Time domain excitation coherent Raman photothermal spectrum microscopic detection system

Abstract

The invention relates to a time domain excitation coherent Raman photo-thermal spectrum microscopic detection system. The system comprises a light source assembly, a scanning assembly and a detection assembly, the light source assembly is used for generating a first excitation pulse, first delay detection laser and first detection laser; the scanning assembly is used for performing copying processing and delay processing on the first excitation pulse to obtain a second excitation pulse and a third excitation pulse with relative delay, and monitoring the relative delay amount between the second excitation pulse and the third excitation pulse by using the first delay detection laser; the detection assembly is used for performing coherent Raman excitation on the to-be-detected sample by adopting the second excitation pulse and the third excitation pulse, so that the to-be-detected sample generates a thermo-optic effect; and the processor is also used for adjusting or converting the first detection laser so as to detect the thermo-optic effect, and determining the coherent Raman scattering spectrum of the to-be-detected sample based on the thermo-optic effect and the relative delay amount. By utilizing the system, the measurement accuracy and the spectral resolution of the coherent Raman spectrum can be improved.

Description

Technical Field

[0001] The present application relates to the field of spectral detection technology, and in particular to a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system. Background Art

[0002] With the development of nonlinear spectroscopy technology, coherent Raman scattering spectroscopy has become an important technology for specific detection of biochemical molecules, and has important application value in the fields of medicine, biology, chemistry, and materials science.

[0003] In traditional technology, coherent Raman photothermal microscopy detection technology can be realized based on spectral focusing and photothermal microscopy imaging.

[0004] However, this method may introduce non-resonance-induced exothermic effects (e.g., relaxation decay exothermicity of two-photon absorption) and has limited spectral resolution (the line width at the natural broadening limit cannot be measured), thus affecting the measured coherent Raman scattering spectrum. Summary of the invention

[0005] Based on this, it is necessary to provide a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system that can improve the measurement accuracy and spectral resolution of coherent Raman photothermal spectroscopy in response to the above technical problems.

[0006] In a first aspect, the present application provides a time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system, the system comprising a light source component, a scanning component and a detection component;

[0007] A light source assembly, used to generate a first excitation pulse, a first delayed detection laser and a first detection laser;

[0008] A scanning component is used to perform a copy process and a delay process on the first excitation pulse to obtain a second excitation pulse and a third excitation pulse with relative delay, and to monitor the relative delay between the second excitation pulse and the third excitation pulse by using a first delay detection laser;

[0009] The detection component is used to use the second excitation pulse and the third excitation pulse to perform coherent Raman excitation on the sample to be tested so that the sample to be tested produces a thermo-optical effect; it is also used to adjust or transform the first detection laser to detect the thermo-optical effect, and determine the coherent Raman scattering spectrum of the sample to be tested based on the thermo-optical effect and the relative delay.

[0010] In one embodiment, the detection assembly includes a backscatter detection assembly, which includes a first detection laser adjustment component, a first detection laser beam splitter component, a first excitation component, and a detection component;

[0011] A first detection laser adjustment member is used to adjust the axial focus position of the first detection laser at the sample to be measured to obtain a second detection laser;

[0012] A first detection laser beam splitter is used to inject the second detection laser into the first excitation component;

[0013] A first excitation component is used to focus the second excitation pulse and the third excitation pulse onto the sample to be tested, so that the sample to be tested generates a thermo-optical effect, and detects the thermo-optical effect based on the second detection laser to obtain a third detection laser;

[0014] The detection element is used to determine the coherent Raman scattering spectrum of the sample to be tested based on the third detection laser.

[0015] In one embodiment, the first excitation assembly includes a first objective lens, a first collecting lens, a first filter, and a first reflecting mirror;

[0016] A first reflecting mirror is used to reflect the second detection laser so that the second detection laser passes through the first filter and enters the first collecting mirror;

[0017] A first collecting mirror is used to collect and tightly focus the second detection laser onto the sample to be tested to obtain a fourth detection laser;

[0018] The first objective lens is used to collect and tightly focus the second excitation pulse and the third excitation pulse onto the sample to be tested, and obtain a fourth excitation pulse and a fifth excitation pulse respectively, wherein the fourth excitation pulse and the fifth excitation pulse excite coherent Raman oscillation of the sample to be tested, so that the sample to be tested generates a thermo-optical effect; the fourth detection laser interacts with the thermo-optical effect to obtain a fifth detection laser;

[0019] The first collecting mirror is further used to collect the fifth detection laser to obtain the sixth detection laser;

[0020] The first filter is also used to filter out residual excitation pulses in the sixth detection laser to obtain a third detection laser;

[0021] The first reflecting mirror is also used to reflect the third detection laser to the detection element.

