Photoacoustic Microscopy Imaging System and Imaging Method for Deep Imaging

Abstract

The present invention discloses a photoacoustic microscopy imaging system and an imaging method for large-depth imaging. The system includes a signal acquisition device and an imaging processing terminal. The signal acquisition device includes: a pulsed laser, a first beam splitter, a first polarizer, a first objective lens, a second beam splitter, a second polarizer, a second objective lens, a helium-neon laser, a polarizer, a dimming glass slide, a first filter, a second filter, a prism, a coupling medium, a first analyzer, a second analyzer, a differential detector, a band-pass filter, and an amplifier. The above photoacoustic microscopy imaging system outputs light beams based on a first optical path and a second optical path, realizes high-throughput and high-resolution focusing of the light beams on the sample through bidirectional excitation of the light beams, and uses a phase-type total internal reflection sensor as a photoacoustic wave detector. By analyzing the change in the refractive index of the coupling medium caused by the photoacoustic signal, broadband and high-sensitivity detection of photoacoustic waves is achieved, greatly improving the imaging quality of the sample.

Description

Technical Field

[0001] The present invention relates to the technical field of optical microscopy imaging, and in particular, to a photoacoustic microscopy imaging system and an imaging method for large-depth imaging. Background Art

[0002] Photoacoustic microscopy imaging technology refers to a means of optical excitation and acoustic detection. By utilizing the optical absorption characteristics of pigment substances in tissue samples for pulsed lasers, light energy is converted into heat energy. Due to the instantaneous thermoelastic effect of the tissue, ultrasonic waves are released. This ultrasonic signal is detected by an ultrasonic detector, and its ultrasonic flight time provides the depth position where the pigment substance is located. By combining an appropriate sample scanning method, a three-dimensional morphological structure image of the sample can be obtained.

[0003] After more than a decade of development, photoacoustic microscopy imaging technology has shown great application potential in fields such as oncology and neuroscience. However, traditional photoacoustic microscopy imaging technology still has many defects to be overcome. First, due to the strong optical attenuation characteristics of tissues for lasers, the penetration depth of photoacoustic microscopy imaging technology is usually about 1 millimeter. This makes it difficult for photoacoustic microscopy imaging technology to accurately obtain the optical absorption characteristics of deep tissues. Second, traditional piezoelectric ultrasonic transducers widely used for photoacoustic signal detection are limited by their own physical properties, with a narrow detection bandwidth (generally only dozens of megahertz), resulting in the longitudinal resolution of photoacoustic microscopy being limited to dozens of micrometers, unable to accurately reflect the depth position of optical absorption substances, affecting the accuracy of depth positioning, and causing serious distortion of three-dimensional images. In addition, the sensitivity of piezoelectric ultrasonic transducers is usually about several hundred pascals, which results in low contrast and poor image quality in photoacoustic imaging, making it difficult to identify tissue samples of small sizes due to the poor imaging quality of the obtained images. Therefore, the existing photoacoustic microscopy imaging technology has problems with poor imaging quality. Summary of the Invention

[0004] Embodiments of the present invention provide a photoacoustic microscopy imaging system and an imaging method for large-depth imaging, aiming to solve the problem of poor imaging quality existing in the existing photoacoustic microscopy imaging technology.

