A method for measuring the difference in effective attenuation coefficients of photons with different wavelengths in tissue

Abstract

The present invention discloses a method for measuring the difference in effective attenuation coefficients of photons with different wavelengths in tissues, which relates to the fields of biomedicine and optics and includes the following steps: preparing surface-enhanced Raman scattering probe nanoparticles coated with a mesoporous silica layer; preparing a surface-enhanced Raman scattering probe contrast agent, ultrasonically dispersing the contrast agent, and injecting a certain amount into a quartz tube; measuring and recording the Raman spectrum of the contrast agent; placing the tissue sample to be measured on a stage, burying the quartz tube containing the contrast agent in the tissue sample, and performing Raman detection; analyzing the characteristic Raman spectra obtained at different burial depths of the quartz tube to obtain the difference in effective attenuation coefficients of Raman photons with different wavelengths propagating in the tissue sample. The present invention combines the multi-peak characteristics of the Raman spectrum contrast agent, and a single Raman spectrum contrast agent can be used to achieve the measurement between Raman photons of multiple wavelengths. The measurement method is simple, low in cost, and wide in application scope.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and optics, and particularly to a method for measuring the difference in effective attenuation coefficients of photons with different wavelengths in tissues. Background Art

[0002] Knowledge of the optical properties of biological tissues is crucial in biomedical and optical research, as it is the basis for designing effective devices and methods or planning treatment regimens, and is also crucial for the interpretation of diagnostic measurements. In-depth understanding of the optical properties of various biological tissues can serve as a starting point for creating accurate light propagation models or testing the capabilities of methods / systems for retrieving optical properties. Further, it is also of great significance to determine the difference in effective attenuation coefficients of photons with different wavelengths propagating in tissues. For example, spectroscopy-based depth estimation techniques strongly rely on prior knowledge of tissue optical properties, especially the prior knowledge of the difference in effective attenuation coefficients of photons with different wavelengths propagating in tissues. The determination of these parameters can help doctors quickly, non-invasively, and accurately predict the depth of lesions.

[0003] Traditional methods for measuring the optical properties of biological tissues are diffuse spectroscopy (including diffuse reflection and diffuse transmission light), usually using spatial frequency domain or time domain systems. Spatial frequency domain detection is a method that can irradiate tissues with sinusoidal patterns having different spatial frequencies and retrieve tissues at multiple effective source-detector separations. By spatially resolving the diffuse reflection of a turbid medium irradiated with different spatial frequencies, the tissue optical properties are restored. Time domain detection is based on time-resolved detection and processes the collected signals using a time-correlated single-photon counting board. Both require complex equipment and computational methods and are sensitive to small instrument deviations. Moreover, the results obtained by traditional methods for measuring the optical properties of biological tissues are usually the scattering and absorption properties of tissues, and further calculations are still required to obtain the difference in effective attenuation coefficients of photons with different wavelengths propagating in tissues.

[0004] In summary, measuring the optical properties of tissues plays an extremely important role in biomedical and optical research. However, the existing testing methods are complex, time-consuming, and sensitive to instrument deviations, making them difficult to promote and popularize. Moreover, the results obtained are usually the scattering and absorption properties of tissues, rather than directly obtaining the difference in effective attenuation coefficients of photons with different wavelengths propagating in tissues.

[0005] Therefore, those skilled in the art are committed to developing a simple and rapid method to directly measure the difference in effective attenuation coefficients of photons with different wavelengths propagating in tissues. Summary of the Invention

[0006] In view of the above defects of the prior art, the technical problem to be solved by the present invention is that the existing methods for measuring the optical properties of tissues are complex and time-consuming, sensitive to instrument deviations, difficult to popularize and apply, and the obtained results are usually the scattering and absorption characteristics of tissues, rather than directly obtaining the difference in attenuation coefficients of photons with different wavelengths propagating in tissues.

