Method for determining subcutaneous detection depth of Raman spectrum system

Abstract

The invention relates to a method for determining subcutaneous detection depths of a Raman spectrum system. The Raman spectrum system has a plurality of detection depths. The method comprises the steps that a plurality of Raman spectrums of a to-be-detected area of a testee are collected through the Raman spectrum system, and the multiple Raman spectrums correspond to the multiple detection depths; for the plurality of Raman spectrums, calculating the ratio of the signal intensity of the first characteristic peak to the signal intensity of the second characteristic peak of each Raman spectrum, and selecting a first spectrum from the plurality of Raman spectrums based on the ratio; acquiring an optical coherence tomography (OCT) image of the to-be-detected area, and determining the depth of a basement membrane (DEJ) of the to-be-detected area; determining the detection depth corresponding to the first spectrum as the DEJ depth; and repeating the steps to determine all the detection depths.

Description

Technical Field

[0001] The present application relates to the field of spectral detection, and in particular to a method for determining the subcutaneous detection depth of a Raman spectroscopy system. Background Art

[0002] Raman spectroscopy-based biomolecule detection is a rapid, sensitive, highly specific, label-free and non-invasive analytical technique that is widely used in clinical diagnosis and biomedical research. However, there are still many challenges in achieving the detection of biomolecules hidden under the skin. The first and foremost challenge is whether Raman spectroscopy can reach the area where the biomolecules to be detected are located under the skin.

[0003] The penetration depth of Raman spectroscopy in the skin is affected by many factors. The strong scattering and absorption of photons by skin tissue severely limits the penetration depth of Raman spectroscopy in living tissues. The high scattering coefficient of epidermal tissue causes most photons to be scattered in the surface layer. The detection depth of Raman spectroscopy in the skin is affected by the excitation wavelength. Long-wavelength photons penetrate deeper into the skin than short-wavelength photons, but the intensity of their Raman signals will also weaken. The penetration depth of Raman spectroscopy in the skin is also closely related to the laser power. The possibility of detecting deep Raman scattered photons increases with the increase of laser power, but excessive laser power can cause damage to the sample. At the same time, the depth of the detectable Raman spectrum is also affected by the detector sensitivity of the Raman spectroscopy system.

[0004] Therefore, the subcutaneous detection depth of Raman spectroscopy is affected by many factors. Quantitative characterization of the detection depth of Raman spectroscopy can provide more powerful support for clinical diagnosis and biomedical research, but there is no mature method at present. Summary of the invention

[0005] The present application proposes a method for determining the subcutaneous detection depth of a Raman spectroscopy system, so as to characterize the detection depth of the Raman spectroscopy when targeting biological tissues in different parts. Wherein, the Raman spectroscopy system has multiple detection depths. The method comprises the following steps: collecting multiple Raman spectra of the subject's test area through the Raman spectroscopy system, wherein the multiple Raman spectra correspond to multiple detection depths; for the multiple Raman spectra, calculating the ratio of the signal intensity of the first characteristic peak of each Raman spectrum to the signal intensity of the second characteristic peak, and based on the ratio, selecting the first spectrum from the multiple Raman spectra; collecting an optical coherence tomography (OCT) image of the test area to determine the basement membrane (DEJ) depth of the test area; determining the detection depth corresponding to the first spectrum as the DEJ depth; repeating the above steps to determine all the detection depths.

[0006] Optionally, the method further includes preprocessing the multiple Raman spectra, including: removal of cosmic rays and fluorescence background, band selection and normalization, so as to obtain the multiple Raman spectra with high signal-to-noise ratio.

[0007] Optionally, the wave number region of the first characteristic peak is 1230 cm -1 ~1250cm -1 The wave number region of the second characteristic peak is 1310 cm -1 ~1330cm -1 .

[0008] Optionally, the wave number of the first characteristic peak is 1240 cm -1 The wave number of the second characteristic peak is 1320cm -1 .

[0009] Optionally, selecting a first spectrum from the plurality of Raman spectra based on the ratio comprises:

[0010] The Raman spectrum corresponding to the turning point where the ratio changes from greater than 1 to less than 1 is determined as the first spectrum.

[0011] Optionally, the plurality of Raman spectra are acquired by a transcutaneous detection mode.

[0012] Optionally, the subcutaneous detection range of the Raman spectroscopy system is 0 microns to 1000 microns, and the resolution is 50 microns to 100 microns.

