A mirror-based a-scan spectral imaging instrument and method of imaging

Abstract

The application discloses a kind of A-scan spectral imaging instruments based on mirror and its imaging method, it is related to optical detection technical field.The instrument includes excitation light source module, light beam adjusting module, dimension mapping module, detection module and data processing module;Excitation light source module provides excitation light, light beam adjusting module is converted into the class Bessel beam focal line satisfying axial coverage;Dimension mapping module is mapped as transverse detectable light signal to sample axial light signal;Detection module can simultaneously obtain multiple different depth light signals in single exposure;After data processing module is corrected and smoothed by multiple steps, the quantitative analysis of sample component depth distribution is realized by component analysis algorithm.The application breaks through the traditional point-by-point scanning framework, measurement speed is significantly improved, suitable for layered sample such as biological tissue and polymer film and the like detection, with the advantages of simple structure, controllable cost, strong practicability.

Description

Technical Field

[0001] This invention belongs to the field of optical detection technology, specifically relating to an A-scan spectral imaging instrument based on a reflector and its imaging method. Background Technology

[0002] Layered structures are a typical structural morphology, commonly found in biological tissues such as skin and artificial materials such as polymer films. Their chemical composition and molecular properties vary with depth. Confocal Raman microscopy, with its non-invasive, label-free, and high depth resolution characteristics, has become an important tool for measuring the depth-resolved Raman spectra of layered samples.

[0003] However, traditional confocal Raman microscopy requires axial point-by-point scanning by moving the sample or objective lens to acquire depth-resolution Raman spectra, which limits the measurement speed. For samples such as biological tissues, spontaneous Raman scattering efficiency is low and easily interfered with by fluorescence signals. Single-point Raman spectroscopy measurements often take tens of seconds to several minutes. For example, when analyzing the penetration of sodium dodecyl sulfate (SDS) in ex vivo skin, scanning 10 depth positions takes 30 minutes. As the number of measurement points increases, the cumulative exposure time prolongs, and the risk of photodamage to the sample increases significantly.

[0004] To address these issues, existing technologies have attempted various improvements: using electro- or acoustically modulated variable-focus lenses to achieve axial focus modulation, but this remains fundamentally axial scanning, resulting in limited efficiency gains; converting lateral scanning to axial focusing via remote focusing is still limited by scanning speed; utilizing Airy beam characteristics for depth-resolved imaging requires complex data post-processing and cannot directly measure spectral characteristics at different depths; and optical needle microscopy based on spatial light modulators has low light energy utilization, which to some extent restricts its application in low signal-to-noise ratio Raman spectroscopy. Furthermore, in existing technologies, mirrors are mostly used to adjust light intensity, such as optimizing slit light intensity through mirror angle calibration or using off-axis parabolic mirrors for repeated laser focusing, but none of these involve the technical concept of mapping the sample's axial dimension signal to the lateral dimension, making it impossible to achieve snapshot-style parallel acquisition of multi-depth Raman spectra.

[0005] Therefore, there is an urgent need to develop a Raman spectroscopy imaging technique that does not require axial scanning, has a fast measurement speed, high resolution, and high light utilization rate to meet the needs of depth resolution detection of layered samples. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an A-scan spectral imaging instrument and its imaging method based on a reflector. The instrument achieves axial-lateral dimension mapping through a reflector assembly and combines it with Bessel-like beam focal line illumination to achieve snapshot-style parallel acquisition of multi-depth Raman spectra, thus solving the problems of slow measurement speed and high risk of sample optical damage in traditional techniques.

[0007] In a first aspect, the present invention proposes an A-scan spectral imaging instrument based on a reflector, comprising an excitation source module, a beam adjustment module, a dimension mapping module, and a detection module; the excitation source module is used to provide excitation light; the beam adjustment module is optically connected to the excitation source module and is used to adjust the excitation light into a Bessel-like beam focal line, the Bessel-like beam focal line forming an illumination area within the sample that satisfies the axial coverage range; the dimension mapping module is disposed in the optical path between the sample and the detection module, and includes a reflector assembly, used to receive light signals generated at different axial depths of the sample and map the light signals at different depths into light signals detectable in the lateral dimension; the detection module is optically connected to the dimension mapping module and is used to receive the light signals detectable in the lateral dimension, acquiring multiple light signals at different depths simultaneously in a single exposure.

[0008] Preferably, the reflector assembly includes a planar reflector that maps linear light signals in the axial dimension to linear light signals in the lateral dimension.

[0009] More preferably, the plane mirror forms a 45° angle with the optical axis.

[0010] More preferably, the entrance of the detection module is a linear slit, or a shape adapted to the linear optical signal.

[0011] Preferably, the reflector assembly includes a conical reflector that maps the linear light signal in the axial dimension to a ring-shaped light signal in the lateral dimension.

[0012] More preferably, the apex angle of the conical reflector is 90°.

[0013] More preferably, the entrance of the detection module is ring-shaped, or has a shape adapted to the ring-shaped optical signal.

[0014] More preferably, the reflector assembly includes a second conical reflector, which further maps the annular light signal in the lateral dimension into a linear light signal in the lateral dimension.

[0015] More preferably, the entrance of the detection module is a linear slit, or a shape adapted to the linear optical signal.

[0016] More preferably, the optical signal is a Raman scattering signal, and the reflector assembly maps the axial linear Raman scattering signal into a lateral linear optical signal or a ring optical signal that can be directly measured by the detection module.

[0017] Preferably, the excitation source module uses a laser with a center wavelength of 532nm; the beam adjustment module includes a first plano-convex lens, a second plano-convex lens, a pinhole, a conical lens, and a first achromatic lens connected in sequence by optical paths; the combination of the first and second plano-convex lenses is used to magnify the beam diameter of the excitation light by 6-10 times, the pinhole has a diameter of 30μm and is used to eliminate laser speckle and spatial noise; the conical lens is used to convert the expanded laser beam into a ring beam, and the first achromatic lens has a focal length of 100mm and is used to focus the ring beam.

[0018] Further preferably, the beam adjustment module further includes a dichroic mirror and a first objective lens. The dichroic mirror is disposed in the optical path between the first achromatic lens and the first objective lens, and is used to reflect the annular beam focused by the first achromatic lens to the first objective lens. The first objective lens is used to focus the annular beam onto the sample to form the Bessel-like beam focal line. The numerical aperture of the first objective lens is 0.95, and the magnification is 40x. The full width at half maximum (FWHM) of the Bessel-like beam focal line is 140-145 μm.

[0019] More preferably, the beam adjustment module includes a first plano-convex lens, a second plano-convex lens, and a pinhole connected in sequence via optical paths; the combination of the first and second plano-convex lenses is used to magnify the beam diameter of the excitation light by 6-10 times, and the pinhole has a diameter of 30 μm to eliminate laser speckle and spatial noise; the conical lens is disposed between the second plano-convex lens and the first achromatic lens to convert the expanded laser beam into a ring beam, which is then focused into the sample by the first achromatic lens and the first objective lens to form a Bessel-like beam focal line.

[0020] More preferably, the optical path connection sequence of the beam adjustment module is as follows: the excitation light source module, the first plano-convex lens, the second plano-convex lens, the pinhole, the conical lens, the first achromatic lens, the dichroic mirror, the first objective lens, and the sample; the numerical aperture of the first objective lens is 0.95, and the magnification is 40x; the full width at half maximum (FWHM) of the Bessel-like beam focal line is 140-145 μm.

