Deep detection system and method based on large light spot spatial offset Raman probe and application of deep detection system and method based on large light spot spatial offset Raman probe

Abstract

The invention discloses a deep detection system and method based on a large light spot space offset Raman probe and application. The deep detection system comprises a handheld probe shell, a laser light source, a light spot amplifier, a sample table, a Raman optical fiber probe, a spectrograph and a data processing module. The handheld probe shell is used for fixing the laser light source, the light spot amplifier and the Raman optical fiber probe into a whole, namely a large light spot space offset Raman probe is arranged on the upper side of the sample table, and the Raman optical fiber probe is electrically connected with the spectrograph and the data processing module respectively; the laser light source emits laser in parallel through the light spot amplifier, a light spot with the diameter of at least 1 cm is obtained on the surface of a sample to be detected, and the Raman optical fiber probe is located at the edge of the light spot. The laser safety is met, the detection depth in the biological tissue is large, and focuses in the tissues with different depths can be detected on the premise that the distance between the light spot center and the Raman collection probe is not adjusted.

Description

Technical Field

[0001] The present invention relates to the field of molecular imaging, and in particular to a deep detection system, method and application based on a large-spot spatially offset Raman probe. Background Art

[0002] Cancer seriously threatens the health and life safety of humans and animals, and is one of the diseases with a high mortality rate. At present, surgical resection is still one of the most effective methods for treating cancer. Complete radical resection of the tumor and achieving the desired R0 resection requirements can enable patients with malignant tumors to achieve long-term survival rates as much as possible. (In 1978, the American Joint Committee on Cancer (AJCC) first introduced the concept of residual tumor grading (R grading), that is, residual tumor grading is an indicator for evaluating the residual status of tumors after resection of malignant tumors, expressed as complete tumor resection (R0), microscopic residual (R1) and macroscopic residual tumor (R2). Today, R grading has been widely used in the evaluation of resection of malignant tumors such as gastric cancer, colorectal cancer and non-small cell lung cancer.) If residual tumor tissue or cells are left after surgery, it will lead to local implantation recurrence and even distant metastasis. In clinical practice, due to tumor infiltration and the presence of microscopic lesions, the tumor margins are usually blurred, and it is difficult for doctors to judge by naked eyes alone, making it difficult for most tumor surgical resections to achieve radical R0 resection, and residual lesions are prone to recurrence.

[0003] Molecular imaging intraoperative navigation technology can accurately detect lesions and define the boundaries of tumors, helping to completely remove tumors while protecting normal tissue structures as much as possible. Optical detection methods have great advantages in real-time, high sensitivity, and non-ionizing radiation in surgical navigation applications. Optical-based molecular imaging navigation has been successfully applied in human lung cancer, brain glioma, breast cancer, liver cancer, and colorectal cancer surgeries. However, due to the strong scattering and absorption effects of biological tissues on photons, the imaging depth of the currently widely used indocyanine green two-dimensional intraoperative fluorescence navigation is usually only a few millimeters. Therefore, we hope to use Raman technology to develop optical detection and navigation technology with high detection depth and safety.

[0004] However, Raman optical detection technology faces two major bottlenecks: detection depth and laser safety. First, due to the strong scattering and absorption effect of biological tissue on photons, the tissue penetration depth of photons is low. The currently widely used backscattering Raman detection technology can usually only detect Raman probe nanoparticles in tissues a few millimeters thick, and cannot detect deep tumor lesions (such as >1 cm deep in tissue). Secondly, traditional Raman detection often uses too high laser power to obtain a deeper detection depth, because increasing laser power is a direct way to improve the signal-to-noise ratio and detection depth, but this will lead to an increase in the rate of energy release per unit area, making the power density far exceed the maximum permissible clinical laser irradiation dose (Maximum Permeable Exposure, MPE), causing biological tissue damage.

[0005] Spatially offset Raman spectroscopy (SORS) separates the laser light source from the Raman fiber probe (which is used to receive Raman signals) with a certain distance (i.e., spatial offset) between the two, so as to collect the deep Raman signals of the sample while effectively eliminating the influence of the surface material signals. The basic principle is that since the migration direction of photons inside the medium is random, compared with the surface layer, the deep Raman photons generated inside are easier to migrate laterally during the diffusion process. Therefore, the Raman spectra collected at different spatial offsets contain Raman photon information of different depth layers.

