolution photoacoustic microscopy imaging device using confocal differential multi-wavelength probe light according to the present invention.

[0032] Figure 3 This is a schematic diagram of the super-resolution photoacoustic microscopy imaging device of the present invention, which uses confocal differential modulation of a single-wavelength probe light.

[0033] Figure 4 This invention relates to a super-resolution photoacoustic microscopy imaging method based on confocal difference.

[0034] Figure 5 This invention provides a comparison of the PARS images obtained using the confocal differential super-resolution imaging method with the imaging effects without using the super-resolution method. Detailed Implementation

[0035] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will understand that the invention can be implemented in other embodiments without these specific details. In other instances, detailed descriptions of well-known systems, apparatuses, circuits, and methods are omitted so as not to obscure the description of the invention with unnecessary detail.

[0036] The imaging principle of the super-resolution non-contact photoacoustic microscopy system based on confocal differential can be explained by the probe spot spread function (PSF). In PARS, the effective PSF of the system is related to both the excitation and probe spot spread functions. This patent improves PARS resolution by designing dual probe paths with probes of different PSF sizes without changing the excitation light, and then using a confocal differential method to compress the size of the probe PSF. Using this method, higher resolution PARS images can be obtained while keeping the intrinsic signal unchanged.

[0037] The principle of "confocal differential" for compressing the probe beam PSF is as follows: In this invention, the key to implementing differential is obtaining probe beam PSFs of different sizes. The smaller PSF is... det-small (x,y) mainly contains focal plane light and a small amount of defocused light; a large PSF is called a large PSF. det-big (x,y) contains not only focal plane light but also more defocused light than the smaller PSF; the difference between these two PSFs is... det-big (x,y)-PSF det-small (x,y), i.e., PSF sub (x, y) mainly contains the defocused light at the edges. By subtracting the defocused light portion from the smaller PSF, a smaller PSF can be obtained without losing most of the effective signal of the focal plane, thus balancing resolution and signal-to-noise ratio. To prevent excessive negative intensity in the subtracted image from causing image distortion, this invention proposes to introduce a parameter α to adjust the difference size. The selection of α should maintain a balance between the resolution improvement and image distortion after difference, and should be determined based on specific experimental conditions. Finally, this invention proposes that the differential expression of the PSF be expressed in the following form:

[0038] PSF sub (x,y)=PSF det-small (x,y)-α(PSF det-big (x,y)-PSF det-small (x,y)) (1)

[0039] In optical imaging systems, due to the diffraction limit, an ideal object point becomes an Airy disk, or Gaussian point spread function, after passing through the system. Two Gaussian PSF curves were plotted through theoretical calculations, and a suitable parameter α was selected for difference analysis. Both the original curve and the difference results are as follows... Figure 1 As shown. The narrower the full width at half maximum (FWHM) of the PSF curve, the higher the system resolution. Figure 1 As can be seen, the PSFsub curve after subtraction is significantly narrower than the PSFdet-small intensity curve, indicating a significant improvement in resolution and proving the feasibility of the differential concept in this patent.

[0040] like Figure 2 As shown, this embodiment provides a confocal differential super-resolution PARS microscopy imaging device using multi-wavelength probe light. The imaging device comprises an excitation light module 1, a probe light module 2, a first beam splitting module 3, a beam combining module 4, a scanning focusing module 5, a data acquisition and image processing module 6, a test object module 7, and a variable module 9. The beam combining module 4 includes a first beam combining module 41 and a second beam combining module 42. The data acquisition and image processing module 6 includes a first photodetector 611, a second photodetector 612, a data acquisition card 62, and a computer 63.

[0041] Excitation light module 1 is used to generate pulsed excitation light.

[0042] Detection optical module 2 is used to generate wavelength λ det1 The first probe light and wavelength is λ det2 The second probe light; the first probe light and the second probe light are combined at the second beam combiner 42 after passing through the first beam splitter 3 respectively; the combined probe light is then transmitted to the first beam combiner 41 to be combined with the pulse excitation light for a second beam combiner.