[0021] Preferably, the dimension mapping module further includes a second achromatic lens, a third achromatic lens, a long-pass filter, a polarizing beam splitter cube, a quarter-wave plate, a second objective lens, and a tube lens; the second objective lens is used to refocus the Raman scattering signal generated by the sample, the numerical aperture of the second objective lens is 0.95, and the magnification is 40x; the second achromatic lens and the third achromatic lens form a 4f structure, used for optical path relay of the Raman scattering signal, and the second achromatic lens and the third achromatic lens are aspherical achromatic lenses.

[0022] Preferably, the optical path connection relationship of the dimension mapping module is as follows: sample, first objective lens, second achromatic lens, third achromatic lens, long-pass filter, polarization beam splitter cube, quarter-wave plate, second objective lens, the mirror assembly, tube lens, and the detection module; the second achromatic lens and the third achromatic lens form a 4f structure for optical path relay of Raman scattering signal; the mirror assembly is set on the working plane of the second objective lens, and the numerical aperture of the second objective lens is 0.95 and the magnification is 40x.

[0023] More preferably, the reflector assembly is disposed on the working plane of the second objective lens, mapping the linear light in the axial dimension into linear light or ring light in the lateral dimension that can be directly measured by the detection module; the tube lens has a focal length of 180mm and is disposed between the polarization beam splitter cube and the detection module, for focusing the light signal onto the detection module.

[0024] More preferably, the dimension mapping module further includes two long-pass filters of different types, which are disposed in the optical path between the first objective lens and the second achromatic lens to filter out the excitation light and avoid interference of the excitation light with the detection of the Raman scattering signal.

[0025] Preferably, the detection module includes a spectrometer and a CCD detector; the slit width of the spectrometer is set to 70-90 μm for dispersing the transversely detectable light signal; the CCD detector is used to receive the dispersed light signal and convert it into Raman spectral data; the data processing module's processing includes dark background correction, wavelength correction, cosmic ray removal, frame summation, fluorescence baseline correction, depth attenuation effect correction, and spectral smoothing, wherein the cosmic ray removal uses a median filter, the fluorescence baseline correction uses a wavelet transform algorithm, and the spectral smoothing uses a 4th-order SG filter with a window number of 15-19.

[0026] Preferably, it also includes a data processing module; the data processing module quantitatively analyzes the component distribution at different depths of the sample using a biochemical component analysis algorithm.

[0027] Secondly, embodiments of the present invention provide a mirror-based A-scan spectral imaging method, implemented using the aforementioned mirror-based A-scan spectral imaging instrument, the method comprising the following steps: S1: Excitation light is provided by the excitation light source module, and the excitation light is adjusted to a Bessel-like beam focal line by the beam adjustment module, so that the Bessel-like beam focal line illuminates the sample and forms an illumination area that meets the axial coverage range inside the sample. S2: The sample generates light signals under the illumination of the focal line of the Bessel-like beam. Through the optical path transmission in the dimension mapping module and the action of the reflector assembly, the light signals at different depths along the sample axis are mapped into light signals that can be detected in the lateral dimension. The reflector assembly includes a plane reflector or a conical reflector. S3: The detection module is used to receive the detectable light signals in the lateral dimension, and the light signals at multiple different depths of the sample are acquired in a single exposure.

[0028] Preferably, in step S2: when the reflector assembly is a plane reflector at a 45° angle to the optical axis, the transversely detectable light signal is a linear light signal, which is compatible with the detection module whose entrance is a linear slit; When the reflector assembly is a conical reflector with a 90° apex angle, the detectable light signal in the lateral dimension is a ring-shaped light signal, which is suitable for a detection module with a ring-shaped inlet or a shape that matches the ring-shaped light signal; alternatively, the ring-shaped light signal in the lateral dimension can be mapped into a linear light signal in the lateral dimension via a second conical reflector, which is suitable for a detection module with a linear slit inlet or a shape that matches the linear light signal.

[0029] Preferably, the process of adjusting the excitation light to a Bessel-like focal line by the beam adjustment module in step S1 includes: magnifying the beam diameter of the excitation light by 6-10 times through a combination of a first plano-convex lens and a second plano-convex lens; eliminating laser speckle and spatial noise through a small aperture with a diameter of 30μm; converting the expanded laser beam into a ring beam through a conical lens; focusing it through a first achromatic lens with a focal length of 100mm; reflecting it through a dichroic mirror to a first objective lens with a numerical aperture of 0.95 and a magnification of 40x; and focusing the ring beam inside the sample through the first objective lens to form an illumination area with an axial coverage range of 100-150μm.

[0030] Preferably, the dimension mapping module in step S2 consists of a 4f structure composed of a second achromatic lens and a third achromatic lens, which realizes the relay of optical signals between the first objective lens and the second objective lens. After the excitation light is filtered out by a long-pass filter, the Raman scattering signal is refocused through the second objective lens with a numerical aperture of 0.95 and a magnification of 40. The mirror assembly maps the linear light in the axial dimension into linear light or ring light in the lateral dimension that can be directly measured by the detection module. In step S3, the detection module disperses the optical signal through a spectrometer with a slit width of 70-90 μm, which is received by the CCD detector and converted into Raman spectral data.

[0031] Preferably, in step S3, the spectra of the sample at multiple different depths are acquired simultaneously in a single exposure.

[0032] Preferably, the method further includes a data processing step S4: processing the spectrum through a data processing module and using a biochemical component analysis algorithm to quantitatively analyze the component distribution at different depths of the sample.

[0033] Preferably, the data processing module in step S4 includes the following steps: first, performing dark background correction and wavelength correction on the Raman spectral data, and then performing spectral acquisition; the spectral data processing steps are, in sequence, cosmic ray removal, frame summation, fluorescence baseline correction, depth attenuation effect correction, and spectral smoothing; specifically, it includes: using a median filter to remove cosmic rays, performing frame summation to enhance signal intensity, removing the fluorescence baseline using a wavelet transform algorithm, correcting the depth attenuation effect using the uniform axial fluorescence intensity distribution curve obtained from a fluorescence microscope slide, and finally performing spectral smoothing using a 4th-order SG filter with a window number of 15-19; the sample is a layered biological tissue, including ex vivo pig skin and mouse skin.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The measurement speed is greatly improved: the axial-lateral dimension mapping is achieved through the reflector assembly, eliminating the need for axial mechanical scanning. Multiple Raman spectra at different depths can be acquired in parallel in a single exposure. The measurement speed is more than 40 times faster than the traditional confocal Raman technology, which greatly shortens the sample exposure time and reduces the risk of light damage.

[0035] (2) Optimized balance between resolution and light utilization: The system's axial resolution can reach 4μm, which is significantly improved compared to the existing snapshot-type depth resolution scheme, and can meet the fine analysis requirements of the depth dimension of layered samples. Through the coordinated design of Bessel-like beam focal line illumination and mirror dimension mapping, coupled with the precise matching of high numerical aperture objectives and optical element parameters, the light loss during signal transmission is effectively reduced, and the light utilization is guaranteed. It can stably capture weak Raman scattering signals, solving the core pain points of low resolution and insufficient light utilization that are common in existing snapshot-type schemes.