[0005] In a first aspect, an embodiment of the present invention provides a photoacoustic microscopy imaging system for large-depth imaging. The system includes a signal acquisition device and an imaging processing terminal. The signal acquisition device includes: a pulsed laser, a first beam splitter, a first polarizer, a first objective lens, a second beam splitter, a second polarizer, a second objective lens, a helium-neon laser, a polarizer, a dimming glass slide, a first filter, a second filter, a prism, a coupling medium, a first analyzer, a second analyzer, a differential detector, a band-pass filter, and an amplifier. The first beam splitter is disposed downstream of the pulsed laser to split the laser output by the pulsed laser through the first beam splitter. The first polarizer and the first objective lens are disposed in a first optical path between the first beam splitter and the three-dimensional moving stage, and the second polarizer and the second objective lens are disposed in a second optical path between the first beam splitter and the three-dimensional moving stage. The three-dimensional moving stage is used to place a sample to be imaged, the coupling medium covers the sample to be imaged, and the prism is disposed on the coupling medium. The light beams output from the first optical path and the second optical path irradiate the sample to be imaged from the upper and lower sides respectively to obtain a two-dimensional photoacoustic detection signal. The polarizer and the dimming glass slide are disposed in a detection optical path between the helium-neon laser and the prism. The detection light beam output from the dimming glass slide is incident on the prism and undergoes total internal reflection on the bottom surface of the prism. The second beam splitter is disposed at the exit position where the detection light beam is reflected by the prism to split the detected light output through the second beam splitter. The first analyzer and the first filter are disposed in a first feedback optical path between the second beam splitter and the differential detector, and the second analyzer and the second filter are disposed in a second feedback optical path between the second beam splitter and the differential detector. The two detection ports of the differential detector are respectively used to input a first detection light beam output from the first filter and a second detection light beam output from the second filter. The differential detector is electrically connected to the imaging processing terminal through the band-pass filter and the amplifier to output a differential detection signal to the imaging processing terminal, and the imaging processing terminal processes the differential detection signal and the two-dimensional photoacoustic detection signal to obtain a display image of the sample to be imaged.

[0006] For the photoacoustic microscopy imaging system for large-depth imaging, the prism is a trapezoidal prism.

[0007] For the photoacoustic microscopy imaging system for large-depth imaging, the refractive index of the prism is greater than 1.3.

[0008] For the photoacoustic microscopy imaging system for large-depth imaging, the dimming glass slide includes a 1 / 2 glass slide and a 1 / 4 glass slide.

[0009] The photoacoustic microscopy imaging system for large-depth imaging, wherein a first confocal lens is disposed between the first polarizer and the first objective lens; a second confocal lens is disposed between the second polarizer and the second objective lens.

[0010] The photoacoustic microscopy imaging system for large-depth imaging, wherein a long-focus lens is further disposed between the dimming glass slide and the prism.

[0011] The photoacoustic microscopy imaging system for large-depth imaging, wherein the coupling medium is physiological saline, distilled water or deionized water.

[0012] On the other hand, an embodiment of the present invention further provides a photoacoustic microscopy imaging method for large-depth imaging, wherein the photoacoustic microscopy imaging method for large-depth imaging is applied to the above-mentioned photoacoustic microscopy imaging system for large-depth imaging, and the method includes:

[0013] Turn on the pulsed laser to output an adjustable pulsed laser with a specific pulse width and a specific wavelength;

[0014] After the adjustable pulsed laser is split by the first beam splitter, one part of the light beam is adjusted to a linearly polarized light beam by the first optical path and irradiates the sample to be imaged from above, and the other part of the light beam is adjusted to a linearly polarized light beam by the second optical path and irradiates the sample to be imaged from below; the polarization directions of the two light beams irradiating the sample to be imaged are different;

[0015] The two light beams perform two-dimensional plane scanning on the sample to be imaged and detect the change in the refractive index of the coupling medium caused by the photoacoustic wave generated during scanning, so as to obtain a two-dimensional photoacoustic detection signal;

[0016] Turn on the helium-neon laser to output a continuous laser, and after being adjusted to a detection light beam by the detection optical path, the continuous laser is incident on the prism, and the polarized light beam undergoes total internal reflection on the bottom surface of the prism and is split by the second beam splitter;

[0017] One part of the detection light beam output by the second beam splitter is input to one detection port of the differential detector through the first feedback optical path, and the other part of the detection light beam is input to the other detection port of the differential detector through the second feedback optical path;

[0018] The differential detector performs differential detection on the light beams from the first feedback optical path and the second feedback optical path and outputs a differential detection signal to the band-pass filter;

[0019] The band-pass filter filters the noise of the differential detection signal and then outputs it to the amplifier for amplification to obtain a detection amplified signal;

[0020] The imaging processing terminal acquires the detected amplified signal and the two-dimensional photoacoustic detection signal and performs three-dimensional image stacking processing to obtain a display image corresponding to the sample to be imaged.

