A CARS spectral imaging apparatus and method

Abstract

The application belongs to the field of molecular structure detection, and discloses a CARS spectral imaging device and method, which comprises a femtosecond laser pulse oscillator; the femtosecond laser pulse oscillator is used for generating incident laser; wherein the incident laser is linearly polarized ultrashort pulse laser; the incident laser is sequentially irradiated on a sample to be measured through a first reflector, a prism pair, a second reflector, an ultrasharp long-pass filter, a fourth reflector, a first polarizer and a polarizing objective, and forms a CARS signal; the CARS signal is transmitted through a transmission part of the sample to be measured, and forms a selected reflected light through a second polarizer, a condenser lens, a sharp short-pass filter and a trap wave filter, which is used for microscopic imaging; wherein the polarization directions of the first polarizer and the second polarizer are orthogonally arranged; through the physical suppression mechanism of the orthogonal polarizer on the background noise, the application realizes the synergistic optimization of system simplification, cost reduction and imaging efficiency improvement, and effectively meets the long-time, high-speed dynamic imaging detection requirements of living cells in vivo.

Description

Technical Field

[0001] This invention belongs to the field of molecular structure detection technology, and relates to Raman scattering microscopy imaging technology, and in particular to a CARS spectral imaging device and method. Background Technology

[0002] Coherent Anti-Stokes Raman scattering Scattering (CARS) is a nonlinear coherent spectral imaging technique. Compared to spontaneous Raman scattering, CARS outputs light signals with intensity 5-6 orders of magnitude higher, resulting in higher signal discrimination and stronger detection sensitivity. It has wide applications in biomedicine, especially in live-cell imaging. Traditional CARS techniques are generally based on multi-beam or multi-source excitation schemes or single-beam excitation schemes optimized by pulse shapers. Multi-beam or multi-source excitation schemes require meeting the frequency components of pump photons, Stokes photons, and probe illumination. This not only involves numerous hardware devices and complex optical path layouts but also necessitates precise spatial alignment of all excitation beams, resulting in extremely low tolerance for optical path adjustments. While single-beam excitation schemes optimized by pulse shapers can reduce the number of light sources, pulse shapers are high-precision optical devices with complex internal optical path algorithms and high device costs. Furthermore, existing single-beam schemes generally suffer from signal-to-noise ratio deficiencies, with background noise during imaging being much stronger than the effective resonant CARS signal, significantly reducing imaging accuracy.

[0003] To overcome the drawbacks of traditional CARS technology, such as cumbersome multi-beam optical paths and high pulse shaper costs, researchers proposed a lightweight single-beam CARS improvement scheme that optimizes the optical path by incorporating a notch filter. This scheme modulates the original laser spectrum using a notch filter, generating notch features within the native laser spectrum. The corresponding CARS imaging spectrum simultaneously replicates these spectral features. Due to the compact size of the notch filter, it can be directly mounted on the outside of the galvanometer scanner to achieve kilohertz-level high-frequency laser modulation, avoiding the space-consuming problem of traditional large modulation equipment. Simultaneously, utilizing the inherent characteristics of the CARS resonant signal—that the effective resonant signal is only bound to the notch center frequency—the non-resonant stray signals generated during imaging are treated as a reference local oscillation signal. Combined with lock-in amplification technology, irrelevant non-resonant signals are filtered out, while the weak effective resonant signal is simultaneously amplified. This simplifies the hardware optical path and initially optimizes the imaging signal-to-noise ratio. Compared to the two traditional mainstream schemes, the overall optical path structure and equipment assembly difficulty are significantly reduced.

[0004] Currently, while single-beam CARS solutions based on notch filters simplify the optical path architecture and eliminate reliance on pulse shapers, they still suffer from cumbersome operation procedures, low imaging efficiency, and difficulty in meeting the actual detection needs of long-term, high-speed dynamic imaging of live cells. Specifically, from a hardware perspective, the manufacturing process of high-precision narrowband notch filters is demanding, and the procurement cost of optical components is high. Even with the addition of matching optical path calibration accessories, the overall cost of the imaging system remains expensive. Furthermore, the optical assembly structure still contains redundant components, and the system integration needs improvement. From a signal processing perspective, optimization... The current approach cannot suppress background noise at its source. The stray background noise generated by single-beam imaging is still much higher than the effective resonant CARS signal. At present, it can only rely on acquiring two sets of differentiated CARS spectra in stages, then performing spectral difference calculations, and superimposing multi-level digital filtering algorithms to extract the effective Raman spectrum. In practical applications of microscopic imaging, it is necessary to complete high-frequency laser modulation in advance, and then carry out complex data post-processing procedures such as multiple rounds of phase-locked loop calculations, spectral noise reduction, and data fitting. This not only prolongs the imaging time, but also introduces algorithm calculation errors, further reducing the accuracy of spectral extraction and imaging stability. Summary of the Invention

[0005] To address the technical problems existing in the prior art, the present invention provides a CARS spectral imaging device and method to solve the technical problems of the single-beam CARS scheme based on notch filter optimization, which has a cumbersome operation process, low imaging efficiency, and difficulty in adapting to the actual detection needs of long-term, high-speed dynamic imaging of live cells.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a CARS spectral imaging device, including a femtosecond laser pulse oscillator; the femtosecond laser pulse oscillator is used to generate incident laser; wherein, the incident laser is a linearly polarized ultrashort pulse laser; The incident laser beam sequentially passes through the first reflecting mirror, the prism pair, the second reflecting mirror, the ultra-steep long-pass filter, the fourth reflecting mirror, the first polarizer, and the polarizing objective lens to illuminate the sample under test, thus forming a CARS signal. The CARS signal passes through the transmission portion of the sample under test, and then through a second polarizer, a condenser lens, a steep short-pass filter, and a notch filter to form selectively reflected light for microscopic imaging. The polarization directions of the first polarizer and the second polarizer are set orthogonally.

[0007] Furthermore, the center wavelength of the linearly polarized ultrashort pulse laser is 780-805nm, the bandwidth is 10-60nm, the repetition frequency is 10-100MHz, and the pulse width is 10-100fs.