[0006] Traditional SORS systems use Gaussian light sources, and the light spots are usually small (hundreds of microns), which makes the power density far exceed the MPE, which may cause permanent tissue damage and there are laser safety issues. In addition, as the spatial offset between the laser light source and the Raman fiber probe increases, the depth of the detectable lesion increases. Keller's research group has determined that a spatial offset of 3.5mm can detect lesions at a depth of 2mm. However, when using a Gaussian light source, as the spatial offset increases, the efficiency of the Raman fiber probe in collecting photons decays logarithmically, which limits the spatial offset (usually less than 4mm), resulting in limited detection depth. In this case, the spatial offset between the laser light source and the Raman fiber probe needs to be dynamically adjusted according to the depth of the object being measured. By changing the spatial offset, the Raman signal of the object being measured at different depth layers can be obtained; however, in actual surgery, the depth of the lesion is unknown, so the traditional SORS system is not suitable for similar surgical scenarios.

[0007] Secondly, the traditional SORS system separates the laser light source and the Raman fiber probe, and the entire system is not convenient for point-by-point moving detection. The current SORS detection research on ex vivo tissue mainly achieves point-by-point detection by moving the tissue, but this is not convenient for doctors to operate and is not suitable for actual surgical applications. Summary of the invention

[0008] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is that the power intensity of the laser dose does not meet the requirements of clinical safety, the operation is inconvenient, the tissue penetration depth is insufficient, the detection depth range is limited, and it is difficult to be applied clinically.

[0009] To achieve the above-mentioned object, the present invention provides a deep detection system based on a large-spot spatial offset Raman probe, comprising a handheld probe housing, a laser light source, a spot amplifier, a sample stage, a Raman fiber probe, a spectrometer and a data processing module;

[0010] The handheld probe housing fixes the laser light source, the spot amplifier and the Raman fiber probe into a whole, that is, a large spot spatial offset Raman probe, which is arranged on the upper side of the sample stage, and the Raman fiber probe is electrically connected to the spectrometer and the data processing module respectively;

[0011] The laser light source emits laser light in parallel through a spot amplifier, and the laser light source can obtain a spot with a diameter of at least 1 cm on the surface of the sample to be tested. The Raman optical fiber probe is located at the edge of the spot.

[0012] The large spot spatial offset Raman technology in the present invention reduces the laser power per unit area by increasing the spot diameter (centimeter level) compared to the traditional Gaussian laser (diameter is only micrometer level), meets the laser safety requirements, and is conducive to the advancement of this technology to clinical safety testing.

[0013] In a preferred embodiment of the present invention, the sample to be tested is placed horizontally on the sample stage, and the handheld probe housing and the Raman fiber optic probe fixed therein are attached to the surface of the sample to be tested and vertically oriented to the sample stage when in use.

[0014] In the present invention, the laser light source and the Raman fiber optic probe can be integrated into a small surgical probe using a handheld probe housing, and the Raman fiber optic probe is fixed to the edge of the laser spot, which is convenient for doctors to operate real-time detection and scanning, meeting the convenience of operation. The two-dimensional position of the detected object in the sample to be tested can be judged by the strength of the collected Raman signal.

[0015] In another preferred embodiment of the present invention, the Raman fiber optic probe is located outside the laser spot, and has a spatial offset of 0 to 10 mm from each point in the spot. Preferably, the spatial offset between the Raman fiber optic probe and the center of the spot is 4 to 6 mm, for example, 4 mm, 4.5 mm, 5 mm, 5.5 mm or 6 mm.

[0016] In the present invention, since the diameter of the spot of the excitation light source is large enough, when the Raman fiber optic probe is placed at the edge of the spot, the spatial offset varies from 0 to the spot diameter. The maximum spatial offset can reach 1 cm, and the detection depth is high. At the same time, the spatial offset of the traditional Gaussian laser-based SORS system is fixed, and it is more advantageous only for the detection of Raman photons in a specific depth layer, so it is necessary to continuously adjust the spatial offset to detect Raman photons in different depth layers. In the present invention, a large spot is used as the excitation light source, and the range of spatial offset is large. Raman photon information of different depth layers can be obtained at the same time, so as to detect lesions in a larger depth range. The two-dimensional imaging deviation is small (usually not more than 2 mm).