[0043] The scanning focusing module 5 confocals the received secondary beam of combined light onto the test object module 7.

[0044] The first detection light reflected by the test object module 7 passes through the scanning focusing module 5, the beam combining module 4, and the first beam splitting module 3, and is then received by the first photodetector 611 in the data acquisition and image processing module 6, thereby realizing the detection of photoacoustic signals.

[0045] The second probe light reflected by the test object module 7 is received by the variable module 9 after passing through the scanning focusing module 5, the beam combining module 4 and the first beam splitting module 3.

[0046] The variable module 9 is used to modulate the point spread function of the received second probe light and input it into the second photodetector 612 to realize the detection of photoacoustic signals.

[0047] Data acquisition card 62 acquires the first photoacoustic image signal obtained by the first photodetector 611 and the second photoacoustic image signal obtained by the second photodetector 612, and transmits them to computer 63 for differential processing to obtain a super-resolution image. Specifically:

[0048] I sub (x,y)=I det2 (x,y)-α(I det1 (x,y)-I det2 (x,y)) (2)

[0049] Among them, I det1 (x,y) corresponds to the photoacoustic image obtained by the first photodetector 611, I det2 (x,y) corresponds to the photoacoustic image obtained by the second photodetector 612, and α is an adjustment parameter used to adjust the difference size.

[0050] Furthermore, the excitation light module 1 includes a pulsed laser 11, a synchronous triggering module 12, and a beam shaping and amplification system 13, used to generate a photoacoustic signal to excite the sample. Even further, the synchronous triggering module 12 includes a second beam splitting module 121 and a third photodetector 122, providing a trigger signal for signal acquisition.

[0051] Furthermore, after the excitation light is emitted from the pulsed laser 11, it is split into two paths by the second beam-splitting module 121 (splitting ratio 9:1) of the synchronous triggering module 12. 10% of the light is received by the third photodetector 122 to generate a trigger signal; the remaining excitation light passes through the beam shaping and amplification system 13 composed of two plano-convex lenses and a pinhole, and is then combined with the probe light at the first beam-combining module 41. Under the condition of multi-wavelength light co-path, it is focused on the test object module 7 by the achromatic objective lens 51 of the scanning focusing module 5 and the three-dimensional scanning system 52 to excite the initial sound pressure.

[0052] Furthermore, the beam shaping and amplification system 13 employs a transmission optical path composed of dual plano-convex lenses and pinholes to optimize the spot quality of the excitation light.

[0053] Furthermore, the detection optical module 2 includes a first superluminescent diode 211, a first beam expander 221, a second superluminescent diode 212, and a second beam expander 222; the first superluminescent diode 211 is used to generate wavelength λ. det1 The first detection light, and the second super-radiative diode 212 are used to generate a wavelength of λ. det2 The second probe light, where λ det1 >λ det2 .

[0054] Furthermore, the first beam splitting module 3 includes a first polarization beam splitter 311, a first quarter-wave plate 321, a second polarization beam splitter 312, and a second quarter-wave plate 322. After the first probe light is emitted, it passes through the first beam expander 221, the first polarization beam splitter 311, and the first quarter-wave plate 321, and is converted from linearly polarized light to circularly polarized light. After the second probe light is emitted, it passes through the second beam expander 222, the second polarization beam splitter 312, and the second quarter-wave plate 322, and is converted from linearly polarized light to circularly polarized light. The two probe beams are combined in the second beam combining module 42 and then transmitted to the first beam combining module 41 to be combined with the excitation light.

[0055] Furthermore, the scanning focusing module 5 includes an achromatic objective lens 51 and a three-dimensional scanning system 52; the light combined by the beam combining module 4 is co-focused on the sample module 7 by the achromatic objective lens 51 and the three-dimensional scanning system 52 of the scanning focusing module 5, thereby realizing three-dimensional scanning of the optical focus on the sample.