[0008] Furthermore, the prism is used to perform primary dispersion compensation on the incident light of the second mirror, and also to perform secondary dispersion compensation on the reflected light of the second mirror. The prism pair includes a first prism and a second prism, which are sequentially disposed between the first reflector and the second reflector.

[0009] Furthermore, the operating wavelengths of both the first and second prisms are 600-900nm.

[0010] Furthermore, it also includes a third reflector, which is positioned between the first prism and the ultra-steep long-pass filter.

[0011] Furthermore, the sample to be tested is mounted on a two-dimensional precision translation stage; the first prism, the second prism, the polarizing objective lens, and the condenser lens are respectively mounted on a one-dimensional precision translation stage; the first mirror, the second mirror, the third mirror, and the fourth mirror are respectively mounted on a two-dimensional adjustable mirror frame; and the first polarizer and the second polarizer are respectively mounted on an angle-adjustable rotation controller.

[0012] Furthermore, the magnification of the polarizing objective lens is 10-100x, and the numerical aperture is 0.2-1.2.

[0013] Furthermore, it also includes a photomultiplier tube, a lens, and a spectrometer; the reflected light is selected to enter the photomultiplier tube for microscopic imaging; the transmitted light passes through the lens and enters the spectrometer for spectral measurement.

[0014] Furthermore, the focal length of the lens is 30-100mm.

[0015] The present invention also provides a CARS spectral imaging method, utilizing the aforementioned CARS spectral imaging device; the imaging method includes: The femtosecond laser pulse oscillator is activated, and the positions of the first mirror, prism pair, second mirror, ultra-steep long-pass filter, fourth mirror, first polarizer, polarizing objective, second polarizer, condenser lens, and steep short-pass filter are adjusted to maximize the spectral intensity of the CARS signal. Among these adjustments, the position of the first polarizer is adjusted to minimize the background noise in the CARS signal. Select the reflection wavelength of the notch filter to determine the characteristic vibration mode of the sample under test; The measurement signals output during the microscopic imaging process are recorded, and the output measurement signals are processed to obtain the microscopic imaging results of the sample under test.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The CARS spectral imaging device provided by this invention achieves synergistic optimization of system simplification, cost reduction, and imaging efficiency improvement through the physical suppression mechanism of background noise by orthogonal polarizers, effectively meeting the needs of long-term, high-speed dynamic imaging and detection of live cells. Specifically, it uses linearly polarized ultrashort pulse laser as the incident laser and introduces a first polarizer and a second polarizer with orthogonal polarization directions. Utilizing the inherent optical characteristics of CARS background noise polarization sensitivity and effective resonant Raman signal polarization stability, it removes and eliminates non-resonant stray background noise from the optical path source. It eliminates the need for expensive optical components such as galvanometer scanners, high-precision narrowband notch filters, and lock-in amplifiers, significantly simplifying the hardware optical path structure and eliminating... In addition to redundant system components, the system improves equipment integration. On the one hand, it eliminates the costs of purchasing high-precision optical components and calibrating optical paths, reduces the difficulty of overall system construction and debugging, avoids the optical power loss caused by laser penetration of notch filters, and optimizes laser utilization efficiency. On the other hand, it eliminates the errors introduced by algorithm calculations, improves the accuracy of Raman spectroscopy extraction and imaging stability, and avoids the limitation of imaging speed on galvanometer scanning rate, increasing the single-pixel imaging speed to within 50μs. This is about 20 times faster than the existing notch-optimized single-beam CARS imaging speed, and is only limited by the signal-to-noise ratio. It balances lightweight equipment, detection economy, and imaging timeliness, and can be efficiently adapted to the dynamic microscopic detection scenarios of live cells in the biomedical field.

[0017] The CARS spectral imaging method provided by this invention possesses all the advantages of the aforementioned CARS spectral imaging device. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 A schematic diagram of the CARS spectral imaging device provided in the embodiment; Figure 2 The CARS spectrum results for different incident laser polarization angles are shown in the example. Figure 3 This is a CARS microscopic image of the rat brain in the example.

[0020] Among them, 1 is a femtosecond laser pulse oscillator, 2 is a first reflecting mirror, 3 is a first prism, 4 is a second prism, 5 is a second reflecting mirror, 6 is a third reflecting mirror, 7 is an ultra-steep long-pass filter, 8 is a fourth reflecting mirror, 9 is a first polarizer, 10 is a polarizing objective lens, 11 is the sample to be tested, 12 is a second polarizer, 13 is a condenser lens, 14 is an ultra-steep short-pass filter, 15 is a notch filter, 16 is a photomultiplier tube, 17 is a lens, and 18 is a spectrometer. Detailed Implementation

[0021] To make the technical problems, technical solutions, and beneficial effects solved by this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0022] Before describing the specific embodiments of this application, some of the technical terms involved in the embodiments of this application are explained as follows: Coherent anti-Stokes Raman scattering (CARS) is a four-wave mixing process involving the pump light, Stokes light, probe light, and anti-Stokes photons generated by the interaction of these three photons with sample molecules. It is a third-order nonlinear optical process; specifically, it utilizes a frequency of ω... p The pump light excites the sample molecules to a virtual energy level, using a frequency of ω. S The Stokes light then excites the sample molecules to a vibrational energy level; where the natural vibrational frequency Ω of the sample molecules is equal to the difference between the frequencies of the pump light and the Stokes light, i.e., Ω = ω. p -ω S Then, using a frequency of ω pr The probe light excites the sample molecules to a higher virtual energy level. The sample molecules, being unstable, return to their ground state, releasing energy with a frequency of ω. aS The anti-Sstokes photon; where the intrinsic vibrational frequency Ω of the sample molecule is equal to the difference between the frequencies of the anti-Sstokes photon and the probe light, i.e., Ω = ω. aS -ω pr .

[0023] This invention provides a CARS spectral imaging device, including a femtosecond laser pulse oscillator 1; the femtosecond laser pulse oscillator 1 is used to generate incident laser; wherein, the incident laser is a linearly polarized ultrashort pulse laser; the incident laser sequentially passes through a first reflecting mirror 2, a prism pair, a second reflecting mirror 5, an ultra-steep long-pass filter 7, a fourth reflecting mirror 8, a first polarizer 9, and a polarizing objective lens 10 to irradiate the sample 11 under test, and forms a CARS signal; the CARS signal passes through the transmission portion of the sample 11 under test, and then through a second polarizer 12, a condenser lens 13, an ultra-steep short-pass filter 14, and a notch filter 15 to form selectively reflected light and transmitted light; wherein, the selectively reflected light is used for microscopic imaging, and the transmitted light is used for spectral measurement; wherein, the polarization directions of the first polarizer 9 and the second polarizer 12 are orthogonally arranged.