[0007] To achieve the above object, the present invention provides a method for measuring the difference in effective attenuation coefficients of photons with different wavelengths in tissues, and the method includes the following steps:

[0008] S101: Prepare surface-enhanced Raman scattering probe nanoparticles, and the surface-enhanced Raman scattering probe nanoparticles are coated with a mesoporous silica layer;

[0009] S103: Prepare a surface-enhanced Raman scattering probe contrast agent, ultrasonically disperse the surface-enhanced Raman scattering probe contrast agent, and inject a certain amount into a quartz tube;

[0010] S105: Measure and record the Raman spectrum of the surface-enhanced Raman scattering probe contrast agent;

[0011] S107: Place the tissue sample to be measured on the stage, and bury the quartz tube containing the contrast agent into the tissue sample for Raman detection;

[0012] S109: Analyze the characteristic Raman spectra obtained by detection at different burial depths of the quartz tube to obtain the difference in effective attenuation coefficients of Raman photons with different wavelengths propagating in the tissue sample.

[0013] Further, the step S101 includes the following steps:

[0014] S1011: Prepare a surface-enhanced Raman scattering probe solution with a core-shell structure containing Raman signal molecules;

[0015] S1012: Centrifuge and redisperse the surface-enhanced Raman scattering probe in a cetylammonium chloride solution;

[0016] S1013: Add a NaOH solution to adjust the pH value of the cetylammonium chloride solution to 10 - 11;

[0017] S1014: Keep the temperature of the cetylammonium chloride solution at 30°C, then add a methanol solution containing tetraethyl orthosilicate three times, 50 - 70 μL each time, with a time interval of 30 minutes each time, and continuously stir and react for 17 hours to obtain nanoparticles coated with a 10 - 15 nm mesoporous silica layer;

[0018] S1015: Centrifuge and wash the surface-enhanced Raman scattering probe nanoparticles, and uniformly disperse them in absolute ethanol.

[0019] Furthermore, the surface-enhanced Raman scattering probe nanoparticles adopt a gold-silver substrate and are combined with Raman signal molecules.

[0020] Furthermore, the Raman signal molecule includes one of IR-780, IR-783, DTTC, Cy5, R6G, and CV Raman signal molecules.

[0021] Furthermore, the concentration of the surface-enhanced Raman scattering probe solution is 0.6 nmol / L, the quantity of the cetylammonium chloride solution is 5 mL, the concentration of the cetylammonium chloride solution is 0.004 mol / L, the concentration of the NaOH solution is 0.1 mol / L, and the content of tetraethyl orthosilicate in the methanol solution is 5%.

[0022] Furthermore, in step S103, the surface-enhanced Raman scattering probe nanoparticles are uniformly dispersed in physiological saline to prepare the surface-enhanced Raman scattering probe contrast agent. The concentration of the surface-enhanced Raman scattering probe contrast agent is 0.2 - 0.6 nmol / L, the quantity of the surface-enhanced Raman scattering probe contrast agent injected into the quartz tube is 20 - 30 μL, and the inner diameter of the quartz tube is 1 mm.

[0023] Furthermore, step S107 includes the following steps:

[0024] S1071: Use a laser diffused beam to irradiate the lower surface of the tissue sample from bottom to top;

[0025] S1072: Use a Raman fiber optic probe to detect characteristic Raman signals on the upper surface of the tissue sample and record the characteristic Raman signals;

[0026] S1073: Change the buried depth of the quartz tube, and use a Raman fiber optic probe to detect and record the characteristic Raman signals on the upper surface of the tissue sample.

[0027] Furthermore, the wavelength of the laser diffused beam is 785 nm, the laser diffused beam is a circular light spot, the diameter of the circular light spot is 0.5 - 1.7 cm, and the power density of the laser is 0.022 - 1.01 W / cm 2 .

[0028] Furthermore, step S109 includes the following steps:

[0029] S1091: Use the Raman characteristic peaks of the Raman signal molecules to construct different Raman peak pairs;

[0030] S1092: Establish a relationship graph between the natural logarithm of the intensity ratio of each Raman peak pair and the embedding depth of the surface-enhanced Raman scattering probe contrast agent;

[0031] S1093: Perform a linear fitting on the natural logarithm of the intensity ratio of the Raman peak pair and the embedding depth of the surface-enhanced Raman scattering probe contrast agent. The fitting slope obtained is the difference in the effective attenuation coefficients of the two different-wavelength photons corresponding to this Raman peak pair when propagating in the tissue sample.