[0056] Furthermore, the excitation light, the first probe light, and the second probe light are reflected by the test object module 7, and the circular polarization state of the first probe light and the second probe light is converted from left-handed (right-handed) circular polarization state to right-handed (left-handed). The reflected excitation light, the first probe light, and the second probe light return along the original optical path, pass through the scanning focusing module 5, and reach the first beam combining module 41. The excitation light is split with the first probe light and the second probe light at the first beam combining module 41, thus avoiding the influence of the excitation light on the collected signal.

[0057] Furthermore, the first and second probe beams are split after traveling from the first beam combiner module 41 to the second beam combiner module 42, and the two beams return along their original optical paths. After passing through the first quarter-wave plate 321, the first probe beam is converted from circularly polarized light to linearly polarized light perpendicular to its original polarization direction. It then passes through the first polarization beam splitter 311 and exits along the direction perpendicular to the original optical path, entering the first photodetector 611 for signal acquisition. After passing through the second quarter-wave plate 322, the second probe beam is converted from circularly polarized light to linearly polarized light perpendicular to its original polarization direction. It then passes through the second polarization beam splitter 312 and exits along the direction perpendicular to the original optical path, entering the second photodetector 612 for signal acquisition. This process avoids mutual interference between the two probe beams and achieves separation of the original optical path and the reflected optical path, ensuring the acquisition of the PARS signal.

[0058] Furthermore, variable module 9 is implemented using pinholes.

[0059] like Figure 3 As shown, this embodiment also provides a super-resolution photoacoustic microscopy imaging device using confocal differential modulation of a single probe light with variable pinhole modulation. This device has only one photodetector 61 within the data acquisition module 6, and a variable module 9 is inserted in front of the photodetector. Two scanning images are performed by changing the size of the variable module. To ensure a certain difference in noise and signal between the two photoacoustic images corresponding to the variable module 9, and to significantly improve the image resolution after differential imaging, the difference between the two images of the variable module 9 cannot be too small.

[0060] The imaging device comprises an excitation light module 1, a detection light module 2, a first beam splitting module 3, a beam combining module 4, a scanning focusing module 5, a data acquisition and image processing module 6, a test object module 7, and a variable module 9; the data acquisition and image processing module 6 includes a first photodetector 61, a data acquisition card 62, and a computer 63.

[0061] Excitation light module 1 is used to generate pulsed excitation light.

[0062] Detection optical module 2 is used to generate wavelength λ det1 The first probe light; after passing through the first beam splitting module 3, the first probe light is combined with the excitation light generated by the excitation light module 1 at the beam combining module 4.

[0063] The scanning focusing module 5 confocals the received combined beam onto the test object module 7, thereby achieving three-dimensional scanning of the test object with the optical focus on it.

[0064] The probe light reflected by the test object module 7 is received by the variable module 9 after passing through the scanning focusing module 5, the beam combining module 4 and the first beam splitting module 3.

[0065] The variable module 9 modulates the point spread function of the received probe light and inputs it into the first photodetector 61 to detect the photoacoustic signal. The PARS AC voltage signal acquired by the first photodetector 61 is digitally sampled by the data acquisition card 62 and transmitted to the computer 63 for image reconstruction. After the first image acquisition is completed, the size of the variable module 9 is changed, and image acquisition is repeated. A super-resolution image is obtained by differential processing of the photoacoustic images obtained from the two scans, specifically:

[0066] I sub (x,y)=I det-small (x,y)-α(I det-big (x,y)-I det-small (x,y)) (3)

[0067] Among them, I det-big (x,y) corresponds to the photoacoustic image obtained when the size of variable module 9 is large, I det-small (x,y) corresponds to the photoacoustic image obtained when the size of variable module 9 is small, and α is an adjustment parameter used to adjust the difference size.

