A method for lesion depth localization in heterogeneous tissue

Abstract

The present invention discloses a method for locating the depth of lesions in heterogeneous tissues, which relates to the field of lesion detection and includes the following steps: Step 1, preparing surface-enhanced Raman scattering probe nanoparticles; Step 2, preparing a surface-enhanced Raman scattering probe contrast agent and ultrasonically dispersing the surface-enhanced Raman scattering probe contrast agent; measuring and recording the Raman spectrum of the surface-enhanced Raman scattering probe contrast agent; Step 3, placing a biological sample to be tested on a stage, and injecting the surface-enhanced Raman scattering probe contrast agent into the biological sample for Raman detection; Step 4, analyzing the detected Raman spectrum, constructing and solving an overdetermined set of equations, calculating the thickness of various homogeneous tissues, and adding them together to obtain the lesion depth. This method can detect deep tumors in heterogeneous tissues and accurately determine the lesion depth, making it have the advantages of convenient acquisition, no need for three-dimensional reconstruction, real-time and fast, and good specificity.

Description

Technical Field

[0001] The present invention relates to the field of lesion detection, and in particular to a method for locating the depth of lesions in heterogeneous tissues. Background Art

[0002] Most lesions are deeply embedded in the human body, ranging from several centimeters to tens of centimeters below the body surface. To determine the location of lesions, current clinical procedures begin with preoperative or intraoperative medical imaging to obtain information about lesion depth. This information is crucial for clinical diagnostic applications, ranging from designing treatment strategies to surgical planning and guidance. Therefore, the detection and depth estimation of deep lesions are crucial for clinical therapeutics.

[0003] However, a major challenge in intraoperative tumor detection is determining the depth of the lesion based on the acquired signals. Currently, fluorescence-based lesion localization methods include time-resolved measurement or fluorescence tomography, but these methods often require complex equipment and cannot meet the requirements for rapid, real-time localization during surgery.

[0004] Surface-enhanced Raman spectroscopy (SERS) involves the highly enhanced Raman scattering of molecules adsorbed on metal nanostructures due to surface plasmon excitation and surface chemical effects. This allows for single-molecule and single-particle detection with exceptionally high sensitivity. Compared to fluorescence spectra, which are broad, prone to overlap, and susceptible to background interference from endogenous fluorophores in biological tissue, Raman spectroscopy offers exceptional specificity and excellent discrimination from background. By adsorbing Raman molecules onto the surfaces of roughened precious metal particles, surface-enhanced Raman spectroscopy probe nanomaterials with excellent specificity and sensitivity can be prepared. SERS probes have garnered widespread attention due to their susceptibility to photobleaching, immunity to interference from autofluorescence in biological tissue, and simplified sample preparation, making them promising bioimaging contrast agents. Compared to fluorescence, their unique fingerprint-like pattern provides exceptionally high specificity and an ultra-narrow half-width peak. Notably, SERS probes have now achieved centimeter-level depth detection, with Raman signals from embedded SERS probes being detected through depths of up to 14 cm of biological tissue. Based on the above outstanding advantages, SERS probes have very broad prospects in the detection and imaging of deep biomedical lesions.

[0005] Researchers have also explored some methods based on Raman spectral intensity values ​​to estimate the depth of lesion phantoms in biological tissues. For example, water in biological tissues has an absorption peak at 950nm, so Raman peaks close to this wavelength will be greatly attenuated, and the peak value is related to the length of the transmission path. However, the above-mentioned depth prediction methods rely on the existence of water absorption peaks. The water content of each tissue varies greatly. The method of examining tissue thickness with a single factor is ineffective for heterogeneous complex models and is not universal. In addition, some researchers have proposed to determine the depth of the lesion based on the relationship between Raman intensity and tissue thickness, but this method requires the Raman intensity of the lesion to be determined in advance and cannot be applied in real clinical scenarios. In short, existing methods almost all rely on prior calibration models and can only be used in homogeneous tissues. They are almost ineffective for predicting lesions in thick heterogeneous tissue complex media.

