Super-resolution non-contact photoacoustic microscopy device and method based on confocal difference

Abstract

The invention is applicable to the field of super-resolution non-contact photoacoustic microscopic imaging, provides a super-resolution non-contact photoacoustic microscopic device and method based on confocal difference in order to solve the problem of low imaging resolution of a PARS microscope in the prior art, and provides two detection light path structures for realizing the design of a 'confocal difference' PARS system. And super-resolution PARS imaging is realized on the premise of ensuring that an endogenous photoacoustic signal is not changed. The invention provides an effective new strategy in the aspect of realizing super-resolution photoacoustic microscopic imaging, which is helpful to break through the dilemma that the existing photoacoustic microscopic imaging system is limited by the resolution capability and promote the application and development of the optical imaging field.

Description

Technical Field

[0001] The patent of this invention belongs to the field of optical microscopy, and in particular relates to a super-resolution non-contact photoacoustic microscopy device and method based on confocal differential. Background Art

[0002] Photoacoustic microscopy (PAM), a functional imaging method based on optical absorption and ultrasound, has been widely studied in recent years. However, traditional PAM relies on contact detection using an ultrasonic transducer coupled to a liquid, limiting its application in contact-sensitive applications. As an alternative, photoacoustic remote sensing (PARS), based on the photoelastic effect, enables non-contact detection of photoacoustic signals and produces high-quality images, becoming a powerful tool in biomedical research and industrial non-destructive testing. In PARS microscopy, the excitation light and the probe light operate in a confocal state. When the excitation light strikes an absorber, the absorber converts the light energy into heat, causing thermal expansion and generating an initial acoustic pressure in the megapascal range (photoacoustic effect). This high initial acoustic pressure modulates the refractive index at the absorption site, with greater initial pressure leading to more pronounced refractive index changes (photoelastic effect). This refractive index change, in turn, affects reflectivity. By measuring changes in the reflected intensity of the probe light, the absorption of the excitation light by the absorber can be detected, thereby obtaining image information of the target location. Based on the above-mentioned "photoacoustic-optical" theory, incoherent PARS avoids the phase noise of coherent detection in principle and can obtain high-quality images with high sensitivity and high resolution.

[0003] High-resolution PAM is of great research significance for accurately reflecting cell tissue structure and monitoring physiological and pathological processes. Improving imaging resolution is an important research direction for PAM microscopy. The PARS system relies on the optical focusing of the objective lens to achieve resolution in the micron to submicron range. Its lateral and axial resolutions are both limited by the optical diffraction limit (currently, a lateral resolution of 2.1μm and an axial resolution of 43μm have been achieved under 532nm excitation light, and a lateral resolution of 2.3μm and 1.6μm, and an axial resolution of 32.27μm have been achieved under 1064nm excitation light). How to further improve the imaging resolution of PARS microscopes and even break through the diffraction limit to achieve super-resolution PARS imaging still requires further research.

[0004] The resolution capability of an optical imaging system is related to its point spread function (PSF). The point spread function is the energy distribution of an ideal geometric point after passing through the optical system, and the smaller the system point spread function, the higher the resolution. In PARS, the effective point spread function of the system is related to the excitation light point spread function and the detection light point spread function. The existing optical super-resolution imaging technology is realized based on the modulation of the PSF, and if the idea of modulating the PSF is applied to the PARS system, the imaging resolution of the PARS is optimized by modulating the PSF of the excitation light or the detection light using a suitable PSF processing method, and then a PARS image with clearer details and higher contrast can be obtained.

[0005] Based on the above background, the present patent proposes a super-resolution non-contact photoacoustic microscopic device and method based on confocal difference, which improves the imaging resolution of the PARS microscope and realizes super-resolution non-contact photoacoustic imaging. SUMMARY

[0006] In order to overcome the problem of low imaging resolution of the PARS microscope in the prior art, the present application proposes a super-resolution non-contact photoacoustic microscopic device and method based on confocal difference, which provides two detection light path structures to realize the design of the PARS system based on "confocal difference", and realizes super-resolution PARS imaging under the premise of ensuring the invariable endogenous photoacoustic signal.