[0068] Furthermore, the excitation light module 1 includes a pulsed laser 11, a synchronous triggering module 12, and a beam shaping and amplification system 13, used to generate a photoacoustic signal to excite the sample. Even further, the synchronous triggering module 12 includes a second beam splitting module 121 and a second photodetector 122, providing a trigger signal for signal acquisition.

[0069] Furthermore, after the excitation light is emitted from the pulsed laser 11, it is split into two paths by the second beam-splitting module 121 (splitting ratio 9:1) of the synchronous triggering module 12. 10% of the light is received by the second photodetector 122 to generate a trigger signal; the remaining excitation light passes through the beam shaping and amplification system 13 composed of two plano-convex lenses and a pinhole, and is then combined with the probe light at the beam-combining module. Under the condition that the excitation light and the probe light are on the same path, the light is focused onto the test object module 7 by the achromatic objective lens 51 of the scanning focusing module 5 and the three-dimensional scanning system 52 to excite the initial sound pressure.

[0070] Furthermore, the beam shaping and amplification system 13 employs a transmission optical path composed of dual plano-convex lenses and pinholes to optimize the spot quality of the excitation light.

[0071] Furthermore, the detection optical module 2 includes a superluminescent diode 21 and a beam expander 22; the superluminescent diode 21 is used to generate different wavelengths λ. det1 The detection light.

[0072] Furthermore, the first beam splitting module 3 includes a polarization beam splitter 31 and a quarter-wave plate 32. After the probe light is emitted, it passes through the first beam expander 22, the polarization beam splitter 31, and the quarter-wave plate 32, and is converted from linearly polarized light to circularly polarized light. Then, the probe light is transmitted to the beam combining module 4 and combined with the excitation light.

[0073] Furthermore, the scanning focusing module 5 includes an achromatic objective lens 51 and a three-dimensional scanning system 52; the light combined by the beam combining module 4 is co-focused on the sample module 7 by the achromatic objective lens 51 and the three-dimensional scanning system 52 of the scanning focusing module 5, thereby realizing three-dimensional scanning of the optical focus on the sample.

[0074] Furthermore, variable module 9 is implemented using pinholes.

[0075] Both of these devices achieve super-resolution PARS imaging through a dual-path probe light design. While ensuring that the intrinsic photoacoustic signal remains unchanged, they improve the resolution of the original photoacoustic image, which is expected to broaden the application scope of photoacoustic imaging and promote the practical application of PARS technology.

[0076] Based on the two devices mentioned above, this invention also proposes a super-resolution PARS microscopy imaging method based on confocal difference, such as... Figure 4 As shown.

[0077] Specifically, the following steps are included:

[0078] Step S1: Select the excitation light signal.

[0079] Specifically, based on the absorption band of the imaging target, a short-pulse laser with a strong absorption wavelength is selected as the excitation light. For example, a 266nm short-pulse laser is selected to excite nucleic acids, a 532nm short-pulse laser to excite hemoglobin, and a 1064nm short-pulse laser to excite melanin. After selecting the excitation light wavelength, the pulse laser is focused onto the position to be measured using an objective lens, thereby exciting the imaging target to generate a photoacoustic signal.

[0080] Step S2: Select continuous laser beams with different point spread functions as probe beams.

[0081] Specifically, since obtaining probe light with different PSF sizes is crucial for confocal differential super-resolution photoacoustic imaging, continuous probe light with different PSF sizes can be obtained by selecting different wavelengths or modulating the PSF of a single wavelength. When using different wavelengths to change the PSF size, multiple continuous lasers with weak absorption wavelengths of the imaging target should be selected as probe light to avoid the probe light affecting the photoacoustic signal of the imaging target, ensuring that the refractive index change caused by the photoacoustic effect can be accurately captured by the probe light and reflected in the reflected signal of the probe light. Commonly used probe light wavelengths include 405nm, 830nm, and 1310nm. Alternatively, a variable-diameter precision pinhole can be used to modulate a probe light of a certain wavelength to obtain probe light with different PSF sizes.