[0006] Therefore, technicians in this field are committed to developing a method for localizing the depth of lesions in heterogeneous tissues, so as to detect deep tumors in heterogeneous tissues and accurately determine the depth of lesions, which has the advantages of convenient acquisition, no need for three-dimensional reconstruction, real-time and fast, and good specificity. Summary of the Invention

[0007] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to conveniently detect deep tumors in heterogeneous tissues in real time and accurately determine the depth of the lesion.

[0008] To achieve the above object, the present invention provides a method for locating the depth of a lesion in a heterogeneous tissue, comprising the following steps:

[0009] Step 1, preparing surface enhanced Raman scattering probe nanoparticles;

[0010] Step 2: preparing a surface-enhanced Raman scattering probe contrast agent, ultrasonically dispersing the surface-enhanced Raman scattering probe contrast agent; measuring and recording the Raman spectrum of the surface-enhanced Raman scattering probe contrast agent;

[0011] Step 3: placing the biological sample to be tested on a stage, and injecting the surface-enhanced Raman scattering probe contrast agent into the biological sample to perform Raman detection;

[0012] Step 4: Analyze the Raman spectrum obtained by detection, construct and solve an overdetermined set of equations, calculate the thickness of various homogeneous tissues, and add them up to obtain the lesion depth.

[0013] Furthermore, the surface-enhanced Raman scattering probe nanoparticles are coated with a mesoporous silica layer.

[0014] Furthermore, step 1 includes: preparing 5mL 0.6nmol / L of a surface-enhanced Raman probe with a core-shell structure containing a Raman signal molecule, centrifuging and redispersing it in 5mL 0.004mol / L hexadecylammonium chloride solution, adding 0.1mol / L NaOH solution to adjust the pH value of the solution to 10-11; maintaining the solution temperature at 30°C, and then adding 70μL of a methanol solution containing 5% tetraethyl orthosilicate three times, each time with a time interval of 30 minutes, and continuing to stir the reaction for 17 hours to obtain nanoparticles coated with a 15nm mesoporous silica layer; centrifuging and washing, and evenly dispersing it in anhydrous ethanol, thereby obtaining surface-enhanced Raman probe nanoparticles.

[0015] Furthermore, step 2 includes: uniformly dispersing the surface-enhanced Raman probe nanoparticles in physiological saline to prepare a 0.6 nmol / L surface-enhanced Raman probe contrast agent; measuring the Raman spectrum of the pure surface-enhanced Raman probe contrast agent; ultrasonically dispersing the particles and injecting 30-200 μL into a heterogeneous tissue sample.

[0016] Furthermore, step 3 includes: using a diffused beam of 785nm laser wavelength, with a spot diameter of 0.5-1.7cm and a power density of 0.2-1.01W / cm 2 The laser is irradiated from bottom to top on the lower surface of the heterogeneous tissue sample.

[0017] Furthermore, the step 3 further comprises: using a Raman fiber optic probe to detect characteristic Raman signals on the upper surface of the inhomogeneous tissue sample.

[0018] Furthermore, in step 4, a set of overdetermined equations is constructed by selecting multiple spectral peak pairs in the characteristic Raman signal.

[0019] Furthermore, the overdetermined system of equations is as follows:

[0020]

[0021] Among them, I1, I2, I3, I4 represent different Raman peaks, X1, X2, ..., X n It represents the thickness of various types of tissues that Raman photons propagate through before reaching the collection surface and detection probe; Δμ is the attenuation coefficient of each spectral peak pair in different tissues.

[0022] Furthermore, the step 4 further includes: pre-calculating the absorption coefficient μ according to the reported absorption coefficient μ a and the effective scattering coefficient μ s 'To deduce or related test methods to obtain Δμ.

[0023] Furthermore, the formula for calculating the depth of the lesion in step 4 is: d = X1 + X2 + ... + Xn .