[0017] In another preferred embodiment of the present invention, the spot amplifier includes a beam expander and a collimator.

[0018] In another preferred embodiment of the present invention, the sample to be tested contains a surface enhanced Raman spectroscopy probe contrast agent, and the surface enhanced Raman spectroscopy probe contrast agent includes Raman probe nanoparticles, and the Raman probe nanoparticles are located in the test area of ​​the sample to be tested.

[0019] The method of combining large spot spatial offset Raman technology with surface enhanced Raman spectroscopy probe contrast agent can realize the non-invasive positioning detection of the above-mentioned lymph nodes, which has the advantages of real-time, non-invasive and no ionizing radiation. Doctors can non-invasively determine the two-dimensional positioning of the lesion, the lymph node resection process has small wounds, the patient suffers less harm, the prognosis is fast, and the complications are small.

[0020] In another preferred embodiment of the present invention, the Raman probe nanoparticles include a nanoparticle core, a Raman reporter molecule intermediate layer, a silver shell, and a protective layer wrapped around the nanoparticles;

[0021] The Raman reporter molecule intermediate layer includes organic fluorescent dye molecules;

[0022] The protective layer comprises any one of silicon dioxide, polyethylene glycol or polylactic acid or a combination of at least two thereof;

[0023] The particle size of the surface enhanced Raman spectroscopy probe contrast agent ranges from 20 to 200 nm, for example, it can be 20 nm, 50 nm, 100 nm, 150 nm, or 200 nm.

[0024] The surface-enhanced Raman probe contrast agent can enter the lymphatic system through intradermal injection via the reflux of lymph fluid, and passively target the sentinel lymph nodes and secondary lymph nodes; and the surface-enhanced Raman probe contrast agent can actively target the tumor by binding to specific molecules or receptors on the surface of tumor cells via surface-modified antibodies, ligands, DNA and other substances.

[0025] In another preferred embodiment of the present invention, the laser power of the laser light source is 200-210 mW, for example, 200 mW, 202 mW, 204 mW, 206 mW, 208 mW or 210 mW, and the laser power per unit area is 0.2-0.3 W / cm -2 , for example, it can be 0.2W / cm -2 , 0.22W / cm -2 , 0.24W / cm -2 , 0.25W / cm -2 , 0.2645W / cm -2 The laser power of the laser light source is the laser power of the Raman fiber probe.

[0026] In another preferred embodiment of the present invention, the Raman fiber optic probe comprises a fiber bundle consisting of collecting optical fibers;

[0027] The diameter of the Raman optical fiber probe does not exceed 3 mm.

[0028] In a second aspect, the present invention provides a method for a deep layer detection system based on a large spot spatial offset Raman probe as described above, the method comprising:

[0029] The sample to be tested is placed horizontally on the sample stage, and the handheld probe housing and the Raman fiber probe fixed therein are attached to the surface of the sample to be tested and are vertically oriented to the sample stage;

[0030] The laser light source emits laser light which is amplified by the spot amplifier and then irradiated vertically onto the sample to be tested. The Raman fiber optic probe collects the Raman signal passing through the sample to be tested and then transmits the signal to the spectrometer and data processing module for processing.

[0031] The collected Raman spectra are preprocessed by removing the baseline, and the characteristic peaks of the Raman probe nanoparticles are selected in the processed spectrum. By comparing the strengths of the characteristic peaks at different positions, it is believed that the projection point at the position with the strongest characteristic peak is the location of the lesion.

[0032] In a third aspect, the present invention provides a use of the deep detection system based on the large spot spatial offset Raman probe, and the deep detection system based on the large spot spatial offset Raman probe is used for precision medical detection.