[0082] Step S3: Perform confocal scanning of the probe light from step S2 and the excitation light from step S1.

[0083] Specifically, throughout the entire confocal differential super-resolution PARS microscopy process, the pulsed excitation light and the multiple probe light are always in a confocal state. To achieve this multi-beam focusing effect, a series of dichroic mirrors with different cutoff wavelengths are used for optical path design, which can realize the beam combining of multiple probe light and single excitation light. After passing through the beam scanning device and achromatic objective lens, the effect of confocalization of multiple probe light and excitation light can be achieved, realizing multi-path confocal detection of the same photoacoustic process and ensuring the consistency of the intrinsic signal.

[0084] Step S4: Obtain the reflection signals of the multi-path probe light.

[0085] Specifically, by modulating the polarization state of the probe light and rationally designing the polarization modulation device and beam combiner, the separate acquisition of multiple probe light reflection signals is achieved. The specific method is as follows: First, a half-wave plate is used to adjust the polarization state of the probe light to the transmission polarization direction of the polarization beam splitter. After the probe light passes through the polarization beam splitter, a quarter-wave plate is used to convert the linear polarization state of the probe light into a circular polarization state (assuming it is now a positive circular polarization state). Then, probe light of different wavelengths is introduced into a dichroic mirror for beam combining, and then incident on the target through the objective lens. At this point, due to the half-wave loss, the polarization state of the reflected light from the probe light changes to a reverse circular polarization state. The reflected light returns along the original path, and after reaching the quarter-wave plate again, the polarization state of the reflected light becomes the polarization direction of the polarization beam splitter. The reflecting surface of the polarization beam splitter forms a 45° angle with the light path, and the reflected light exits at a 90° angle with the original light path and is received by a photodetector, acquiring the reflection signal of the probe light. Each wavelength of probe light has its reflection signal received by an independent photodetector, achieving simultaneous acquisition of multiple probe light reflection signals.

[0086] Step S5: Differential calculation is used to obtain the super-resolution PARS image.

[0087] Specifically, differential calculation is performed on the acquired PSF images of different sizes. The specific calculation process is as follows: select two PSF images of different sizes, subtract the two to obtain an image that only contains edge defocus signals, and then use the smaller PSF image to subtract the image containing only edge defocus signals to further remove the defocus signals of the smaller PSF image, thereby obtaining an image with a smaller PSF and realizing the acquisition of super-resolution PARS images.

[0088] The principle of "confocal differential" for compressing the probe beam PSF is as follows: In this invention, the prerequisite for implementing differential is to obtain probe beam PSFs of different sizes, one large and one small. The smaller PSF is referred to as PSF. det-small (x,y) mainly contains focal plane light and a small amount of defocused light; a large PSF is called a large PSF. det-big (x,y) contains not only focal plane light but also more defocused light than the smaller PSF; the difference between these two PSFs (PSF) det-big (x,y)-PSF det-small (x,y)), i.e., PSF sub (x,y) mainly contains out-of-focus light. By subtracting the out-of-focus portion from a smaller PSF, a smaller PSF can be obtained without losing most of the effective signal of the focal plane, thus balancing resolution and signal-to-noise ratio. To prevent excessive negative intensity in the subtracted image from causing image distortion, a parameter α is introduced to adjust the difference size. The selection of α should maintain a balance between the increased resolution after difference and image distortion, and should be determined based on specific experimental conditions. Finally, the differential expression of the PSF is expressed in the following form:

[0089] PSF sub (x,y)=PSF det-small (x,y)-α(PSF det-big (x,y)-PSF det-small (x,y)) (4)

[0090] In optical imaging systems, due to the diffraction limit, an ideal object point becomes an Airy disk, or Gaussian point spread function, after passing through the system.