[0032] Further, the linear equation obtained by performing a linear fitting on the natural logarithm of the intensity ratio of the Raman peak pair and the embedding depth of the surface-enhanced Raman scattering probe contrast agent is:

[0033]

[0034] Among them, is the natural logarithm of the ratio of two spectral peaks in the Raman spectrum, and Δμ 21 is the difference in the effective attenuation coefficients of the Raman photons of the two spectral peaks in the Raman spectrum when propagating in the tissue. d is the depth of the Raman source, b is the natural logarithm of the original intensity ratio of the spectral peak pair of the pure Raman spectrum contrast agent, σ is the cross-section parameter of Raman scattering, D is the photon diffusion coefficient, and λ1 and λ2 are the wavelengths of the two spectral peaks.

[0035] In a preferred embodiment of the present invention, compared with the prior art, the present invention has the following beneficial technical effects:

[0036] 1. It can measure the difference in the effective attenuation coefficients of different-wavelength photons in various tissues;

[0037] 2. The method of the present invention combines the multi-peak characteristics of the Raman spectrum contrast agent, and one Raman spectrum contrast agent can achieve the measurement between multiple-wavelength Raman photons;

[0038] 3. The measurement method of the present invention uses a Raman spectrum contrast agent, which has the characteristics of strong specificity, good stability, not easy to occur photobleaching, and can avoid tissue background interference, etc. This helps to achieve the measurement of the difference in the effective attenuation coefficients of different-wavelength photons with high accuracy;

[0039] 4. The equipment adopted by the present invention is simple, the acquisition is convenient, the cost is low, and the applicable range is wide.

[0040] The following will further illustrate the concept, specific structure and technical effects generated by the present invention in conjunction with the drawings to fully understand the purpose, features and effects of the present invention. Description of the Drawings

[0041] Figure 1 is a schematic diagram of the measurement steps of a preferred embodiment of the present invention;

[0042] Figure 2 It is a linear relationship diagram of the natural logarithm of the intensity ratio of spectral peaks to the difference in the effective attenuation coefficients of photons at two wavelengths in a preferred embodiment of the present invention;

[0043] Figure 3 It is a schematic structural diagram of a core-shell structure surface-enhanced Raman scattering probe in a preferred embodiment of the present invention;

[0044] Figure 4 It is a transmission electron microscope characterization diagram of a core-shell structure surface-enhanced Raman scattering probe in a preferred embodiment of the present invention;

[0045] Figure 5 It is a Raman spectrum diagram of a core-shell structure surface-enhanced Raman scattering probe in a preferred embodiment of the present invention;

[0046] Figure 6 It is a diagram of the difference in the effective attenuation coefficients of different Raman peaks propagating in porcine adipose tissue in a preferred embodiment of the present invention. Detailed implementation manners

[0047] The following introduces multiple preferred embodiments of the present invention with reference to the accompanying drawings of the specification to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0048] In the accompanying drawings, components with the same structure are denoted by the same numerical reference signs, and components with similar structures or functions everywhere are denoted by similar numerical reference signs. The size and thickness of each component shown in the drawings are arbitrarily shown, and the present invention does not limit the size and thickness of each component. To make the illustration clearer, the thickness of some components in the drawings is appropriately exaggerated.

[0049] As Figure 1 shown, a method for measuring the difference in the effective attenuation coefficients of photons with different wavelengths in tissue provided by an embodiment of the present invention includes the following steps:

[0050] S101: Prepare surface-enhanced Raman scattering probe nanoparticles, and the surface-enhanced Raman scattering probe nanoparticles are coated with a mesoporous silica layer. The surface-enhanced Raman scattering probe nanoparticles adopt a gold-silver substrate and are combined with Raman signal molecules.

[0051] Among them, the above steps further include the following sub-steps:

[0052] S1011: Prepare a surface-enhanced Raman scattering probe solution with a core-shell structure containing Raman signal molecules. The Raman signal molecules include one of IR-780, IR-783, DTTC, Cy5, R6G, and CV Raman signal molecules. The quantity of the surface-enhanced Raman scattering probe solution is 5 mL, and the concentration of the surface-enhanced Raman scattering probe solution is 0.6 nmol / L.

[0053] S1012: Centrifuge and redisperse the surface-enhanced Raman scattering probe in 5 mL of a cetylammonium chloride solution with a concentration of 0.004 mol / L.

[0054] S1013: Add 0.1 mol / L of NaOH solution to adjust the pH value of the cetylammonium chloride solution to 10 - 11.