[0007] The present application provides a confocal difference super-resolution PARS microscopic imaging device using multi-wavelength detection light, which comprises an excitation light module, a detection light module, a first beam splitting module, a beam combining module, a scanning focusing module, a data acquisition and image processing module, a sample module, and a variable module; the beam combining module comprises a first beam combining module and a second beam combining module; the data acquisition and image processing module comprises 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 light module is used to generate first detection light with a wavelength of λ det1 and second detection light with a wavelength of λ det2 ; the first detection light and the second detection light are respectively subjected to beam splitting by the first beam splitting module and then subjected to beam combining at the second beam combining module; the combined detection light is then transmitted to the first beam combining module for secondary beam combining with the pulsed excitation light.

[0010] The scanning focusing module focuses the received secondary beam combined light on the sample module.

[0011] The first detection light reflected by the object to be measured module passes through the scanning and focusing module, the beam combining module and the first beam splitting module and is received by the first photodetector in the data acquisition and image processing module to detect the photoacoustic signal.

[0012] The second detection light reflected by the object to be measured module passes through the scanning and focusing module, the beam combining module and the first beam splitting module and is then received by the variable module.

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

[0014] The data acquisition card collects 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] The present invention also provides a confocal differential super-resolution photoacoustic microscopy imaging device using variable pinhole modulation of a single-path probe beam. This device incorporates a single photodetector within a data acquisition module, with a variable module inserted in front of the photodetector. The device performs two scanning imaging operations by varying the size of the variable module. To ensure a certain difference in noise and signal between the two imaging operations, significantly improving image resolution after differential analysis, the difference between the two scans of the variable module must be sufficient.

[0018] The imaging device includes an excitation light module, a detection light module, a first beam splitting module, a beam combining module, a scanning and 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 photoelectric detector, a data acquisition card, and a computer.

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

[0020] The detection light module is used to generate a wavelength of λ det1 the first detection light passes through the first beam splitting module and is then combined with the excitation light generated by the excitation light module at the beam combining module.

[0021] The scanning focusing module focuses the received combined light on the object module, so as to realize three-dimensional scanning of the optical focus on the object.

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

[0023] The variable module is used for modulating the point spread function of the received probe light and inputting into the first photodetector, so as to realize detection of the photoacoustic signal. The PARS alternating voltage signals collected by the first photodetector are digitally sampled by the data acquisition card and transmitted into the computer for image reconstruction. After completing the first image acquisition, the size of the variable module is changed, and the image acquisition is repeated. The photoacoustic images obtained by the two times of scanning are subjected to difference processing, so as to obtain the super-resolution image, specifically:

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

[0025] Wherein, I det-big (x,y) corresponds to the photoacoustic image obtained when the size of the variable module is large, I det-small (x,y) corresponds to the photoacoustic image obtained when the size of the variable module is small, and a is an adjustment parameter for adjusting the difference size.

[0026] The present application provides a kind of " confocal difference " technology for improving the resolution of PARS microscopic imaging system.The core of the technology is to compress the PSF size of probe light beam, and its principle is simple and efficient, and it is theoretically applicable to all waveband PARS microscopic imaging systems.The present application provides an effective new strategy in realizing super-resolution photoacoustic microscopic imaging, which helps to break through the dilemma of existing photoacoustic microscopic imaging system limited by resolution, and promotes the application and development of optical imaging field.

[0027] The present application provides a variety of PARS " confocal difference " system designs based on " photoacoustic-elastic light " theory, realizes full-optical non-contact super-resolution photoacoustic imaging, and can be combined with other optical imaging technologies to form a multi-modal high-resolution optical imaging system, providing structural and functional information at cell or molecular level.