[0024] Compared with the existing technical solutions, the beneficial technical effects of the present invention include at least:

[0025] 1. The present method, combined with surface-enhanced Raman probe contrast agents, can predict the depth of probe-enriched lesions in tissue in real time. Data analysis is performed directly alongside existing Raman detection, eliminating the need for additional equipment or prior knowledge of the lesion. The method utilizes simple equipment, facilitates data collection, and is fast and real-time.

[0026] 2. The method of the present invention is applicable not only to lesions in homogeneous tissues, but also to lesions in heterogeneous tissues.

[0027] 3. The method of the present invention uses a highly sensitive surface-enhanced Raman spectroscopy probe contrast agent, which has high sensitivity, good stability, is not prone to photobleaching, and can avoid tissue background interference, etc., and can achieve precise positioning of the tumor site and stable imaging.

[0028] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of laser acting on homogeneous tissue;

[0030] Figure 2 is the relationship between Raman intensity and Raman shift;

[0031] Figure 3 It is a linear relationship diagram between the natural logarithm of the spectral peak intensity ratio in homogeneous tissue and the depth of the lesion;

[0032] Figure 4 is the relationship between Raman intensity and Raman shift;

[0033] Figure 5 It is a depth positioning relationship diagram of different Raman peak pairs in homogeneous tissue;

[0034] Figure 6 Schematic diagram of the effect of contrast agents on heterogeneous tissues;

[0035] Figure 7 It is a graph showing the relationship between the natural logarithm of the spectral peak-to-intensity ratio and the depth of the lesion in heterogeneous tissue;

[0036] Figure 8 It is a flow chart of the method steps;

[0037] Figure 9 This is a schematic diagram of the structure of a core-shell surface enhanced Raman probe;

[0038] Figure 10 This is a transmission electron microscopy characterization of the core-shell structure surface enhanced Raman probe;

[0039] Figure 11 This is the Raman spectrum of the core-shell structure surface enhanced Raman probe;

[0040] Figure 12 is the predicted depth map of the surface-enhanced Raman probe in heterogeneous ex vivo tissue. DETAILED DESCRIPTION

[0041] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0042] Since biological tissues have different attenuation rates for photons of different wavelengths, when Raman photons are emitted from the lesion and propagate a certain distance to reach the tissue surface, the intensity of the spectrum peaks of different wavelengths changes differently. Therefore, when a specific pair of Raman spectrum peaks is selected, the natural logarithm of the intensity ratio will be correlated with the depth of the signal source, such as Figure 1-3 shown.

[0043] Here, the radiative transfer equation is used to simulate the process of photon propagation in uniform tissue, laying the theoretical foundation. First, assuming that a continuous laser source is incident from one side of the tissue and the Raman signal is collected from the opposite side of the tissue, the corresponding steady-state diffusion approximation model is:

[0044]

[0045] This represents the light intensity at any point (x, y, z) at a distance r from the light source. Wherein, the radius of the illumination beam on the sample is a, the center of the illumination beam on the sample is the origin (0, 0, 0), I0 is the brightness of the light source, D is the photon diffusion coefficient, μ eff is the effective attenuation coefficient of the tissue, which can be obtained from the absorption coefficient μ a and the effective scattering coefficient μ s' The lesion containing the SERS probe is regarded as a point light source. When the laser excites the lesion at (x0, y0, z0), the distance from the lesion is The Raman intensity at the (x, y, z) position is:

[0046]

[0047] and Denote the diffusion coefficient and effective attenuation coefficient of Raman photons, respectively. C and σ represent the concentration of Raman probes and the surface-enhanced Raman scattering cross section, respectively. Therefore, assuming that the lesion depth (i.e., the linear distance from the lesion to the collection surface) is d, for the transmission detection method, combining equations (1) and (2), the Raman intensity of the collection surface can be obtained as

[0048]