[0024] In the above embodiments, a linearly polarized ultrashort pulse laser is used as the incident laser. Utilizing the linear polarization characteristics of the incident laser, the first polarizer purifies the polarization state of the incident laser, making it highly pure linearly polarized light. When the CARS signal is transmitted through the sample under test, the second polarizer is orthogonally set to the first polarizer. Because the effective resonant CARS signal and the non-resonant background noise have different polarization response characteristics—that is, the effective resonant CARS signal maintains the same polarization direction as the incident light, while the polarization state of the background noise undergoes depolarization or changes due to the randomness of the nonlinear optical process—the second polarizer can... It can selectively transmit effective resonant signals and significantly attenuate background noise, thereby suppressing interference from non-resonant stray signals at the source through a physical filtering mechanism. This physical filtering mechanism completely replaces the multi-stage signal processing link in the traditional solution, which relies on notch filters for spectral modulation, galvanometer scanners for high-frequency laser modulation, and lock-in amplifiers for complex demodulation operations. It not only eliminates expensive components such as high-precision notch filters, high-speed galvanometers, and lock-in amplifiers, reducing hardware procurement and system integration costs, but also avoids power loss caused by laser reflection on the notch filter, thus improving the efficiency of excitation light utilization.

[0025] More importantly, since background noise is physically eliminated at the optical path level, there is no need for cumbersome post-processing steps such as acquiring two sets of differentiated CARS spectra in stages and performing difference calculations and multi-level digital filtering. The spectral extraction process is completed in one step, eliminating the errors introduced by algorithm calculations and significantly improving the accuracy of spectral measurement and imaging stability. At the same time, the imaging speed is no longer limited by the mechanical response frequency of the galvanometer scanner, and the imaging time of a single pixel is shortened to less than 50μs, which is about 20 times faster than the traditional single-beam CARS microscopy imaging based on notch filtering. It can meet the actual detection needs of long-term, high-speed dynamic imaging of live cells and has significant practical value in the field of rapid biomedical imaging.

[0026] The following specific embodiments further explain and illustrate the CARS spectral imaging device provided by the present invention: Example As attached Figure 1 As shown, this embodiment provides a CARS spectral imaging device, including a femtosecond laser pulse oscillator 1, a first reflector 2, a prism pair, a second reflector 5, a third reflector 6, an ultra-steep long-pass filter 7, a fourth reflector 8, a first polarizer 9, a polarizing objective lens 10, a sample to be tested 11, a second polarizer 12, a condenser lens 13, an ultra-steep short-pass filter 14, a notch filter 15, a photomultiplier tube 16, a lens 17, and a spectrometer 18.

[0027] Femtosecond laser pulse oscillator 1 is used to emit incident laser light; the incident laser light is a linearly polarized ultrashort pulse laser. Explain, a linearly polarized ultrashort pulse laser refers to a near-infrared laser with a fixed electric field vibration direction and a pulse time domain width on the order of femtoseconds. It has extremely high instantaneous peak power and can efficiently excite the third-order nonlinear coherent anti-Stokes Raman scattering effect of sample molecules. At the same time, the average output power is low, so it will not rapidly burn biological samples. It should be noted that the femtosecond laser pulse oscillator 1 is the only laser source of the entire device, which generates an ultrashort pulse linearly polarized incident light source adapted to the excitation of CARS molecules, realizing CARS excitation based on a single beam, and simplifying the system architecture from the light source level.

[0028] Optionally, the center wavelength of the linearly polarized ultrashort pulse laser is 780-805nm, the bandwidth is 10-60nm, the repetition frequency is 10-100MHz, and the pulse width is 10-100fs. Explained, the center wavelength refers to the wavelength corresponding to the peak energy distribution of the femtosecond laser spectrum, the bandwidth refers to the full width at half maximum (FWHM) of the laser spectrum, the repetition frequency refers to the total number of pulses output per second by the laser, and the pulse width refers to the duration of a single laser pulse. In this embodiment, by limiting the parameter range of the linearly polarized ultrashort pulse laser, the device construction cost can be effectively controlled while ensuring the excitation intensity of the CARS signal, and it is also suitable for long-term low-phototoxicity imaging experiments of live cells. Preferably, the linearly polarized ultrashort pulse laser uses a near-infrared laser with a center wavelength of 790nm and a bandwidth of 15nm, a repetition frequency of 80MHz, and a pulse width of 10fs, to utilize the high peak power of the ultra-steep pulse to achieve low-average-power CARS excitation, thereby reducing phototoxicity to biological samples.

[0029] The incident laser beam is reflected sequentially by the first reflecting mirror 2 and enters the prism pair for first dispersion compensation. After first dispersion compensation, the laser beam enters the second reflecting mirror 5 and is reflected by the second reflecting mirror 5 again for second dispersion compensation. After second dispersion compensation, the laser beam passes sequentially through the third reflecting mirror 6, the ultra-steep long-pass filter 7, the fourth reflecting mirror 8, the first polarizer 9, and the polarizing objective lens 10 before illuminating the sample 11 to be tested, forming a CARS signal.

[0030] Explanatoryly, the incident laser passes through the first reflecting mirror 2, the prism pair, the second reflecting mirror 5, the third reflecting mirror 6, the ultra-steep long-pass filter 7, the fourth reflecting mirror 8, the first polarizer 9, and the polarizing objective lens 10 to achieve optical path reversal, dispersion compensation, stray light filtering, polarization purification, and focused excitation of the incident laser, so as to shape the original broadband laser into a focused beam adapted to the laser of the sample 11 under test.