[0028] The present application is expected to realize full-optical non-contact three-dimensional super-resolution photoacoustic microscopic imaging, which can provide strong technical support for PARS technology in intraoperative rapid pathology, early cancer diagnosis and monitoring, etc. biomedical clinical applications, and promote PARS technology to realize ultra-high resolution nondestructive testing and high-precision identification of complex structure materials in industry, and expand its application in aviation and other industrial fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

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

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

[0032] Figure 3 This is a schematic structural diagram of a super-resolution photoacoustic microscopy imaging device using confocal differential modulation of a single-wavelength probe light according to the present invention;

[0033] Figure 4 The present invention is a super-resolution photoacoustic microscopy method based on confocal difference;

[0034] Figure 5 The PARS image obtained by the confocal differential super-resolution imaging method of the present invention is compared with the imaging effect without using the super-resolution method. DETAILED DESCRIPTION

[0035] In the following description, specific details such as particular system structures and techniques are provided for purposes of illustration, not limitation, to facilitate a thorough understanding of the embodiments of the present invention. However, it will be apparent to those skilled in the art that the present invention may be practiced in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices, circuits, and methods are omitted so as not to obscure the description of the present invention with unnecessary detail.

[0036] The imaging principle of super-resolution non-contact photoacoustic microscopy systems based on confocal differential imaging can be explained by the detection light point spread function. In PARS, the system's effective point spread function is related to the excitation light point spread function and the detection light point spread function. This patent improves the resolution of PARS by designing a dual-path detection optical path. Without changing the excitation light, the detection light has different PSF sizes. The confocal differential method is then used to compress the size of the detection light PSF. This method can achieve higher-resolution PARS images while ensuring that the intrinsic signal remains unchanged.

[0037] The principle of "confocal differential" for compressing the detection light PSF is as follows: In the present invention, the key to implementing differential is to obtain a large and a small size of the detection light PSF. Among them, the small PSF is the PSF det-small (x, y) mainly contains the focal plane light and a small amount of out-of-focus light; the large PSF is PSF det-big (x,y) contains not only the in-focus light but also more out-of-focus light than the smaller PSF; the difference PSF of these two PSFs is det-big (x,y)-PSF det-small (x,y), also known as PSF sub (x, y) mainly contains edge out-of-focus light. By subtracting the out-of-focus light portion from a small PSF, a smaller PSF can be obtained without losing most of the effective signal in the focal plane, thereby taking into account both resolution and signal-to-noise ratio. In order to prevent excessive negative intensity from appearing in the subtracted image and causing image distortion, the present invention proposes to introduce a parameter α to adjust the difference size. The selection of α should maintain a balance between the improved resolution and image distortion after the difference, and is obtained based on specific experimental conditions. Finally, the present invention proposes that the differential formula of PSF is 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 existence of diffraction limit, an ideal object point is an Airy disk after passing through the system, that is, a Gaussian point spread function. Through theoretical calculation, two Gaussian PSF curves are drawn and the appropriate parameter α is selected for difference. The original curve and the difference result are as follows Figure 1 As shown. The narrower the FWHM of the PSF curve is, the higher the system resolution is. Figure 1 It can be seen that the PSFsub curve after subtraction is significantly narrower than the PSFdet-small intensity curve, which means that the resolution has been significantly improved, proving the feasibility of the differential idea of ​​this patent.

[0040] like Figure 2 As shown, this embodiment provides a confocal differential super-resolution PARS microscopy imaging device using multi-wavelength detection light, the imaging device includes an excitation light module 1, a detection light module 2, a first beam splitting module 3, a beam combining module 4, a scanning and 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] The excitation light module 1 is used to generate pulsed excitation light.

[0042] Detection light module 2 is used to generate a wavelength of λ det1 The first probe light has a wavelength of λ det2 the first detection light and the second detection light are respectively combined at the second beam combining module 42 after passing through the first beam splitting module 3; the combined detection light is then transmitted to the first beam combining module 41 for secondary combination with the pulsed excitation light.

[0043] The scanning and focusing module 5 co-focuses the received secondary combined light beams onto the object to be measured module 7 .