[0049] λ0 is the incident laser wavelength (known), λ i is the wavelength corresponding to the peak of the outgoing Raman spectrum (known). T is the tissue thickness (unknown), and d is the depth of the lesion (unknown). Select two peaks, corresponding to wavelengths λ1 and λ2, then the difference in the effective tissue attenuation coefficient of the two wavelengths is

[0050] Δμ 21 =μ eff (λ2)-μ eff (λ1)#(4)

[0051] Combining equations (3) and (4), the natural logarithm of the intensity ratio of the two peaks in the collected Raman spectrum is

[0052]

[0053] in

[0054] This is a linear equation of the form y=ax+b, and the slope of the linear function is Δμ 21 It is the difference in the effective attenuation coefficients of two wavelengths of Raman photons propagating in tissue. b can be calculated experimentally by testing the spectrum of the pure SERS probe when there is no tissue coverage (i.e. when d = 0). The ratio of the two Raman peak pairs is = b. Therefore, the only unknown in the linear equation (5) is the lesion depth d. For a single lesion in a homogeneous tissue, it can be known from formula (5) that the lesion depth in the homogeneous tissue can be determined based on one of the Raman peak pairs. Figure 1-3 The figure shows the linear relationship between the natural logarithm of the spectral peak-to-intensity ratio and the depth of the lesion in homogeneous tissue.

[0055] According to the above calculation, the Raman peak contrast value It is linearly related to the depth of the SERS probe, and the effective attenuation coefficient difference of photons of different wavelengths propagating in tissue is different. Therefore, the slope and intercept of the linear model (5) calculated for different Raman peak pairs are different. Figure 4-5 Shown are the depth localizations of different Raman peak pairs in homogeneous tissue.

[0056] Since Raman spectra always present multiple peaks, and the peak width is usually less than a few nanometers, this makes Raman spectra fully capable of meeting multi-peak high-throughput applications, and can be combined with Raman multi-spectral peak pairs to achieve lesion depth prediction. For the detection scenario of heterogeneous tissue models (i.e., a single lesion is superimposed by multiple homogeneous tissues), multiple spectral peak pairs can be selected to construct an overdetermined set of equations for solution, and the thickness of various homogeneous tissues can be calculated and added to the total thickness of the tissue above the lesion, thereby determining the lesion depth, such as Figure 6-7 As shown. The overdetermined equations are constructed as follows:

[0057] The overdetermined system of equations is constructed as follows:

[0058]

[0059] Where I1, I2, I3, I4 represent different Raman peaks, X1, X2, ..., X n represents the thickness of various types of tissues that Raman photons propagate through before reaching the collection surface and detection probe. The calculation of this set of equations requires the difference in attenuation coefficients of each spectral peak pair in different tissues, Δμ, which can be calculated in advance based on the reported absorption coefficient μ a and the effective scattering coefficient μ s 'To infer or obtain related test methods. It is only necessary to measure the Raman spectrum and calculate the depth of the lesion based on the natural logarithm of the spectrum peak ratio. The depth of the SERS probe can be obtained as:

[0060] d=X1+X2+…+X n

[0061] Based on the above principle, the present invention provides a method for locating the depth of lesions in heterogeneous tissues, such as Figure 8 As shown, the following steps are included:

[0062] Step 1. Prepare 5mL0.6nmol / L of a surface-enhanced Raman probe with a core-shell structure containing a Raman signal molecule, centrifuge and redisperse it in 5mL of 0.004mol / L hexadecylammonium chloride solution, add 0.1mol / L NaOH solution to adjust the pH value of the solution to 10-11; maintain the solution temperature at 30°C, then add 70μL of a methanol solution containing 5% tetraethyl orthosilicate three times, each time with a time interval of 30 minutes, continue stirring and reacting for 17h to obtain nanoparticles coated with a 15nm mesoporous silica layer; centrifuge and wash, and evenly disperse in anhydrous ethanol to obtain surface-enhanced Raman probe nanoparticles.