[0021] The photoacoustic microscopy imaging method for large-depth imaging, wherein the polarization directions of the two light beams irradiating the sample to be imaged are perpendicular to each other.

[0022] The photoacoustic microscopy imaging method for large-depth imaging, wherein the detection light beam output by the light modulation glass sheet is an elliptically polarized light having different phase differences in the vertical component and the horizontal component.

[0023] An embodiment of the present invention provides a photoacoustic microscopy imaging system and an imaging method for large-depth imaging. The system includes a signal acquisition device and an imaging processing terminal. The signal acquisition device includes: a pulsed laser, a first beam splitter, a first polarizer, a first objective lens, a second beam splitter, a second polarizer, a second objective lens, a helium-neon laser, a polarizer, a light modulation glass sheet, a first filter, a second filter, a prism, a coupling medium, a first analyzer, a second analyzer, a differential detector, a band-pass filter, and an amplifier. The above-mentioned photoacoustic microscopy imaging system for large-depth imaging outputs light beams based on a first optical path and a second optical path, and realizes high-throughput and high-resolution focusing of the light beams on the sample through bidirectional excitation light beams, which not only increases the imaging depth of the sample but also ensures the efficient detection of photoacoustic signals; based on the total internal reflection of the prism, the photoacoustic waves generated by the detection light beam after the laser excites the sample interact with the evanescent field during propagation in the coupling medium, thereby causing a phase change of the detection light beam. The change in the light intensity of the detection light beam is detected by a differential detector, realizing highly sensitive detection of broadband photoacoustic waves, and this solution can transmit the excitation light to the tissue sample with high optical transmittance, thereby enabling bidirectional excitation of pulsed lasers, increasing the photoacoustic imaging depth, and greatly improving the imaging quality of the sample. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 It is a schematic structural diagram of the photoacoustic microscopy imaging system for large-depth imaging provided by the embodiment of the present invention;

[0026] Figure 2 It is a schematic flow diagram of the photoacoustic microscopy imaging method for large-depth imaging provided by the embodiment of the present invention;

[0027] Figure 3Schematic diagram of the effect of the photoacoustic microscopy imaging system for large-depth imaging provided by the embodiments of the present invention. Detailed implementation manners

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] It should be understood that when used in this specification and the appended claims, the terms "comprises" and "comprising" indicate the presence of the described features, wholes, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or their combinations.

[0030] It should also be understood that the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in the specification of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" are intended to include the plural forms.

[0031] It should be further understood that the term "and / or" used in the specification of the present invention and the appended claims refers to any combination and all possible combinations of one or more of the related listed items, and includes these combinations.

[0032] In this embodiment, please refer to Figure 1 , Figure 1Schematic structural diagram of a photoacoustic microscopy imaging system for large-depth imaging provided by an embodiment of the present invention. As shown in the figure, an embodiment of the present invention provides a photoacoustic microscopy imaging system for large-depth imaging. The system includes a signal acquisition device and an imaging processing terminal 20. The signal acquisition device includes: a pulsed laser 1, a first beam splitter 2, a first polarizer 11, a first objective lens 12, a second beam splitter 3, a second polarizer 21, a second objective lens 22, a helium-neon laser 4, a polarizer 5, a dimming glass sheet 6, a first filter 31, a second filter 41, a prism 7, a coupling medium 8, a first analyzer 32, a second analyzer 42, a differential detector 9, a band-pass filter 18, and an amplifier 19. The first beam splitter 2 is arranged downstream of the pulsed laser 1 to split the laser output by the pulsed laser 1 through the first beam splitter 2. The first polarizer 11 and the first objective lens 12 are arranged in the first optical path between the first beam splitter 2 and the three-dimensional moving stage 101, and the second polarizer 21 and the second objective lens 22 are arranged in the second optical path between the first beam splitter 2 and the three-dimensional moving stage 101. Among them, a first confocal lens 13 is arranged between the first polarizer 11 and the first objective lens 12; a second confocal lens 23 is arranged between the second polarizer 21 and the second objective lens 22. The three-dimensional moving stage 101 is used to place the sample to be imaged 10, the coupling medium 8 covers the sample to be imaged 10, and the prism 7 is arranged on the coupling medium 8. The light beam output from the first optical path and the light beam output from the second optical path irradiate the sample to be imaged 10 from the upper and lower sides respectively to obtain a two-dimensional photoacoustic detection signal.