[0055] S1014: Keep the temperature of the cetylammonium chloride solution at 30 °C, and then add a methanol solution containing 5% tetraethyl orthosilicate in three portions, 50 - 70 μL each time, with a time interval of 30 minutes between each addition, and continuously stir and react for 17 hours to obtain nanoparticles coated with a 10 - 15 nm mesoporous silica layer.

[0056] S1015: Centrifuge and wash the surface-enhanced Raman scattering probe nanoparticles, and uniformly disperse them in absolute ethanol.

[0057] S103: Prepare a surface-enhanced Raman scattering probe contrast agent, ultrasonically disperse the surface-enhanced Raman scattering probe contrast agent, and inject a certain quantity into a quartz tube.

[0058] Uniformly disperse the surface-enhanced Raman scattering probe nanoparticles in physiological saline to make a surface-enhanced Raman scattering probe contrast agent with a concentration of 0.2 - 0.6 nmol / L. The quantity of the surface-enhanced Raman scattering probe contrast agent injected into the quartz tube is 20 - 30 μL, and the inner diameter of the quartz tube is 1 mm.

[0059] S105: Measure and record the Raman spectrum of the surface-enhanced Raman scattering probe contrast agent.

[0060] S107: Place the tissue sample to be tested on the stage, and bury the quartz tube containing the contrast agent in the tissue sample for Raman detection.

[0061] This step includes the following sub-steps:

[0062] S1071: Use a laser diffused beam to irradiate the lower surface of the tissue sample from the bottom up.

[0063] S1072: Detect the characteristic Raman signal on the upper surface of the tissue sample using a Raman fiber optic probe, and record the characteristic Raman signal;

[0064] S1073: Change the embedding depth of the quartz tube, and use a Raman fiber optic probe to detect and record the characteristic Raman signal on the upper surface of the tissue sample.

[0065] In the above steps, the wavelength of the laser diffused beam used is 785 nm, the laser diffused beam is a circular spot with a diameter of 0.5 - 1.7 cm, and the power density of the laser is 0.022 - 1.01 W / cm 2 .

[0066] S109: Analyze the characteristic Raman spectra obtained at different embedding depths of the quartz tube to obtain the difference in the effective attenuation coefficients of Raman photons with different wavelengths propagating in the tissue sample.

[0067] When analyzing the characteristic Raman spectra obtained at different embedding depths, the following sub-steps are included:

[0068] S1091: Use the Raman characteristic peaks of the Raman signal molecules to construct different Raman peak pairs;

[0069] S1092: Establish a relationship graph between the natural logarithm of the intensity ratio of each Raman peak pair and the embedding depth of the surface-enhanced Raman scattering probe contrast agent;

[0070] S1093: Perform a linear fit on the natural logarithm of the intensity ratio of the Raman peak pair and the embedding depth of the surface-enhanced Raman scattering probe contrast agent. The obtained fitting slope is the difference in the effective attenuation coefficients of the two different wavelength photons corresponding to this Raman peak pair when propagating in the tissue sample.

[0071] The linear equation obtained by performing a linear fit on the natural logarithm of the intensity ratio of the Raman peak pair and the embedding depth of the surface-enhanced Raman scattering probe contrast agent is:

[0072]

[0073] Among them, is the natural logarithm of the ratio of two spectral peaks in the Raman spectrum, Δμ 21 is the difference in the effective attenuation coefficients of the Raman photons of two spectral peaks in the Raman spectrum when propagating in the tissue, d is the depth of the Raman source, b is the natural logarithm of the original intensity ratio of the spectral peak pair of the pure Raman spectrum contrast agent, σ is the cross-section parameter of Raman scattering, D is the photon diffusion coefficient, and λ1 and λ2 are the wavelengths of the two spectral peaks.

[0074] In view of the fact that the existing methods for measuring the optical properties of tissues are complex, time-consuming, sensitive to instrument deviations, difficult to popularize and apply, and the results obtained are usually the scattering and absorption characteristics of tissues, rather than directly obtaining the difference in attenuation coefficients of photons with different wavelengths propagating in tissues, the present invention proposes a method for measuring the difference in effective attenuation coefficients of photons with different wavelengths in tissues. Compared with the prior art, the present invention has the following beneficial technical effects:

[0075] 1. It can measure the difference in effective attenuation coefficients of photons with different wavelengths in various tissues;

[0076] 2. The method of the present invention combines the multi-peak characteristics of Raman spectroscopic contrast agents, and a single Raman spectroscopic contrast agent can achieve the measurement between multiple wavelength Raman photons;

[0077] 3. The measurement method of the present invention uses Raman spectroscopic contrast agents, which have the characteristics of strong specificity, good stability, not easily undergoing photobleaching, and can avoid tissue background interference, which helps to achieve the measurement of the difference in effective attenuation coefficients of photons with different wavelengths with high accuracy;

[0078] 4. The equipment adopted by the present invention is simple, easy to collect, low in cost, and wide in application range.