[0063] Step 2: uniformly dispersing the obtained surface-enhanced Raman probe nanoparticles in physiological saline to prepare a surface-enhanced Raman probe contrast agent; and measuring the Raman spectrum of the pure surface-enhanced Raman probe contrast agent.

[0064] Step 3: Place the tissue sample to be tested on the stage and inject the ultrasonically dispersed surface-enhanced Raman probe contrast agent into the heterogeneous tissue for Raman detection. Laser light is irradiated from the bottom up onto the lower surface of the heterogeneous tissue sample. A Raman fiber probe is used to detect the characteristic Raman signal on the upper surface of the heterogeneous tissue sample.

[0065] Step 4: Analyze the test results. This includes: selecting multiple peak pairs in the characteristic Raman signal, constructing an overdetermined set of equations, solving them, calculating the thickness of various homogeneous tissues, and adding them together to obtain the total thickness of the tissue above the lesion, thereby determining the depth of the lesion. The overdetermined set of equations is constructed as follows:

[0066]

[0067] The depth of the lesion is thus obtained as:

[0068] d=X1+X2+…+X n

[0069] Example 1:

[0070] In this embodiment, Figure 9 The figure shows the schematic diagram of the structure of the core-shell surface enhanced Raman probe; Figure 10 The figure shows the transmission electron microscope characterization of the core-shell structure surface enhanced Raman probe; Figure 11 Shown is the Raman spectrum of the core-shell structure surface enhanced Raman probe.

[0071] Step 1: Prepare 5mL0.6nmol / L of a surface-enhanced Raman probe with a core-shell structure containing the IR-780 Raman signal molecule, centrifuge, and redisperse it in 5mL of 0.004mol / L hexadecylammonium chloride solution, add 0.1mol / L NaOH solution to adjust the pH value of the solution to 10-11; maintain the solution temperature at 30°C, and then add 70μL of a methanol solution containing 5% tetraethyl orthosilicate three times, with an interval of 30 minutes between each addition, and continue stirring the reaction for 17h to obtain nanoparticles coated with a 15nm mesoporous silica layer; centrifuge and wash, and evenly disperse in anhydrous ethanol.

[0072] Step 2: The surface-enhanced Raman probe nanoparticles obtained above are uniformly dispersed in physiological saline to prepare a 0.6 nmol / L surface-enhanced Raman probe contrast agent; the Raman spectrum of the pure surface-enhanced Raman probe contrast agent is measured; the particles are ultrasonically dispersed and 30 μL is injected into a heterogeneous tissue composed of stacked isolated pig muscle and fat tissue of different thicknesses.

[0073] Step 3: Place the tissue sample to be tested on the stage for Raman detection. Use a diffuse beam of 785nm laser wavelength, a circular spot with a diameter of 1.7cm, and a power density of 0.26W / cm 2 Laser is irradiated from bottom to top on the lower surface of the in vitro heterogeneous tissue. A Raman fiber probe is used to detect characteristic Raman signals on the upper surface of the in vitro heterogeneous tissue.

[0074] Step 4: Analyze the test results.

[0075] The experimental test obtained the characteristic Raman spectrum of the Raman signal molecule IR-780 in the surface enhanced Raman probe (such as Figure 6 ), among which, characteristic peaks (520, 931, 1203, 1369, 1523, 1580 cm-1) are selected, and 9 pairs of Raman peaks (520 / 1369, 931 / 1369, 1203 / 1369, 520 / 1580, 931 / 1580, 1203 / 1580, 520 / 1523, 931 / 1523, 1203 / 1523) are combined to construct an overdetermined system of equations and solve them.