[0036] (3) Simple structure and controllable cost: The core components are conventional optical elements (plane mirror, conical mirror, objective lens, etc.), which do not require expensive spatial light modulators and are easy to mass-produce and industrialize.

[0037] (4) Wide range of applications: It is suitable for layered samples, such as the hierarchical structure characterization of polymer films, the study of drug penetration mechanism of biological tissues (ex vivo pig skin, mouse skin, etc.), and the in-depth analysis of tissue components. It has important application value in the fields of biomedicine, materials science, and pharmaceutical engineering. Attached Figure Description

[0038] The accompanying drawings are included to provide a further understanding of the embodiments and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments and, together with the description, serve to explain the principles of the invention. Other embodiments and many anticipated advantages of the embodiments will be readily recognized as they become better understood through reference to the following detailed description. Elements in the drawings are not necessarily to scale. The same reference numerals refer to corresponding similar parts.

[0039] Figure 1 This is an architectural diagram of a mirror-based A-scan spectral imaging instrument according to an embodiment of the present invention; Figure 2 This is a system schematic diagram of an A-scan spectral imaging instrument based on a mirror, according to an embodiment of the present invention. Figure 3 This is a system schematic diagram of an A-scan spectral imaging instrument based on a conical mirror with a 90° apex angle, according to an embodiment of the present invention. Figure 4 The following are test images of a Bessel-like beam focal line irradiating a rhodamine solution in an embodiment of the present invention: (a) is a focal line XZ plane image, and (b) is a fluorescence intensity distribution along the Z direction. Figure 5 This is a schematic flowchart of an A-scan spectral imaging method based on a reflector according to an embodiment of the present invention; Figure 6The following are system imaging point spread function calibration diagrams for embodiments of the present invention: (a) is the theoretically calculated axial point spread function xz plane image, (b) is the xz plane image of a 500nm quantum dot microsphere, and (c) is the axial PSF curve. Figure 7 Raman spectra of isolated pig skin and subcutaneous fat as an embodiment of the present invention: (a) is a photograph of the pig skin to be tested; (b) is a reference spectrum of the skin and fat; (c) is a representative spectrum at different depths; and (d) is a curve showing the percentage contribution of skin and fat at different depths. Figure 8 The following are the results of measuring SDS permeation in mouse skin according to an embodiment of the present invention: (a) is a HE staining image of mouse skin, and (b) is a reference spectrum of mouse skin and SDS. Figure 9 To illustrate the use of A-scan microscopy to acquire Raman spectra of mouse skin in control and experimental groups in an embodiment of the present invention, (a)-(b) show the depth-resolved Raman spectra of mouse skin in control and experimental groups acquired in A-scan mode; (c)-(d) show the reference depth-resolved Raman spectra obtained by scanning longitudinal sections of mouse skin in control and experimental groups point by point; where the gray area represents the standard deviation and the vertical gray dashed line represents a specific Raman shift; (e) shows the depth distribution of SDS in mouse skin acquired in A-scan mode; and (f) shows the reference depth distribution of SDS in mouse skin acquired in point-by-point scanning mode. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0042] Layered structures are a typical structural morphology, commonly found in biological tissues such as skin and artificial materials such as polymer films. Their chemical composition and molecular properties vary with depth. Confocal Raman microscopy, due to its non-invasive, label-free, and high depth resolution characteristics, can measure depth-resolved Raman spectra in layered samples such as skin, tablet drugs, and polymer films. However, when measuring depth-resolved Raman spectra, confocal Raman microscopy requires point-by-point scanning along the depth direction by moving the sample or objective lens, thus limiting the measurement speed. For some samples, especially biological tissues, spontaneous Raman scattering efficiency is low and fluorescence signal interference is strong, and single-point Raman spectroscopy measurements typically take tens of seconds to several minutes. For example, when analyzing the permeation of sodium dodecyl sulfate (SDS) in ex vivo skin, confocal Raman scanning was performed at 10 depth locations, with each point measured for 3 minutes, for a total time of 30 minutes. Franzen et al., using confocal Raman microscopy to investigate the distribution of caffeine in ex vivo human skin, measured 100 points at each location, with each point measured for 10 seconds. As the number of measurement points increases, the cumulative exposure time lengthens, thereby increasing the risk of photodamage to the sample. Therefore, improving the measurement speed of depth-resolution spectra in Raman microscopy has become an urgent problem to be solved.

[0043] To overcome this limitation, several methods have been applied in optical imaging technology. The introduction of electro- or acoustically modulated variable-focus lenses and deformable mirrors allows for axial modulation of the focal point without mechanical movement. However, this strategy focuses on increasing the axial focus switching speed without avoiding axial scanning, thus offering limited improvement in spectral measurement efficiency, and the optical modulation devices used are generally expensive. A similar approach is to convert axial scanning into faster lateral scanning.

[0044] Chakraborty et al. converted lateral scanning into aberration-free axial focusing within a 55µm range using remote focusing, but this was still limited by scanning speed. In addition, several beam modulation methods have been used for depth-resolved imaging. Depth-resolved imaging can also be achieved using a stereo V-shaped point spread function formed by two tilted excitation beams, with an axial imaging range of approximately 40µm, but post-processing is still required to obtain the depth-resolved image.

[0045] Utilizing the characteristics of Airy beams is also a technique for depth-resolved imaging. The self-bending property of mirrored Airy beams is used to reconstruct depth-resolved images, but image reconstruction relies on manual matching of information points, and the imaging range is limited to 32 µm. Subsequently, the use of multifocal sampling and sparse-constrained deconvolution algorithms further optimized the method, achieving automatic 3D image reconstruction and extending the field of view to 40 µm.

[0046] While these methods have been shown to improve measurement speed in depth-resolved imaging, they require complex computational processing and cannot directly measure the spectral characteristics at different depth locations, thus limiting their direct application to depth-resolved Raman spectroscopy measurements. Motion-based fluorescence signals collected by a needle microscope are modulated into an Airy beam using a spatial light modulator, mapping axial intensity information to a lateral distribution to achieve snapshot-like depth-resolved imaging. However, this method suffers from low light utilization and faces significant challenges when measuring weak Raman signals. Furthermore, wavefront modulation of the collected fluorescence signal can induce linear lateral shifts in the light signals at different axial positions, focusing them onto a one-dimensional fiber array to achieve snapshot-like imaging. Depth-sensitive Raman microscopy combining conical lenses and fiber optic components has also been reported; this method can simultaneously acquire Raman signals at different depths in a single acquisition. Although these methods all achieve snapshot-like measurements, they are all limited by low light utilization and axial resolution.

[0047] In this embodiment of the invention, we developed a snapshot-type depth-resolution Raman spectroscopy technique, namely A-scan Raman spectroscopy. By using a plane mirror to map axial information to lateral information, Raman signals at different depths can be directly acquired without axial scanning. This system can simultaneously acquire Raman spectra at multiple different depth positions in a single illumination. We systematically evaluated the depth resolution capability of this system using a thin film model and an ex vivo porcine skin model, and further evaluated the transdermal penetration of SDS in mouse skin, verifying the application potential of this method in skin absorption and penetration studies.