[0013] Optionally, the Raman spectroscopy system comprises:

[0014] Confocal Raman spectroscopy system, or micro spatial offset Raman spectroscopy (μSORS) system, or multi-channel micro spatial offset Raman scattering spectroscopy (mμSORS) system.

[0015] Based on the Raman spectral characteristics of the obvious stratification of epidermis and dermis in the skin, this application innovatively proposes that the above-mentioned subjects whose skin thickness has been characterized by OCT act as a "scale", and then all "scales" are determined by multiple subjects with different skin thicknesses. The method proposed in this application can be used to characterize the depth of subcutaneous Raman spectroscopy detection. Compared with the existing subcutaneous detection using Raman spectroscopy systems, this application can:

[0016] (1) Improve detection accuracy: By accurately characterizing the subcutaneous detection depth of the Raman spectroscopy system, the molecules to be detected can be more accurately located and detected, thereby accurately obtaining the characteristic information of the subcutaneous molecules to be detected, reducing the interference of other information, and improving the reliability of the detection results;

[0017] (2) Promote medical applications: By optimizing the subcutaneous detection depth of the Raman spectroscopy system, its matching with the subcutaneous depth of each target biological molecule can be clarified, and Raman spectroscopy technology can be applied to in situ non-destructive detection of target samples or tissues in vivo, further expanding its application areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the principle of detecting signals at different subcutaneous depths by the mμSORS Raman spectroscopy system according to an embodiment of the present application.

[0019] Figure 2 It is a flow chart of a method for determining the subcutaneous detection depth of a Raman spectroscopy system according to an embodiment of the present application.

[0020] Figure 3 It is a signal preprocessing flow chart according to an implementation manner of the present application.

[0021] Figure 4 It is a schematic diagram of the Raman spectrum collected according to the implementation mode of the present application.

[0022] Figure 5 Schematic diagram of the depth of human skin and basement membrane according to an embodiment of the present application.

[0023] Figure 6 (a) is a graph showing depth and signal intensity of multiple-region OCT images according to an embodiment of the present application.

[0024] Figure 6 (b) is a graph showing the mean signal intensity versus subcutaneous depth according to an embodiment of the present application.

[0025] Figure 7 (a) is a schematic diagram of the DEJ depth of the skin of a subject according to an embodiment of the present application.

[0026] Figure 7 (b) is a graph of subcutaneous depth and signal strength of a subject according to an embodiment of the present application.

[0027] Figure 7 (c) is a "ruler" consisting of the value of the detection depth of the subject determined according to the embodiment of the present application.

[0028] Figure 7 (d) is a Raman spectrum diagram of a subject according to an embodiment of the present application.

[0029] Figure 7 (e) is a schematic diagram of a subject according to an embodiment of the present application. DETAILED DESCRIPTION

[0030] Next, the method proposed in this application is described in detail with reference to the accompanying drawings.

[0031] In the present application, a multi-channel micro spatial offset Raman scattering spectroscopy (mμSORS) system is taken as an example to illustrate the method according to the present application.

[0032] Figure 1 Schematic diagram of the principle of detecting subcutaneous signals at different depths by the mμSORS Raman spectroscopy system according to the embodiment of the present application. The mμSORS Raman spectroscopy system detects Raman spectroscopy signals at different skin depths through multiple groups of optical fiber channels with different offset distances, such as Figure 1 As shown in the figure, after the excitation light interacts with the skin, a large number of Raman photons are scattered. The mμSORS system sets different numbers of optical fiber channels at different offset distances (offset 1, offset 2, offset 3, offset 4, ... offset n) from the laser excitation point on the sample surface. The larger the above displacement, the more contribution of deeper samples in the obtained Raman signal, that is, the signals collected by different offsets come from different depths under the skin, that is, the detection depths as shown in the figure are D1, ..., D i ,...,D n Multiple Raman spectra.

[0033] Normally, the depth that can be detected by each offset can be determined based on theoretical calculations. However, due to the strong scattering and absorption of photons by skin tissue, the penetration depth of Raman spectroscopy in living tissues is severely limited. Therefore, when performing subcutaneous detection on human skin, the actual depth detected by each offset is not based on the depth value calculated theoretically. Therefore, it is necessary to use the method proposed in this application to characterize the actual detection depth of the Raman spectroscopy system during subcutaneous detection.