[0076]

[0077] The left side of the equation represents the natural logarithm of the intensity ratio of each Raman peak pair (520 / 1369, 931 / 1369, 1203 / 1369, 520 / 1580, 931 / 1580, 1203 / 1580, 520 / 1523, 931 / 1523, 1203 / 1523). Different columns in the matrix represent different embedding depths of the surface-enhanced Raman probe contrast agent, and different rows represent different Raman peak pairs. The first matrix on the right side of the equation is the effective attenuation coefficient difference (Δμ) matrix. The first column represents the difference in the effective attenuation coefficient of each Raman peak pair in porcine adipose tissue, the second column represents the difference in the effective attenuation coefficient of each Raman peak pair in porcine muscle tissue, and the third column is the intercept. The predicted depth of the SERS probe can be obtained as:

[0078]

[0079] The final SERS probe depth prediction results are as follows Figure 12 As shown in Figure 3, the predicted depth is highly consistent with the actual depth, with a prediction error of only 8.35%.

[0080] Example 2

[0081] Step 1: Prepare 5mL0.6nmol / L of a surface-enhanced Raman probe with a core-shell structure containing the IR-783 Raman signal molecule, centrifuge, and redisperse it in 5mL0.004mol / L hexadecylammonium chloride solution, add 0.1mol / L NaOH solution to adjust the pH value of the solution to 10-11; maintain the solution temperature at 30°C, and then add 70μL of a methanol solution containing 5% tetraethyl orthosilicate three times, each time with a time interval of 30 minutes, and continue stirring the reaction for 17h to obtain nanoparticles coated with a 15nm mesoporous silica layer; centrifuge and wash, and evenly disperse in anhydrous ethanol.

[0082] Step 2: The surface-enhanced Raman probe nanoparticles obtained above are uniformly dispersed in physiological saline to prepare a 0.6 nmol / L surface-enhanced Raman probe contrast agent, ultrasonically dispersed and 30 μL is injected into a quartz tube with a diameter of 1 mm; the Raman spectrum of the pure surface-enhanced Raman probe contrast agent is measured; and the quartz tube is buried in ex vivo porcine adipose tissue.

[0083] Step 3: Place the tissue sample to be tested on the stage for Raman detection. Use a diffuse beam of 785nm laser wavelength, a circular spot with a diameter of 0.5cm, and a power density of 1.01W / cm 2 The laser was irradiated from bottom to top on the lower surface of the isolated porcine adipose tissue. The characteristic Raman signal was detected on the upper surface of the isolated porcine adipose tissue using a Raman fiber probe.

[0084] Step 4: Analyze the test results.

[0085] Example 3

[0086] Step 1: Prepare 5mL0.6nmol / L of a surface-enhanced Raman probe with a core-shell structure containing a DTTC Raman signal molecule, centrifuge, and redisperse it in 5mL of 0.004mol / L hexadecylammonium chloride solution, add 0.1mol / L NaOH solution to adjust the pH value of the solution to 10-11; maintain the solution temperature at 30°C, and then add 5% tetraethyl orthosilicate methanol solution three times, 50μL each time, with an interval of 30 minutes between each addition, and continue stirring the reaction for 17h to obtain nanoparticles coated with a 10nm mesoporous silica layer; centrifuge and wash, and evenly disperse in anhydrous ethanol.

[0087] Step 2: The surface-enhanced Raman probe nanoparticles obtained above are uniformly dispersed in physiological saline to prepare a 1 nmol / L surface-enhanced Raman probe contrast agent; the Raman spectrum of the pure surface-enhanced Raman probe contrast agent is measured; and 200 μL of the ultrasonically dispersed 1 nmol / L surface-enhanced Raman probe contrast agent is injected into normal mice via the tail vein.

[0088] Step 3: 24 hours after injection, the sample to be tested was placed on the stage and Raman detection was performed on the mouse abdomen. A parallel beam of 785nm laser wavelength was used, the spot was a circular shape with a diameter of 1.5cm, and the power density was 0.2W / cm 2 The mouse lay flat on the detection platform under anesthesia, and the laser was irradiated on the back from bottom to top. A Raman fiber optic probe was used to detect the characteristic Raman signal on the upper side of the abdomen, and the probe was moved step by step for scanning.

[0089] Step 4: Analyze the detection and imaging results.