[0031] Specifically, the first reflecting mirror 2, the second reflecting mirror 5, the third reflecting mirror 6, and the fourth reflecting mirror 8 serve as optical path steering elements, flexibly adjusting the overall size of the optical path and enabling miniaturized placement of the device. The prism pair acts as dispersion compensation elements, used to counteract the pulse broadening caused by laser transmission through the polarizing objective lens 10 and the lens 17. Specifically, the prism pair performs primary dispersion compensation on the incident light from the second reflecting mirror 5 and secondary dispersion compensation on the reflected light from the second reflecting mirror 5. The ultra-steep long-pass filter 7 is used to filter out the output of the femtosecond laser pulse oscillator 1. The short-wavelength stray light below the target wavelength in the emission spectrum is eliminated to remove baseline noise caused by stray light; the first polarizer 9 performs polarization purification on the incident laser to ensure that the laser incident on the sample 11 is high-purity linearly polarized light, providing a basis for subsequent orthogonal polarization noise reduction; the polarizing objective 10 focuses the parallel incident laser into a micron-sized spot to accurately irradiate the surface of the sample and excite molecules to generate CARS scattering signals; the polarizing objective 10 integrates a polarization transmission structure, which can completely preserve the purified linearly polarized excitation light without destroying the laser polarization state.

[0032] It should be noted that femtosecond laser pulses will generate group velocity dispersion when passing through various optical lenses and objectives. After the pulse time domain width is widened, the peak power decreases, which directly weakens the CARS nonlinear signal intensity. Therefore, a prism pair is set in the optical path to perform dispersion compensation before and after. Natural light or partially polarized light that has not been polarized will significantly increase the non-resonant background noise. By introducing the first polarizer 9 to pre-purify the polarization state of the incident laser, the orthogonal polarization noise reduction structure at the back end can play its role.

[0033] It is worth noting that the third reflector 6 is positioned between the first prism 3 and the ultra-steep long-pass filter 7. Specifically, after the laser passes through the prism pair in the reverse direction via the second reflector 5, it is incident on the third reflector 6. The third reflector 6 completes the optical path redirection and is incident on the ultra-steep long-pass filter 7. The third reflector 6, as an optical path redirection element, flexibly adjusts the direction of the optical path, which can compress the overall lateral length of the optical path, making the entire device smaller and realizing the miniaturization design of the device. At the same time, it accurately corrects the angle of the laser incident filter, improves the stray light filtering effect, and reduces background noise.

[0034] The CARS signal passes through the transmission portion of the sample 11, then through the second polarizer 12, the condenser lens 13, and the steep short-pass filter 14. After reflection and transmission through the notch filter 15, it forms selectively reflected light and transmitted light. The selectively reflected light is used for microscopic imaging, and the transmitted light is used for spectral measurement. Specifically, the selectively reflected light enters the photomultiplier tube 16 for microscopic imaging, and the transmitted light passes through the lens 17 and enters the spectrometer 18 for spectral measurement.

[0035] Explanatoryly, the CARS signal passes through the transmission portion of the sample 11, and then through the second polarizer 12, the condenser lens 13, and the steep short-pass filter 14. After reflection and transmission through the notch filter 15, it forms selective reflected light and transmitted light, thereby collecting the transmitted CARS signal of the sample 11, filtering out residual excitation laser in layers, and splitting the imaging optical path and the spectral measurement optical path through the notch filter 15 to simultaneously complete the molecular imaging and Raman spectral acquisition and detection functions.

[0036] Specifically, the scattered signal transmitted from the sample 11 is first orthogonally polarized and denoised by the second polarizer 12, which filters out most of the non-resonant background noise in advance; the focusing lens 13 converges the divergent transmitted scattered signal into a parallel beam, improving the efficiency of subsequent filtering and signal collection; the ultra-steep short-pass filter 14 is used to completely filter out the residual original pump-excited laser, retaining only the shorter wavelength CARS signal; the notch filter 15 realizes beam splitting of the optical path, the reflected light path is sent to the photomultiplier tube to be converted into an electrical signal for microscopic imaging, and the transmitted light path is coupled into the spectrometer through the focusing lens to collect complete Raman scattering spectral data.

[0037] In one possible implementation, the prism pair includes a first prism 3 and a second prism 4, which are sequentially arranged between the first reflector 2 and the second reflector 5. It should be noted that the laser is incident through the first reflector 2, passes through the first prism 3 and the second prism 4 in sequence to complete one dispersion pre-compensation, and is reflected after reaching the second reflector 5. It then passes through the second prism 4 and the first prism 3 in the opposite direction again to complete a second precise dispersion correction.

[0038] In this embodiment, the femtosecond laser dispersion loss can be corrected without an additional pulse shaper by using a reciprocating double dispersion compensation prism pair structure, eliminating the need for expensive pulse shaping hardware, simplifying the optical path structure, and stably improving the resonance CARS signal intensity. Specifically, the first prism 3 and the second prism 4 are arranged sequentially in the reciprocating optical path between the first reflector 2 and the second reflector 5. The laser passes through the first prism 3 and the second prism 4 twice, in both the forward and reverse directions. The superposition of the two dispersion compensations can completely offset the pulse broadening caused by the subsequent polarizing objective lens 10 and the condenser lens 13. The laser pulse reaching the surface of the sample 11 is restored to the original narrow pulse width state, maximizing the nonlinear excitation efficiency.

[0039] Explanatoryly, dispersion compensation refers to the use of a prism to disperse light and counteract the group velocity dispersion generated during laser propagation through lenses and objective optical elements, thus eliminating pulse temporal broadening. Group velocity dispersion is an optical phenomenon where different frequencies of light propagate at different speeds when a femtosecond laser passes through a transparent optical medium, causing narrow pulses to be broadened and peak power to decrease.

[0040] Preferably, the first prism 3 and the second prism 4 both operate at wavelengths of 600-900nm. By limiting the operating wavelength range of the prisms, the incident laser band of the matching device is matched, reducing laser power loss. At the same time, they naturally filter short-wavelength stray light, eliminating the need for additional stray light blocking components and simplifying the number of optical path components.

[0041] In one possible implementation, the polarization directions of the first polarizer 9 and the second polarizer 12 are orthogonally set. Explanation: relying on orthogonally polarized polarizers, the inherent non-resonant background noise of CARS detection can be directly suppressed from an optical level, replacing the traditional circuit modulation phase-locked loop noise reduction scheme. The first polarizer 9 is set at the front end of the incident excitation light path to fix the polarization transmission axis of the incident laser. The second polarizer 12 is set at the rear end of the sample scattering signal collection light path and rotates its own transmission axis to be completely perpendicular to the first polarizer 9 to form an orthogonal polarization structure.

