A deep detection system, method and application based on a large-spot spatially offset Raman probe

Abstract

The present invention discloses a deep-layer detection system, method, and application based on a large-spot spatially offset Raman probe. The system comprises 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 secures the laser light source, spot amplifier, and Raman fiber probe into a single unit, namely, a large-spot spatially offset Raman probe, which is disposed 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 the spot amplifier, producing a spot with a diameter of at least 1 cm on the surface of the sample to be tested. The Raman fiber probe is positioned at the edge of the spot. The present invention ensures laser safety and has a large detection depth within biological tissue. It can detect lesions within tissues at different depths without adjusting the distance between the spot center and the Raman collection probe.

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 poses a serious threat to the health and safety of humans and animals, and is one of the diseases with the highest mortality rates. Currently, surgical resection remains one of the most effective treatments for cancer. Complete and radical resection of the tumor, achieving the desired R0 resection, maximizes the long-term survival rate for patients with malignant tumors. (In 1978, the American Joint Committee on Cancer (AJCC) first introduced the concept of residual tumor grading (R-grading). Residual tumor grading is an indicator used to assess residual tumor after resection of a malignant tumor, expressed as complete resection (R0), microscopic residual tumor (R1), and macroscopic residual tumor (R2). Today, R-grading is widely used in the assessment of resections for malignant tumors such as gastric cancer, colorectal cancer, and non-small cell lung cancer.) Residual tumor tissue or cells after surgery can lead to local recurrence and even distant metastasis. In clinical practice, tumor margins are often blurred due to tumor infiltration and the presence of microscopic lesions, making them difficult to assess visually. This makes it difficult for most surgical resections to achieve a radical R0 resection, and residual tumors 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 the huge advantages of real-time, high sensitivity, and non-ionizing radiation in surgical navigation applications. Optical-based molecular imaging navigation has been successfully applied in human surgeries for lung cancer, brain glioma, breast cancer, liver cancer, and colorectal cancer. However, due to the strong scattering and absorption effects of biological tissue 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 backscattered Raman detection technology can usually only detect Raman probe nanoparticles in tissues a few millimeters thick, and cannot detect deep tumor lesions (such as tissues >1 cm deep). Secondly, traditional Raman detection often uses excessively 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, this will lead to an increase in the rate of energy release per unit area, making the power density far exceed the maximum permissible exposure (MPE) of clinical lasers, causing damage to biological tissues.

[0005] Spatially offset Raman spectroscopy (SORS) separates the laser source and the Raman fiber probe (which receives the Raman signal) by a certain distance (i.e., a spatial offset). This allows the collection of Raman signals from deep within the sample while effectively eliminating the influence of surface material signals. The basic principle is that, because photons migrate randomly within a medium, Raman photons generated deep within the medium are more likely to migrate laterally during diffusion than those generated at the surface. Therefore, Raman spectra collected at different spatial offsets contain information about Raman photons from different depths.

[0006] Traditional SORS systems use Gaussian light sources, which typically have a small spot size (hundreds of microns), resulting in a power density far exceeding the MPE. This can cause permanent tissue damage and raises concerns about laser safety. Furthermore, as the spatial offset between the laser source and the Raman fiber probe increases, the depth of the detectable lesion increases. Keller's group previously determined that a spatial offset of 3.5 mm could detect lesions at a depth of 2 mm. However, when using a Gaussian light source, the efficiency of the Raman fiber probe in collecting photons decreases logarithmically with increasing spatial offset, limiting the spatial offset (typically less than 4 mm) and, consequently, limiting the detection depth. In this scenario, the spatial offset between the laser source and the Raman fiber probe needs to be dynamically adjusted based on the depth of the object being measured. By varying the spatial offset, Raman signals from different depths of the object can be obtained. However, in actual surgical procedures, the lesion depth is unknown, making traditional SORS systems unsuitable for such scenarios.

[0007] Secondly, traditional SORS systems separate the laser light source and Raman fiber probe, making it difficult to move the entire system for point-by-point detection. Current SORS research on ex vivo tissue primarily relies on moving the tissue to achieve point-by-point detection, but this is inconvenient for doctors and unsuitable 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 clinical safety requirements, the operation is inconvenient, the tissue penetration depth is insufficient, and the detection depth range is limited, making it 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 an integral body, namely the large spot spatial offset Raman probe, which 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;

[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 measured. The Raman fiber probe is located at the edge of the spot.

[0012] The large-spot spatially offset Raman technology in this invention reduces the laser power per unit area by increasing the spot diameter (centimeter-level) compared to traditional Gaussian lasers (diameter only micrometer-level), meets laser safety requirements, and facilitates the advancement of this technology towards 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 perpendicular to the sample stage when in use.