[0091] The following example uses the first type of device, namely multi-wavelength probe light, as a case study.

[0092] In this example, a 1064nm pulsed laser is selected as the excitation source 11 of the excitation light module 1. After the excitation light is emitted from the laser 11, it is split into two paths by the second beam splitting module 121 (beam splitting ratio 9:1) of the synchronous triggering module 12. 10% of the light is received by the third photodetector 122 to generate a trigger signal; the remaining excitation light passes through the beam shaping and amplification module 13, which consists of two plano-convex lenses and a pinhole, and is then combined with the probe light at the first beam combining module 41. Under the condition of multi-wavelength light co-path, it is focused onto the test object module 7 by the scanning focusing module 5 to excite the initial sound pressure.

[0093] The detection light module 2 uses two superluminescent diodes, 211 and 212, with wavelengths of 1310nm and 830nm respectively, as dual-wavelength detection beams. The 1310nm detection beam, after being emitted, passes through the first beam expander 221, the first polarization beam splitter 311 of the first beam splitter module 3, and the first quarter-wave plate 321, converting from linearly polarized light to circularly polarized light. The 830nm detection beam, after being emitted, passes through the second beam expander 222, the second polarization beam splitter 312 of the first beam splitter module 3, and the second quarter-wave plate 322, converting from linearly polarized light to circularly polarized light. The two detection beams are combined in the second beam combiner module 42, then transmitted to the first beam combiner module 41 where they are combined with the excitation beam. Finally, the scanning focusing module 5 confociens the beams onto the sample module 7, achieving a three-dimensional scan of the sample with the optical focus.

[0094] When the 1310nm probe light reflected from the sample passes through the first quarter-wave plate 321 in the first beam-splitting module 3, it is converted into linearly polarized light with a polarization direction perpendicular to the outgoing polarization direction. This light is then reflected by the first polarization beam splitter 311 and received by the first photodetector 611, thus detecting the photoacoustic signal. Similarly, when the 830nm probe light reflected from the sample passes through the second quarter-wave plate 322 in the first beam-splitting module 3, it is converted into linearly polarized light with a polarization direction perpendicular to the outgoing polarization direction. This light is reflected by the second polarization beam splitter 312, and after the PSF of the 830nm probe light is modulated by the adjustable module 9, it is received by the second photodetector 612, thus detecting the photoacoustic signal. The PARS AC voltage signals acquired by the first photodetector 611 and the second photodetector 612 are digitally sampled by the data acquisition card 62 and transmitted to the computer 63 for image differential reconstruction.

[0095] The super-resolution image can be obtained by performing image processing on the photoacoustic image under the 830nm probe light and the photoacoustic image under the 1310nm probe light using the difference method represented by Equation (1). Figure 5 The imaging results of axial scanning of a 6μm tungsten wire are shown. It can be seen that the differential image removes the interference of edge signals at non-focal positions, and the axial resolution of the system is improved, proving the feasibility of the technology proposed in this patent.

[0096] It should be understood that the sequence number of each step in the above embodiments does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0097] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the above-described division of functional units and modules is merely an example. In practical applications, the above functions can be assigned to different functional units and modules as needed, that is, the internal structure of the device can be divided into different functional units or modules to complete all or part of the functions described above. The functional units and modules in the embodiments can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit. Furthermore, the specific names of the functional units and modules are only for easy differentiation and are not intended to limit the scope of protection of this application. The specific working process of the units and modules in the above system can be referred to the corresponding process in the foregoing method embodiments, and will not be repeated here.

[0098] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0099] Those skilled in the art will recognize that the units and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementations should not be considered beyond the scope of this invention.

[0100] In the embodiments provided by this invention, it should be understood that the disclosed apparatus / terminal devices and methods can be implemented in other ways. For example, the apparatus / terminal device embodiments described above are merely illustrative. For instance, the division of modules or units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be through some interfaces; the indirect coupling or communication connection between devices or units may be electrical, mechanical, or other forms.

