Method for determining subcutaneous detection depth of raman spectroscopy system

Abstract

The present application relates to a method for determining a subcutaneous detection depth of a Raman spectroscopy system, the Raman spectroscopy system having a plurality of detection depths. The method comprises: acquiring a plurality of Raman spectra in a region of interest in a subject by the Raman spectroscopy system, wherein the plurality of Raman spectra correspond to the plurality of detection depths; calculating, for the plurality of Raman spectra, a ratio of the signal intensity of a first characteristic peak to the signal intensity of a second characteristic peak of each Raman spectrum, and selecting, on the basis of the ratio, a first spectrum from the plurality of Raman spectra; acquiring an optical coherence tomography (OCT) image of the region of interest, and determining a dermal-epidermal junction (DEJ) depth of the region of interest; determining the detection depth corresponding to the first spectrum as the DEJ depth; and repeating the described steps to determine all the detection depths.

Description

Methods for determining the subcutaneous detection depth of a Raman spectroscopy system Technical Field

[0001] This 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 Technology

[0002] Raman spectroscopy-based biomolecular detection is a rapid, sensitive, highly specific, label-free, and non-invasive analytical technique widely used in clinical diagnosis and biomedical research. However, detecting biomolecules hidden under the skin still faces many challenges, the most prominent being whether Raman spectroscopy can reach the region where the target biomolecule is located.

[0003] The penetration depth of Raman spectroscopy in skin is influenced by numerous factors. The strong scattering and absorption of photons by skin tissue severely limits the penetration depth of Raman spectra in living tissue. The high scattering coefficient of epidermal tissue means that most photons are scattered at the surface. The detection depth of Raman spectroscopy in skin is affected by the excitation wavelength; longer wavelength photons penetrate deeper into the skin than shorter wavelength photons, but the intensity of their Raman signal is correspondingly weakened. The penetration depth of Raman spectroscopy in skin is also closely related to laser power. The possibility of detecting deep Raman scattered photons increases with increasing laser power, but excessively high laser power can damage the sample. Furthermore, the detectable depth of Raman spectra is also affected by the detector sensitivity of the Raman spectroscopy system.

[0004] Therefore, the subcutaneous detection depth of Raman spectroscopy is influenced by a combination of factors. Quantitatively characterizing the detection depth of Raman spectroscopy could provide stronger support for clinical diagnosis and biomedical research, but currently there are no mature methods available. Summary of the Invention

[0005] This application proposes a method for determining the subcutaneous detection depth of a Raman spectroscopy system to characterize the detection depth of Raman spectra when targeting biological tissues at different sites. The Raman spectroscopy system has multiple detection depths. The method includes the following steps: acquiring multiple Raman spectra of a test area of ​​a subject using the Raman spectroscopy system, wherein the multiple Raman spectra correspond to multiple detection depths; for each of the multiple Raman spectra, calculating the ratio of the signal intensity of a first characteristic peak to the signal intensity of a second characteristic peak of each Raman spectrum, and selecting a first spectrum from the multiple Raman spectra based on the ratio; acquiring 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 plurality of Raman spectra, including: removal of cosmic rays and fluorescent background, band selection and normalization, to obtain the plurality of Raman spectra with high signal-to-noise ratio.

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

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

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

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

[0011] Optionally, the multiple Raman spectra can be acquired via transdermal detection mode.

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

[0013] Optionally, the Raman spectroscopy system includes:

[0014] Confocal Raman spectroscopy system, or micro spatial shift Raman spectroscopy (μSORS) system, or multichannel micro spatial shift Raman scattering spectroscopy (mμSORS) system.

[0015] Based on the discovered Raman spectral characteristics of distinct epidermal and dermal stratification in the skin, this application innovatively proposes using subjects whose skin thickness has been characterized by OCT as a "scale," and then having multiple subjects with different skin thicknesses determine all the "scales." The method proposed in this application can be used to characterize the depth of subcutaneous Raman spectroscopy detection. Compared to 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 located and detected more precisely, thereby accurately obtaining the characteristic information of the molecules to be detected under the skin, reducing interference from 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 biomolecule can be clarified, realizing the application of Raman spectroscopy technology to in vivo in situ non-destructive detection of target samples or tissues, and further expanding its application fields. Attached Figure Description

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

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

[0020] Figure 3 is a flowchart of signal preprocessing according to an embodiment of this application.

[0021] Figure 4 is a schematic diagram of the Raman spectrum collected according to the embodiments of this application.

[0022] Figure 5 is a schematic diagram of the depth of human skin and basement membrane according to an embodiment of this application.