[0033] The pulsed laser 1 can be a Nd:YAG (Neodymium-doped Yttrium Aluminium Garnet; Nd:Y3Al5O12, yttrium aluminum garnet crystal) pulsed laser. The pulsed laser can be used to generate picosecond-level photoacoustic excitation light. The first beam splitter 2 is used to divide the pulsed laser output by the pulsed laser into two beams, one beam is transmitted through the first optical path, and the other beam is transmitted through the second optical path. The polarizer is used to adjust the polarization direction of the incident beam, the lens is used for focusing or collimation, and the objective lens is used for focusing the beam. The pulsed laser in the first optical path passes through the first polarizer 11 and outputs linearly polarized light. The linearly polarized light in the first optical path can be expanded by a 4F optical system composed of a pair of confocal lenses. The linearly polarized light in the first optical path is reflected by the mirror to change the beam direction and enters the first objective lens 12 from top to bottom, passes through the prism 7 and the coupling medium 8, and then is focused and irradiated on the surface of the sample 10 to be imaged. The pulsed laser in the second optical path passes through the second polarizer 21 and outputs linearly polarized light. The linearly polarized light in the second optical path can be expanded by a 4F optical system composed of a pair of confocal lenses. The linearly polarized light in the second optical path is reflected by the mirror to change the beam direction and enters the second objective lens 22 from bottom to top, and is focused and irradiated on the back of the sample 10 to be imaged. Thus, a two-way excitation light illumination method is realized to simultaneously excite the sample from above and below the sample to be imaged to generate photoacoustic signals.

[0034] In the detection optical path between the helium-neon laser 6 and the prism 7, the polarizer 5 and the beam adjusting glass sheet 6 are provided; the detection beam output by the beam adjusting glass sheet 6 is incident on the prism 7 and undergoes total internal reflection on the bottom surface of the prism 7; the second beam splitter 3 is arranged at the output position where the detection beam is reflected by the prism 7 to split the detected light output; in the first feedback optical path between the second beam splitter 3 and the differential detector 9, the first analyzer 32 and the first filter 31 are provided, and in the second feedback optical path between the second beam splitter 3 and the differential detector 9, the second analyzer 42 and the second filter 41 are provided; the two detection ports provided by the differential detector 9 are respectively used to input the first detection beam output by the first filter 31 and the second detection beam output by the second filter 41. Specifically, the beam adjusting glass sheet 6 includes a 1 / 2 glass sheet 61 and a 1 / 4 glass sheet 62. Specifically, a long-focus lens 71 is further provided between the beam adjusting glass sheet 6 and the prism 7. Among them, the coupling medium 8 can be normal saline, distilled water or deionized water. In actual use, distilled water is preferably selected, and deionized water is selected in the optimal embodiment. Specifically, the long-focus lens 71 can be used to slightly focus the detection beam output by the beam adjusting glass sheet 6.

[0035] More specifically, the prism 7 is a trapezoidal prism. Among them, the refractive index of the prism 7 is greater than 1.3. Based on the prism 7 and the coupling medium 8, a TIR (total internal reflection) photoacoustic detector can be constructed. In a specific embodiment, the prism 7 can be set as an isosceles trapezoidal prism, so as to improve the symmetry of the light beam propagating in the prism 7 and improve the accuracy of subsequent imaging. In addition, the prism can also be set as a prism with other angles, such as a regular hexagonal prism, etc. The refractive index of the prism 7 can be set to be greater than 1.3. A reflection interface is formed between the prism 7 (optically dense medium) and the coupling medium 8 (optically sparse medium). When the incident light is incident on the interface at a specific angle, total internal reflection will occur when the incident angle is greater than the critical angle. For example, a prism can be made of K9 glass with a refractive index n = 1.51509. In order to streamline the optical path design, a special-shaped trapezoidal prism can be customized using K9 glass (n K9 = 1.51509) so that the incident light beam can enter and exit parallelly, and it is ensured that the incident angle at this time is exactly the total internal reflection angle. The specific effect is as shown in Figure 3 shown, Figure 3 in which ∠A is 114.66°, ∠B is 155.34°, the upper side length L1 of the trapezoidal prism is 12.74 mm, the lower side length L2 is 40 mm, the total height H1 is 10 mm, and the side height H2 is 3.743 mm.