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

[0045] The second detection light reflected by the object to be measured module 7 passes through the scanning and focusing module 5 , the beam combining module 4 and the first beam splitting module 3 and is then received by the variable module 9 .

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

[0047] The data acquisition card 62 collects 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. 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 trigger module 12, and a beam shaping and amplification system 13, which are used to generate a photoacoustic signal to excite the sample. Furthermore, the synchronous trigger module 12 includes a second beam splitter module 121 and a third photodetector 122, which provide a trigger signal for signal acquisition.

[0051] Furthermore, after emitting from the pulsed laser 11, the excitation light is split into two paths by the second beam splitter module 121 (splitting ratio 9:1) of the synchronous trigger module 12. Ten percent 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, consisting of two plano-convex lenses and a pinhole. It is then combined with the detection light at the first beam combining module 41. Under the condition of multi-wavelength light co-path, it is focused on the object under test module 7 through the achromatic objective lens 51 and three-dimensional scanning system 52 of the scanning and focusing module 5, and used to stimulate the initial sound pressure.

[0052] Furthermore, the beam shaping and amplification system 13 adopts a transmission light path composed of a double plano-convex lens and a pinhole to optimize the spot quality of the excitation light.

[0053] Furthermore, the detection light module 2 includes a first super luminescent diode 211, a first beam expander 221, a second super luminescent diode 212, and a second beam expander 222; the first super luminescent diode 211 is used to generate a wavelength of λ det1 The second superluminescent diode 212 is used to generate a first detection light with 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, where it 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, where it is converted from linearly polarized light to circularly polarized light. The two probe light 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 and 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 cofocused on the object to be measured module 7 through the achromatic objective lens 51 and the three-dimensional scanning system 52 of the scanning and focusing module 5, thereby realizing three-dimensional scanning of the optical focus on the sample.

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

[0057] Furthermore, the first and second probe lights travel from the first beam combining module 41 to the second beam combining module 42, where they are split and return along their original optical paths. The first probe light passes through the first quarter-wave plate 321, converting it from circularly polarized light to linearly polarized light perpendicular to its original polarization direction. It then passes through the first polarization beam splitter 311, exiting in a direction perpendicular to its original optical path, and enters the first photodetector 611 for signal collection. The second probe light passes through the second quarter-wave plate 322, converting it from circularly polarized light to linearly polarized light perpendicular to its original polarization direction. It then passes through the second polarization beam splitter 312, exiting in a direction perpendicular to its original optical path, and enters the second photodetector 612 for signal collection. This prevents the two probe lights from interfering with each other, while achieving separation between the original optical path and the reflected optical path, ensuring PARS signal collection.

[0058] Furthermore, the variable module 9 is implemented by a pinhole.

[0059] like Figure 3 As shown, this embodiment also provides a confocal differential super-resolution photoacoustic microscopy imaging device using variable pinhole modulation of a single-path probe beam. This device includes a single photodetector 61 within a data acquisition module 6. A variable module 9 is inserted in front of the photodetector, and two scanning imaging operations are performed by varying 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 during the two imaging operations, and to significantly improve the resolution of the image after differentiation, the difference between the two different measurements of the variable module 9 must be considerable.

[0060] The imaging device includes an excitation light module 1, a detection light module 2, a first beam splitting module 3, a beam combining module 4, a scanning and 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] The excitation light module 1 is used to generate pulsed excitation light.

[0062] Detection light module 2 is used to generate a wavelength of λ det1 the first detection light passes through the first beam splitting module 3 and then is combined with the excitation light generated by the excitation light module 1 at the beam combining module 4.

[0063] The scanning and focusing module 5 co-focuses the received combined light beams on the object to be measured module 7, thereby achieving three-dimensional scanning of the object to be measured with the optical focus.

[0064] The detection light reflected by the object to be measured module 7 passes through the scanning and focusing module 5 , the beam combining module 4 and the first beam splitting module 3 and is then received by the variable module 9 .