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

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

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

[0026] Figure 7(b) is a graph of subcutaneous depth versus signal intensity of a subject according to an embodiment of this application.

[0027] Figure 7(c) is a “ruler” constructed from the detection depth values ​​of the subjects determined according to the embodiments of this application.

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

[0029] Figure 7(e) is a schematic diagram of a subject according to an embodiment of this application. Detailed Implementation

[0030] The method proposed in this application will now be described in detail with reference to the accompanying drawings.

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

[0032] Figure 1 is a schematic diagram illustrating the principle of the mμSORS Raman spectroscopy system for detecting signals at different subcutaneous depths according to an embodiment of this application. The mμSORS Raman spectroscopy system detects Raman spectral signals at different skin depths through multiple sets of fiber optic channels with different offset distances (offsets). As shown in Figure 1, after the excitation light interacts with the skin, a large number of Raman photons are scattered. The mμSORS system sets different numbers of fiber optic 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 offset, the greater the contribution from deeper samples in the obtained Raman signal. That is, the signals collected at different offsets originate from different subcutaneous depths, as shown in the figure, where the detection depths are D1, ..., D... i ... D n Multiple Raman spectra.

[0033] Typically, the detectable depth of each offset can be determined theoretically. However, the strong scattering and absorption of photons by skin tissue severely limits the penetration depth of Raman spectroscopy in living tissue. Therefore, when performing subcutaneous detection on human skin, the actual depth detected by each offset is not the theoretically calculated value. Thus, the method proposed in this application is needed to characterize the actual detection depth of the Raman spectroscopy system during subcutaneous detection.

[0034] Those skilled in the art will understand that, in addition to the mμSORS Raman spectroscopy system, the method according to this application is also applicable to confocal Raman spectroscopy systems that acquire spectra in batches by gradient defocusing, or micro spatial shift Raman spectroscopy (μSORS) systems that acquire spectra in batches by micrometer-level gradient spatial shifting.

[0035] Figure 2 is a flowchart of the method for determining the subcutaneous detection depth of a Raman spectroscopy system according to this application.

[0036] In step 201, multiple Raman spectra of the test area of ​​the subject are acquired using a Raman spectroscopy system. These multiple Raman spectra correspond to multiple detection depths, preferably using a non-invasive transdermal detection mode. Based on practical needs, the detection depth range needs to cover the epidermis and dermis, approximately 0 μm to 1000 μm. The detection step size is determined by the resolution of the Raman spectroscopy system, approximately 50 μm to 100 μm. Typically, the quality of the Raman spectral signal acquired by the Raman spectroscopy system is insufficient, affecting subsequent processes; therefore, preprocessing of the Raman spectrum is necessary. As shown in Figure 3, the signal diagrams at different stages of preprocessing are as follows: First, the fluorescence background in the acquired signal is removed using second-order air PLS, retaining only the Raman spectral signal. Then, the desired band in the Raman spectrum is selected (e.g., the band region 1150-1400 cm⁻¹) to process and consider the Raman spectrum in this band. Next, cosmic rays need to be removed to smooth the signal and eliminate sharp peaks. The smoothed multiple Raman spectra are then normalized, for example, according to 1450 cm⁻¹. -1 The peak area is normalized. Then, the peaks are arranged from top to bottom according to the detection depth from shallow to deep to obtain the desired high signal-to-noise ratio Raman spectrum. It is understood that since this application only requires the signal intensity ratio at the corresponding wavenumber position of the Raman spectrum, the above preprocessing will not affect the detection results. Furthermore, in one embodiment, the Raman spectroscopy acquisition time is 4 minutes, and to avoid saturation, the specific parameters are set to 8 seconds × 30 spectra. When using the aforementioned mμSORS Raman spectroscopy system, due to the multi-channel design, four Raman spectra at different subcutaneous depths can be obtained in a single spectral acquisition.

[0037] Figure 4 shows examples of multiple acquired Raman spectra, with the vertical axis representing signal intensity and the horizontal axis representing wavenumber, D1, D... i-1 D i D i+1 D n This represents different detection depths, with the detection depth gradually increasing; the curve represents the Raman spectral signal corresponding to the detection depth. It is understandable that, although in existing Raman spectroscopy systems, detection depths D1, D2, and D3 represent different detection depths, the curve represents the Raman spectral signal corresponding to each detection depth. i-1 D i D i+1 D n While the scale has different values, the original scale values ​​are not applicable when the Raman spectroscopy system is used for subcutaneous detection in this application. Therefore, the detection depths D1 and D2 in this application are... i-1、 D i D i+1 D nThese are merely identifiers (similar to offset 1, offset 2, offset 3, offset 4, ... offset n in the aforementioned mμSORS Raman spectroscopy system), and are not considered to have specific numerical values ​​in this step. The purpose of this application is to enable the Raman spectroscopy system to obtain the detection depths D1 and D2 when performing subcutaneous detection. i-1 D i D i+1 D n The corresponding scale value is applicable to subcutaneous testing.