[0036] In this embodiment, the detection of photoacoustic signals uses phase-type total internal reflection sensing technology. After the continuous laser is adjusted by the polarizer 5 and the light-adjusting glass sheet 6, elliptically polarized light is obtained. The polarized light beam is slightly focused by the long-focus lens 71 and incident on the prism 7, and total internal reflection occurs on the bottom surface of the prism 7. After the reflected light beam is adjusted by the lens, it is divided into two beams of light by the second beam splitter 3. The two beams of light are respectively transmitted to the two detection ports of the differential detector through the first feedback optical path and the second feedback optical path.

[0037] Adopting a bidirectional excitation light illumination method to simultaneously excite the sample to generate photoacoustic signals from above and below the sample to be imaged, and at the same time using a phase-mode total internal reflection optical surface wave sensor to realize the detection of photoacoustic signals, the photoacoustic microscopy technology can observe the microscopic morphological structure characteristics in a large depth range without labeling, which is beneficial to accurately obtaining the physiological / pathological information of tissues.

[0038] The differential detector 9 is electrically connected to the imaging processing terminal 20 through the band-pass filter 18 and the amplifier 19 to output a differential detection signal to the imaging processing terminal 20. The imaging processing terminal 20 processes the differential detection signal and the two-dimensional photoacoustic detection signal to obtain the display image of the sample to be imaged.

[0039] During the photoacoustic imaging process, the sample 10 to be imaged is fixed on a three-dimensional moving stage 101, so that two light beams are focused on the sample 10 to be imaged for two-dimensional plane scanning to obtain the two-dimensional photoacoustic detection signals required for the three-dimensional image stack. The photoacoustic waves generated during scanning are coupled by a coupling medium (aqueous solution), which in turn causes a change in the refractive index of the coupling medium. By detecting this change, the detection of photoacoustic signals can be achieved.

[0040] The technical method in this embodiment has the following characteristics: 1. Establish a two-way excitation light illumination method to simultaneously excite photoacoustic signals from the upper and lower sides of the biological sample, achieving a significant increase in the depth of photoacoustic imaging. 2. Establish a photoacoustic detection method based on phase-mode total internal reflection (TIR) optical surface wave sensing to achieve high-sensitivity and broadband photoacoustic signal detection, improve the depth resolution of photoacoustic microscopy imaging technology, and accurately locate the depth position of the microstructure of the sample to be observed. At the same time, this sensor has the characteristic of optical transparency, allowing two-way illumination of the photoacoustic excitation beam.

[0041] Compared with the traditional photoacoustic microscopy imaging technology, the two-way excitation light illumination method can increase the depth of photoacoustic imaging by up to two times at most; the photoacoustic detection method based on phase-type optical surface wave sensing increases the detection bandwidth to hundreds of megahertz, and the detection sensitivity increases to the level of dozens of pascals, which is significantly better than the piezoelectric ultrasonic transducer. The two innovative technologies are integrated into the new photoacoustic microscopy imaging system, achieving simultaneous improvement in imaging depth and longitudinal resolution, so that high-longitudinal-resolution and large-depth imaging of biological tissues can be performed, providing a reliable technical means for observing the three-dimensional morphological structure of thick tissue samples.