[0048] In a first aspect, embodiments of the present invention disclose an A-scan spectral imaging instrument based on a reflector, such as... Figure 1 As shown, it includes an excitation light source module 11, a beam adjustment module 21, a dimension mapping module 31, and a detection module 41; Excitation light source module 11 is used to provide excitation light; The beam adjustment module 21 is optically connected to the excitation source module 11 and is used to adjust the excitation light into a Bessel-like beam focal line, which forms an illumination area inside the sample that meets the axial coverage range. In one specific embodiment, the excitation light source module 11 uses a laser with a center wavelength of 532nm; the beam adjustment module 21 includes a first plano-convex lens, a second plano-convex lens, a pinhole, a conical lens, and a first achromatic lens connected in sequence by optical paths; the combination of the first and second plano-convex lenses is used to magnify the beam diameter of the excitation light by 6-10 times, the pinhole diameter is 30μm, and it is used to eliminate laser speckle and spatial noise; the conical lens is used to convert the expanded laser beam into a ring beam, and the first achromatic lens has a focal length of 100mm, and it is used to focus the ring beam.

[0049] The beam adjustment module 21 also includes a dichroic mirror and a first objective lens. The dichroic mirror is disposed in the optical path between the first achromatic lens and the first objective lens and is used to reflect the ring beam focused by the first achromatic lens to the first objective lens. The first objective lens is used to focus the ring beam onto the sample to form a Bessel-like beam focal line. The numerical aperture of the first objective lens is 0.95 and the magnification is 40 times. The full width at half maximum (FWHM) of the Bessel-like beam focal line is 140-145 μm.

[0050] Preferably, the beam adjustment module includes a first plano-convex lens, a second plano-convex lens, and a pinhole connected in sequence via optical paths; the combination of the first and second plano-convex lenses is used to magnify the beam diameter of the excitation light by 6-10 times, and the pinhole diameter is 30μm, used to eliminate laser speckle and spatial noise; the conical lens is disposed between the second plano-convex lens and the first achromatic lens, used to convert the expanded laser beam into a ring beam, which is then focused into the sample by the first achromatic lens and the first objective lens to form a Bessel-like beam focal line.

[0051] In one specific embodiment, the optical path connection sequence of the beam adjustment module is as follows: excitation source module, first plano-convex lens, second plano-convex lens, pinhole, conical lens, first achromatic lens, dichroic mirror, first objective lens, and sample; the numerical aperture of the first objective lens is 0.95, and the magnification is 40x; the full width at half maximum (FWHM) of the Bessel-like beam focal line is 140-145μm.

[0052] The dimension mapping module 31 is set in the optical path between the sample and the detection module 41, and includes a reflector assembly. It is used to receive light signals generated at different depths along the sample axis and map the light signals at different depths into light signals that can be detected in the lateral dimension.

[0053] Preferably, the reflector assembly includes a planar reflector that maps the linear light signal in the axial dimension to a linear light signal in the lateral dimension. In this embodiment, the planar reflector forms a 45° angle with the optical axis. The entrance to the detection module is a linear slit or a shape adapted to the linear light signal.

[0054] Preferably, the reflector assembly includes a conical reflector that maps the linear light signal in the axial dimension to a ring-shaped light signal in the lateral dimension. In this embodiment, the apex angle of the conical reflector is 90°. The entrance of the detection module is ring-shaped or has a shape adapted to the ring-shaped light signal.

[0055] More preferably, the reflector assembly includes a second conical reflector, which further maps the annular light signal in the lateral dimension into a linear light signal in the lateral dimension.

[0056] More preferably, the entrance to the detection module is a linear slit or a shape adapted to the linear optical signal.

[0057] It should be noted that, in this embodiment of the invention, the shape of the entrance of the detection module is designed to effectively receive the corresponding optical signal and reduce optical loss. For example, when receiving a linear optical signal, the entrance can be a linear slit, or a rectangular slit, elliptical slit, or other shape that matches the contour of the linear optical signal; when receiving a ring-shaped optical signal, the entrance can be ring-shaped, or an arc-shaped opening, or other shape that matches the contour of the ring-shaped optical signal, all of which can achieve the detection objective of this invention.

[0058] In this embodiment, the optical signal is a Raman scattering signal. The reflector assembly maps the linear Raman scattering signal in the axial dimension into a linear or ring-shaped optical signal in the lateral dimension that can be directly measured by the detection module.

[0059] Preferably, the optical path connection of the dimension mapping module is as follows: sample, first objective lens, second achromatic lens, third achromatic lens, long-pass filter, polarization beam splitter cube, quarter-wave plate, second objective lens, mirror assembly, tube lens, and detection module; the second achromatic lens and the third achromatic lens form a 4f structure, which is used to relay the Raman scattering signal in the optical path; the mirror assembly is set at the working plane of the second objective lens, and the numerical aperture of the second objective lens is 0.95 and the magnification is 40 times.

[0060] Specifically, the dimension mapping module 31 also includes a second achromatic lens, a third achromatic lens, a long-pass filter, a polarizing beam splitter cube, a quarter-wave plate, a second objective lens, a mirror assembly, and a tube lens. The second objective lens is used to refocus the Raman scattering signal generated by the sample. The numerical aperture of the second objective lens is 0.95, and the magnification is 40x. The second and third achromatic lenses form a 4f structure, which is used to relay the Raman scattering signal in the optical path. The second and third achromatic lenses are aspherical achromatic lenses. The polarizing beam splitter cube and the quarter-wave plate are disposed between the third achromatic lens and the second objective lens. The mirror assembly is disposed at the working plane of the second objective lens and is used to map the axial dimension light signal into a transversely detectable light signal. The tube lens has a focal length of 180mm and is disposed between the polarizing beam splitter cube and the detection module 41 to focus the light signal onto the detection module 41.

[0061] The dimension mapping module 31 also includes two long-pass filters of different types, which are set in the optical path between the first objective lens and the second achromatic lens to filter out the excitation light and avoid interference of the excitation light on the detection of the Raman scattering signal.

[0062] The detection module 41 is optically connected to the dimension mapping module 31 and is used to receive transversely detectable light signals, acquiring multiple light signals at different depths in a single exposure.

[0063] In a preferred embodiment, the sample also includes a data processing module; the data processing module uses a biochemical component analysis algorithm to quantitatively analyze the component distribution at different depths of the sample.

[0064] The data processing module is used to process Raman spectra and quantitatively analyze the component distribution at different depths of the sample using biochemical composition analysis algorithms.

[0065] The detection module 41 includes a spectrometer and a CCD detector; the slit width of the spectrometer is set to 70-90 μm to disperse the transversely detectable light signal; the CCD detector is used to receive the dispersed light signal and convert it into Raman spectral data.

[0066] The data processing module includes dark background correction, wavelength correction, cosmic ray removal, frame summation, fluorescence baseline correction, depth attenuation effect correction, and spectral smoothing. Cosmic ray removal uses a median filter, fluorescence baseline correction uses a wavelet transform algorithm, and spectral smoothing uses a 4th-order SG filter with a window of 15-19 points.

[0067] Figure 2 This is a schematic diagram of the optical path in Embodiment 1 of the present invention, wherein the laser is the excitation source, the first objective is the excitation objective, the second objective is the signal collection objective, the tilting mirror is the mirror assembly of the dimension mapping module, and the spectrometer and the CCD detector constitute the detection module.