[0038] In step 202, for multiple Raman spectra, the ratio of the signal intensity of the first characteristic peak to the signal intensity of the second characteristic peak in each Raman spectrum is calculated, and based on this ratio, a first spectrum is selected from the multiple Raman spectra. Human skin has a clear layered structure between the dermis and epidermis. The boundary between the dermis and 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, as shown in Figure 5. Therefore, the Raman spectrum of the dermis has a stronger signal near the first characteristic peak, for example, at 1230 cm⁻¹. -1 ~1250cm -1 The wavenumber region is preferably 1240 cm⁻¹. -1 Wavenumber (as shown by I1 in Figure 4); the Raman spectrum of the epidermis has a stronger signal near the second characteristic peak, for example, it could be 1310 cm⁻¹. -1 1330cm -1 The wavenumber region is preferably 1320 cm⁻¹. -1 Wavenumber (as shown by I2 in Figure 4). Once the first and second characteristic peaks are determined, the signal intensity at the first and second characteristic peaks of each Raman spectrum, and the ratio R between the two signal intensities, can be determined. By determining multiple inflection points of the ratio R at multiple detection depths, the Raman spectrum corresponding to the ratio R at the inflection point can be determined as the first spectrum. According to embodiments of this application, an inflection point where the ratio changes from greater than 1 to less than 1 is preferred for the ratio R, i.e., ratio R ≈ 1. For example, referring to Figure 4, the signal intensity ratio R of the Raman spectrum corresponding to Di+1 is greater than 1, D... i-1 The signal intensity ratio R of the corresponding Raman spectra is less than 1, while the signal intensity ratio R of the Raman spectra corresponding to Di is approximately 1. Therefore, the Raman spectrum corresponding to Di is determined as the first spectrum.

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

[0040] Calculate the mean p of the signal strength across all regions. mean This serves as the basis for in-depth DEJ analysis:

[0041] p mean The second minimum point is the DEJ depth. Figure 6(a) shows the curves of depth and signal intensity of OCT images of multiple regions, and Figure 6(b) shows the mean signal intensity p obtained after calculating the mean signal intensity of multiple OCT images in Figure 6(a). mean The curve comparing the subcutaneous depth to the DEJ depth shows that the second minimum point is the DEJ depth, with the shaded area representing the standard deviation of the DEJ. It is understood that the area collected in step 203 is the same as that in step 201.

[0042] In step 204, the detection depth corresponding to the first spectrum is determined as the DEJ depth. Step 202 obtained the first spectrum Di for the current subject, and step 203 obtained the DEJ depth for the current subject. Therefore, during subcutaneous testing, the detection depth Di corresponding to the first spectrum can be determined as the DEJ depth obtained in step 203; that is, the scale value corresponding to the detection depth Di is the corresponding DEJ depth. Thus, the actual value of a detection depth Di in the Raman spectroscopy system during subcutaneous testing is determined.

[0043] In step 205, steps 201-204 above are repeated to determine all detection depths. After determining one detection depth of the Raman spectroscopy system for subcutaneous detection using one subject, the above steps are repeated with different subjects to determine the actual values ​​of all detection depths in the Raman spectroscopy system for subcutaneous detection. The overall results are shown in Figures 7(a)-(e). It is understood that if the same detection depth is determined after changing subjects, other subjects are used to determine other detection depths.

[0044] The detection results using the mμSORS system according to the method described in this application are shown in Figure 7. Figure 7(e) shows a schematic diagram of the four subjects, and Figure 7(a) shows schematic diagrams of the skin DEJ depth of the four subjects.