[0101] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0102] Furthermore, the functional units in the various embodiments of the present invention can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit.

[0103] If the integrated module / unit is implemented as a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, all or part of the processes in the methods of the above embodiments can also be implemented by a computer program instructing related hardware. The computer program can be stored in a computer-readable storage medium, and when executed by a processor, it can implement the steps of the various method embodiments described above. The computer program includes computer program code, which can be in the form of source code, object code, executable files, or certain intermediate forms. The computer-readable medium can include: any entity or device capable of carrying the computer program code, recording media, USB flash drives, portable hard drives, magnetic disks, optical disks, computer memory, read-only memory (ROM), random access memory (RAM), electrical carrier signals, telecommunication signals, and software distribution media, etc. It should be noted that the content included in the computer-readable medium can be appropriately added or removed according to the requirements of legislation and patent practice in the jurisdiction. For example, in some jurisdictions, according to legislation and patent practice, computer-readable media do not include electrical carrier signals and telecommunication signals.

[0104] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A confocal differential super-resolution PARS microscopy imaging device employing multi-wavelength probe light, characterized in that, The imaging device is used for horizontal and vertical imaging. The imaging device consists of an excitation light module (1), a probe light module (2), a first beam splitting module (3), a beam combining module (4), a scanning focusing module (5), a data acquisition and image processing module (6), a test object module (7), and a variable module (9). The beam combining module (4) includes a first beam combining module (41) and a second beam combining module (42); the data acquisition and image processing module (6) includes a first photodetector (611), a second photodetector (612), a data acquisition card (62), and a computer (63); The excitation light module (1) is used to generate pulsed excitation light; The detection optical module (2) is used to generate a wavelength of The first detection light and wavelength are The second detection light, in which... > ; The scanning focusing module (5) confocals the received secondary beam of combined light onto the test object module (7); The first detection light reflected by the test object module (7) is received by the first photodetector (611) in the data acquisition and image processing module (6) after passing through the scanning focusing module (5), the beam combining module (4) and the first beam splitting module (3), thereby realizing the detection of photoacoustic signals; The first beam splitting module (3) includes a first polarization beam splitter (311), a first quarter-wave plate (321), a second polarization beam splitter (312), and a second quarter-wave plate (322). After the first probe light is emitted, it passes through the first beam expander (221), the first polarization beam splitter (311), and the first quarter-wave plate (321) and is converted from linearly polarized light to circularly polarized light. After the second probe light is emitted, it passes through the second beam expander (222), the second polarization beam splitter (312), and the second quarter-wave plate (322) and is converted from linearly polarized light to circularly polarized light. The two probe lights are combined in the second beam combining module (42) and then transmitted to the first beam combining module (41) to be combined with the excitation light. The scanning focusing module (5) confocals the received secondary combined light onto the test object module (7) to realize the three-dimensional scanning of the optical focus on the sample. The second probe light reflected by the test object module (7) is received by the variable module (9) after passing through the scanning focusing module (5), the beam combining module (4) and the first beam splitting module (3); The variable module (9) is used to modulate the point spread function of the received second probe light and input it into the second photodetector (612) to realize the detection of photoacoustic signals; The data acquisition card (62) acquires the first photoacoustic image signal obtained by the first photodetector (611) and the second photoacoustic image signal obtained by the second photodetector (612), and transmits them to the computer (63) for differential processing to obtain a super-resolution image.

2. The confocal differential super-resolution PARS microscopy imaging device using multi-wavelength probe light according to claim 1, wherein the differential processing specifically comprises: （1） in, The photoacoustic image obtained by the first photodetector (611) The photoacoustic image obtained by the second photodetector (612) is α, which is an adjustment parameter used to adjust the difference size.