[0042] Please refer to Figure 2 , Figure 2 which is a schematic flow chart of the photoacoustic microscopy imaging method for large-depth imaging provided by the embodiment of the present invention. The embodiment of the present invention also provides a photoacoustic microscopy imaging method for large-depth imaging, wherein the photoacoustic microscopy imaging method for large-depth imaging is applied to the above-mentioned photoacoustic microscopy imaging system for large-depth imaging, as Figure 2 shown, and the method includes steps S110 - S180.

[0043] S110. Turn on the pulsed laser to output an adjustable pulsed laser with a specific pulse width and a specific wavelength.

[0044] Specifically, in this embodiment, the pulsed laser can generate tunable pulsed laser with a specific wavelength ranging from 150 to 400 nm. The pulse width of the generated pulsed laser is adjustable, and the specific pulse width ranges from 50 to 900 picoseconds. For a certain specific tissue sample, tunable pulsed laser with a specific wavelength of 266 nm and a specific pulse width of 800 ps (picoseconds) can be used. The wavelength of the photoacoustic excitation light is not limited to 266 nm. For different samples, the wavelength can be changed to maximize the absorption coefficient of the sample and generate the strongest photoacoustic signal.

[0045] S120. After the tunable pulsed laser is split by the first beam splitter, one part of the light beam is adjusted to a linearly polarized light beam by the first optical path and irradiates the sample to be imaged from above, and the other part of the light beam is adjusted to a linearly polarized light beam by the second optical path and irradiates the sample to be imaged from below; the polarization directions of the two light beams irradiating the sample to be imaged are different.

[0046] The two light beams irradiating the sample to be imaged are not necessarily linearly polarized light perpendicular to each other, as long as the polarization directions are different, that is, they can be any two light beams with different vibration directions (elliptical or circularly polarized light is acceptable), aiming for the highest signal and the maximum imaging depth. In the optimal embodiment, the polarization directions of the two light beams irradiating the sample to be imaged can be set to be perpendicular to each other.

[0047] S130. The two light beams perform two-dimensional planar scanning on the sample to be imaged and detect the change in the refractive index of the coupling medium caused by the photoacoustic wave generated during scanning, obtaining a two-dimensional photoacoustic detection signal.

[0048] S140. Turn on the helium-neon laser to output continuous laser, which is adjusted to a detection light beam by the detection optical path and then incident on the prism. The polarized light beam undergoes total internal reflection on the bottom surface of the prism and is split by the second beam splitter.

[0049] The wavelength of the continuous laser output by the helium-neon laser can be 450 - 750 nm. When detecting a certain sample to be imaged, the helium-neon laser can be controlled to output continuous laser with a wavelength of 632.8 nm. Among them, the detection light beam output by the light control glass sheet is an elliptically polarized light with different phase differences in the vertical component and the horizontal component. The detection light wavelength is not limited to 632.8 nm either, and different detection light wavelengths correspond to different excitation angles.

[0050] S150. One part of the detection light beam output by the second beam splitter is input to one detection port of the differential detector through the first feedback optical path, and the other part of the detection light beam is input to the other detection port of the differential detector through the second feedback optical path.

[0051] S160. The differential detector performs differential detection on the light beams from the first feedback optical path and the second feedback optical path to obtain a differential detection signal and outputs it to the band-pass filter.

[0052] S170. The band-pass filter filters the noise of the differential detection signal and then outputs it to the amplifier for amplification to obtain a detected amplified signal.

[0053] S180. The imaging processing terminal acquires the detected amplified signal and the two-dimensional photoacoustic detection signal and performs three-dimensional image stacking processing to obtain a display image corresponding to the sample to be imaged.

[0054] In the specific application process, the imaging processing terminal can be electrically connected to the pulsed laser and the helium-neon laser respectively, so that corresponding parameters can be input through the imaging processing terminal to adjust the pulsed laser output by the pulsed laser and the continuous laser output by the helium-neon laser. In addition, the imaging processing terminal can also be electrically connected to the three-dimensional moving stage, so that corresponding moving parameters can be input through the imaging processing terminal to control the precision electric three-dimensional moving stage to perform three-dimensional movement. The imaging processing terminal can be a desktop computer, a laptop computer, a tablet computer and other terminal devices that can be used for data and image processing.