[0065] The variable module 9 is used to modulate the point spread function of the received detection light and input it into the first photodetector 61 to detect the photoacoustic signal. The PARS AC voltage signal collected by the first photodetector 61 is digitally sampled by the data acquisition card 62 and transmitted to the computer 63 for image reconstruction. After completing the first image acquisition, the size in the variable module 9 is changed and the image acquisition is repeated. The photoacoustic images obtained by the two scans are subjected to differential processing to obtain a super-resolution image, 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 variable module 9 is larger in size, I det-small (x, y) corresponds to the photoacoustic image obtained when the size of the 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 trigger module 12, and a beam shaping and amplification system 13, which are used to generate a photoacoustic signal to excite the sample. Furthermore, the synchronous trigger module 12 includes a second beam splitting module 121 and a second photodetector 122, which provide a trigger signal for signal acquisition.

[0069] Furthermore, after emitting from the pulsed laser 11, the excitation light is split into two paths by the second beam splitter module 121 (splitting ratio 9:1) of the synchronous trigger 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, consisting of two plano-convex lenses and a pinhole, and is then combined with the detection light at the beam combining module. With the excitation and detection light co-pathing, it is focused on the object under test module 7 through the achromatic objective lens 51 and three-dimensional scanning system 52 of the scanning and focusing module 5, where it is used to stimulate the initial sound pressure.

[0070] Furthermore, the beam shaping and amplification system 13 adopts a transmission light path composed of a double plano-convex lens and a pinhole to optimize the spot quality of the excitation light.

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

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

[0073] Furthermore, the scanning and 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 cofocused on the object to be measured module 7 through the achromatic objective lens 51 and the three-dimensional scanning system 52 of the scanning and focusing module 5, thereby realizing three-dimensional scanning of the optical focus on the sample.

[0074] Furthermore, the variable module 9 is implemented by a pinhole.

[0075] Both of the above devices achieve super-resolution PARS imaging through a dual-path detection light design. While ensuring that the endogenous photoacoustic signal remains unchanged, the resolution of the original photoacoustic image is improved. This is expected to broaden the application scope of photoacoustic imaging and promote the practical application of PARS technology.

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

[0077] The specific steps include:

[0078] Step S1, selecting an excitation light signal.

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

[0080] Step S2: Select continuous lasers with different point spread functions as detection light.

[0081] Specifically, since obtaining probe light with different PSF sizes is the key to confocal differential super-resolution photoacoustic imaging, continuous light probe light with different PSF sizes can be obtained by selecting different wavelengths or performing PSF modulation on a single wavelength. When using different wavelengths to achieve PSF size changes, continuous lasers with multiple imaging target weak absorption wavelengths should be selected as probe light to avoid the probe light from affecting the imaging target photoacoustic signal and to ensure 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, 1310nm, etc. A precision pinhole with variable diameter can also be used to modulate a certain wavelength of probe light to obtain probe light with different PSF sizes.

[0082] Step S3, performing confocal scanning on the detection light in step S2 and the excitation light in step S1.

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

[0084] Step S4: Acquire reflection signals of the multi-path detection lights.

[0085] Specifically, by modulating the polarization state of the probe light and rationally designing the polarization modulation and beam combining components, the separate acquisition of multi-path 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 to a circular polarization state (assuming it is in the forward circular polarization state at this time). Subsequently, probe lights of different wavelengths are introduced into a dichroic mirror for beam combining and are incident normally on the imaging target through the objective lens. At this time, due to half-wave loss, the polarization state of the reflected probe light is converted to the 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 returns to the reflection polarization direction of the polarization beam splitter. The reflective surface of the polarization beam splitter is at a 45° angle to the optical path, and the reflected light is emitted at a 90° angle to the original optical path and received by a photodetector to obtain the reflected signal of the probe light. Each wavelength of probe light is received by a separate photodetector, achieving the simultaneous acquisition of the reflected signals of multiple probe lights.