[0068] Figure 3 This is a schematic diagram of the optical path in Embodiment 2 of the present invention. In this embodiment, the laser is the excitation source, the first objective is the excitation objective, the second objective is the signal collection objective, the conical reflector 1 and the conical reflector 2 are the reflector components of the dimension mapping module, the third objective is the signal focusing objective, the sample is the layered sample to be tested, and the spectrometer and the CCD detector constitute the detection module.

[0069] Specifically, the A-scan spectral imaging instrument based on a reflector is set up as follows: A schematic diagram of A-scan Raman microscopy is shown below. Figure 2 and Figure 3 As shown. Figure 2 Lens 1 and 2 are plano-convex lenses, lens 3 is an achromatic lens, lens 4 and 5 are achromatic aspherical lenses, the pinhole is a small hole, and the tilting mirror is a D-shaped mirror tilted at 45 degrees. Figure 3 Conical reflector 1 and conical reflector 2 are conical reflectors with a vertex angle of 90 degrees.

[0070] The system uses a 532 nm laser (MW-GL-532, Changchun LaserOptoelectronics Technology, Changchun, Jilin, China) as the excitation source. The laser beam is adjusted by a combination of two plano-convex lenses, namely lens 1 (f = 25.4 mm, LA1951-AB, Thorlabs, Newton, MA, USA) and lens 2 (f = 200 mm, LA1708-AB, Thorlabs, Newton, MA, USA), which magnifies the laser beam diameter by approximately 8 times. A 30 µm diameter pinhole (P30W, Thorlabs, Newton, MA, USA) is used to eliminate laser speckle and other spatial noise.

[0071] To generate a Bessel-like beam, the expanded laser beam is converted into a ring beam by a conical lens (AX255-A, Thorlabs, Newton, MA, USA). This ring beam is focused by an achromatic lens 3 (f = 100mm, AC254-100-AB-ML, Thorlabs, Newton, MA, USA) and reflected by a dichroic mirror (RT532rdc, Chroma, BellowsFalls, VT, USA), finally focusing onto the working plane of objective lens 1 (UPLXAPO 40X / 0.95NA, Olympus, Tokyo, Japan). A Bessel-like focal line is formed inside the sample, as shown in Figure 4(a).

[0072] To evaluate the focal line length, a quartz cuvette containing 0.1 mg / mL rhodamine solution was placed below objective lens 1, and a 20x long working distance objective lens (CFI60 TU Plan Epi ELWD 20 X / 0.4NA, Nikon, Tokyo, Japan) was placed to the side of the cuvette for focusing the focal line image. The results are as follows: Figure 4 As shown in (b), the full width at half maximum (FWHM) value of the focal length is 143 micrometers.

[0073] The sample is fixed on an XYZ three-axis displacement stage for adjusting the sample position. The X and Z axes are moved by two electric displacement stages (MT1 / M-Z9, Thorlabs, Newton, MA, USA), and the Y axis is moved by a mechanical displacement stage (LNR25M / M, Thorlabs, Newton, MA, USA).

[0074] The system is also equipped with a white light imaging module ( Figure 2 (Not shown in the schematic diagram) is used to assist in adjusting the sample position. The Raman signal from the sample, along with background light such as fluorescence and excitation light, will return along the original optical path until it reaches the dichroic mirror and two long-pass filters (LP03-532RU-25, Semrock, Rochester, NY, USA and RET537lp, Chroma, Bellows Falls, VT, USA), where the excitation light is filtered out.

[0075] Two aspherical achromatic lenses 4 and 5 (APAC18, Newport, Irvine, CA, USA) form a 4f structure for optical path relay. Objective lens 2 (UPLXAPO 40X / 0.95NA, Olympus, Tokyo, Japan) refocuses the light from the sample, with the top of the converged focal line coinciding with the reflecting surface of the D-shaped mirror (PFD10-03-P01, Thorlabs, Newton, MA, USA).

[0076] The D-shaped mirror is tilted at 45 degrees relative to the optical axis of objective lens 2. For objective lens 2, the light source is a line image from the lateral plane, which is due to the reflection from the D-shaped mirror converting the axial virtual image into a lateral real image. However, due to the tilt angle of the D-shaped mirror, the light cone received by objective lens 2 will be clipped, which will affect the effective numerical aperture of objective lens 2 and thus the axial resolution of the system. Figure 3 The 90-degree apex cone-shaped mirror converts the axial virtual image into a transverse annular real image. Compared to the 45-degree tilted D-shaped mirror, this improves light utilization, but the cone-shaped clipping problem still exists. Subsequently, the light is focused through a tube lens (f = 180mm, SWTLU-C, Olympus, Tokyo, Japan) onto the slit of a spectrometer (IsoPlane 160, Princeton Instruments, Trenton, NJ, USA), with a slit width set to 80 μm. The beam passing through the slit is dispersed by a diffraction grating and then projected onto a CCD (PIXIS 256, Princeton Instruments, Trenton, NJ, USA) for detection.

[0077] Figure 4 Tests were performed on rhodamine solution irradiated by a laser focal line. (a) XZ plane image of the focal line, (b) fluorescence intensity distribution along the Z direction, with the red area representing the full width at half maximum (FWHM). The scale bar is 10 micrometers.

[0078] Secondly, embodiments of the present invention also disclose a mirror-based A-scan spectral imaging method, implemented based on the aforementioned mirror-based A-scan spectral imaging instrument, such as... Figure 5 As shown, the method includes the following steps: S1: Excitation light is provided by the excitation light source module, and the excitation light is adjusted to a Bessel-like beam focal line by the beam adjustment module, so that the Bessel-like beam focal line illuminates the sample and forms an illumination area that meets the axial coverage range inside the sample. In a specific embodiment, the process of adjusting the excitation light into a Bessel-like beam focal line in step S1 includes: magnifying the beam diameter of the excitation light by 6-10 times through a combination of a first plano-convex lens and a second plano-convex lens; eliminating laser speckle and spatial noise through a small aperture with a diameter of 30μm; converting the expanded laser beam into a ring beam through a conical lens; focusing the ring beam through a first achromatic lens with a focal length of 100mm; reflecting the ring beam through a dichroic mirror to a first objective lens with a numerical aperture of 0.95 and a magnification of 40; and focusing the ring beam onto the sample through the first objective lens to form a Bessel-like beam focal line with a full width at half maximum (FWHM) of 140-145μm, thus forming an illumination area with an axial coverage range of 100-150μm inside the sample.

[0079] S2: The sample generates light signals under the illumination of the focal line of a Bessel-like beam. Through the optical path transmission in the dimension mapping module and the action of the mirror assembly, the light signals at different depths along the sample axis are mapped into light signals that can be detected in the lateral dimension. The mirror assembly includes a plane mirror or a conical mirror. Preferably, in step S2: when the reflector assembly is a plane reflector at a 45° angle to the optical axis, the transversely detectable light signal is a linear light signal, which is compatible with the detection module whose entrance is a linear slit. When the reflector assembly is a conical reflector with a 90° apex angle, the detectable light signal in the lateral dimension is a ring-shaped light signal, which is suitable for a detection module with a ring-shaped inlet or a shape that matches the ring-shaped light signal; alternatively, the ring-shaped light signal in the lateral dimension can be mapped into a linear light signal in the lateral dimension via a second conical reflector, which is suitable for a detection module with a linear slit inlet or a shape that matches the linear light signal.

