A method for studying tissue hardness based on substrate photoacoustic and application thereof

Abstract

The application provides a kind of based on substrate photoacoustic tissue hardness research method and application.Black tape or mark sign with strong optical absorption is pasted or drawn on the slide as substrate absorption, then the target object to be detected hardness is pasted on the slide, the marker is imaged using photoacoustic microscopic imaging, and the corresponding elastic information of the target object is analyzed by analyzing the time difference of photoacoustic signal arrival.The application inversely deduces the related elastic information of the target object by the transmission time difference of substrate (strong absorption) photoacoustic signal in different detection target objects, without considering the optical absorption of the target object itself, and is suitable for viscoelasticity research of non-optical absorption or weak absorption material.The application can ensure the uniformity of the thickness of the tissue sample during the slicing process, and the combination of the substrate photoacoustic method makes the measurement result more accurate.The method provided by the application is simple and fast in operation, has wide application prospect, and can be used for obtaining the elastic information of transparent non-detection wavelength optical absorption material and biological tissue.

Description

Technical Field

[0001] This invention relates to the field of photoacoustic imaging, and more particularly to a method and application for studying tissue stiffness based on substrate photoacoustics. Background Technology

[0002] Cancer is one of the most common diseases today, with extremely high recurrence and mortality rates. During its evolution, a tumor undergoes changes in multiple structural and functional aspects, including angiogenesis, changes in blood oxygen saturation, and tissue elasticity. Many studies have found that tumor evolution is accompanied by changes in the microvascular network at the tumor site; simultaneously, the increased oxygen consumption required for angiogenesis leads to a hypoxic environment and the accumulation of oxides at the tumor site; furthermore, changes in pH and stiffness / elasticity parameters occur at the tumor site. Comprehensive research on tumor tissue and aberrant tissue is crucial for understanding these underlying mechanisms.

[0003] Currently, research on mechanical properties such as hardness and elasticity parameters can be mainly divided into macroscopic and microscopic cellular scale studies. A common method for studying the hardness of organisms from a macroscopic perspective is palpation by doctors, who typically assess the hardness of corresponding areas to determine the presence of lesions. However, this diagnostic method is highly dependent on the doctor's experience, leading to inaccurate or delayed diagnoses, and it is limited to superficial lesions. For the examination of soft tissues and organs within the body, ultrasound elastography is used clinically to indirectly reflect changes in soft tissue hardness. Different tissues have different elastic coefficients, resulting in varying degrees of deformation under external pressure, leading to temporal differences in the received ultrasound signals. Further information extraction and image processing methods are used to ultimately reflect the differences in soft tissue hardness within the body through images. However, ultrasound elastography itself has low contrast; additionally, early, slight changes in the hardness of organs may lead to inaccurate diagnoses due to the insufficient sensitivity of ultrasound elastography. For the study of hardness at the microscopic scale, such as in cells, atomic force microscopy has made significant contributions. Atomic force microscopy (AFM) utilizes probes attached to its probe to contact the sample surface. Due to variations in sample hardness, the probes bend and deflect to varying degrees; this deformation is then detected and analyzed. AFM uses the degree of probe deformation to indicate the hardness of the sample. However, the main problems with AFM are its small imaging range, slow speed, and significant dependence on the probe itself.

[0004] Photoacoustic imaging, as a novel imaging modality, utilizes the absorption of pulsed light by biological tissues, causing localized temperature rise and thermoelastic expansion. This converts the strongly scattered light within the tissue into weakly scattered ultrasound at a fraction of one-thousandth of the original intensity. Combining the advantages of optical contrast and ultrasonic resolution, it is currently the only medium capable of simultaneously acquiring comprehensive imaging information of tissues at the morphological, functional, and molecular levels. Furthermore, it enables multi-dimensional imaging from microscopic to macroscopic scales, providing a promising strategy for overcoming the aforementioned challenges. Current research on the effects of photoacoustic imaging on tissue elasticity has been reported. Yue et al. established a physical model of tissue viscoelasticity and photoacoustic signal time delay based on photoacoustic viscoelastic imaging and successfully verified the relationship between the viscoelasticity of different samples and the photoacoustic signal time delay.