[0033] The present invention has at least the following beneficial technical effects:

[0034] 1. The deep detection system based on the large spot spatial offset Raman probe provided by the present invention obtains a laser light source with a diameter of at least 1 cm through a spot amplifier, reduces the unit laser power density, and ultimately reaches the standard of clinical laser safety dose, meeting the premise of system detection of laser safety.

[0035] 2. The deep layer detection system based on the large spot spatial offset Raman probe provided by the present invention uses a handheld probe housing to fix the laser light source, spot amplifier and Raman fiber probe into a whole, that is, a large spot spatial offset Raman probe. The system of the present invention has a simple structure, convenient operation, and wide environmental adaptability, and can achieve accurate positioning and stable imaging of lesions in different scenarios.

[0036] 3. The deep detection system, method and use based on the large spot spatial offset Raman probe provided by the present invention are based on the spatial offset Raman technology, and a large spot with a diameter of at least 1 cm is obtained through the spot amplifier, and the Raman fiber probe is fixed at the edge of the spot to obtain a spatial offset of at least 1 cm, thereby improving the detection depth of the lesion. In the present invention, the spatial offset of the Raman fiber probe and each point in the spot is 0 to 10 mm, and the spatial offset has a large range of variation. The information of lesions in different depth layers can be obtained at the same time, and the intensity difference of the Raman signal from different depth layers is small, which realizes the detection of lesions in a large depth range. The Raman signal collected by the present invention decays smoothly along the moving direction of the probe. When detecting the lesion, the signal of the nanoparticles at a certain position from the probe can also be detected, and it is not easy to miss the tiny lesion; when imaging the lesion, the number of sampling points can be reduced and the sampling rate can be increased. The present invention has the advantages of large detection depth, large detection depth range when the spatial offset is not changed, high detection sensitivity and high imaging efficiency.

[0037] 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

[0038] Figure 1 It is a structural schematic diagram of a deep layer detection system based on a large spot spatial offset Raman probe according to a preferred embodiment of the present invention;

[0039] Figure 2 It is a schematic structural diagram of a core-shell structure surface enhanced Raman spectroscopy probe contrast agent according to a preferred embodiment of the present invention;

[0040] Figure 3 This is a transmission electron microscope characterization image of a core-shell structure surface enhanced Raman spectroscopy probe contrast agent of a preferred embodiment of the present invention;

[0041] Figure 4 It is a Raman spectrum diagram of a core-shell structure surface enhanced Raman spectroscopy probe contrast agent of a preferred embodiment of the present invention;

[0042] Figure 5 It is a detection effect diagram of different laser spots at different depths obtained based on Monte Carlo simulation according to different embodiments of the present invention.

[0043] Figure 6 This is a diagram showing the effect of a deep detection system based on a large-spot spatial offset Raman probe of a preferred embodiment of the present invention on tracing sentinel lymph nodes with a surface-enhanced Raman probe contrast agent injected into a living New Zealand rabbit.

[0044] in:

[0045] 1-handheld probe housing, 2-laser light source, 3-beam expander, 4-collimator, 5-sample to be tested, 6-surface enhanced Raman spectroscopy probe contrast agent, 7-sample stage, 8-Raman fiber probe, 9-spectrometer, 10-data processing module. DETAILED DESCRIPTION

[0046] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is 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.

[0047] In the drawings, components with the same structure are indicated by the same numerical reference numerals, and components with similar structures or functions are indicated by similar numerical reference numerals. 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. In order to make the illustration clearer, the thickness of the components is appropriately exaggerated in some places in the drawings.

[0048] like Figure 1 As shown, the present invention provides a deep detection system based on a large spot spatial offset Raman probe, characterized in that it includes a handheld probe housing 1, a laser light source 2, a spot amplifier, a sample stage 7, a Raman fiber probe 8, a spectrometer 9 and a data processing module 10;

[0049] The handheld probe housing 1 fixes the laser light source 2, the spot amplifier and the Raman fiber probe 8 as a whole, and is arranged on the upper side of the sample stage 7. The Raman fiber probe 8 is connected to the spectrometer 9, and further electrically connected to the data processing module 10;

[0050] The laser light source 2 emits laser light in parallel through a spot amplifier to obtain a spot with a diameter of at least 1 cm on the surface of the sample to be tested, and the Raman fiber probe 8 is located at the edge of the spot.