[0080] In a specific embodiment, the dimension mapping module in step S2 consists of a 4f structure composed of a second achromatic lens 4 and a third achromatic lens 5, which realizes the relay of optical signals between the first objective lens and the second objective lens. After the excitation light is filtered out by a long-pass filter, the Raman scattering signal is re-converged into a 4f structure by the second objective lens with a numerical aperture of 0.95 and a magnification of 40 for optical path relay. The mirror assembly maps the linear light in the axial dimension into linear light or ring light in the lateral dimension that can be directly measured by the detection module.

[0081] S3: Utilize the detection module to receive transversely detectable light signals and acquire Raman spectra of multiple different depths of the sample in a single exposure. In this step, the detection module disperses the light signal using a spectrometer with a slit width of 70-90 μm, which is then received by a CCD detector and converted into Raman spectral data.

[0082] Preferably, in step S3, the spectra of the sample at multiple different depths are acquired simultaneously in a single exposure.

[0083] In a preferred embodiment, a data processing step S4 is also included; S4: The Raman spectrum is processed by the data processing module, and the component distribution at different depths of the sample is quantitatively analyzed using a biochemical component analysis algorithm.

[0084] In a specific embodiment, the data processing module's processing procedure includes: first, performing dark background correction and wavelength correction on the Raman spectral data, and then performing spectral acquisition; the spectral data processing steps are, in sequence, cosmic ray removal, frame summation, fluorescence baseline correction, depth attenuation effect correction, and spectral smoothing. Specifically, the process includes: using a median filter to remove cosmic rays, performing frame summation to enhance signal intensity, removing the fluorescence baseline using a wavelet transform algorithm, correcting the depth attenuation effect using the uniform axial fluorescence intensity distribution curve obtained from a fluorescence microscope slide, and finally using a 4th-order SG filter with a window number of 15-19 for spectral smoothing; the samples are layered biological tissues, including ex vivo pig skin and mouse skin.

[0085] The sample preparation method of the present invention is described in detail below.

[0086] Female C57BL6 / J mice were used to establish an ex vivo skin model. Mice were housed in a university laboratory animal center under specific pathogen-free conditions, provided with standard feed and water, and kept in a 12-hour light-dark cycle. Mice were 9-12 weeks old. The experimental procedures were approved by the animal ethics committee.

[0087] These mice were divided into two equal groups: the control group (unexposed to the drug) and the experimental group (exposed to SDS). Before the experiment, the hair on the backs of the mice was shaved using a hair clipper and depilatory cream. The next day, a solution of 25 mg / ml (w / v) of SDS was prepared using phosphate-buffered saline (PBS). 50 μL of this solution was applied to the hairless back skin of the experimental group, covering an area of ​​approximately 20 mm × 20 mm, and left exposed for 8 hours. The mice were then anesthetized and euthanized, and a 20 mm × 20 mm piece of skin was harvested. The harvested skin was stored at 4°C for no more than 24 hours before measurement. The control group mice were kept in the same environment and for the same duration as the experimental group, except that they were not exposed to SDS.

[0088] Target regions were marked in skin samples for A-scan measurements to obtain the distribution of SDS along the skin axis. Subsequently, two adjacent skin samples (approximately 10 mm × 2 mm) were excised from adjacent locations of the target regions and placed in an Optimal Cutting Temperature Compound (OCT). After freezing at -20°C, longitudinal sections of the skin with thicknesses of 100 μm and 16 μm were excised from the skin tissue surface using a microtome (CM1950, Leica, Wetzlar, Germany). The 100 μm section, attached to an aluminum-coated glass slide, was immersed in PBS solution for 15 minutes to remove the OCT. This section was used for longitudinal scanning of drug distribution to help validate the depth resolution capability of the system. The 16 μm section was used to prepare HE-stained sections to provide a reference for the physiological structure of the skin.

[0089] The data processing method of this invention is further described below: spectral data acquisition and storage are performed using Lightfield 6.17 (Princeton Instrument, Trenton, NJ, USA).

[0090] After completing dark background and wavelength correction in Lightfield, spectral acquisition was performed. The spectral data processing steps were, in sequence, cosmic ray removal, frame summation, fluorescence baseline correction, depth attenuation effect correction, and spectral smoothing. Except for fluorescence baseline correction and spectrum plotting, the rest were implemented using MATLAB 2023b (MathWorks, Natick, MA, USA). Cosmic rays were removed using a median filter, and fluorescence was removed using a wavelet transform algorithm. The effect of depth attenuation was corrected using a uniform axial fluorescence intensity distribution curve obtained from a fluorescence microscope slide (FSK4, Thorlabs, Newton, MA, USA). Spectral smoothing used an SG filter, order 4, with a window size of 17.

[0091] In addition, Biochemical Component Analysis (BCA) is used to analyze the changes in the proportion of biochemical components at different depth locations, thereby quantitatively evaluating the system's depth resolution. BCA is a spectral decomposition algorithm based on least squares regression. This algorithm treats the Raman spectrum to be analyzed as a linear combination of the spectra of various known biochemical components. By fitting the data, the relative contribution of each component to the spectrum to be analyzed can be obtained, and the changes of each biochemical component can be directly and quantitatively revealed.

[0092] The specific experimental results are as follows. System point spread function test: In order to evaluate the depth resolution capability of the device, the system's point spread function was calculated, estimated, and experimentally measured.

[0093] As Figure 2 As shown, all objectives in the optical system are high-NA objectives. Traditional scalar diffraction theory is not applicable to high-NA objective systems. Furthermore, the scalar Debye theory neglects the vector nature of light, leading to inaccurate predictions of the point spread function (PSF). Vector diffraction theory considering electromagnetic wave polarization is used to accurately calculate the system's PSF. When calculating the PSF, the observation point... electric field at The calculation is performed using vector Kirchhoff integrals. Because this system uses a high-NA objective lens, the distance between the observation point and the focal point is much smaller than the focal length, and the Fresnel number of the system is much greater than 1. Therefore, the Kirchhoff integral can be approximated as the Debye integral, and the electric field... The wavefront electric field of the exit pupil surface of the distal imaging objective 2 can be obtained. Integrating, we obtain the following expression: ; Where k, , , respectively, are the wavenumber and wavelength in a medium with a refractive index of n, and f is the distance from the focal point to the wavefront. It is a unit vector perpendicular to the wavefront. The wavelength parameter settings correspond to the maximum emission spectrum of the quantum dot microspheres used, the objective numerical aperture is 0.95, and the mirror tilt angle is set to 45 degrees. The system PSF obtained from the simulation calculation is as follows: Figure 6 As shown.

[0094] Furthermore, 500nm quantum dot microspheres were embedded in an optical adhesive for use in the PSF of the experimental measurement system. Based on the system's 40x magnification, the spectrometer slit was set to 20 micrometers, and the spectrometer grating was set to 0-order for CCD imaging. The measurement results are as follows: Figure 6 As shown in (b). Figure 6 (c) Shows the full width at half maximum (FWHM) calculated theoretically and measured practically. The theoretically calculated FWHM of the system's PSF along the axial direction is 3.98 micrometers, and the experimental result is 4.06 micrometers, which is consistent with the theoretical calculation.