[0086] Step S5: performing differential calculation to obtain a super-resolution PARS image.

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

[0088] The principle of "confocal differential" for compressing the detection light PSF is as follows: In the present invention, the premise of implementing differential is to obtain a large and a small size of the detection light PSF. Among them, the small PSF is the PSF det-small (x, y) mainly contains the focal plane light and a small amount of out-of-focus light; the large PSF is PSF det-big (x,y) contains not only the in-focus light but also more out-of-focus light than the smaller PSF; the difference between these two PSFs (PSF det-big (x,y)-PSF det-small (x,y)), which is PSF sub (x,y) mainly contains edge out-of-focus light. By subtracting the out-of-focus light from a small PSF, a smaller PSF can be obtained without losing most of the effective signal in the focal plane, thus taking into account both resolution and signal-to-noise ratio. In order to prevent excessive negative intensity from appearing in the subtracted image and causing image distortion, a parameter α is introduced to adjust the difference size. The selection of α should maintain a balance between the improved resolution and image distortion after the difference, and should be obtained based on the specific experimental conditions. Finally, the difference formula of the PSF is expressed as follows:

[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 existence of diffraction limit, an ideal object point is an Airy disk after passing through the system, that is, a Gaussian point spread function.

[0091] The following takes the specific implementation of the first device, i.e., multi-wavelength detection light, as an example.

[0092] In this example, a 1064nm pulsed laser is used as the excitation light source 11 of the excitation light module 1. After emitting from the laser 11, the excitation light is split into two paths by the second beam splitter 121 (splitting ratio 9:1) of the synchronous trigger 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, consisting of two plano-convex lenses and a pinhole. It is then combined with the detection light at the first beam combining module 41. Under the condition of multi-wavelength light co-path, it is focused on the object under test module 7 by the scanning focusing module 5 to stimulate the initial sound pressure.

[0093] The detection light module 2 uses superluminescent diodes 211 and 212 with two wavelengths of 1310nm and 830nm as dual-wavelength detection light. After the 1310nm detection light is emitted, it passes through the first beam expander 221, the first polarization beam splitter 311 of the first beam splitting module 3, and the first quarter-wave plate 321, and is converted from linearly polarized light to circularly polarized light. After the 830nm detection light is emitted, it passes through the second beam expander 222, the second polarization beam splitter 312 of the first beam splitting module 3, and the second quarter-wave plate 322, and is converted from linearly polarized light to circularly polarized light. The two beams of detection light 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. Finally, they are cofocused on the object to be measured module 7 by the scanning focusing module 5, realizing three-dimensional scanning of the optical focus on the sample.

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

[0095] The obtained photoacoustic image under the 830nm detection light and the photoacoustic image under the 1310nm detection light are processed using the difference method represented by formula (1) to obtain a super-resolution image. 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 in non-focus positions, and the axial resolution of the system is improved, proving that the technology proposed in this patent is feasible.

[0096] It should be understood that the size of the serial number of each step in the above embodiments does not mean the order of execution, and the execution order of each process should be determined according to its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the application.

[0097] It can be clearly understood by those skilled in the art that, for the convenience and brevity of description, only the above-mentioned division of each functional unit and module is exemplified, and in actual application, the above-mentioned functions can be completed by different functional units and modules according to needs, that is, the internal structure of the device is divided into different functional units or modules to complete all or part of the functions described above. Each functional unit and module in the embodiment can be integrated in one processing unit, or each unit can be physically present separately, or two or more units can be integrated in one unit. The above-mentioned integrated unit can be realized in the form of hardware or software function unit. In addition, the specific name of each functional unit and module is only for the convenience of mutual distinction, and does not limit the protection scope of the application. The specific working process of the unit and module in the above system can be referred to the corresponding process in the foregoing method embodiments, which will not be described here.

[0098] In the above embodiments, the description of each embodiment has its own emphasis, and the parts not described or recorded in detail in a certain embodiment can be referred to the related description of other embodiments.