[0005] Currently, traditional ultrasonic viscoelastic imaging has low resolution, while atomic force microscopy (AFM) is complex to detect mechanical properties and is only applicable to microscopic materials. Furthermore, traditional methods require perfectly uniform sample thickness to ensure that the photoacoustic signal delay is due to inconsistent viscoelasticity within the sample itself, rather than errors caused by inconsistent distances between the ultrasonic transducer and the sample. Existing photoacoustic viscoelasticity research methods also have limitations. For example, they can only detect substances with inherent optical absorption and are unsuitable for substances with weak or no optical absorption at the test wavelength. In such cases, the illumination wavelength cannot generate a photoacoustic signal, and further analysis based on the absorbed photoacoustic signal is impossible.

[0006] Therefore, existing technologies still have shortcomings. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method and application for studying tissue hardness based on substrate photoacoustics, which aims to solve the problem that current photoacoustic viscoelasticity research methods can only detect targets with optical absorption, and are not applicable to targets with no absorption or weak absorption.

[0008] The technical solution of the present invention is as follows:

[0009] A method for studying tissue stiffness based on substrate photoacoustics, comprising the following steps:

[0010] 1.1 Provide a glass slide on which the optical absorption substrate is marked;

[0011] 1.2 Section the tissue to be tested and place the cut tissue section sample on the optical absorption substrate position marked in advance on the glass slide;

[0012] 1.3 The tissue section sample provided in step 1.2 was imaged using a photoacoustic microscope, and the obtained photoacoustic signals were analyzed.

[0013] The method for studying tissue stiffness based on substrate photoacoustics, wherein step 1.1, the specific step of marking the optical absorption substrate on the glass slide, is as follows:

[0014] Draw a uniform long line in the middle of the slide with a marker, or cover the middle of the slide evenly with black tape. The middle of the slide is the optical absorption substrate in photoacoustic imaging.

[0015] The method for studying tissue stiffness based on substrate photoacoustics, wherein in step 1.2, tissue sections of a fixed thickness are cut from the tissue to be tested using frozen sections.

[0016] The method for studying tissue stiffness based on substrate photoacoustics, wherein step 1.3, the specific steps for analyzing the obtained photoacoustic signal, are as follows:

[0017] The structural information of the tissue section sample is analyzed by observing the maximum amplitude projection view; the corresponding elastic information of the tissue section sample is analyzed by detecting the time difference of photoacoustic signal arrival.

[0018] An application of a substrate photoacoustic-based method for studying tissue stiffness, wherein any of the methods described above are applied to the study of tissue stiffness and viscoelasticity.

[0019] The application of the substrate photoacoustic-based tissue stiffness research method is specifically applied to the study of tissue stiffness and viscoelasticity in biological tissues and substances with no or weak optical absorption.

[0020] The application of the substrate photoacoustic-based tissue stiffness research method is particularly applicable to the study of biological tissue boundary delineation or fibrosis degree.

[0021] Beneficial Effects: This invention provides a method and application for studying tissue hardness based on substrate photoacoustics. Substrate photoacoustics refers to using the difference in photoacoustic signals of the carrier of the target object, rather than the target object itself, to study the hardness information of the target object. In this invention, a black tape or marker with strong optical absorption is affixed or drawn on a glass slide as a substrate for absorption. The target object whose hardness needs to be tested is then attached to the slide. Photoacoustic microscopy is used to image the marker. This allows for the capture of structural information of the tissue section by analyzing the maximum amplitude projection view, and also allows for the analysis of the elastic information of the tissue section sample by analyzing the difference in the arrival time of the photoacoustic signals. This invention determines the hardness information of different tissues by analyzing the delay of the photoacoustic signal, and infers the relevant elastic information by using the time difference of the transmission of the substrate (strong absorption) photoacoustic signal in different target objects. Therefore, it does not require consideration of whether the target object itself has optical absorption, overcoming the shortcomings of traditional photoacoustic viscoelasticity studies that require optical absorption of the tissue sample. It is applicable to viscoelasticity studies of materials with no or weak optical absorption, and has particularly promising applications in the viscoelasticity studies of transparent tissues. Meanwhile, the slicing process of this invention ensures uniform tissue sample thickness, eliminating the need to consider inaccurate calculations caused by varying tissue thicknesses affecting the distance from the ultrasound transducer. Combined with the substrate photoacoustic method, this results in more accurate measurements. Furthermore, the method provided by this invention is simple and rapid to operate, with broad application prospects. It can be used not only for studying viscoelastic parameters of common biological tissues but also for rapid, real-time tissue boundary delineation and for refining the degree of liver cirrhosis, liver fibrosis, and pulmonary fibrosis, among other things. Attached Figure Description

[0022] Figure 1 A schematic diagram illustrating the principle and steps of the substrate photoacoustic-based tissue stiffness research method provided in this embodiment of the invention.