[0079] The following will describe the present invention in detail in conjunction with the preferred embodiments of the present invention.

[0080] Due to the scattering and absorption characteristics, photons are attenuated when propagating in tissues, and photons with different wavelengths are attenuated to different degrees. After the Raman photons emitted by the contrast agent propagate through the tissue, the intensities of different Raman peaks are attenuated differently, and the difference in their attenuation is controlled by the difference in the effective attenuation coefficients of Raman photons with each wavelength propagating in the tissue. Selecting a specific pair of Raman spectral peaks, the natural logarithm of the intensity ratio will be correlated with the difference in the effective attenuation coefficients of the photons of these two spectral peaks, as shown in the appendix Figure 2 as shown.

[0081] Here, we use the radiative transfer equation to simulate the process of photon propagation in tissues to lay a theoretical foundation. First, assume that a continuous laser source is incident from one side of the tissue, and then Raman signals are collected from the opposite side of the tissue. Then, the corresponding steady-state diffusion approximation model is:

[0082]

[0083] This represents the light intensity at any point (x, y, z) at a distance r from the light source. Among them, 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 light source brightness, D is the photon diffusion coefficient, μ eff is the effective attenuation coefficient of the tissue, and can all be obtained from the absorption coefficient μ a and the effective scattering coefficient μ sCalculated. The signal source containing the Raman spectroscopic contrast agent is embedded in the tissue. Regarding the Raman source as a point light source, when the Raman source at (x0, y0, z0) is excited by a laser, the Raman light intensity at the position (x, y, z) with a distance from the Raman source is:

[0084]

[0085] Among them, and respectively represent the diffusion coefficient and the effective attenuation coefficient of Raman photons. C and σ represent the concentration of the Raman contrast agent and the Raman scattering cross section respectively. Therefore, assuming that the depth of the Raman source (i.e., the straight-line distance from it 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

[0086]

[0087] λ0 is the incident laser wavelength, λ i is the wavelength corresponding to the spectral peak of the outgoing Raman spectrum, T is the thickness of the turbid medium, and d is the depth of the Raman source. The above four parameters are all known. Select two spectral peaks with corresponding wavelengths λ1 and λ2 respectively, then the difference between the effective attenuation coefficients of the tissue at the two wavelengths is

[0088] Δμ 21 = μ eff (λ2) - μ eff (λ1) (4)

[0089] Combining equations (3) and (4), in the collected Raman spectrum, the natural logarithm of the ratio of the two spectral peaks is

[0090]

[0091] Among them,

[0092] This is a linear equation in the form of y = ax + b. The natural logarithm of the intensity ratio of the spectral peak pair in the linear function The depth d and the intercept b (i.e., the natural logarithm of the original intensity ratio of the spectral peak pair of the pure Raman spectroscopic contrast agent) are both known. Therefore, there is only one unknown in this linear equation, that is, the difference Δμ between the effective attenuation coefficients of the photons of the two spectral peaks propagating in the medium. During the transmission Raman measurement process, according to the natural logarithm of the intensity ratio of the spectral peak pair obtained by measurement and the depth d, construct a relationship diagram between the two and perform linear fitting. The obtained fitting slope is Δμ.

[0093] Example 1

[0094] Step 1: Prepare 5 mL of a surface-enhanced Raman scattering probe with a core-shell structure containing IR-780 Raman signal molecules at a concentration of 0.6 nmol / L (as shown in Attachment Figure 3 and Attachment Figure 4 ). Centrifuge and redisperse it in 5 mL of a 0.004 mol / L cetylammonium chloride solution. Add 0.1 mol / L NaOH solution to adjust the pH of the solution to 10 - 11. Keep the solution temperature at 30 °C, then add a methanol solution containing 5% tetraethyl orthosilicate in three portions, 70 μL each time, with a 30-minute interval between each addition. Continue stirring and reacting for 17 hours to obtain nanoparticles coated with a 15-nm mesoporous silica layer. Centrifuge and wash, and uniformly disperse in absolute ethanol.