[0022] In one embodiment, the first detection laser adjustment member includes a second reflector, a first lens, and a second lens;

[0023] A second reflector is used to reflect the first detection laser to the first lens; the optical axes of the first lens and the second lens are located on the same straight line;

[0024] The first lens and the second lens are used to adjust the axial focus position of the first detection laser at the sample to be measured to obtain the second detection laser.

[0025] In one embodiment, the detection assembly includes a forward scatter detection assembly, which includes a second detection laser adjustment member, a second excitation member, and a detection member;

[0026] A second detection laser adjustment member is used to adjust the axial focus position of the first detection laser at the sample to be measured to obtain a second detection laser;

[0027] A second excitation component is used to focus the second excitation pulse and the third excitation pulse onto the sample to be tested, so that the sample to be tested generates a thermo-optical effect, and detects the thermo-optical effect based on the second detection laser to obtain a third detection laser;

[0028] The detection element is used to determine the coherent Raman scattering spectrum of the sample to be tested based on the third detection laser.

[0029] In one embodiment, the second excitation assembly includes a second objective lens, a second collecting lens, a second filter, and a third reflecting mirror;

[0030] The second objective lens is used to collect and tightly focus the second detection laser, the second excitation pulse and the third excitation pulse onto the sample to be tested, and obtain the fourth detection laser, the fourth excitation pulse and the fifth excitation pulse respectively. The fourth excitation pulse and the fifth excitation pulse excite the coherent Raman oscillation of the sample to be tested, so that the sample to be tested generates a thermo-optical effect; the fourth detection laser interacts with the thermo-optical effect to obtain the fifth detection laser;

[0031] a second collecting mirror, used for collecting the fifth detection laser to obtain a sixth detection laser;

[0032] A second filter is used to filter out residual excitation pulses in the sixth detection laser to obtain a third detection laser;

[0033] The third reflecting mirror is also used to reflect the third detection laser to the detection element.

[0034] In one embodiment, the second detection laser adjustment element includes a third lens and a fourth lens;

[0035] The third lens and the fourth lens are used to adjust the axial focus position of the first detection laser at the sample to be measured to obtain the second detection laser.

[0036] In one embodiment, the detection assembly includes a Bessel beam detection assembly, which includes a second detection laser beam splitter, a first Bessel beam transformation assembly, a third excitation assembly, a fourth reflector, and a detection element;

[0037] The second detection laser beam splitter is used to split the first detection laser to obtain the first reference arm detection laser and the first measurement arm detection laser;

[0038] A first Bessel beam conversion component is used to convert the first measuring arm detection laser into a Bessel beam, and adjust the axial focus position of the first measuring arm detection laser at the sample to be measured to obtain a second measuring arm detection laser;

[0039] a third excitation component, used for focusing the second measuring arm detection laser, the second excitation pulse and the third excitation pulse onto the sample to be measured, so as to generate a thermo-optical effect on the sample to be measured, and detecting the thermo-optical effect based on the second measuring arm detection laser to obtain a third measuring arm detection laser;

[0040] A fourth reflector is used to reflect the first reference arm detection laser so that the first reference arm detection laser returns to the second detection laser beam splitter to obtain the second reference arm detection laser;

[0041] The second detection laser beam splitter is also used to combine the third measuring arm detection laser and the second reference arm detection laser to obtain a combined detection laser.

[0042] The detection component is used to determine the coherent Raman scattering spectrum of the sample to be tested based on the combined beam detection laser.

[0043] In one embodiment, the third excitation assembly includes a third objective lens, a third collecting lens, a third filter, a second Bessel beam transformation assembly, and a fifth reflecting mirror;

[0044] The third objective lens is used to collect and tightly focus the second excitation pulse, the third excitation pulse and the second measuring arm detection laser onto the sample to be measured, and obtain the fourth excitation pulse, the fifth excitation pulse and the fourth measuring arm detection laser respectively. The fourth excitation pulse and the fifth excitation pulse excite the coherent Raman oscillation of the sample to be measured, so that the sample to be measured produces a thermo-optical effect; the fourth measuring arm detection laser interacts with the thermo-optical effect to obtain the fifth measuring arm detection laser;

[0045] a third collecting mirror, used for collecting the detection laser of the fifth measuring arm to obtain the detection laser of the sixth measuring arm;