[0034] Those skilled in the art will appreciate that, in addition to the mμSORS Raman spectroscopy system, the method according to the present application is also applicable to a confocal Raman spectroscopy system that collects spectra in batches by means of gradient defocusing, or a micro spatial offset Raman spectroscopy (μSORS) system that collects spectra in batches by means of micron-level gradient spatial offset.

[0035] Figure 2 The present invention is a flow chart of a method for determining the subcutaneous detection depth of a Raman spectroscopy system according to the present application.

[0036] In step 201, a plurality of Raman spectra of the test area of ​​the subject are collected by a Raman spectroscopy system, wherein the plurality of Raman spectra corresponding to the plurality of detection depths preferably adopts a non-invasive transcutaneous detection mode. Based on actual needs, the range of the detection depth needs to cover the human epidermis and dermis, which is about 0 microns to 1000 microns, and the detection step length is determined by the resolution of the Raman spectroscopy system, which is about 50 microns to 100 microns. Usually, the quality of the Raman spectroscopy signal collected by the Raman spectroscopy system is not good enough, which will affect the subsequent process, so the Raman spectrum needs to be preprocessed. Figure 3 As shown in the signal diagram at different stages of preprocessing, first, the fluorescence background in the collected signal is removed by the second order of air PLS, and only the Raman spectrum signal is retained. Then, the required band in the Raman spectrum is selected (for example, the band area 1150-1400cm-1) to process and consider the Raman spectrum of this band. Next, cosmic rays need to be removed to remove the peaks in the signal and smooth it. The smoothed multiple Raman spectra are normalized, for example, according to 1450cm -1 The peak area is normalized. Then, the desired Raman spectrum with a high signal-to-noise ratio can be obtained by arranging the detection depth from shallow to deep from top to bottom. It can be understood that since the present application only requires the signal intensity ratio of the corresponding wavenumber position of the Raman spectrum, the above preprocessing will not affect the detection results. In addition, in one embodiment, the Raman spectrum acquisition time is 4 minutes, and in order to avoid saturation, the specific parameters are set to 8 seconds × 30 spectra. When the aforementioned mμSORS Raman spectroscopy system is used, due to the multi-channel design, Raman spectra at 4 different subcutaneous depths can be obtained in one spectrum acquisition.

[0037] Figure 4 The vertical axis is the signal intensity, and the horizontal axis is the wave number. i-1 , D i , D i+1 , D n That is, it represents different detection depths, and the detection depth gradually increases, and the curve is the Raman spectrum signal corresponding to the detection depth. It can be understood that although in the existing Raman spectrum system, the detection depths D1 and D i-1 , D i , D i+1 , D n As a scale, there are different scale values, but in this application, when the Raman spectroscopy system is used for subcutaneous detection, the original scale value is not applicable. i-1 , D i , D i+1 , D nIt is only used as an identification symbol (which can be understood as offset 1, offset 2, offset 3, offset 4, ... offset n in the aforementioned mμSORS Raman spectroscopy system), and is not considered to have a specific value in this step. The purpose of this application is to enable the Raman spectroscopy system to obtain the detection depth D1, D2 when performing subcutaneous detection. i-1 , D i , D i+1 , D n Applicable to the corresponding scale value for subcutaneous detection.

[0038] In step 202, for a plurality of Raman spectra, the ratio of the signal intensity of the first characteristic peak to the signal intensity of the second characteristic peak of each Raman spectrum is calculated, and based on the ratio, the first spectrum is selected from the plurality of Raman spectra. There is a clear layered structure between the dermis and the epidermis of human skin. The junction between the dermis and the epidermis is called the basement membrane (DEJ). Above the basement membrane is the epidermis mainly composed of cells, and below the basement membrane is the dermis rich in collagen. Figure 5 As shown. Therefore, the Raman spectrum of the dermis has a stronger signal near the first characteristic peak, for example, 1230cm -1 ~1250cm -1 The wave number region is preferably 1240 cm -1 Wave number (such as Figure 4 The Raman spectrum of the epidermis has a stronger signal near the second characteristic peak, for example, 1310 cm -1 ~1330cm -1 The wave number region is preferably 1320 cm -1 Wave number (such as Figure 4 As shown in I2). After the first characteristic peak and the second characteristic peak are determined, the signal intensity of each Raman spectrum at the first characteristic peak and the signal intensity of the second characteristic peak, and the ratio R of the two signal intensities can be determined. By determining the turning points of multiple ratios R at multiple detection depths, the Raman spectrum corresponding to the ratio R at the turning point can be determined as the first spectrum. According to an embodiment of the present application, the turning point from greater than 1 to less than 1 is preferred as the ratio R, that is, the ratio R≈1. For example, referring to Figure 4 , D i+1 The corresponding Raman spectrum signal intensity ratio R is greater than 1, D i-1 The corresponding Raman spectrum signal intensity ratio R is less than 1, and the ratio D i The corresponding Raman spectrum signal intensity ratio R≈1, so D i The corresponding Raman spectrum is determined as the first spectrum.