[0095] Figure 6 Calibrate the spread function for the system's imaging points. Figure 6 In (a), using a 0.95NA objective lens, assuming the light signal is unpolarized light with a wavelength of 560nm, the theoretically calculated axial point spread function xz plane image is shown. Figure 6 (b) Measurement of the xz plane image of a quantum dot microsphere with a diameter of 500 nm. Figure 6 (c) Axial PSF curves. The red curve represents the theoretically calculated PSF curve, and the black dots represent the measured results of quantum dot microspheres with an axial diameter of 500 nm. The scale bar is 5 micrometers.

[0096] Experimental testing of isolated pig skin samples: The isolated pig skin samples were obtained from pork belly purchased from a local market.

[0097] According to the American National Laser Usage Standard (ANSI Z136.1), for lasers with a center wavelength of 532 nm, the range is 10 to 3 × 10⁻⁶. 4 Within the effective time of s, based on the limiting aperture of 3.5 mm, the maximum laser exposure to the skin is 0.2 W / cm². 2 To verify the system's depth resolution in skin tissue, such as... Figure 7 As shown in (a), the pig skin tissue was obliquely cut to gradually thin the upper layer of skin tissue, and the test point was selected in the transition zone between the skin tissue and subcutaneous fat. A 1 MPE laser was used to measure the Raman spectrum at different depths by irradiating the test point from the top. Each frame had an exposure time of 50 s, for a total of 3 frames, with a cumulative exposure time of 150 s.

[0098] First, measurements were taken at the upper left pure skin area and the lower right pure adipose tissue area using an A-scan spectrometer. The spectrum at the first depth position (i.e., Z = 0µm) was selected as the reference Raman spectrum for the skin tissue and subcutaneous adipose tissue, respectively. Figure 7 As shown in (b), biomolecules in skin tissue exhibit different molecular vibrational modes under laser irradiation, and the resulting Raman characteristic peaks serve as identifiers to distinguish different biological tissues. Among them, 1075 cm⁻¹... -1 (CC stretch), 1268cm -1 (CH bending), 1300 cm -1 (CH2 twisting), 1450 cm -1 (CH2 bending), 1652 cm -1 (C = O stretch) and 1751 cm -1 (C = O stretch) is associated with adipose tissue. The Raman characteristic peak of skin is observed at 1268 cm⁻¹. -1 (CH bending), 1450 cm -1 (CH2 bending) and 1658 cm -1 (Amide I). 1658 cm observed in the skin -1 It is usually more than 1652 cm observed in fat. -1 Peak width, and at 1300 cm -1 and 1751 cm -1 At the peak, the peak value of the fat spectrum is stronger than that of the skin. Then, at the test point containing the upper skin layer and the lower fat layer ( Figure 7 Depth-resolution spectral acquisition was performed at the location marked by the black arrow in (a), and spectra from 5 depth locations were selected, such as... Figure 7 As shown in (c), qualitative observations revealed that the spectrum at deeper locations increased with depth, specifically at 1652 cm⁻¹. -1 The peak is narrower, 1450 cm -1 The peak relative intensity is stronger. Furthermore, Figure 7 Some peaks in (c) include 1075 cm. -1 and 1751 cm -1 It was not clearly observed, and there were extraneous peaks in the spectrum. This may be due to the thicker skin tissue at the measurement location, or it may be affected by factors such as the autofluorescence, dark current, and readout noise of the skin tissue, resulting in a low signal-to-noise ratio of the Raman spectral signal.

[0099] Figure 7 To measure the Raman spectra of isolated pig skin and subcutaneous fat using depth-resolution Raman microscopy. Figure 7 (a) is a photograph of the isolated pig skin to be tested. The upper left and lower right of the test site are the skin (including the epidermis and dermis) and subcutaneous adipose tissue, respectively. Figure 7(b) shows the reference spectra of the pig skin surface measured from the left side of the measurement point and the exposed subcutaneous fat on the right side, where the black curve is the subcutaneous fat reference spectrum and the red curve is the skin reference spectrum. Figure 7 (c) Representative spectra at five specific depths were selected from Raman spectra obtained at different depths from the measurement points. The gray dashed lines represent specific Raman shifts. Figure 7 Percentage contribution of skin and fat at different depths (d). The black curve represents the trend of skin weight with depth, and the red curve represents the trend of fat weight with depth.

[0100] To further validate the system's depth resolution in ex vivo porcine skin samples, Raman spectra of skin and fat were used as references for component quantification based on the BCA method. Test spectra at different depths were treated as linear combinations of the skin and fat reference spectra. All spectra were normalized, and the sum of the weighting coefficients for the reference spectra was scaled to 100%. Figure 7 As shown in (d), the weight of skin gradually decreases and the weight of fat gradually increases with increasing depth. The changing trends of skin and fat are similar to the measurement results of Su et al., indicating the effectiveness of the system in Raman depth resolution of biological tissues.

[0101] Mouse ex vivo skin measurement: To verify the effectiveness of the system in measuring skin permeability, mouse skin tissue, including control and experimental groups, was used for Raman testing.

[0102] HE-stained sections of skin samples were used to quantify the physiological structure of the skin, such as... Figure 8 As shown in (a), mouse skin structure includes the epidermis, dermis, and subcutaneous tissue. The dermis contains hair follicles and hair roots. The thicknesses of the epidermis and dermis are approximately 20 μm and 310 μm, respectively. Laser irradiation was performed from the skin surface along the depth direction, with a laser exposure of 1 MPE. Each group consisted of two mice, and two skin samples were obtained. Each skin sample had four measurement locations, and each measurement location was repeatedly sampled five times. Three frames were acquired each time, each frame lasting 30 seconds, for a total time of 90 seconds, obtaining Raman spectra at 21 different depths. Compared to the 30-minute process reported by Mao et al. for obtaining Raman spectra at 10 depth locations in the SDS skin penetration experiment, the method proposed in this paper increases the measurement speed of depth-resolution spectra by more than 40 times. The reference Raman spectra of mouse skin and SDS drug are shown below. Figure 8 As shown in (b).

[0103] Figure 8 In the middle (a), there is a HE staining image of mouse skin with a scale bar of 100 micrometers. In the image, Epi stands for Epidermis, Der stands for Dermis, Hypo stands for Hypodermis, and HF stands for Hair follicle. Figure 8In the middle (b), the Raman spectra of mouse skin and SDS are shown. The black curve is the spectrum of mouse skin, and the red curve is the spectrum of SDS drug.

[0104] The Raman characteristic peaks of the skin are mainly at 1080 cm⁻¹. -1 (CC stretch), 1266 cm -1 (Amide III), 1305cm -1 (CH2 twisting), 1448 cm -1 (CH2 deformation), 1657 cm -1 (Amide I), 2856 cm -1 (CH2symmetric stretch), 2890 cm -1 (CH2 Asymmetric stretch), 2929 cm -1 (CH2 Asymmetricstretch), 3010 cm -1 (CH stretch). The characteristic peak of SDS is at 1074 cm⁻¹. -1 1128 cm -1 1296 cm -1 1448cm -1 2847 cm -1 2879 cm -1 .