[0055] The photoacoustic microscopy imaging method for large-depth imaging in this embodiment has the following characteristics: 1. Establish a bidirectional excitation optical path to increase the imaging depth. This method utilizes the efficient focusing and conduction of the excitation light beam by optical elements and the optical transparency of the ultrasonic detector, enabling the bidirectional excitation light beam to be focused on the tissue sample with high throughput and high resolution, which not only increases the imaging depth of the tissue sample but also ensures the efficient detection of photoacoustic signals. 2. Photoacoustic signal detection based on a total internal reflection phase TIR sensor. After the laser excites the sample, the generated photoacoustic wave propagates in the coupling medium and interacts with the evanescent field, thereby changing the phase change of the detected light. The change in polarization light intensity is detected and analyzed by the differential detection method, realizing the highly sensitive detection of broadband photoacoustic waves. In addition, the sensor has the characteristic of optical transparency, enabling the excitation light to be transmitted to the tissue sample with high optical transmittance, so that bidirectional excitation of pulsed laser can be realized and the photoacoustic imaging depth can be increased. The above design realizes the large-depth excitation and efficient detection of photoacoustic signals of biological tissues, enabling the new photoacoustic microscopy imaging technology to accurately observe the three-dimensional microscopic morphological structure of tissue samples.

[0056] In an embodiment of the present invention, there is provided a photoacoustic microscopy imaging system and an imaging method for large-depth imaging. The system includes a signal acquisition device and an imaging processing terminal. The signal acquisition device includes: a pulsed laser, a first beam splitter, a first polarizer, a first objective lens, a second beam splitter, a second polarizer, a second objective lens, a helium-neon laser, a polarizer, a dimming glass slide, a first filter, a second filter, a prism, a coupling medium, a first analyzer, a second analyzer, a differential detector, a band-pass filter, and an amplifier. The above-mentioned photoacoustic microscopy imaging system for large-depth imaging outputs light beams based on a first optical path and a second optical path, and realizes high-throughput and high-resolution focusing of the light beams on the sample through bidirectional excitation of the light beams, which not only increases the imaging depth of the sample but also ensures the efficient detection of photoacoustic signals; based on the total internal reflection of the prism, after the laser excites the sample, the photoacoustic waves generated by the detection beam interact with the evanescent field during propagation in the coupling medium, thereby causing a phase change of the detection beam. The change in the light intensity of the detection beam is detected by a differential detector, realizing highly sensitive detection of broadband photoacoustic waves. Moreover, this solution can transmit the excitation light to the tissue sample with high optical transmittance, thereby enabling bidirectional excitation of the pulsed laser, increasing the photoacoustic imaging depth, and greatly improving the imaging quality of the sample.

[0057] As described above, the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of various equivalent modifications or substitutions, and these modifications or substitutions should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A photoacoustic microscopy imaging system for large-depth imaging, characterized in that, The system includes a signal acquisition device and an imaging processing terminal. The signal acquisition device includes: a pulsed laser, a first beam splitter, a first polarizer, a first objective lens, a second beam splitter, a second polarizer, a second objective lens, a helium-neon laser, a polarizer, a dimming glass slide, a first filter, a second filter, a prism, a coupling medium, a first analyzer, a second analyzer, a differential detector, a band-pass filter, and an amplifier; The first beam splitter is arranged downstream of the pulsed laser to split the laser output by the pulsed laser through the first beam splitter; The first polarizer and the first objective lens are arranged in the first optical path between the first beam splitter and the three-dimensional moving stage, and the second polarizer and the second objective lens are arranged in the second optical path between the first beam splitter and the three-dimensional moving stage; The three-dimensional moving stage is used to place the sample to be imaged, the coupling medium covers the sample to be imaged, and the prism is arranged on the coupling medium; the light beams output from the first optical path and the second optical path irradiate the sample to be imaged from the upper and lower sides respectively to obtain a two-dimensional photoacoustic detection signal; The polarizer and the dimming glass slide are arranged in the detection optical path between the helium-neon laser and the prism; the detection light beam output from the dimming glass slide is incident on the prism and undergoes total internal reflection on the bottom surface of the prism; The second beam splitter is arranged at the exit position where the detection light beam is reflected by the prism to split the detected light beam output through the second beam splitter; The first analyzer and the first filter are arranged in the first feedback optical path between the second beam splitter and the differential detector, and the second analyzer and the second filter are arranged in the second feedback optical path between the second beam splitter and the differential detector; the two detection ports arranged on the differential detector are respectively used for inputting the first detection light beam output from the first filter and the second detection light beam output from the second filter; The differential detector is electrically connected to the imaging processing terminal through the band-pass filter and the amplifier to output a differential detection signal to the imaging processing terminal, and the imaging processing terminal processes the differential detection signal and the two-dimensional photoacoustic detection signal to obtain a display image of the sample to be imaged.