[0023] Figure 2 This is a schematic diagram of the standardized analysis results of the tissue stiffness research method based on substrate photoacoustics provided in an embodiment of the present invention.

[0024] Figure 3 This is a schematic diagram illustrating the results of applying the substrate photoacoustic-based tissue hardness research method provided in this embodiment of the invention to the study of tissue hardness and viscoelasticity. Detailed Implementation

[0025] This invention provides a method and application for studying tissue stiffness based on substrate photoacoustics. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0026] This invention provides a method for studying tissue stiffness based on substrate photoacoustics, comprising the following steps:

[0027] S10. Provide a glass slide and mark the optical absorption substrate on the glass slide;

[0028] S20. Slice the tissue to be tested and place the cut tissue slices on the optical absorption substrate position marked in advance on the glass slide.

[0029] S30. Image the tissue section sample provided in step S20 using a photoacoustic microscope, and analyze the obtained photoacoustic signal.

[0030] The tissue stiffness research method based on substrate photoacoustics provided in this invention aims to overcome many shortcomings of traditional photoacoustic methods. Traditional methods require the target object to have optical absorption to generate a detectable photoacoustic signal, making them unsuitable for targets with no or weak absorption, especially transparent and non-absorbing targets. For example, tissue sections are typically only tens to hundreds of micrometers thick, appearing transparent to the naked eye, and exhibiting very weak optical absorption. Traditional photoacoustic methods are insufficient to generate a sufficiently strong photoacoustic signal for further analysis, making it difficult to extract the characteristics of the tissue section. Currently, commonly used tissue sections mainly include paraffin sections and frozen sections. Paraffin section preparation involves numerous procedures and steps, requiring several days to complete one cycle, which is time-consuming; frozen sections are relatively faster, but staining is required to determine boundaries similar to tumors, and the staining process is also cumbersome and time-consuming. Therefore, to overcome and solve the shortcomings of traditional photoacoustic methods, this invention designs a novel photoacoustic method for acquiring elasticity information of transparent substances and biological tissues with no detection wavelength optical absorption.

[0031] Figure 1The diagram illustrates the principle and steps of the substrate photoacoustic method for studying tissue hardness in this invention. Substrate photoacoustics refers to using the difference in photoacoustic signals from the carrier of the target object, rather than the target object itself, to study the hardness information of the target object. In this invention, a black adhesive tape or marker with strong optical absorption is first affixed or drawn on a glass slide as a substrate absorber. Then, the target object whose hardness needs to be tested is directly placed on the strongly absorbing carrier. The relevant elasticity information is inferred by the time difference in the transmission of the substrate (strong absorption) photoacoustic signal in different target objects. The principle is based on the different propagation speeds of the photoacoustic signals generated by the substrate absorption, rather than the absorption of the material itself, in different target objects, resulting in different arrival times of the photoacoustic signals at the transducer. The elastic parameters of the target object are inferred by using the time difference of the photoacoustic signals. Therefore, it is not necessary to consider whether the target object itself has optical absorption, and it can be applied to transparent tissues or weakly absorbing target objects. Furthermore, since the position of the substrate is the same from the receiving end, the operation is relatively simple, and there is no need to consider how to ensure sample thickness. Due to its simple and rapid operation, it holds promise for research on rapid real-time intraoperative tumor tissue boundary delineation and liver fibrosis degree.

[0032] In some embodiments, step S10, specifically marking the optical absorption substrate on the slide, involves drawing a uniform long line in the middle of the slide with a marker, or uniformly covering the middle of the slide with black tape. The middle of the slide is the optical absorption substrate in photoacoustic imaging. Figure 1 As shown, markings are made on the glass slide using a marker or black tape to serve as the optical absorption substrate in photoacoustic imaging. Therefore, this method does not require the tissue or sample to have optical absorption properties.