[0099] Those skilled in the art can realize that the units and algorithm steps of each example described in combination with the embodiments disclosed herein can be realized by electronic hardware or a combination of computer software and electronic hardware. Whether the functions are executed in hardware or software depends on the specific application and design constraints of the technical solution. Professional technicians can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of the present application.

[0100] In the embodiments provided by the present application, it should be understood that the disclosed device / terminal equipment and method can be implemented by other ways. For example, the above-mentioned device / terminal equipment embodiments are only schematic, for example, the division of the modules or units is only a logical function division, and there can be another division way in actual implementation, for example, a plurality of units or components can be combined or integrated into another system, or some features can be ignored or not executed. In addition, the coupling or direct coupling or communication connection between the shown or discussed mutual ones can be indirect coupling or communication connection through some interfaces, devices or units, and can be electrical, mechanical or other forms.

[0101] The units described as separate components may or may not be physically separate, and the components displayed as units may or may not be physical units, i.e. may be located in one place, or may be distributed on multiple network units. Part or all of the units can be selected according to actual needs to achieve the purpose of the embodiment scheme.

[0102] In addition, each functional unit in each embodiment of the present application can be integrated in one processing unit, or each unit can exist physically, or two or more units can be integrated in one unit. The integrated unit can be realized in the form of hardware or in the form of a software functional unit.

[0103] The integrated module / unit, if realized in the form of a software functional unit and sold or used as an independent product, can be stored in a computer readable storage medium. Based on this understanding, all or part of the processes in the above-mentioned embodiment methods can also be completed by a computer program instructing related hardware, and the computer program can be stored in a computer readable storage medium. The computer program can implement the steps of each method embodiment when executed by a processor. The computer program includes computer program code, which can be in the form of source code, object code, executable files or some intermediate forms, etc. The computer readable medium can include any entity or device capable of carrying the computer program code, recording medium, U disk, mobile hard disk, magnetic disk, optical disk, computer memory, read-only memory (ROM), random access memory (RAM), electrical carrier signal, telecommunication signal and software distribution medium, etc. It should be noted that the contents included in the computer readable medium can be appropriately increased or decreased according to the requirements of legislation and patent practice in the jurisdiction, for example, in some jurisdictions, according to legislation and patent practice, the computer readable medium does not include electrical carrier signals and telecommunication signals.

[0104] The above-described embodiments are only used to illustrate the technical solutions of the present application, rather than limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should be included in the protection scope of the present application.

Claims

1. A confocal differential super-resolution PARS microscopic imaging device using multi-wavelength detection light, the imaging device comprising an excitation light module (1), a detection light module (2), a first beam splitting module (3), a beam combining module (4), a scanning and 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 light module (2) is used to generate a wavelength of λ det1 The first probe light has a wavelength of λ det2 The first detection light and the second detection light are respectively combined at the second beam combining module (42) after passing through the first beam splitting module (3); the combined detection light is then transmitted to the first beam combining module (41) for secondary beam combining with the pulsed excitation light; The scanning and focusing module (5) co-focuses the received secondary combined light beams onto the object to be measured module (7); The first detection light reflected by the object to be measured module (7) passes through the scanning and focusing module (5), the beam combining module (4) and the first beam splitting module (3) and is received by the first photodetector (611) in the data acquisition and image processing module (6), thereby realizing the detection of the photoacoustic signal; The second detection light reflected by the object to be measured module (7) passes through the scanning and focusing module (5), the beam combining module (4) and the first beam splitting module (3) and is then received by the variable module (9); The variable module (9) is used to modulate the point spread function of the received second detection light and input the modulated point spread function into the second photodetector (612) to detect the photoacoustic signal. 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 detection light according to claim 1, wherein the differential processing is specifically: I sub (x,y)=I det2 (x,y)-α(I det1 (x,y)-I det2 (x,y)) (1) in, 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.