[0051] The spot amplifier is composed of a beam expander 3 and a collimator 4, which is used to amplify the spot diameter of the Gaussian laser emitted from the laser light source 2 and make the laser emit in parallel. The beam expander 3 makes the emitted laser have a small angle deviation, and spots of different diameters can be obtained according to the distance of the laser emission.

[0052] like Figure 2-4As shown, the surface enhanced Raman spectroscopy probe contrast agent comprises Raman probe nanoparticles, wherein the Raman probe nanoparticles comprise a nanoparticle core, a Raman reporter molecule intermediate layer, a silver shell and a protective layer wrapped on the nanoparticles;

[0053] The Raman reporter molecule intermediate layer includes organic fluorescent dye molecules;

[0054] The protective layer comprises any one of silicon dioxide, polyethylene glycol or polylactic acid or a combination of at least two thereof;

[0055] The particle size of the surface enhanced Raman spectroscopy probe contrast agent ranges from 20 to 200 nm. When applied to biological imaging, it has the advantages of strong signal and good repeatability, and can achieve highly sensitive and quantitative target detection.

[0056] like Figure 5 As shown in the figure, the detection effect of the spot diameter on the objects to be tested at different depths in the biological tissue in the large spot spatial offset Raman detection system is simulated according to the Monte Carlo simulation. Figure A shows the simulated model, the horizontal axis is the position of the Raman fiber probe, and the object to be tested is fixed at the position X = 0mm. On the surface of the biological tissue, the large spot spatial offset Raman probe is moved with a step length of 1mm. The vertical axis is the intensity of the collected Raman signal, which is proportional to the number of collected Raman photons. In Figure B, the laser spot diameter is small, and the difference in the intensity of the Raman photon signal at different depth layers is large, which means that it only has an advantage in signal detection at a specific depth layer. In Figure C, the spot diameter is increased to 1cm, and the difference in the intensity of the Raman photon signal from different depth layers becomes smaller, which means that Raman photon information within a larger depth range can be detected. At the same time, as the spot diameter increases, the attenuation of the Raman signal along the moving direction of the Raman fiber probe becomes more gentle, which means that the signal of nanoparticles at a certain distance from the probe can also be detected, and it is not easy to miss tiny lesions.

[0057] like Figure 6 As shown, the above-mentioned imaging system of the present invention adopts a large spot spatial offset Raman detection device system to collect deep Raman photons, realizes a deep penetration detection method, and combines with a highly sensitive surface-enhanced Raman spectroscopy probe contrast agent to realize Raman optical detection of deep tumors, which is applied to the lesion positioning of the surface-enhanced Raman spectroscopy probe contrast agent injected into the living New Zealand rabbit. Figure A is to inject 150μL of surface-enhanced Raman spectroscopy probe contrast agent around the second pair of nipples of the rabbit, massage for 1 hour, and wait for the surface-enhanced Raman spectroscopy probe contrast agent to flow along the lymphatic vessels to the vicinity of the sentinel lymph node. Then, based on experience, a point-by-point Raman scan is performed on the skin surface near the sentinel lymph node. Figure B is based on the information obtained from the Raman scan, with the Raman characteristic peak (1203cm -1) intensity, and the two-dimensional heat map drawn. The smaller the grayscale, the greater the signal intensity. Figure C is based on the point with the smallest grayscale in the two-dimensional heat map of Figure B, judging that the point is the projection point of the sentinel lymph node on the skin surface. Figure D is to operate from the projection point, remove the skin, muscle and other tissues covering the sentinel lymph node layer by layer, and finally expose the sentinel lymph node (marked with a circle), realizing the non-invasive positioning and surgical guidance of the sentinel lymph node traced by the surface-enhanced Raman spectroscopy probe contrast agent.