[0046] A third filter is used to filter out residual excitation pulses in the detection laser of the sixth measuring arm to obtain the detection laser of the seventh measuring arm;

[0047] A second Bessel beam conversion component is used to convert the seventh measuring arm detection laser into a Bessel beam to obtain an eighth measuring arm detection laser; and is also used to convert the eighth measuring arm detection laser to restore the eighth measuring arm detection laser to an original beam form to obtain a ninth measuring arm detection laser;

[0048] The fifth reflector is used to return the ninth measuring arm detection laser to its original path, so that the ninth measuring arm detection laser again passes through the mirror-symmetrical process experienced by the first measuring arm detection laser to the ninth measuring arm detection laser in turn, so as to obtain the third measuring arm detection laser before the second detection laser beam splitter.

[0049] In one embodiment, the first Bessel beam transformation assembly includes a first conical lens and a fifth lens;

[0050] A first conical lens is used to transform the first measuring arm detection laser into a Bessel beam to obtain the middle measuring arm detection laser;

[0051] The fifth lens is used to transform the detection laser of the middle measuring arm to form an annular line light source on the front focal plane of the third objective lens to obtain the detection laser of the second measuring arm.

[0052] In one embodiment, the second Bessel beam transformation assembly includes a sixth lens and a second conical lens;

[0053] a sixth lens, used for transforming the seventh measuring arm detection laser into a Bessel beam to obtain the eighth measuring arm detection laser;

[0054] The second conical lens is used to transform the eighth measuring arm detection laser to restore it to the original light beam form to obtain the ninth measuring arm detection laser.

[0055] In one embodiment, the detection member includes a seventh lens and a first photodetector;

[0056] a seventh lens, used for focusing the third detection laser or the combined-beam detection laser onto the detection target surface of the first photodetector;

[0057] The first photoelectric detector is used to detect the signal intensity of the third detection laser or the combined beam detection laser, and to analyze the coherent Raman scattering spectrum of the sample to be tested by Fourier transform.

[0058] In one of the embodiments, the light source assembly includes a femtosecond pulse generating device, a femtosecond pulse splitting device, a single wavelength continuous laser generating device, a first beam splitter and a first dichroic mirror;

[0059] A femtosecond pulse generating device, used to generate original pulses;

[0060] A femtosecond pulse spectrometer, used for performing spectrometry and energy control processing on the original pulse to obtain a redundant pulse and a first excitation pulse;

[0061] A single-wavelength continuous laser generating device for generating original laser;

[0062] A first beam splitter is used to split the original laser to obtain a first delayed detection laser and a first detection laser;

[0063] The first dichroic mirror is used to combine the first excitation pulse and the first delayed detection laser; wherein the first excitation pulse and the first delayed detection laser then enter the scanning component, and the first detection laser then enters the detection component.

[0064] In one embodiment, the scanning assembly includes a second beam splitter, a first delay assembly, a second delay assembly, a second dichroic mirror, and a delay detection assembly;

[0065] A second beam splitter is used to perform replication processing on the first excitation pulse and the first delayed detection laser to obtain a first replicated excitation pulse, a second replicated excitation pulse, a first replicated delayed detection laser, and a second replicated delayed detection laser;

[0066] A first delay component is used to reflect the first replica excitation pulse and the first replica delayed detection laser to obtain a second excitation pulse and a second delayed detection laser;

[0067] A second delay component is used to reflect and delay scan the second replica excitation pulse and the second replica delayed detection laser to obtain a third excitation pulse and a third delayed detection laser;

[0068] The second beam splitter is further used to perform beam combining processing on the second excitation pulse and the third excitation pulse; and is also used to perform the same beam combining processing on the second delayed detection laser and the third delayed detection laser;

[0069] A second dichroic mirror is used to separate the excitation pulse and the delayed detection laser, and is also used to combine the excitation pulse and the detection laser in the detection assembly;

[0070] The delay detection component is used to detect the relative delay between the second excitation pulse and the third excitation pulse; the relative delay between the second delay detection laser and the third delay detection laser is used to characterize the relative delay between the second excitation pulse and the third excitation pulse.