[0045] Figure 7(b) shows the relationship between subcutaneous depth and signal intensity for four subjects. The four subplots in Figure 7(b) correspond to DEJ depths from top to bottom as d. I =270μm, d II =370μm, d III =430μm and d IV =620μm; Figure 7(c) shows the scale formed by the detection depth values ​​of the four subjects; Figure 7(d) shows the Raman spectra of the four subjects. The Raman spectra of each sub-figure in Figure 7(d) from top to bottom correspond to detection depths D1, D2, D3, and D4, respectively, and the corresponding signal intensity ratios are R1, R2, R3, and R4, respectively. Among them, R2, R3, and R4 of volunteer I are all greater than 1, and R1≈1. Therefore, the subcutaneous Raman spectrum detected by the offset 1 of the mμSORS system comes from the vicinity of its DEJ, that is, D1≈d I =270μm; For volunteer II, R1<1, R3 and R4 are both greater than 1 and R2≈1, therefore the subcutaneous Raman spectrum detected by offset 2 of the mμSORS system comes from the vicinity of its DEJ, i.e., D2≈dh=370μm; For volunteer III, R1 and R2 are less than 1, R4>1 and R3≈1, therefore the subcutaneous Raman spectrum detected by offset 3 of the mμSORS system comes from the vicinity of its DEJ, i.e., D3≈d III =430μm; R1, R2, and R3 of volunteer IV are all less than 1, and R4≈1, therefore the subcutaneous Raman spectrum detected by offset 4 of the mμSORS system originates from its vicinity of DEJ, i.e., D4≈d IV =620 μm. Therefore, the subcutaneous detection depth of the aforementioned mμSORS system can be characterized as follows: offset 0 has a detection depth of 0, offset 1 has a detection depth of approximately 270 μm, offset 2 has a detection depth of 370 μm, offset 3 has a detection depth of 430 μm, and offset 4 has a detection depth of 620 μm. It should be understood that the above is merely an example, and the same method can be used to determine the subcutaneous detection depth for any Raman spectroscopy system.

[0046] The above description illustrates the implementation of this application through specific embodiments. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Although the description of this application is presented in conjunction with preferred embodiments, this does not mean that the features of this invention are limited to these embodiments. Furthermore, to avoid confusion or obscuring the focus of this application, some specific details will be omitted in the description. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0047] Furthermore, the various operations will be described as multiple discrete operations in a manner most conducive to understanding the illustrative embodiments; however, the order of description should not be construed as implying that these operations must depend on the order. In particular, these operations do not need to be performed in the order presented.

[0048] Unless the context otherwise specifies, the terms “contains,” “has,” and “includes” are synonyms. The phrase “A / B” means “A or B.” The phrase “A and / or B” means “(A and B) or (A or B).”

[0049] In the accompanying drawings, certain structural or methodological features are shown in a specific arrangement and / or order. However, it should be understood that such a specific arrangement and / or order may not be necessary. In some embodiments, these features may be arranged in a manner and / or order different from that shown in the illustrative drawings. Furthermore, the inclusion of structural or methodological features in a particular figure does not imply that such features are required in all embodiments, and in some embodiments, these features may be omitted or may be combined with other features.

[0050] It should be understood that although terms such as "first," "second," etc., may be used herein to describe various units or data, these units or data should not be limited by these terms. These terms are used merely to distinguish one feature from another. For example, without departing from the scope of the exemplary embodiments, a first feature may be referred to as a second feature, and similarly, a second feature may be referred to as a first feature.

[0051] It should be noted that in this specification, similar reference numerals and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0052] Although the invention has been illustrated and described with reference to certain preferred embodiments thereof, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims

1. A method of determining a subcutaneous probing depth of a Raman spectroscopy system having a plurality of probing depths, the method comprising: Comprising: acquiring a plurality of Raman spectra of a region of interest of a subject by the Raman spectroscopy system, wherein the plurality of Raman spectra correspond to a plurality of probing depths; for the plurality of Raman spectra, calculating a ratio of signal intensity of a first characteristic peak and 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 region of interest, and determining a depth of the Dejerine-Andre-Thomas membrane (DEJ) of the region of interest; determining the probing depth corresponding to the first spectrum as the DEJ depth; repeating the above steps to determine all the probing depths.

2. The method of claim 1, wherein, Further comprising pre-processing the plurality of Raman spectra, including cosmic ray removal, fluorescence background removal, wavelength band selection, and normalization, to obtain the plurality of Raman spectra with high signal-to-noise ratio.

3. The method of claim 2, wherein, the first characteristic peak is in a wave number region of 1230 cm -1 ~ 1250 cm -1 , and the second characteristic peak is in a wave number region of 1310 cm -1 ~ 1330 cm -1 .

4. The method of claim 3, wherein, the first characteristic peak has a wave number of 1240 cm -1 the second characteristic peak has a wave number of 1320 cm -1 .

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

6. The method of claim 5, wherein, The plurality of Raman spectra are acquired by a transcutaneous probing mode.

7. The method of claim 6, wherein, The Raman spectroscopy system has a range of 0-1000 micrometers and a resolution of 50-100 micrometers for subcutaneous probing.

8. The method of claim 7, wherein, The Raman spectroscopy system comprises: a confocal Raman spectroscopy system, or a micro spatially offset Raman spectroscopy (μSORS) system, or a multi-channel micro spatially offset Raman scattering spectroscopy (mμSORS) system.

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