[0095] Step 2: Uniformly disperse the surface-enhanced Raman scattering probe nanoparticles obtained above in physiological saline to prepare a 0.6 nmol / L surface-enhanced Raman scattering probe contrast agent. Ultrasonically disperse it and take 30 μL to inject into a quartz tube with an inner diameter of 1 mm. Measure and record the Raman spectrum of the pure surface-enhanced Raman scattering probe contrast agent.

[0096] Step 3: Place the ex vivo porcine adipose tissue sample to be tested on the stage, and bury the quartz tube containing the contrast agent into the tissue. Record the embedding depth and perform Raman detection. Use a diffused beam with a laser wavelength of 785 nm, a circular spot with a diameter of 1.7 cm, and a power density of 0.26 W / cm 2 . The laser irradiates the lower surface of the ex vivo porcine adipose tissue from bottom to top. Use a Raman fiber optic probe to detect and record the characteristic Raman signal of the contrast agent on the upper surface of the ex vivo porcine adipose tissue. Change the embedding depth of the contrast agent, and use a Raman fiber optic probe to detect and record the characteristic Raman spectrum of the contrast agent on the upper surface of the ex vivo porcine adipose tissue.

[0097] Step 4: Analyze the characteristic Raman spectra obtained at different embedding depths of the contrast agent. Record the natural logarithm of the intensity ratio of different Raman peak pairs, and establish a relationship graph with the depth of the contrast agent. Perform linear fitting on the relationship graph between the natural logarithm of the intensity ratio of Raman peak pairs and the depth. The obtained fitting slope is the difference in the effective attenuation coefficients of the two different wavelength photons corresponding to this Raman peak pair when propagating in porcine adipose tissue.

[0098] The experimental results are as shown in Attachment Figure 6 . Using the Raman characteristic peaks (520, 931, 1203, 1369, 1523, 1580 cm -1 ) of the Raman signal molecule IR-780 in the surface-enhanced Raman scattering probe (as shown in Attachment Figure 5As shown in the figure, different Raman peak pairs (520 / 1369, 520 / 1523, 931 / 1369, 931 / 1523, 931 / 1580, 1203 / 1369, 1203 / 1523, 1203 / 1580) were constructed, and the relationship between the natural logarithm of the intensity ratio of each Raman peak pair and the depth of the surface enhanced Raman scattering contrast agent was established. Then, a linear fit was performed on the relationship between the two, and the fitting slope obtained was the difference in the effective attenuation coefficient of Raman photons of different wavelengths propagating in the tissue.

[0099] Example 2

[0100] Step 1: Prepare 5mL 0.6nmol / L of a core-shell structured surface enhanced Raman scattering probe containing IR-783 Raman signal molecules, centrifuge and redisperse in 5mL 0.004mol / L hexadecyl ammonium chloride solution, add 0.1mol / L NaOH solution to adjust the pH value of the solution to 10-10.5; keep the solution temperature at 30°C, then add 5% tetraethyl orthosilicate methanol solution three times, 50uL each time, each time interval of 30 minutes, continue stirring and reacting for 17 hours, and obtain nanoparticles coated with a 10nm mesoporous silica layer; centrifuge and wash, and evenly disperse in anhydrous ethanol.

[0101] Step 2: The surface-enhanced Raman scattering probe nanoparticles obtained above are uniformly dispersed in physiological saline to prepare a 0.6nmol / L surface-enhanced Raman scattering probe contrast agent, ultrasonically dispersed and 30uL is injected into a quartz tube with an inner diameter of 1mm; the Raman spectrum of the pure surface-enhanced Raman scattering probe contrast agent is measured and recorded.

[0102] Step 3: Place the in vitro pig muscle tissue sample to be tested on the stage, and embed the quartz tube containing the contrast agent into the tissue, record the embedding depth and perform Raman detection. Use a diffuse beam of 785nm laser wavelength, the spot is a circle with a diameter of 0.5cm, and the power density is 1.01W / cm 2 The laser was irradiated from bottom to top on the lower surface of the isolated pig muscle tissue. The characteristic Raman spectrum of the contrast agent was detected and recorded on the upper surface of the isolated pig muscle tissue using a Raman fiber probe; the embedding depth of the contrast agent was changed, and the characteristic Raman spectrum of the contrast agent was detected and recorded on the upper surface of the isolated pig muscle tissue using a Raman fiber probe.