[0042] It should be noted that orthogonal polarization refers to the strict 90° angle between the transmission axes of the two polarizers. The non-resonant scattering noise generated by the linearly polarized excitation light has the same polarization state as the incident laser and will be completely blocked by the orthogonally placed second polarizer 12. However, the polarization state of the molecular resonance CARS signal changes and can be partially transmitted through the second polarizer 12 for acquisition. In the existing technology, non-resonant background noise cannot be eliminated by filters and can only be separated by high-frequency modulation and lock-in amplifier circuit algorithms. Circuit processing will significantly increase the imaging time. Orthogonal polarization optical noise reduction relies on pure optical elements to filter noise in real time. It does not require circuit modulation or multiple spectral difference calculations. After signal acquisition, the pure resonance Raman signal can be directly read.

[0043] As attached Figure 2 As shown, attached Figure 2 The figure shows the CARS spectra for different incident laser polarization angles; from the appendix... Figure 2 As can be seen, as the polarization angle increases, the background noise gradually decreases and is eventually completely suppressed. At the same time, the CARS resonance signal characteristics gradually emerge, and finally only the required CARS resonance signal characteristics remain.

[0044] In one possible implementation, the first reflector 2, the second reflector 5, the third reflector 6, and the fourth reflector 8 are respectively mounted on a two-dimensional adjustable mirror frame; wherein, the two-dimensional adjustable mirror frame is used to correct the collimation of the laser beam path and ensure that the laser beam is transmitted stably along the designed beam path; optionally, the two-dimensional adjustable mirror frame includes two sets of independent fine-tuning knobs for horizontal and vertical adjustment to continuously fine-tune the reflector angles.

[0045] In one possible implementation, the first prism 3, the second prism 4, the polarizing objective 10, the condensing lens 13, and the lens 17 are respectively mounted on a one-dimensional precision translation stage. The one-dimensional precision translation stage is used to adjust the axial position of the optical elements along the optical path, optimizing laser dispersion compensation, focus, and signal coupling efficiency. By independently controlling the axial position of the corresponding optical elements through the one-dimensional precision translation stage, the optical path debugging is optimized step-by-step, allowing for rapid identification of the optimal element position with the highest signal intensity, thus reducing the difficulty of optical path debugging. Explained, the first prism 3 and the second prism 4 are each equipped with a one-dimensional precision translation stage, enabling adjustment of the axial distance between the two prisms to continuously change the dispersion compensation amount. The polarizing objective 10 is equipped with a one-dimensional precision translation stage, enabling adjustment of the axial distance between the polarizing objective 10 and the sample 11 under test, accurately focusing the laser spot. The condensing lens 13 is equipped with a one-dimensional precision translation stage, enabling adjustment of the focal point for the scattered signal. The lens 17 is equipped with a one-dimensional precision translation stage, enabling optimization of fiber coupling efficiency.

[0046] In one possible implementation, the first polarizer 9 and the second polarizer 12 are respectively mounted on an angle-adjustable rotation controller; wherein, the angle-adjustable rotation controller is used to precisely control the rotation angle of the transmission axis of the first polarizer 9 or the second polarizer 12 to achieve orthogonal polarization matching, maximize the suppression of non-resonant background noise, thereby ensuring that the orthogonal polarization noise reduction structure can continuously and stably exert the best noise suppression effect.

[0047] In one possible implementation, the polarizing objective 10 has a magnification of 10-100x and a numerical aperture of 0.2-1.2. By selecting a wide range of magnifications and numerical apertures, it can be compatible with various biological sample imaging and detection needs, making the equipment more versatile and eliminating the need to replace the entire optical path. The wide magnification range of 10-100x covers two mainstream detection scenarios: panoramic biological sections and microcellular areas. The numerical aperture of 0.2-1.2 is suitable for different light transmission conditions of thin and thick sections. The high numerical aperture objective can collect more weak scattered signals and improve the imaging signal-to-noise ratio, while the low magnification objective has a faster scanning speed and is suitable for rapid large-area screening.

[0048] In one possible implementation, the sample to be tested 11 is mounted on a two-dimensional precision translation stage. The two-dimensional precision translation stage has two orthogonal axial independent stepping drives to carry the sample to complete a full-area planar scan, and works with the objective lens to achieve two-dimensional microscopic imaging acquisition. Explained, the sample to be tested 11 is fixed inside the sample slot of the translation stage, and the two-dimensional precision translation stage drives the sample to complete stepping movement in the X and Y axes. For each step, the photomultiplier tube 16 simultaneously acquires a set of imaging signals, and finally generates complete two-dimensional matrix imaging data, realizing fully automatic full-area scanning of the sample without the need for manual adjustment of the sample position. The scanning area and stepping distance can be customized by software to adapt to the imaging needs of biological slices of different sizes.

[0049] In one possible implementation, the focal length of lens 17 is 30-100mm; wherein, the lens is used to focus and couple the CARS signal transmitted by the notch filter to the optical fiber input end of the spectrometer; by limiting the focal length range of lens 17, the optical fiber coupling efficiency and the safety of optical fiber use are balanced, ensuring that the spectrometer stably acquires high-intensity Raman spectral data; preferably, the focal length of lens 17 is 50mm to ensure high optical fiber coupling efficiency of the first spectrometer 16.

[0050] This embodiment also provides a CARS spectral imaging method using the aforementioned CARS spectral imaging device, comprising: activating the femtosecond laser pulse oscillator 1, and adjusting the positions of the first reflecting mirror 2, prism pair, second reflecting mirror 5, ultra-steep long-pass filter 7, fourth reflecting mirror 8, first polarizer 9, polarizing objective 10, second polarizer 12, condenser lens 13, and steep short-pass filter 14 to maximize the spectral intensity of the CARS signal; wherein, adjusting the position of the first polarizer 9 minimizes the background noise in the CARS signal; selecting the reflection wavelength of the notch filter 15 to determine the characteristic vibration mode of the sample 11 under test; recording the measurement signal output during the microscopic imaging process, and performing data processing on the output measurement signal to obtain the microscopic imaging result of the sample 11 under test.