[0014] In the present invention, the laser light source and the Raman fiber probe can be integrated into a small surgical probe using a handheld probe housing, and the Raman fiber 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 determined 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 light spot of the excitation light source is large enough, when the Raman fiber optic probe is placed at the edge of the light spot, the spatial offset varies from 0 to the diameter of the light spot. 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, which is more advantageous only for detecting Raman photons at a specific depth layer. Therefore, it is necessary to continuously adjust the spatial offset to detect Raman photons at different depth layers. In the present invention, a large light spot is used as the excitation light source, and the range of spatial offset is large. Raman photon information at different depth layers can be obtained simultaneously, enabling the detection of lesions at a larger depth range. The two-dimensional imaging deviation is small (usually no 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, which includes Raman probe nanoparticles. The Raman probe nanoparticles are located in the test area of the sample to be tested.

[0019] Combining large-spot spatially offset Raman technology with surface-enhanced Raman spectroscopy probe contrast agents enables the aforementioned noninvasive localization of lymph nodes. This approach offers the advantages of real-time, noninvasive, and ionizing radiation-free detection. Doctors can noninvasively determine the two-dimensional location of lesions, while lymph node removal surgery is minimally invasive, resulting in minimal patient injury, a faster prognosis, and fewer complications.

[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 backflow of lymph fluid, and passively target the sentinel lymph nodes and secondary lymph nodes; moreover, 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 through 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 0.2 W / cm -2 , 0.22 W / cm -2 , 0.24 W / cm -2 , 0.25 W / cm -2 , 0.2645 W / 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 includes a fiber bundle consisting of collecting optical fibers;

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

[0028] In a second aspect, the present invention provides a method for a deep detection system based on a large-spot spatially offset Raman probe, the method comprising:

[0029] Place the sample to be tested horizontally on the sample stage, and attach the handheld probe housing and the Raman fiber probe fixed therein to the surface of the sample to be tested, and face vertically 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 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 were preprocessed by removing the baseline, and the characteristic peaks of the Raman probe nanoparticles were selected in the processed spectrum. By comparing the strengths of the characteristic peaks at different positions, it was determined that the projection point at the position with the strongest characteristic peak was 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 spatially offset Raman probe, wherein the deep detection system based on the large-spot spatially 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-layer detection system based on a large-spot spatially 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, reducing the unit laser power density and ultimately achieving the clinical laser safety dose standard, meeting the prerequisite for system detection of laser safety.

[0035] 2. The deep-layer detection system based on a large-spot spatially offset Raman probe, provided by this invention, utilizes a handheld probe housing to secure a laser light source, a spot amplifier, and a Raman fiber probe into a single unit. This system, known as the large-spot spatially offset Raman probe, boasts a simple structure, convenient operation, and wide adaptability, enabling precise lesion location and stable imaging in a variety of scenarios.

[0036] 3. The present invention provides a deep-layer detection system, method, and use based on a large-spot spatially offset Raman probe. Based on spatially offset Raman technology, a large spot diameter of at least 1 cm is achieved through a spot amplifier. The Raman fiber probe is fixed to the edge of the spot, achieving a spatial offset of at least 1 cm, thereby increasing the depth of lesion detection. The spatial offset between the Raman fiber probe and each point in the spot ranges from 0 to 10 mm, allowing for a wide range of spatial offset variations. This allows for simultaneous acquisition of information on lesions at different depths, while minimizing the difference in Raman signal intensity from different depths, enabling lesion detection over a wide depth range. The Raman signal collected by the present invention decays smoothly along the direction of probe movement. When detecting lesions, signals from nanoparticles at a certain distance from the probe can also be detected, making it less likely to miss tiny lesions. When imaging lesions, the number of sampling points can be reduced, increasing the sampling rate. The present invention offers the advantages of a large detection depth, a wide detection depth range when the spatial offset is unchanged, 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 1 is a schematic structural 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 This 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 according to a preferred embodiment of the present invention;

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

[0042] Figure 5 These are detection effect diagrams 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 tracing sentinel lymph nodes using a surface-enhanced Raman probe contrast agent injected into a living New Zealand rabbit using a deep detection system based on a large-spot spatial offset Raman probe according to a preferred embodiment of the present invention.

[0044] in:

[0045] 1-Handheld probe housing, 2-Laser light source, 3-Beam expander, 4-Collimator, 5-Sample to be measured, 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 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.

[0047] In the drawings, components with identical structures are denoted by the same reference numerals, and components with similar structures or functions are denoted by similar reference numerals. The size and thickness of each component shown in the drawings are arbitrary and are not limited by the present invention. For clarity, the thickness of components in some places in the drawings is appropriately exaggerated.