[0071] The above-mentioned time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system includes a light source component, a scanning component and a detection component; the light source component is used to generate a first excitation pulse, a first delayed detection laser and a first detection laser; the scanning component is used to copy and delay the first excitation pulse to obtain a second excitation pulse and a third excitation pulse with a relative delay, and use the first delayed detection laser to monitor the relative delay between the second excitation pulse and the third excitation pulse; the detection component is used to use the second excitation pulse and the third excitation pulse to perform coherent Raman excitation on the sample to be tested so that the sample to be tested produces a thermo-optical effect; it is also used to adjust or transform the first detection laser to detect the thermo-optical effect, and determine the coherent Raman scattering spectrum of the sample to be tested based on the thermo-optical effect and the relative delay. Since the system uses a combination of time-domain excitation and photothermal detection to perform coherent Raman excitation, signal collection and detection on the sample to be tested, the coherent Raman spectral information of the biochemical substances in the sample to be tested is obtained; therefore, the non-resonance-induced exothermic effect can be suppressed by removing the data near zero delay, and the complete time-domain Raman oscillation relaxation decay curve can be scanned to measure the spectral linewidth of the natural broadening limit, thereby improving the accuracy and spectral resolution of coherent Raman photothermal spectroscopy measurement while ensuring high sensitivity and spatial resolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] In order to more clearly illustrate the technical solutions in the embodiments of the present application or related technologies, the drawings required for use in the embodiments of the present application or related technical descriptions will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0073] Figure 1 A frequency domain principle diagram of transforming the limit femtosecond pulse to excite coherent Raman oscillation in one embodiment;

[0074] Figure 2 A time domain schematic diagram of a thermo-optical effect induced by a single transformation-limited femtosecond pulse exciting coherent Raman oscillation relaxation decay in one embodiment;

[0075] Figure 3 A time domain schematic diagram of a time domain excitation coherent Raman photothermal spectroscopy microscopy detection system in one embodiment;

[0076] Figure 4 A schematic diagram of backscatter detection laser detection of thermo-optical effect in one embodiment;

[0077] Figure 5 A schematic diagram of a forward scattered detection laser detecting a thermo-optical effect in one embodiment;

[0078] Figure 6 A schematic diagram of Bessel beam-Mach-Zehnder interference detection of thermo-optical effect in one embodiment;

[0079] Figure 7 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in one embodiment;

[0080] Figure 8 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in one embodiment;

[0081] Fig. 9 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0082] Fig.10 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0083] Fig.11 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0084] Fig.12 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0085] Fig.13 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0086] Fig.14 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0087] Fig.15 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0088] Fig.16 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0089] Fig.17 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0090] Fig.18 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0091] Fig.19 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0092] Fig. 20 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0093] Fig.21 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0094] Fig. 22 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0095] Fig.23 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0096] Fig.24 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0097] Fig.25 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0098] Fig.26 It is a schematic structural diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0099] Description of reference numerals:

[0100] Light source assembly 10, femtosecond pulse generating device 101, femtosecond pulse spectrometer 102;

[0101] Half-wave plate 1021, polarization beam splitter 1022, light block 1023;

[0102] A single wavelength continuous laser generating device 103, a first beam splitter 104, and a first dichroic mirror 105;

[0103] Scanning component 11, second beam splitter 111, first delay component 112;

[0104] A second delay component 113, a sixth reflector 1131, and a curved mirror 1132;

[0105] Resonant scanning mirror 1133, second dichroic mirror 114, time-delay detection component 115;

[0106] An eighth lens 1151, a second photodetector 1152, and a detection assembly 12;

[0107] Backscatter detection component 121, first detection laser adjustment component 1211;

[0108] A second reflecting mirror 12111, a first lens 12112, and a second lens 12113;

[0109] A first detection laser beam splitter 1212 and a first excitation component 1213;

[0110] A first objective lens 12131, a first collecting lens 12132, and a first filter 12133;

[0111] A first reflector 12134, a forward scattering detection assembly 122;

[0112] A second detection laser adjustment member 1221, a third lens 12211, and a fourth lens 12212;

[0113] A second excitation component 1222, a second objective lens 12221, and a second collecting lens 12222;

[0114] A second filter 12223, a third reflector 12224, and a Bessel beam detection assembly 123;

[0115] A second detection laser beam splitter 1231 and a first Bessel beam transformation component 1232;

[0116] A first conical lens 12321, a fifth lens 12322, and a third excitation component 1233;

[0117] A third objective lens 12331, a third collecting lens 12332, and a third filter 12333;

[0118] A second Bessel beam transformation component 12334, a sixth lens 123341;

[0119] A second conical lens 123342, a fifth reflector 12335, and a fourth reflector 1234;

[0120] Detection element 1214, seventh lens 12141, first photodetector 12142, and sample to be tested 13. DETAILED DESCRIPTION

[0121] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0122] With the development of nonlinear spectroscopy technology, coherent Raman scattering spectroscopy has become an important technology for specific detection of biochemical molecules, and has important application value in the fields of medicine, biology, chemistry, materials science, etc. In traditional technology, coherent Raman photothermal microscopy detection technology can be realized based on spectral focusing and photothermal microscopy imaging.