[0051] Explanatoryly, characteristic vibrational modes refer to the inherent vibrational frequencies of chemical bonds in biomolecules. Different molecules correspond to unique characteristic vibrational wavelengths. By filtering the reflected wavelengths through a notch filter 15, the imaging signal of the target molecule can be extracted individually. In this embodiment, through a process of step-by-step optical path optimization, molecular characteristic wavelength filtering, and fully automated scanning acquisition of imaging signals, the traditional modulation and demodulation circuits and spectral difference calculations are eliminated by relying on orthogonal polarization optical noise reduction. The imaging acquisition process is simplified, the single-pixel imaging speed is greatly improved, and the molecular microscopic imaging detection of biological slices can be completed quickly.

[0052] Specifically, the implementation process of the imaging method involves the following steps: Step S1: Start the femtosecond laser pulse oscillator 1, roughly adjust the optical path components, and align the laser with the optical components; optionally, place the power meter probe at the rear end of the first polarizer 9, adjust the angle of the first polarizer 9 until the power meter value can no longer increase; then, place the power meter probe at the rear end of the second polarizer 12, and adjust the angle of the second polarizer 12 until the power meter value can no longer increase; roughly adjust the relative distance of the prism pair, and adjust the focus of the lens 17 to focus the laser onto the sample 11 to be tested until the CARS signal is observed from the spectrometer 18, while adjusting the position of the reflector and the condenser lens 13 to maximize the CARS spectral intensity measured by the spectrometer 18.

[0053] Explanatoryly, in step S1, by coarsely adjusting the relative distance between the prism pairs and adjusting the focus of lens 17, the laser is focused onto the sample 11 to be tested, which enables the detection element to couple with the collected CARS signal with high efficiency.

[0054] Specifically, the implementation process of step S1 is as follows: Step S101: Adjust the focal position of the polarizing objective 13 using a one-dimensional precision translation stage, and simultaneously adjust the angles of the first mirror 2, the second mirror 5, the third mirror 6, and the fourth mirror using a two-dimensional adjustable frame, thereby adjusting the pitch angle and collimation of the laser.

[0055] Step S102: Adjust the angle between the ultra-steep long-pass filter 7 and the ultra-steep short-pass filter 14, and use the spectrometer 18 to observe the CARS signal to ensure that the incident laser is cleaned up.

[0056] Step S103: Using the angle-adjustable rotation controller, adjust the angle of the second polarizer 12 until the CARS signal observed by the spectrometer 18 no longer increases.

[0057] Step S2: Optimize the relative position of the prism pair so that the dispersion of the laser on the sample 11 to be tested is compensated until the CARS signal can no longer increase, thereby achieving compensation for the group velocity dispersion of the laser caused by passing through the polarizing objective 10.

[0058] Specifically, in step S2, the relative positions of the first prism 3 and the second prism 4 are changed by a one-dimensional precision translation stage, and the change in CARS spectral intensity is observed by the spectrometer 18 until the CARS spectral intensity no longer increases.

[0059] Step S3: Observe the CARS spectral intensity measured by the spectrometer 18. Use a one-dimensional precision translation stage to adjust the distance between the polarizing objective 10 and the sample 11 to be tested until the CARS signal observed by the spectrometer 18 no longer increases, so that the incident laser can pass through the polarizing objective 10 as much as possible.

[0060] Specifically, in step S3, the CARS spectral intensity measured by the spectrometer 18 is observed, and the one-dimensional precision translation stage containing the polarizing objective 10, the condenser lens 13, and the lens 17 is adjusted until the observed CARS signal no longer increases.

[0061] Step S4: Adjust the position of lens 17 and adjust the fiber angle of spectrometer 18 until the CARS signal observed by spectrometer 18 no longer increases, so that the fiber coupling efficiency reaches the maximum value.

[0062] Specifically, in step S4, the two-dimensional adjustment frame of the optical fiber of the spectrometer 18 is adjusted until the CARS signal observed by the spectrometer 18 no longer increases. At this time, the spectrometer 18 and the lens 17 are matched to the optimal position.

[0063] Step S5: Using an angle-adjustable rotation controller, adjust the angle of the second polarizer 12 so that the polarization directions of the first polarizer 9 and the second polarizer 12 are orthogonal. This is to quickly extract the resonant CARS signal for Raman spectroscopy and microscopic imaging by removing the influence of background noise. Specifically, precisely adjust the angle-adjustable rotation controller where the second polarizer 12 is located to rotate 90° so that the background noise is minimized. At this time, the CARS signal observed by the spectrometer 18 is mainly the resonant CARS signal, and the non-resonant CARS signal is cleared.

[0064] Step S6: Prepare rat brain slices as test sample 11, place them on a glass slide, cover them with a coverslip, and seal the gap between the coverslip and the glass slide with nail polish; wherein, the thickness of the rat brain slice is 60μm and the area is 600μm × 600μm.

[0065] Step S7: Select the vibration mode of interest using the notch filter 15; specifically, select the reflection wavelength of the notch filter to determine the characteristic vibration mode of the sample to be tested; optionally, set the reflection wavelength of the notch filter 15 to the preset calculation target, and at the same time move the position of the photomultiplier tube 16 so that the signal voltage / current measured by the photomultiplier tube 16 is maximized.

[0066] Step S8: Record the measurement signals output during the microscopic imaging process, and process the output measurement signals to obtain the microscopic imaging results of the sample under test. Specifically, record the measurement signals of the photomultiplier tube 16, and record the voltage / current values ​​at any point on the sample under test 11. Convert the obtained analog signals into digital signals through the PCI card. Then, use LabVIEW software to record the data and use Matlab to process the data.

[0067] It should be noted that the process of recording the measurement signal of photomultiplier tube 16 is as follows: When the relative distance between the polarizing objective lens 10 and the sample 11 remains constant, a coordinate plane is established with the horizontal plane where the sample 11 is located as the coordinate plane. xoy The coordinate system is used to determine the coordinates of the position of the light spot on the sample 11 to be tested.