3. The confocal differential super-resolution PARS microscopy imaging device using multi-wavelength detection light according to claim 1, wherein the excitation light module (1) comprises a pulsed laser (11), a synchronous trigger module (12) and a beam shaping and amplification system (13), for generating a photoacoustic signal to excite the sample; The synchronous trigger module (12) comprises a second beam splitting module (121) and a third photodetector (122), and provides a trigger signal for signal acquisition.

4. According to the confocal differential super-resolution PARS microscopy imaging device using multi-wavelength detection light according to claim 1, the beam shaping and amplification system (13) uses a transmission light path composed of a double plano-convex lens and a pinhole to optimize the spot quality of the excitation light.

5. The confocal differential super-resolution PARS microscopy imaging device using multi-wavelength detection light according to claim 1, wherein the detection light module (2) comprises 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 a wavelength of λ det1 The second superluminescent diode (212) is used to generate a first detection light with a wavelength of λ det2 The second probe light, where λ det1 >λ det2 .

6. According to the confocal differential super-resolution PARS microscopy imaging device using multi-wavelength detection light according to claim 1, the first beam splitting module (3) includes a polarization beam splitter (31) and a quarter-wave plate (32); after the detection 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 detection light is transmitted to the beam combining module (4) to be combined with the excitation light.

7. The confocal differential super-resolution PARS microscopic imaging device using multi-wavelength detection light according to claim 1, wherein the variable module (9) is realized by a pinhole.

8. A confocal differential super-resolution photoacoustic microscopy imaging device using variable pinhole modulation of single-path detection light, the imaging device comprising an excitation light module (1), a detection light module (2), a first beam splitting module (3), a beam combining module (4), a scanning and 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) comprises a first photodetector (61), a data acquisition card (62), and a computer (63); The excitation light module (1) is used to generate pulsed excitation light; The detection light module (2) is used to generate a wavelength of λ det1 The first detection light passes through the first beam splitting module (3) and is then combined with the excitation light generated by the excitation light module (1) at the beam combining module (4); The scanning and focusing module (5) co-focuses the received combined light beams on the object to be measured module (7), thereby achieving three-dimensional scanning of the optical focus on the object to be measured; The detection light reflected by the object to be measured module (7) passes through the scanning and focusing module (5), the beam combining module (4) and the first beam splitting module (3) and is then received by the variable module (9); The variable module (9) is used to modulate the point spread function of the received detection light and input it into the first photodetector (61) to realize the detection of the photoacoustic signal; the PARS AC voltage signal collected by the first photodetector (61) is digitally sampled by the data acquisition card (62) and transmitted to the computer (63) for image reconstruction; after completing the first image acquisition, the size in the variable module (9) is changed and the image acquisition is repeated; the photoacoustic images obtained by the two consecutive scans are subjected to differential processing to obtain a super-resolution image, specifically: I sub (x,y)=I det-small (x,y)-α(I det-big (x,y)-I det-small (x,y)) (2) in, I det-big (x, y) corresponds to the photoacoustic image obtained when the size of the variable module (9) is large, I det-small (x, y) corresponds to the photoacoustic image obtained when the size of the variable module (9) is small, and α is an adjustment parameter used to adjust the difference size. 9 . The super-resolution photoacoustic microscopy imaging device according to claim 8 , wherein the variable module 9 is implemented by a pinhole.

10. A confocal differential super-resolution PARS microscopy method based on the apparatus of claim 1 or the apparatus of claim 8, comprising the following steps: Step S1, selecting an excitation light signal; Step S2, selecting continuous lasers with different point spread functions as detection light; Step S3, performing confocal scanning on the detection light in step S2 and the excitation light in step S1; Step S4, obtaining reflection signals of the multi-path detection light; Step S5, obtaining a super-resolution PARS image by differential calculation; Specifically, the difference calculation is performed on the acquired PSF images of different sizes. The specific calculation process is as follows: Select two images corresponding to PSFs of different sizes, subtract the two to obtain an image containing only 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 and obtain an image with a smaller PSF.

Images