[0039] In step 203, an optical coherence tomography (OCT) image of the area to be tested is acquired to determine the depth of the basement membrane (DEJ) of the area to be tested. OCT images of the subject are acquired as follows: the light beam is focused on a specific point of the area to be tested on the subject's skin, and reflected light data is collected along the depth direction. Since the OCT field of view is small and there are certain local differences in the DEJ depth of human skin, images need to be acquired at multiple points for the same sample. For example, the refractive index of the system matching the skin is 1.3, and OCT images of 15 areas near the thenar eminence of the palm are acquired, namely p1, p2, p3...p15,

[0040] Calculate the mean signal intensity p of all regions mean , as the basis for DEJ's in-depth analysis:

[0041]

[0042] p mean The second minimum point of is the DEJ depth. Figure 6 (a) is a graph of depth and signal intensity of OCT images in multiple regions. Figure 6 (b) is to calculate Figure 6 The average signal intensity p obtained by averaging the signal intensities of multiple OCT images in (a) mean The curve graph of the depth of the skin and the depth of the skin, from which the second minimum point can be read is the depth of the DEJ, wherein the shaded portion is the standard deviation of the DEJ. It can be understood that the area collected in step 203 is the same as that in step 201.

[0043] In step 204, the detection depth corresponding to the first spectrum is determined as the DEJ depth. i , step 203 obtains the DEJ depth for the current subject. Therefore, when performing subcutaneous detection, the detection depth D corresponding to the first spectrum can be i Determined as the DEJ depth obtained in step 203, that is, the detection depth D i The corresponding scale value is the corresponding DEJ depth. Therefore, a detection depth D in the Raman spectroscopy system during subcutaneous detection is determined. i The actual value of .

[0044] In step 205, the above steps 201-204 are repeated to determine all detection depths. After a detection depth of the Raman spectroscopy system during subcutaneous detection is determined by a subject, the above steps are repeated with different subjects to determine the actual values ​​of all detection depths in the Raman spectroscopy system during subcutaneous detection. The result of the whole process is as follows: Figure 7It is understood that if the same detection depth is determined after replacing the subject, other subjects are replaced to determine other detection depths.

[0045] According to the method described in this application, the detection results of the mμSORS system are as follows: Figure 7 As shown. Figure 7 (e) is a schematic diagram of four subjects. Figure 7 (a) Schematic diagram of skin DEJ depth of four subjects;

[0046] Figure 7 (b) is a graph showing the relationship between subcutaneous depth and signal strength of four subjects. Figure 7 The DEJ depths corresponding to the four subgraphs of (b) are d from top to bottom. I =270μm, d II =370μm, d III =430μm and d IV =620μm; Figure 7 (c) a scale consisting of the values ​​of the detection depth determined by the four subjects; Figure 7 (d) is the Raman spectra of four subjects. Figure 7 (d) The detection depths of the Raman spectra in each sub-figure from top to bottom are D1, D2, D3, and D4, and the corresponding signal intensity ratios are R1, R2, R3, and R4, respectively. R2, R3, and R4 of volunteer I are all greater than 1, R1≈1, so the subcutaneous Raman spectrum detected by offset 1 of the mμSORS system comes from the vicinity of its DEJ, that is, D1≈d I =270μm; R1<1, R3 and R4 are both greater than 1 and R2≈1 for volunteer II. Therefore, the subcutaneous Raman spectrum detected by offset 2 of the mμSORS system comes from the vicinity of its DEJ, that is, D2≈d II =370μm; R1 and R2 of volunteer III are less than 1, R4>1 and R3≈1, so the subcutaneous Raman spectrum detected by offset 3 of the mμSORS system comes from the vicinity of its DEJ, that is, D3≈d III =430μm; R1, R2 and R3 of volunteer IV are all less than 1, and R4≈1, so the subcutaneous Raman spectrum detected by offset 4 of the mμSORS system comes from the vicinity of its DEJ, that is, D4≈d IV=620μm. Therefore, the subcutaneous detection depth of the mμSORS system can be characterized as follows: the detection depth of offset 0 is 0, the detection depth of offset 1 is about 270μm, the detection depth of offset 2 is 370μm, the detection depth of offset 3 is 430μm, and the detection depth of offset 4 is 620μm. It can be understood that the above is only an example, and the same method can be used for any Raman spectroscopy system to determine the subcutaneous detection depth.