[0105] Due to 1657 cm -1 The peak is not affected by the drug and is used as a reference peak. Its maximum intensity is used to correct the intensity of the original spectra of different samples. The Raman spectra of representative skin surface locations in the control and experimental groups are as follows: Figure 9 As shown in (a) and (b). Qualitative observation shows that the spectrum of the experimental group is at 1080 cm⁻¹. -1 1305 cm -1 1448 cm -1 and 2890 cm -1 The spectrum showed a significant enhancement compared to the control group, which may be related to the 1074 cm⁻¹ spectrum derived from SDS. -1 1296 cm -1 1448 cm -1 And 2879 cm -1 The contribution is related to the lateral surface area. For 100-micron thick longitudinal section samples of the experimental and control groups, Raman spectra were measured using a side-by-side point-by-point scanning method, with the step size consistent with the depth interval of direct measurement. The Raman spectra of the control and experimental groups are as follows: Figure 9 As shown in (c) and (d), 1080 cm-1 1305 cm -1 1448 cm -1 and 2890 cm -1 It also showed enhanced intensity, consistent with the results obtained by A-Scan, which demonstrates the accuracy of A-Scan Raman microscopy in deeply resolving the qualitative information contained in Raman spectra.

[0106] Figure 9 Raman spectra of mouse skin from control and experimental groups were obtained using an A-scan microscope. (a)-(b) show the depth-resolved Raman spectra of mouse skin from the control and experimental groups obtained in A-scan mode; (c)-(d) show the reference depth-resolved Raman spectra obtained by scanning longitudinal sections of mouse skin from the control and experimental groups point-by-point. Gray areas represent standard deviations, and vertical gray dashed lines represent specific Raman shifts; (e) shows the depth distribution of SDS in mouse skin obtained in A-scan mode; (f) shows the reference depth distribution of SDS in mouse skin obtained in point-by-point scanning mode. Gray error bars represent standard deviations.

[0107] This invention presents a snapshot-type depth-resolved Raman microscopy technique. By mapping axial dimensional information from the sample to the lateral dimension, Raman signals at different depths can be directly acquired without axial scanning, enabling rapid detection of the sample's depth-resolution spectrum. The system's depth-resolution capability was evaluated through theoretical simulations and microsphere measurements. Furthermore, the depth-resolution capability of this microscope in layered structures was verified using ex vivo pig skin samples. The depth variation trend of the spectrum was consistent with the theory, indicating that the system can exhibit effective depth-resolution capability in scattering media.

[0108] The permeation and absorption experiments of SDS in mouse skin revealed the permeation trend of SDS in the skin, demonstrating the feasibility and effectiveness of this system for depth-resolution Raman spectroscopy in layered samples. Compared with traditional confocal Raman microscopy, this method avoids mechanical movement and refocusing. Compared with previous reports of SDS skin permeation experiments using confocal Raman microscopy, this method increases the measurement speed of depth-resolution spectroscopy by more than 40 times. Therefore, the snapshot-type depth-resolution Raman spectroscopy technique proposed in this invention can achieve rapid, non-destructive depth-resolution Raman spectroscopy measurements of multilayer samples, verifying its application potential in the analysis of multilayer samples.

[0109] The mirror-based A-scan spectral imaging instrument and imaging method described in this invention can be mass-produced and can be applied to the in-depth analysis and detection of layered samples in fields such as biomedicine, materials science, and pharmaceutical engineering, demonstrating significant industrial applicability.

[0110] The above description is merely a preferred embodiment of the present invention and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention is not limited to the specific combination of the above-described technical features, but also includes other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above-described features with (but not limited to) technical features with similar functions disclosed in this invention.

Claims

1. A mirror-based A-scan spectral imaging instrument, characterized in that, It includes an excitation light source module, a beam adjustment module, a dimension mapping module, and a detection module; The excitation light source module is used to provide excitation light; The beam adjustment module is optically connected to the excitation source module and is used to adjust the excitation light into a Bessel-like beam focal line, which forms an illumination area inside the sample that satisfies the axial coverage range. The dimension mapping module is set in the optical path between the sample and the detection module, and includes a mirror assembly for receiving light signals generated at different depths along the sample axis and mapping the light signals at different depths into light signals that can be detected in the lateral dimension. The detection module is optically connected to the dimension mapping module and is used to receive detectable light signals in the horizontal dimension, acquiring multiple light signals at different depths in a single exposure.

2. The mirror-based A-scan spectral imaging instrument according to claim 1, characterized in that, The mirror assembly includes a planar mirror that maps linear light signals in the axial dimension to linear light signals in the lateral dimension.

3. The mirror-based A-scan spectral imaging instrument according to claim 2, characterized in that, The plane mirror forms a 45° angle with the optical axis.

4. The mirror-based A-scan spectral imaging instrument according to claim 2, characterized in that, The entrance to the detection module is a linear slit, or a shape adapted to the linear optical signal.

5. The mirror-based A-scan spectral imaging instrument according to claim 1, characterized in that, The mirror assembly includes a conical mirror that maps linear light signals in the axial dimension to ring-shaped light signals in the lateral dimension.

6. The mirror-based A-scan spectral imaging instrument according to claim 5, characterized in that, The apex angle of the conical reflector is 90°.

7. The mirror-based A-scan spectral imaging instrument according to claim 5, characterized in that, The entrance to the detection module is ring-shaped, or has a shape adapted to the ring-shaped optical signal.

8. The mirror-based A-scan spectral imaging instrument according to claim 5, characterized in that, The reflector assembly includes a second conical reflector, which further maps the annular light signal in the lateral dimension into a linear light signal in the lateral dimension.

9. The mirror-based A-scan spectral imaging instrument according to claim 8, characterized in that, The entrance to the detection module is a linear slit, or a shape adapted to the linear optical signal.

10. An imaging method based on A-scan spectroscopy using a mirror, characterized in that, Based on the mirror-based A-scan spectral imaging instrument as described in any one of claims 1-9, the method includes the following steps: S1: Excitation light is provided by the excitation light source module, and the excitation light is adjusted to a Bessel-like beam focal line by the beam adjustment module, so that the Bessel-like beam focal line illuminates the sample and forms an illumination area that meets the axial coverage range inside the sample. S2: The sample generates light signals under the illumination of the focal line of the Bessel-like beam. Through the optical path transmission of the dimension mapping module and the action of the reflector assembly, the light signals at different depths along the sample axis are mapped into light signals that can be detected in the lateral dimension. The reflector assembly includes a plane reflector or a conical reflector. S3: The detection module is used to receive the detectable light signals in the lateral dimension, and the light signals at multiple different depths of the sample are acquired in a single exposure.

11. The imaging method based on A-scan spectrum using a reflector according to claim 10, characterized in that, In step S2: When the reflector assembly is a plane reflector at a 45° angle to the optical axis, the transversely detectable light signal is a linear light signal, which is compatible with a detection module with a linear slit entrance. When the reflector assembly is a conical reflector with a 90° apex angle, the detectable light signal in the lateral dimension is a ring-shaped light signal, which is suitable for a detection module with a ring-shaped inlet or a shape that matches the ring-shaped light signal; alternatively, the ring-shaped light signal in the lateral dimension can be mapped into a linear light signal in the lateral dimension via a second conical reflector, which is suitable for a detection module with a linear slit inlet or a shape that matches the linear light signal.

Images