[0090] The above describes in detail the preferred embodiments of the present invention. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A method for locating the depth of a lesion in a heterogeneous tissue, characterized in that: The following steps are involved: Step 1, preparing surface enhanced Raman scattering probe nanoparticles; Step 2: preparing a surface-enhanced Raman scattering probe contrast agent, and ultrasonically dispersing the surface-enhanced Raman scattering probe contrast agent; measuring and recording the Raman spectrum of the surface-enhanced Raman scattering probe contrast agent; Step 3: placing the biological sample to be tested on a stage, and injecting the surface-enhanced Raman scattering probe contrast agent into the biological sample to perform Raman detection; Step 4: Analyze the Raman spectrum obtained by detection, construct and solve an overdetermined set of equations, calculate the thickness of various homogeneous tissues, and add them up to obtain the lesion depth.

2. The method for locating the depth of a lesion in a heterogeneous tissue according to claim 1, wherein: The surface enhanced Raman scattering probe nanoparticles are coated with a mesoporous silica layer.

3. The method for locating the depth of a lesion in a heterogeneous tissue according to claim 2, wherein: The step 1 comprises: preparing 5 mL of a 0.6 nmol / L surface-enhanced Raman probe having a core-shell structure containing a Raman signal molecule, centrifuging and redispersing the solution in 5 mL of a 0.004 mol / L hexadecyl ammonium chloride solution, and adding a 0.1 mol / L NaOH solution to adjust the pH value of the solution to 10-11; maintaining the solution temperature at 30° C., and then adding a methanol solution containing 5% tetraethyl orthosilicate three times, 70 μL each time, with a time interval of 30 minutes between each addition, and continuing to stir and react for 17 hours to obtain nanoparticles coated with a 15 nm mesoporous silica layer; and centrifuging and washing the mixture, and uniformly dispersing the mixture in anhydrous ethanol to obtain surface-enhanced Raman probe nanoparticles.

4. The method for locating the depth of a lesion in a heterogeneous tissue according to claim 3, wherein: The step 2 comprises: uniformly dispersing the surface-enhanced Raman probe nanoparticles in physiological saline to prepare a 0.6 nmol / L surface-enhanced Raman probe contrast agent; measuring the Raman spectrum of the pure surface-enhanced Raman probe contrast agent; ultrasonically dispersing the particles and injecting 30-200 μL into a heterogeneous tissue sample.

5. The method for locating the depth of a lesion in a heterogeneous tissue according to claim 4, wherein: Step 3 includes: using a diffused beam of 785 nm laser wavelength, with a spot diameter of 0.5-1.7 cm and a power density of 0.2-1.01 W / cm 2 The laser is irradiated from bottom to top on the lower surface of the heterogeneous tissue sample.

6. The method for locating the depth of a lesion in a heterogeneous tissue according to claim 5, wherein: The step 3 further includes: using a Raman fiber optic probe to detect characteristic Raman signals on the upper surface of the inhomogeneous tissue sample.

7. The method for locating the depth of a lesion in a heterogeneous tissue according to claim 6, wherein: In step 4, a set of overdetermined equations is constructed by selecting multiple spectral peak pairs in the characteristic Raman signal.

8. The method for locating the depth of a lesion in a heterogeneous tissue according to claim 7, wherein: The overdetermined system of equations is as follows: Among them, I1, I2, I3, I4 represent different Raman peaks, X1, X2, ..., X n It represents the thickness of various types of tissues that Raman photons propagate through before reaching the collection surface and detection probe; Δμ is the attenuation coefficient of each spectral peak pair in different tissues.

9. The method for locating the depth of a lesion in a heterogeneous tissue according to claim 8, wherein: The step 4 also includes: pre-calculating the absorption coefficient μ according to the reported absorption coefficient μ a and the effective scattering coefficient μ s 'To deduce or related test methods to obtain Δμ.

10. The method for locating the depth of a lesion in a heterogeneous tissue according to claim 9, wherein: The formula for calculating the depth of the lesion in step 4 is: d = X1 + X2 + ... X n .

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