3. The confocal differential super-resolution PARS microscopy imaging device with multi-wavelength probe light according to claim 1, wherein the excitation light module (1) includes a pulsed laser (11), a synchronous triggering module (12) and a beam shaping and amplification system (13), used to generate photoacoustic signals to excite the sample; The synchronous trigger module (12) includes a second beam splitter module (121) and a third photodetector (122) to provide a trigger signal for signal acquisition.

4. The confocal differential super-resolution PARS microscopic imaging device with multi-wavelength probe light according to claim 3, wherein the beam shaping and amplification system (13) adopts a transmission optical path composed of double plano-convex lenses and pinholes to optimize the spot quality of the excitation light.

5. The confocal differential super-resolution PARS microscopy imaging device using multi-wavelength probe light according to claim 1, wherein the probe light module (2) comprises a first superradiative diode (211), a first beam expander (221), a second superradiative diode (212), and a second beam expander (222); the first superradiative diode (211) is used to generate a wavelength of The first detector light, and the second superluminescent diode (212) are used to generate a wavelength of The second detection light.

6. The confocal differential super-resolution PARS microscopic imaging device with multi-wavelength probe light according to claim 1, wherein the first beam splitting module (3) includes a polarization beam splitter (31) and a quarter-wave plate (32); after the probe light is emitted, it is converted from linearly polarized light to circularly polarized light after passing through the first beam expander (22), the polarization beam splitter (31), and the quarter-wave plate (32); then, the probe light is transmitted to the beam combining module (4) and combined with the excitation light.

7. The confocal differential super-resolution PARS microscopy device with multi-wavelength probe light according to claim 1, wherein the variable module (9) is implemented by a pinhole.

8. According to claim 1, the confocal differential super-resolution PARS microscopic imaging device using multi-wavelength probe light, the excitation light, the first probe light, and the second probe light are reflected by the test object module (7), the circular polarization state of the first probe light and the second probe light is converted from left-handed or right-handed circular polarization state to right-handed or left-handed, the reflected excitation light, the first probe light, and the second probe light return along the original optical path, pass through the scanning focusing module (5), and reach the first beam combining module (41), the excitation light is split with the first probe light and the second probe light at the first beam combining module (41).

9. The confocal differential super-resolution PARS microscopic imaging device with multi-wavelength probe light according to claim 8, wherein the excitation light is split from the first probe light and the second probe light at the first beam combining module (41), and the two beams return along the original optical path respectively; after the first probe light passes through the first quarter-wave plate (321), it is converted from circularly polarized light to linearly polarized light perpendicular to the original polarization direction, and then passes through the first polarization beam splitter (311) and exits along the direction perpendicular to the original optical path, and enters the first photodetector (611) for signal acquisition; after the second probe light passes through the second quarter-wave plate (322), it is converted from circularly polarized light to linearly polarized light perpendicular to the original polarization direction, and then passes through the second polarization beam splitter (312) and exits along the direction perpendicular to the original optical path, and enters the second photodetector (612) for signal acquisition, thereby avoiding mutual interference between the two probe lights and realizing the separation of the original optical path and the reflected optical path.

10. A super-resolution PARS microscopy method based on confocal differential imaging of the apparatus according to any one of claims 1-9, specifically comprising the following steps: Step S1: Select the excitation light signal; Step S2: Select continuous laser beams with different point spread functions as probe beams; Step S3: Perform confocal scanning of the probe light from step S2 and the excitation light from step S1; Step S4: Acquire the reflection signals of the multiple probe beams; Step S5: Differential calculation to obtain super-resolution PARS images; Specifically, differential calculation is performed on the images of different PSF sizes. The specific calculation process is as follows: select images corresponding to two different PSF sizes, subtract the two to obtain an image that only contains edge defocus signals, and then subtract the image containing only edge defocus signals from the smaller PSF image to further remove the defocus signals of the smaller PSF image, thus obtaining an image with a smaller PSF.

Images