[0047] The above specific embodiments illustrate the implementation of the present application, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification. Although the description of the present application will be introduced in conjunction with the preferred embodiment, this does not mean that the features of this invention are limited to the implementation. In addition, in order to avoid confusion or blurring the focus of the present application, some specific details will be omitted in the description. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0048] Furthermore, various operations will be described as multiple discrete operations in a manner that is most helpful for understanding the illustrative embodiments; however, the order of description should not be construed as implying that these operations are necessarily order dependent. In particular, these operations do not need to be performed in the order presented.

[0049] Unless the context dictates otherwise, the terms "comprising," "having," and "including" are synonymous. The phrase "A / B" means "A or B." The phrase "A and / or B" means "(A and B) or (A or B)."

[0050] In the accompanying drawings, some structural or method features are shown in a specific arrangement and / or order. However, it should be understood that such specific arrangement and / or ordering may not be required. In some embodiments, these features may be arranged in a manner and / or order different from that shown in the illustrative drawings. In addition, the inclusion of structural or method features in a specific figure does not mean that such features are required in all embodiments, and in some embodiments, these features may not be included or may be combined with other features.

[0051] It should be understood that although the terms "first", "second", etc. may be used here to describe various units or data, these units or data should not be limited by these terms. These terms are used only to distinguish one feature from another. For example, without departing from the scope of the exemplary embodiment, a first feature may be referred to as a second feature, and similarly, a second feature may be referred to as a first feature.

[0052] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

[0053] While the present invention has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims

1. A method for determining the subcutaneous detection depth of a Raman spectroscopy system, wherein the Raman spectroscopy system has multiple detection depths, characterized in that: include: Collecting a plurality of Raman spectra of the subject's test area by the Raman spectroscopy system, wherein the plurality of Raman spectra correspond to a plurality of detection depths; For the plurality of Raman spectra, calculating a ratio of a signal intensity of a first characteristic peak to a signal intensity of a second characteristic peak of each Raman spectrum, and selecting a first spectrum from the plurality of Raman spectra based on the ratio; Acquiring an optical coherence tomography (OCT) image of the area to be tested to determine the depth of the basement membrane (DEJ) of the area to be tested; Determine the detection depth corresponding to the first spectrum as the DEJ depth; Repeat the above steps to determine all the detection depths.

2. The method according to claim 1, characterized in that The method also includes preprocessing the multiple Raman spectra, including: removing cosmic rays and fluorescence background, band selection and normalization, so as to obtain the multiple Raman spectra with high signal-to-noise ratio.

3. The method according to claim 2, characterized in that The wave number region of the first characteristic peak is 1230 cm -1 ~1250cm -1 The wave number region of the second characteristic peak is 1310 cm -1 ~1330cm -1 .

4. The method according to claim 3, characterized in that The wave number of the first characteristic peak is 1240 cm -1 The wave number of the second characteristic peak is 1320cm -1 .

5. The method according to any one of claims 1 to 4, characterized in that The selecting a first spectrum from the plurality of Raman spectra based on the ratio comprises: The Raman spectrum corresponding to the turning point where the ratio changes from greater than 1 to less than 1 is determined as the first spectrum.

6. The method according to claim 5, characterized in that The plurality of Raman spectra are collected by a transcutaneous detection mode.

7. The method according to claim 6, characterized in that The subcutaneous detection range of the Raman spectroscopy system is 0 micrometers to 1000 micrometers, and the resolution is 50 micrometers to 100 micrometers.

8. The method according to claim 7, characterized in that The Raman spectroscopy system comprises: Confocal Raman spectroscopy system, or micro spatial offset Raman spectroscopy (μSORS) system, or multi-channel micro spatial offset Raman scattering spectroscopy (mμSORS) system.

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