[0103] Step 4: Analyze the characteristic Raman spectra detected at different contrast agent embedding depths. Record the natural logarithm of the intensity ratio of different Raman peaks, and establish a relationship diagram with the depth of the contrast agent. Perform a linear fit on the relationship diagram between the natural logarithm of the intensity ratio of each Raman peak and the depth. The fitting slope obtained is the difference in the effective attenuation coefficients of the two different wavelength photons corresponding to the Raman peak pair when propagating in pig muscle tissue.

[0104] Example 3

[0105] Step 1: Prepare 5 mL of a surface-enhanced Raman scattering probe with a core-shell structure containing CV Raman signal molecules at a concentration of 0.6 nmol / L. Centrifuge and redisperse it in 5 mL of a 0.004 mol / L cetylammonium chloride solution. Add 0.1 mol / L NaOH solution to adjust the pH of the solution to 10 - 11. Keep the solution temperature at 30 °C, and then add a methanol solution containing 5% tetraethyl orthosilicate in three portions, 50 μL each time, with a 30-minute interval between each addition. Continue stirring and reacting for 17 hours to obtain nanoparticles coated with a 10-nm mesoporous silica layer. Centrifuge and wash, and uniformly disperse them in absolute ethanol.

[0106] Step 2: Uniformly disperse the surface-enhanced Raman scattering probe nanoparticles obtained above in physiological saline to prepare a surface-enhanced Raman scattering probe contrast agent at a concentration of 0.2 nmol / L. Ultrasonically disperse it and take 20 μL and inject it into a quartz tube with an inner diameter of 1 mm. Measure and record the Raman spectrum of the pure surface-enhanced Raman scattering probe contrast agent.

[0107] Step 3: Place the ex vivo porcine skin tissue sample to be measured on the stage, and bury the quartz tube containing the contrast agent in the tissue. Record the embedding depth and perform Raman detection. Use a diffused beam with a laser wavelength of 785 nm, a circular spot with a diameter of 1.5 cm, and a power density of 0.022 W / cm 2 . The laser irradiates the lower surface of the ex vivo porcine skin tissue from bottom to top. Use a Raman fiber optic probe to detect and record the characteristic Raman spectrum of the contrast agent on the upper surface of the ex vivo porcine skin tissue; change the embedding depth of the contrast agent, and use a Raman fiber optic probe to detect and record the characteristic Raman spectrum of the contrast agent on the upper surface of the ex vivo porcine skin tissue.

[0108] Step 4: Analyze the characteristic Raman spectra detected at different embedding depths of the contrast agent. Record the natural logarithm of the intensity ratio of different Raman peak pairs, and establish a relationship diagram with the contrast agent depth. Perform linear fitting on the relationship diagram between the natural logarithm of the intensity ratio of each Raman peak pair and the depth, and the obtained fitting slope is the difference in the effective attenuation coefficients of the two different-wavelength photons corresponding to the Raman peak pair when propagating in porcine skin tissue.

[0109] In a preferred embodiment of the present invention, when preparing a surface-enhanced Raman scattering probe with a core-shell structure containing Raman signal molecules, the above Raman signal molecules can also use one of DTTC, Cy5, and R6G Raman signal molecules.

[0110] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field based on the concept of the present invention through logical analysis, reasoning, or limited experiments on the basis of the prior art should fall within the protection scope determined by the claims.