[0068] When the coordinates of the position of the light spot on the sample 11 to be tested y When the value is constant, the pressure applied to the two-dimensional precision translation stage where the sample 11 is located is increased. x Voltage value in the axial direction, light spot x The values ​​were shifted in 1μm increments, and 150 different values ​​were recorded simultaneously. x The signal of the photomultiplier tube at that time.

[0069] when y=y At +1μm, 150 different [samples] were recorded simultaneously again. x The signal from photomultiplier tube 16 at the specified value; to y=y At +150μm, 150 different [samples] were recorded simultaneously again. x The signal from the photomultiplier tube 16 is used to scan a 150μm×150μm region and record all values ​​on the 150×150 two-dimensional matrix grid.

[0070] The CARS spectral imaging device and method described in this embodiment uses a linearly polarized ultrashort pulse laser as the incident laser and sets the polarization directions of the first and second polarizers orthogonally. This improves upon existing single-beam CARS imaging schemes by utilizing the orthogonal polarization principle. The system structure is simplified, hardware costs and laser power loss are significantly reduced, and pure resonance CARS signals can be extracted without complex spectral post-processing. Single-pixel imaging speed is greatly improved, making it suitable for rapid molecular imaging detection scenarios in biomedical live tissue sections. Simultaneously, orthogonal polarization noise reduction enables faster terahertz band CARS microscopy imaging. Specifically, the generated CARS signal intensity can be gradually and systematically increased, and the positions of various CARS spectral detection elements are optimized. Furthermore, during imaging, the CARS signal intensity is increased by adjusting all optical path elements step by step, background noise is significantly suppressed using orthogonal polarizers, the reflection wavelength of the notch filter is selected to lock the sample's characteristic vibration mode, and the output signal of the photomultiplier tube is acquired and processed to obtain the imaging results.

[0071] In this embodiment, linearly polarized ultrashort pulse laser is used as the incident laser, and a polarizer is used to make the incident laser more purely linearly polarized light. The transmission part utilizes the polarization sensitivity of background noise to reduce, and eventually eliminates, the interference of background noise. This eliminates the dependence on galvanometer scanner, notch filter, and lock-in amplifier, simplifies the CARS system, reduces costs, and reduces the power loss of the laser on the notch filter. The use of orthogonal polarization to remove background noise makes CARS spectroscopy and microscopic imaging faster, increasing the single-pixel imaging speed to 50μs.

[0072] Test application instructions; This experimental application description uses the microscopic imaging process of rat brain slices as an example to illustrate the CARS spectral imaging device and method described in the above embodiments, as detailed below: A linearly polarized ultrashort pulse laser with a pulse width of 90 fs is generated using a femtosecond laser pulse oscillator 1. Its center wavelength is 790 nm and its bandwidth is approximately 15 nm. The linearly polarized ultrashort pulse laser first passes through a first reflecting mirror 2, and then through a pair of first prisms 3 and second prisms 4 placed on a one-dimensional precision translation stage for dispersion compensation. After the first dispersion compensation, it is reflected by a second reflecting mirror 5, and then passes through a second prism 4 and a first prism 3 for secondary dispersion compensation. After the secondary dispersion compensation, it passes through a third reflecting mirror 6, and then through an ultra-steep long-pass filter 7. After being reflected by a fourth reflecting mirror 8, it passes through a first polarizer 9 placed on an angle-controllable rotation controller, and then through a polarizing objective lens 10 placed on a one-dimensional precision translation stage. It is then focused onto the sample 11 to be tested, forming a CARS signal. The sample 11 to be tested is placed on a sample slot of a two-dimensional precision translation stage.

[0073] The CARS signal is transmitted through the sample 11, passes through the second polarizer 12 placed on the angle-controllable rotation controller, and then converged by the focusing lens 13 placed on the one-dimensional precision translation stage. The transmitted part is filtered out by the ultra-steep short-pass filter 14. Part of the signal is selectively reflected by the notch filter 15 and guided to the photomultiplier tube 16 for CARS microscopic imaging. The other part is transmitted through the notch filter 15 and guided through the lens 17 into the optical fiber connected to the spectrometer 16 for measuring the CARS spectrum.

[0074] Model descriptions of the above components: The models of the first reflecting mirror 2, the second reflecting mirror 5, the third reflecting mirror 6, and the fourth reflecting mirror 8 are all Thorlabs, UM10-AG-10; the models of the first prism 3 and the second prism 4 are both Thorlabs, AFS-FS; the model of the ultra-steep long-pass filter 7 is Semrock LP01-785RE; the models of the first polarizer 9 and the second polarizer 12 are both Thorlabs, DGL10; the model of the polarizing objective 10 is Newport, 20x, 0.4 NA; the model of the condenser lens 13 is Edmund Optics, 0.5 NA; the model of the ultra-steep short-pass filter 14 is Semrock SP01-785RU; the model of the notch filter 15 is UnaxisBlazers; the model of the photomultiplier tube 16 is Hamamatsu R955; the model of the lens 17 is Thorlabs, AC254-050-AB; and the model of the spectrometer 16 is Jobin Yvon. Triax 320; the model of the one-dimensional precision translation stage is: Thorlabs, PT1B; the model of the two-dimensional precision translation stage is: Mad City Labs, Inc. Nano-Bios; the model of the two-dimensional adjustable frame is: LBTEK AMM-1; the model of the angle-controllable rotation controller is: Thorlabs DDR100.

[0075] Experimental results indicate: In this experimental application manual, when performing CARS microscopy on rat brain slices as the test sample 11, the selection of the reflection wavelength of the notch filter 15 can satisfy the requirements for Raman scattering microscopy imaging at different vibration frequencies of the sample. When the reflection wavelength of the notch filter 15 is 792.5 nm, the imaging results of the rat brain slices are shown in the attached figure. Figure 3 As shown; from the appendix Figure 3 As can be seen from the attached Figure 3 The image shows a CARS microscopic image to avoid the influence of background noise; the white bright spots in the image represent the spatial distribution of proteins in the rat brain; this demonstrates that the method described in this embodiment can achieve rapid CARS resonance signal extraction and microscopic imaging results.