[0033] In some embodiments, in step S20, a tissue section sample of fixed thickness is cut from the tissue to be tested using a frozen section.

[0034] Specifically, a tissue section of fixed thickness is cut from the tissue to be tested using a cryostat. There is no strict requirement for the sample thickness; common cryostat thicknesses of 10 micrometers, 20 micrometers, and 50 micrometers are all suitable. It is only necessary to ensure that different tissues are cut simultaneously. The method provided in this embodiment of the invention can be used to detect transparent tissues, therefore, staining of the tissue sections is unnecessary, and analysis of transparent tissue sections can be quickly achieved directly using cryostat technology. First, the tissue to be tested is cut into sections of fixed thickness using a cryostat, and the tissue sections are placed along the marked lines or the area covered by black tape, such as... Figure 1As shown. During this process, the tissue sections can be positioned at different angles relative to the marker lines or the direction covered by black tape to ensure that all information is captured during imaging. Because the tissue samples are directly cut from a cryostat, the tissue thickness is uniform, thus avoiding the problem of uneven tissue thickness distribution affecting the distance to the ultrasound transducer and leading to inaccurate measurements.

[0035] In some embodiments, the specific steps of analyzing the obtained photoacoustic signal in step S30 are as follows: analyzing the structural information of the tissue section sample by observing the maximum amplitude projection view; and analyzing the corresponding elastic information of the tissue section sample by detecting the time difference of arrival of the photoacoustic signal.

[0036] Specifically, the tissue section sample provided in step S20 is imaged using a photoacoustic microscope. The structural information of the tissue is observed by analyzing the maximum amplitude projection view. This acquisition of structural information is similar to the principle of using an inverted optical microscope, where the structural information is reflected by the varying degrees of penetration of emitted light into different tissues. Furthermore, the elastic information corresponding to the tissue section sample can be analyzed by examining the differences in the arrival time of the photoacoustic signals. Figure 1 As shown, if the photoacoustic signal from tissue 1 arrives before that from tissue 2, it indicates that the photoacoustic signal propagates faster in tissue 1, suggesting that tissue 1 has greater hardness. Specific hardness parameters can be calibrated and quantified. Dual-modal information on structure and hardness can provide more accurate verification information. Especially when the structural information of different parts of a tissue sample is not obvious, due to the sufficiently high sampling rate of the photoacoustic microscopy system, even slight differences in hardness can be reflected in the difference in the arrival time of the photoacoustic signal. Therefore, the method provided by this invention can serve as a powerful auxiliary tool for rapid tissue analysis.

[0037] This invention also provides an application of a substrate photoacoustic method for studying tissue stiffness, which applies any of the methods described above to the study of tissue stiffness and viscoelasticity.

[0038] In some embodiments, the aforementioned substrate photoacoustic-based tissue stiffness research method is applied to the study of tissue stiffness and viscoelasticity in materials and biological tissues with no or weak optical absorption.

[0039] Traditional photoacoustic methods require the target object to have optical absorption to generate a detectable photoacoustic signal, making them unsuitable for transparent, non-absorbing targets. For example, tissue sections, typically only tens to hundreds of micrometers thick, are transparent to the naked eye and have very weak optical absorption. Traditional photoacoustic methods are insufficient to generate a sufficiently strong photoacoustic signal for further analysis. This invention provides a substrate-based photoacoustic method for studying tissue stiffness. It designs a novel photoacoustic method for acquiring elastic information from transparent substances and biological tissues with no or weak optical absorption, applicable to the study of tissue stiffness and viscoelasticity in substances and biological tissues with no or weak optical absorption. The method involves directly attaching the target object to a strongly optically absorbing carrier. The photoacoustic signal, generated by the substrate's absorption rather than the material's own absorption, travels at different speeds in different detection materials, resulting in varying arrival times at the transducer. This time difference is used to infer the elastic parameters of the target object. Furthermore, since the substrate's position relative to the receiver is always the same, the operation is relatively simple and does not require consideration of sample thickness.

[0040] In some embodiments, the aforementioned substrate photoacoustic-based tissue stiffness research method is applied to the study of biological tissue boundary delineation or fibrosis degree.