Claims

1. A method for measuring the difference in effective attenuation coefficients of photons with different wavelengths in tissue, characterized in that, The method includes the following steps: S101: Prepare surface-enhanced Raman scattering (SERS) probe nanoparticles, where the SERS probe nanoparticles are coated with a mesoporous silica layer; S103: Prepare a SERS probe contrast agent, ultrasonically disperse the SERS probe contrast agent, and inject a certain amount into a quartz tube; S105: Measure and record the Raman spectrum of the SERS probe contrast agent; S107: Place the tissue sample to be measured on a stage, and bury the quartz tube containing the contrast agent into the tissue sample for Raman detection; S109: Analyze the characteristic Raman spectra obtained at different burial depths of the quartz tube to obtain the difference in the effective attenuation coefficients of Raman photons with different wavelengths propagating in the tissue sample; Wherein, The SERS probe nanoparticles use a gold-silver substrate and are combined with Raman signal molecules, and the Raman signal molecules include one of IR-780, IR-783, DTTC, Cy5, R6G, and CV Raman signal molecules; By recording the natural logarithm of the intensity ratio of different Raman peak pairs, establish a relationship diagram with the depth of the contrast agent, and perform linear fitting on the relationship diagram between the natural logarithm of the intensity ratio of the Raman peak pair and the depth. The obtained fitting slope is the difference in the effective attenuation coefficients of the two different wavelength photons corresponding to the Raman peak pair when propagating in the tissue; The linear equation obtained by performing linear fitting on the natural logarithm of the intensity ratio of the Raman peak pair and the burial depth of the SERS probe contrast agent is: wherein, is the natural logarithm of the ratio of two spectral peaks in the Raman spectrum, Δμ 21 is the difference in the effective attenuation coefficients of the Raman photons of two spectral peaks propagating in the tissue, d is the depth of the Raman source, and b is the natural logarithm of the ratio of the spectral peak pair of the pure Raman spectrum contrast agent to the original intensity, σ is the cross-sectional parameter of Raman scattering, D is the photon diffusion coefficient, and λ1 and λ2 are the wavelengths of the two spectral peaks.

2. The method according to claim 1, characterized in that, The step S101 includes the following steps: S1011: Prepare a SERS probe solution with a core-shell structure containing Raman signal molecules; S1012: Centrifuge and redisperse the SERS probe in a cetylammonium chloride solution; S1013: Add a NaOH solution to adjust the pH value of the cetylammonium chloride solution to 10 - 11; S1014: Keep the temperature of the cetylammonium chloride solution at 30°C, and then add a methanol solution containing tetraethyl orthosilicate three times, 50 - 70 μL each time, with a time interval of 30 minutes each time, and continuously stir and react for 17 hours to obtain nanoparticles coated with a 10 - 15 nm mesoporous silica layer; S1015: Centrifuge and wash the SERS probe nanoparticles, and uniformly disperse them in absolute ethanol.

3. The method according to claim 2, wherein The concentration of the SERS probe solution is 0.6 nmol / L, the volume of the cetylammonium chloride solution is 5 mL, the concentration of the cetylammonium chloride solution is 0.004 mol / L, the concentration of the NaOH solution is 0.1 mol / L, and the content of tetraethyl orthosilicate in the methanol solution is 5%.

4. The method according to claim 1, characterized in that In the step S103, the surface-enhanced Raman scattering probe nanoparticles are uniformly dispersed in physiological saline to prepare the surface-enhanced Raman scattering probe contrast agent. The concentration of the surface-enhanced Raman scattering probe contrast agent is 0.2 - 0.6 nmol / L, and the amount of the surface-enhanced Raman scattering probe contrast agent injected into the quartz tube is 20 - 30 μL. The inner diameter of the quartz tube is 1 mm.

5. The method according to claim 1, characterized in that, The step S107 includes the following steps: S1071: Use a laser diffused beam to irradiate the lower surface of the tissue sample from bottom to top; S1072: Use a Raman fiber probe to detect the characteristic Raman signal on the upper surface of the tissue sample and record the characteristic Raman signal; S1073: Change the embedding depth of the quartz tube, and use a Raman fiber probe to detect and record the characteristic Raman signal on the upper surface of the tissue sample.

6. The method according to claim 5, wherein The wavelength of the laser diffused beam is 785 nm. The laser diffused beam is a circular light spot, the diameter of the circular light spot is 0.5 - 1.7 cm, and the power density of the laser is 0.022 - 1.01 W / cm 2 .

7. The method according to claim 3, characterized in that, The step S109 includes the following steps: S1091: Construct different Raman peak pairs by using the Raman characteristic peaks of the Raman signal molecules; S1092: Establish a relationship graph between the natural logarithm of the intensity ratio of each Raman peak pair and the embedding depth of the surface-enhanced Raman scattering probe contrast agent; S1093: Perform linear fitting on the natural logarithm of the intensity ratio of the Raman peak pair and the embedding depth of the surface-enhanced Raman scattering probe contrast agent. The obtained fitting slope is the difference in the effective attenuation coefficients of the two different wavelength photons corresponding to the Raman peak pair when propagating in the tissue sample.

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