[0076] The CARS spectral imaging device and method described in this invention, by setting a first polarizer in the incident light path and a second polarizer in the signal collection light path, and setting the polarization directions of the first and second polarizers orthogonally, directly filters out non-resonant background noise in CARS detection using an orthogonal polarization optical structure. This eliminates the need for the high-frequency galvanometer scanner, lock-in amplifier, and matching modulation and demodulation circuit required by traditional methods, significantly reducing the overall hardware procurement cost of the device. Simultaneously, it eliminates laser power loss caused by laser reflection on multi-layer notch-coated filters, resulting in lower overall energy consumption of the optical path. By performing two dispersion compensations through prisms, it corrects the group velocity dispersion generated during femtosecond laser transmission, ensuring stable peak power of the laser pulse reaching the sample and improving the excitation intensity of the CARS resonance signal. It eliminates the need for multiple acquisitions of two sets of spectra for difference calculations, saving complex spectral post-processing steps and making the spectral extraction process simpler and more efficient. Relying on the orthogonal polarization structure to achieve real-time background noise suppression, the imaging process is no longer limited by the galvanometer scanning modulation speed, enabling rapid full-area scanning imaging of large-area biological tissue sections. It can be widely applied in the biomedical field for rapid display of molecular characteristics of living cells and animal tissue sections.

[0077] In this invention, a pair of orthogonally polarized polarizers are used to remove background noise, making the system simpler, reducing costs, and making Raman spectroscopy extraction and CARS microscopy imaging simpler and faster. Specifically, using a pair of orthogonally placed polarizers to remove the inherent background noise of CARS eliminates the need for a complex modulation-demodulation system, greatly simplifying the system, reducing costs, and minimizing power loss caused by laser reflection on the notch filter. Orthogonal polarizers are significantly cheaper than notch filters, galvanometer scanners, and lock-in amplifiers. Furthermore, only optomechanical placement is required, eliminating the need for high-frequency modulation of the laser and complex circuit algorithms, making it more convenient and simpler. In this invention, the interference of background noise is eliminated by using orthogonally placed polarizers, thus eliminating the need for post-processing of the CARS spectrum and multiple measurements for subtraction, making the spectral measurement method simple. Due to the absence of background noise interference, the single-pixel imaging speed can be increased to below 50 μs, which is about 20 times faster than the traditional single-beam CARS microscopy imaging based on notch filtering. It can be applied to rapid imaging in the biomedical field, and this speed is not limited by the galvanometer scanning speed, but only by the signal-to-noise ratio.

[0078] The above embodiments are merely one of the implementation methods for achieving the technical solution of the present invention. The scope of protection claimed by the present invention is not limited to this embodiment, but also includes any variations, substitutions and other implementation methods that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention.

Claims

1. A CARS spectral imaging device, characterized in that, Includes a femtosecond laser pulse oscillator (1); the femtosecond laser pulse oscillator (1) is used to generate incident laser; wherein the incident laser is a linearly polarized ultrashort pulse laser; The incident laser beam is sequentially irradiated onto the sample (11) by the first reflecting mirror (2), the prism pair, the second reflecting mirror (5), the ultra-steep long-pass filter (7), the fourth reflecting mirror (8), the first polarizer (9), and the polarizing objective (10), and a CARS signal is formed. The CARS signal passes through the transmission part of the sample under test (11), and is then processed by the second polarizer (12), condenser lens (13), steep short-pass filter (14) and notch filter (15) to form selectively reflected light for microscopic imaging. The polarization directions of the first polarizer (9) and the second polarizer (12) are orthogonal.

2. The CARS spectral imaging device according to claim 1, characterized in that, The center wavelength of the linearly polarized ultrashort pulse laser is 780-805nm, the bandwidth is 10-60nm, the repetition frequency is 10-100MHz, and the pulse width is 10-100fs.

3. The CARS spectral imaging device according to claim 1, characterized in that, The prism is used to perform primary dispersion compensation on the incident light of the second mirror (5) and secondary dispersion compensation on the reflected light of the second mirror (5). The prism pair includes a first prism (3) and a second prism (4), which are arranged sequentially between the first reflector (2) and the second reflector (5).

4. A CARS spectral imaging device according to claim 3, characterized in that, The operating wavelengths of the first prism (3) and the second prism (4) are both 600-900nm.

5. A CARS spectral imaging device according to claim 3, characterized in that, It also includes a third reflector (6), which is positioned between the first prism (3) and the ultra-steep long-pass filter (7).

6. A CARS spectral imaging device according to claim 5, characterized in that, The sample to be tested (11) is mounted on a two-dimensional precision translation stage; the first prism (3), the second prism (4), the polarizing objective (10) and the condenser lens (13) are mounted on a one-dimensional precision translation stage; the first mirror (2), the second mirror (5), the third mirror (6) and the fourth mirror (8) are mounted on a two-dimensional adjustable mirror frame; the first polarizer (9) and the second polarizer (12) are mounted on an angle-adjustable rotation controller.

7. A CARS spectral imaging device according to claim 1, characterized in that, The polarizing objective (10) has a magnification of 10-100 times and a numerical aperture of 0.2-1.

2.

8. A CARS spectral imaging device according to claim 1, characterized in that, It also includes a photomultiplier tube (16), a lens (17) and a spectrometer (18); the selected reflected light enters the photomultiplier tube (16) for microscopic imaging; the transmitted light passes through the lens (17) and enters the spectrometer (18) for spectral measurement.

9. A CARS spectral imaging device according to claim 1, characterized in that, The focal length of the lens (17) is 30-100mm.

10. A CARS spectral imaging method, characterized in that, Using the CARS spectral imaging apparatus as described in any one of claims 1-9; the imaging method includes: The femtosecond laser pulse oscillator (1) is activated, and the positions of the first mirror (2), prism pair, second mirror (5), ultra-steep long-pass filter (7), fourth mirror (8), first polarizer (9), polarizing objective (10), second polarizer (12), condenser lens (13), and steep short-pass filter (14) are adjusted to maximize the spectral intensity of the CARS signal; among them, the position of the first polarizer (9) is adjusted to minimize the background noise in the CARS signal. Select the reflection wavelength of the notch filter (15) to determine the characteristic vibration mode of the sample (11) to be tested; Record the measurement signals output during the microscopic imaging process, and perform data processing on the output measurement signals to obtain the microscopic imaging results of the sample (11) to be tested.

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