[0058] In a preferred embodiment, during the non-invasive positioning and intraoperative navigation of sentinel lymph nodes in the human body, a commonly used sector scanning technique can be selected to replace the above-mentioned dot scanning, and the large spot spatial offset Raman probe can be used according to the peak 1203cm in the collected Raman spectrum. -1 The signal intensity at the location emits sounds of different frequencies. The greater the signal intensity of the characteristic peak, the greater the sound frequency. During the probe scanning process, the doctor can judge the existence of a sentinel lymph node near the projection point based on the probe sound frequency (for example, we set a threshold for the intensity of the characteristic peak, the detected signal intensity is greater than a certain threshold, and the probe sound frequency is larger), and perform sentinel lymph node resection surgery.

[0059] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians 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 be within the scope of protection determined by the claims.

Claims

1. A deep detection system based on a large spot spatial offset Raman probe, characterized in that: It includes a handheld probe housing, a laser light source, a spot amplifier, a sample stage, a Raman fiber probe, a spectrometer and a data processing module; The handheld probe housing fixes the laser light source, the spot amplifier and the Raman fiber probe as a whole, and is arranged on the upper side of the sample stage. The Raman fiber probe is electrically connected to the spectrometer and the data processing module respectively; The laser light source emits laser light in parallel through a spot amplifier to obtain a spot with a diameter of at least 1 cm on the surface of the sample to be tested, and the Raman optical fiber probe is located at the edge of the spot.

2. The deep layer detection system based on the large spot spatial offset Raman probe as claimed in claim 1, characterized in that: The sample to be tested is placed horizontally on the sample stage. The handheld probe housing and the Raman optical fiber probe fixed therein are attached to the surface of the sample to be tested and are vertically oriented to the sample stage when in use.

3. The deep layer detection system based on the large spot spatial offset Raman probe as claimed in claim 1, characterized in that: The Raman optical fiber probe is located outside the light spot, and has a spatial offset of 0 to 10 mm from each point in the light spot.

4. The deep layer detection system based on the large spot spatial offset Raman probe as claimed in claim 1, characterized in that: The light spot amplifier comprises a beam expander and a collimator.

5. The deep layer detection system based on the large spot spatial offset Raman probe as claimed in claim 1, characterized in that: The sample to be tested contains a surface enhanced Raman spectroscopy probe contrast agent, wherein the surface enhanced Raman spectroscopy probe contrast agent includes Raman probe nanoparticles, and the Raman probe nanoparticles are located in a test area of ​​the sample to be tested; The Raman probe nanoparticles include a nanoparticle core, a Raman reporter molecule intermediate layer, a silver shell and a protective layer wrapped on the nanoparticles.

6. The deep layer detection system based on the large spot spatial offset Raman probe as claimed in claim 5, characterized in that: The Raman reporter molecule intermediate layer includes organic fluorescent dye molecules; The protective layer comprises any one of silicon dioxide, polyethylene glycol or polylactic acid or a combination of at least two thereof; The particle size of the surface enhanced Raman spectroscopy probe contrast agent ranges from 20 to 200 nm.

7. The deep layer detection system based on the large spot spatial offset Raman probe as claimed in claim 1, characterized in that: The laser power of the laser light source is 200-210 mW, and the laser power per unit area is 0.2-0.3 W / cm -2 .

8. The deep layer detection system based on the large spot spatial offset Raman probe as claimed in claim 1, characterized in that: The Raman optical fiber probe includes an optical fiber bundle composed of collecting optical fibers; The diameter of the Raman optical fiber probe does not exceed 3 mm.

9. The method of the deep layer detection system based on the large spot spatial offset Raman probe as claimed in claims 1 to 8, characterized in that: The method comprises: The sample to be tested is placed horizontally on the sample stage, and the handheld probe housing and the Raman fiber probe fixed therein are attached to the surface of the sample to be tested and are vertically oriented to the sample stage; The laser emitted by the light source is amplified by the spot amplifier and then irradiated vertically on the sample to be tested. The Raman fiber optic probe collects the Raman signal emitted by the surface enhanced Raman spectroscopy probe imaging in the sample to be tested, and then transmits the signal to the spectrometer and data processing module for processing.

10. Use of the deep layer detection system based on the large spot spatial offset Raman probe as claimed in claims 1 to 8, characterized in that: The deep detection system based on the large-spot spatially offset Raman probe is used for precision medical detection.

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