[0041] In tumor diagnosis, tissue biopsy is a crucial technique for determining the disease condition. Currently, the most commonly used tissue sections in clinical practice include paraffin sections and frozen sections. Paraffin section preparation involves numerous procedures and steps, requiring several days to complete one cycle, which is too time-consuming to promptly assist doctors in ensuring the resection of tumor boundaries. Frozen sections are relatively faster, but staining is required to determine tumor-like boundaries, and this staining process is also cumbersome and time-consuming. The method provided in this invention uses black adhesive tape or markers with strong optical absorption, affixed / drawn on a glass slide as a substrate for absorption. The target object whose hardness needs to be tested is then attached to the slide. By analyzing the time difference in the transmission of the photoacoustic signal from the substrate (strong absorption) through different target objects, the relevant elastic information is inferred. Based on the hardness difference between normal and abnormal tissues, the location of the tumor boundary can be quickly defined. This method does not require staining of the tissue section, enabling rapid boundary analysis of transparent tissue sections. Furthermore, because the thickness of the section is stable, it does not need to consider the inaccuracy caused by the varying distances of different tissues from the ultrasound transducer. This invention can serve as a powerful auxiliary tool for rapid analysis of biological tissues. Moreover, it is simple and fast to operate and has the potential to be used for rapid real-time delineation of tumor tissue boundaries. It provides a new approach for the precise resection of tumor boundaries without excessive damage to normal tissues and can also be used to further refine studies on conditions such as the degree of cirrhosis and liver fibrosis.

[0042] The following specific embodiments further illustrate the present invention's method for studying tissue stiffness based on substrate photoacoustics and its application:

[0043] Example 1

[0044] The tissue stiffness research method based on substrate photoacoustics provided by this invention was subjected to standardized analysis.

[0045] First, PDMS samples of the same thickness but different hardness were prepared. Six groups of liquid samples with different viscosities were prepared by mixing PDMS samples with solution A and solution B in ratios of 4:1, 6:1, 8:1, 10:1, 12:1, and 14:1, respectively. The six mixed liquid samples were then poured into molds with a thickness of 300 micrometers and dried before demolding, ultimately obtaining six groups of PDMS samples with the same thickness but different hardness. The PDMS samples, solution A, and solution B are all commonly used reagents or materials in the field and can be purchased commercially.

[0046] Then, marker marks were drawn on the glass slide as optical absorption carriers, and six PDMS samples of the same thickness but different hardness were placed side by side on the marks. The marks were then imaged using photoacoustic microscopy.

[0047] Finally, the time difference of arrival of the photoacoustic signal was extracted from the obtained results, and this time difference was successfully used to distinguish PDMS samples with different hardness.

[0048] The results are as follows Figure 2 As shown in the figure, the left image is a standard photoacoustic image, and the right image is a time difference graph of photoacoustic signal arrival. The graph shows that the 4:1 ratio PDMS, which has higher hardness, arrives fastest, while the 14:1 ratio PDMS, which has lower hardness, arrives slowest. This demonstrates that the tissue hardness research method based on substrate photoacoustics provided by this invention can successfully distinguish PDMS samples of different hardnesses using the time difference of photoacoustic signal arrival. It is important to note that since PDMS is a transparent sample, the difference in hardness has little impact on the amplitude of the photoacoustic signal. Therefore, the six sets of standard photoacoustic images of PDMS show little difference, while the corresponding signal arrival time difference can distinguish PDMS samples of different hardnesses. This also indicates that the method of this invention can be used to distinguish feature extraction from transparent, non-absorbing samples, making it applicable to a wider range of scenarios.

[0049] Example 2

[0050] The substrate photoacoustic-based tissue hardness research method provided by this invention is applied to the study of tissue hardness and viscoelasticity.

[0051] In a mouse subcutaneous tumor model, the subcutaneous tumor, along with the surrounding normal skin, was excised. A portion of the tissue was placed on a tray, filled with cryogel, and then cryopreserved using a cryostat. Subsequently, 50-micrometer-thick tissue sections were cut from the cross-section of the tissue and attached to pre-marked markers on a glass slide. The markers were imaged using photoacoustic microscopy, and the results were analyzed using conventional photoacoustic processing and the time difference of arrival of the maximum signal value.