2. The photoacoustic microscopy imaging system for large-depth imaging according to claim 1, wherein The prism is a trapezoidal prism.

3. The photoacoustic microscopy imaging system for deep imaging according to claim 1 or 2, characterized in that, The refractive index of the prism is greater than 1.

3.

4. The photoacoustic microscopy imaging system for large-depth imaging according to claim 3, wherein, The dimming glass slide includes a half-wave plate and a quarter-wave plate.

5. The photoacoustic microscopy imaging system for large-depth imaging according to claim 3, wherein A first confocal lens is arranged between the first polarizer and the first objective lens; a second confocal lens is arranged between the second polarizer and the second objective lens.

6. The photoacoustic microscopy imaging system for large-depth imaging according to claim 3, wherein, A long-focus lens is also arranged between the dimming glass slide and the prism.

7. The photoacoustic microscopy imaging system for deep imaging according to claim 3, wherein, The coupling medium is physiological saline, distilled water or deionized water.

8. A photoacoustic microscopy imaging method for large-depth imaging, characterized in that, The photoacoustic microscopy imaging method for large-depth imaging is applied to the photoacoustic microscopy imaging system for large-depth imaging as described in any one of claims 1-7. The method includes: Turn on the pulsed laser to output an adjustable pulsed laser with a specific pulse width and a specific wavelength; After the adjustable pulsed laser is split by the first beam splitter, a part of the light beam is adjusted into a linearly polarized light beam through the first optical path and irradiates the sample to be imaged from above, and another part of the light beam is adjusted into a linearly polarized light beam through the second optical path and irradiates the sample to be imaged from below; the polarization directions of the two light beams irradiating the sample to be imaged are different; The two light beams perform two-dimensional planar scanning on the sample to be imaged and detect the change in the refractive index of the coupling medium caused by the photoacoustic wave generated during scanning, and obtain a two-dimensional photoacoustic detection signal; Turn on the helium-neon laser to output a continuous laser, which is adjusted into a detection light beam through the detection optical path and then incident on the prism. The polarized light beam undergoes total internal reflection on the bottom surface of the prism and is split by the second beam splitter; A part of the detection light beam output by the second beam splitter is input into one detection port of the differential detector through the first feedback optical path, and another part of the detection light beam is input into the other detection port of the differential detector through the second feedback optical path; The differential detector performs differential detection on the light beams from the first feedback optical path and the second feedback optical path to obtain a differential detection signal and outputs it to the band-pass filter; The band-pass filter filters the noise of the differential detection signal and then outputs it to the amplifier for amplification to obtain a detection amplified signal; The imaging processing terminal acquires the detection amplified signal and the two-dimensional photoacoustic detection signal and performs three-dimensional image stacking processing to obtain a display image corresponding to the sample to be imaged.

9. The photoacoustic microscopy imaging method for large-depth imaging according to claim 8, wherein The polarization directions of the two light beams irradiating the sample to be imaged are perpendicular to each other.

10. The photoacoustic microscopy imaging method for large-depth imaging according to claim 8, characterized in that, The detection light beam output through the light-adjusting glass sheet is an elliptically polarized light beam with different phase differences in the vertical component and the horizontal component.

Images