[0048] like Figure 1 As shown, the present invention provides a deep detection system based on a large spot spatial offset Raman probe, which is characterized by comprising 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 into an integral body, and is disposed 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, obtaining a spot with a diameter of at least 1 cm on the surface of the sample to be measured, and the Raman fiber probe 8 is located at the edge of the spot.

[0051] The spot amplifier, consisting of a beam expander 3 and a collimator 4, is used to amplify the diameter of the Gaussian laser spot emitted from laser source 2 and parallelize the laser beam. The beam expander 3 deflects the emitted laser beam by a small angle, allowing different spot diameters to be obtained depending on the distance the laser beam is emitted.

[0052] like Figure 2-4 As shown, the surface enhanced Raman spectroscopy probe contrast agent includes Raman probe nanoparticles, wherein 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;

[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 used in 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 FIG, the detection effect of the spot diameter on the objects at different depths in the biological tissue in the large spot spatial offset Raman detection system is simulated based on Monte Carlo simulation. Figure 5 (A) shows the simulated model. The horizontal axis represents the position of the Raman fiber probe, with the object being measured fixed at position X = 0 mm. The Raman probe is spatially offset by moving the large spot size in 1 mm steps across the tissue surface. The vertical axis represents the collected Raman signal intensity, which is proportional to the number of collected Raman photons. Figure 5 In (B), the laser spot diameter is small, and the Raman photon signal intensity at different depth layers varies greatly, which means that it has an advantage only in signal detection at specific depth layers. Figure 5 In (C), increasing the spot diameter to 1 cm reduces the difference in Raman photon signal intensity from different depth layers, allowing detection of Raman photon information across a wider depth range. Furthermore, as the spot diameter increases, the attenuation of the Raman signal along the Raman fiber probe's direction of motion becomes more gradual, allowing the detection of signals from nanoparticles at a certain distance from the probe, making it less likely 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, realizing a deep-penetration detection method. Combined with a highly sensitive surface-enhanced Raman spectroscopy probe contrast agent, it realizes Raman optical detection of deep tumors and is applied to localize lesions by injecting surface-enhanced Raman spectroscopy probe contrast agents into living New Zealand rabbits. Figure 6 (A) 150 μL of surface-enhanced Raman spectroscopy (SERS) contrast agent was injected around the second nipple of a rabbit. Massage was then performed for 1 hour, allowing the SERS contrast agent to flow through the lymphatic vessels to the vicinity of the sentinel lymph node. Then, based on empirical evidence, point-by-point Raman scanning was performed on the skin surface near the sentinel lymph node. Figure 6 (B) is the information obtained from Raman scanning, with the Raman characteristic peak (1203 cm -1 ) intensity, the two-dimensional heat map drawn, the smaller the grayscale, the greater the signal intensity. Figure 6 (C) is based on Figure 6 (B) The point with the smallest grayscale in the two-dimensional heat map is judged to be the projection point of the sentinel lymph node on the skin surface. Figure 6 (D) The surgery is performed from the projection point, and the skin, muscle and other tissues covering the sentinel lymph node are removed layer by layer, finally exposing the sentinel lymph node (marked with a circle), thus achieving 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, the commonly used sector scanning technique can be selected to replace the above-mentioned dot scanning, and the large spot spatial offset Raman probe is based on the peak 1203 cm 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 higher 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 higher), and perform sentinel lymph node resection surgery.

[0059] The preferred embodiments of the present invention have been described in detail above. 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 one 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 deep detection system based on a large-spot spatially 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 into an integral 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 measured, and the Raman fiber probe is located at the edge of the spot; The Raman optical fiber probe is located outside the light spot.

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

3. The deep layer detection system based on the large spot spatial offset Raman probe according to claim 1, characterized in that: The light spot amplifier includes a beam expander and a collimator.

4. The deep layer detection system based on the large spot spatial offset Raman probe according to 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 the test area of the sample to be tested; The Raman probe nanoparticles comprise a nanoparticle core, a Raman reporter molecule intermediate layer, a silver shell and a protective layer wrapped around the nanoparticles.

5. The deep detection system based on the large spot spatial offset Raman probe according to claim 4, 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.

6. The deep layer detection system based on the large spot spatial offset Raman probe according to 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 .

7. The deep layer detection system based on the large spot spatial offset Raman probe according to claim 1, characterized in that: The Raman fiber optic probe includes a fiber bundle consisting of collecting optical fibers; The diameter of the Raman fiber optic probe does not exceed 3 mm.

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