[0052] The final result is as follows Figure 3 As shown in the figure, the elastic information (right figure) of different tissues distinguished by the difference in photoacoustic arrival time of the substrate is consistent with their structural information (left figure). Compared with normal tissue, tumor tissue is harder, resulting in faster ultrasound propagation speed and an earlier arrival time of the maximum photoacoustic signal. It can be seen that the method provided by this invention can reflect even slight differences in hardness in the difference in photoacoustic signal arrival time, exhibiting extremely high sensitivity compared to ordinary photoacoustic imaging methods.

[0053] In summary, this invention provides a method and application for studying tissue hardness based on substrate photoacoustics. Substrate photoacoustics refers to using the difference in photoacoustic signals of the carrier of the target object, rather than the target object itself, to study the hardness information of the target object. This invention uses a black adhesive tape or marker with strong optical absorption, affixed or drawn on a glass slide as a substrate absorber. The target object whose hardness needs to be tested is then attached to the slide. Photoacoustic microscopy is used to image the marker. This allows for the capture of structural information of the tissue section by analyzing the maximum amplitude projection view, and also allows for the analysis of the elastic information of the tissue section sample by analyzing the difference in the arrival time of the photoacoustic signals. This invention determines the hardness information of different tissues by analyzing the delay of the photoacoustic signal, and infers the relevant elastic information by using the time difference of the transmission of the substrate (strong absorption) photoacoustic signal in different target objects. Therefore, it does not need to consider whether the target object itself has optical absorption, overcoming the shortcomings of traditional photoacoustic viscoelasticity studies that require optical absorption of the tissue sample. It is applicable to viscoelasticity studies of materials with no or weak optical absorption, and has particularly promising applications in the viscoelasticity studies of transparent tissues. Meanwhile, the slicing process of this invention ensures uniform tissue sample thickness, eliminating the need to consider inaccurate calculations caused by varying tissue thicknesses affecting the distance from the ultrasound transducer. Combined with the substrate photoacoustic method, this results in more accurate measurements. Furthermore, the method provided by this invention is simple and rapid to operate, with broad application prospects. It can be used not only for studying viscoelastic parameters of common biological tissues but also for rapid, real-time tissue boundary delineation and for refining the degree of liver cirrhosis, liver fibrosis, and pulmonary fibrosis, among other things.

[0054] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for studying tissue stiffness based on substrate photoacoustic, characterized in that, Includes the following steps: 1.1 Provide a glass slide on which the optical absorption substrate is marked; 1.2 Section the tissue to be tested and place the cut tissue section sample on the optical absorption substrate position marked in advance on the glass slide; 1.3 The tissue section sample provided in step 1.2 was imaged using a photoacoustic microscope, and the obtained photoacoustic signals were analyzed; Specifically, in step 1.1, the step of marking the optical absorption substrate on the glass slide is as follows: Draw a uniform long line in the middle of the slide with a marker, or cover the middle of the slide evenly with black tape. The middle of the slide is the optical absorption substrate in photoacoustic imaging.

2. The method for studying tissue stiffness based on substrate photoacoustics according to claim 1, characterized in that, In step 1.2, tissue sections of a fixed thickness are cut from the tissue to be tested using frozen sections.

3. The method of claim 1, wherein the substrate-based photoacoustic tissue stiffness study method is characterized by, In step 1.3, the specific steps for analyzing the obtained photoacoustic signal are as follows: The structural information of the tissue section sample is analyzed by observing the maximum amplitude projection view; the corresponding elastic information of the tissue section sample is analyzed by detecting the time difference of photoacoustic signal arrival.

4. Use of a method for studying the hardness of tissue based on substrate photoacoustics, characterized in that, The method described in any one of claims 1-3 is applied to the study of tissue stiffness and viscoelasticity.

5. The use of the method for studying hardness of tissue based on substrate photoacoustic according to claim 4, characterized in that, It is applied to the study of tissue stiffness and viscoelasticity in substances with no or weak optical absorption and biological tissues.

6. The use of the method for studying hardness of tissue based on substrate photoacoustic according to claim 4, characterized in that, It is used for defining the boundaries of biological tissues or studying the degree of fibrosis.

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