[0123] However, the above coherent Raman photothermal spectroscopy technology is based on the excitation strategy of spectral focusing to scan and obtain broadband spectra, which has the following technical limitations. 1) Spectral focusing is difficult to obtain high spectral resolution. Even if the excitation light is strictly isolinear chirped without high-order dispersion, it is impossible to measure the Raman linewidth of the natural broadening limit; 2) The excitation method of spectral focusing may introduce non-resonance-induced exothermic effects (such as relaxation decay exothermicity of two-photon absorption), which will interfere with the measured spectral signal.

[0124] Based on this, it is necessary to provide a time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system to address the above-mentioned technical problems, improve the spectral resolution (theoretically, the Raman peak width of the natural broadening limit can be measured) and avoid the influence of the non-resonance induced exothermic effect. At the same time, in one implementation scheme, based on the Bessel beam-Mach-Zehnder interference principle, a new thermal-optical effect detection scheme is proposed in the field of coherent Raman photothermal spectroscopy, which makes the detection signal more stable and reliable and further improves the spatial resolution of the spectral microscopy system.

[0125] First, the technical principle of the time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system involved in the embodiment of the present application is explained. The embodiment of the present application uses a transform-limited femtosecond pulse to excite the coherent Raman oscillation of the sample to be tested, and its frequency domain principle diagram is as follows Figure 1 As shown, the relatively high-frequency part of the excitation pulse spectrum acts as pump light, and the relatively low-frequency part acts as Stokes light to excite coherent Raman oscillations.

[0126] In the time domain, Figure 2 As shown in (a), after a single excitation pulse is incident on the sample to be tested, the sample molecules are stimulated to generate Raman oscillations Q1 (assuming the oscillation angular frequency is Ω0). After being excited, the intensity of Q1 will relax and decay in the form of an exponential function over time (the decay constant is about picoseconds), while releasing heat. Figure 2 (b), where r represents the distance from the center of the excitation pulse focus. This heat will cause the local temperature distribution near the excitation pulse focus to rise, which in turn causes the change of the local refractive index distribution, that is, the thermo-optic effect induced by the relaxation decay of the coherent Raman oscillation. The time domain principle of the time domain excitation coherent Raman photothermal spectroscopy detection system is shown in Figure 2. Figure 3 As shown, through replication and delay, the fourth excitation pulse and the fifth excitation pulse after a delay of τ are successively incident on the sample to be tested, respectively exciting coherent Raman oscillations Q1 and Q2. Q1 and Q2 interfere after the fifth excitation pulse excites Q2, and the coherent Raman oscillation of the molecule continues in the form of Q1+Q2. The relaxation decay and heat release of the coherent Raman oscillation are also always carried out simultaneously. Ultimately, the amount of heat released depends on the constructive or destructive interference of Q1+Q2, that is, it depends on the relative delay τ between the fourth excitation pulse and the fifth excitation pulse. By scanning the relative delay τ, as Figure 3 As shown in (c) in the figure, the exothermic curve modulated by the Raman oscillation information to be measured can be obtained. Figure 3 As shown in (d, e) in the figure, the thermo-optical effect near the focus of the excitation pulse is also encoded by the heat release curve, and finally carries the Raman oscillation information to be measured with the scanning of the relative delay τ. The thermo-optical effect encoded with the Raman oscillation information to be measured is detected by using a detection laser, and the Raman oscillation information to be measured can be demodulated by Fourier transform to obtain a broadband Raman spectrum.

[0127] For the detection of the above thermo-optical effect, the embodiments of the present application provide three solutions.

[0128] First, as Figure 4 As shown, a backscattering detection method is used. That is, the incident detection laser interacts with the above-mentioned thermo-optical effect to generate backscattered detection laser. Since the above-mentioned thermo-optical effect carries the Raman oscillation information to be measured with the scanning of the relative delay τ, the generated backscattered detection laser will also carry this information. By detecting the relative intensity change of the backscattered detection laser and performing Fourier transform on it, a broadband Raman spectrum can be obtained. It should be noted that the detection laser can use a continuous laser with a shorter wavelength than the excitation pulse to obtain a smaller diffraction-limited spot to improve the spatial resolution.

[0129] Second, if Figure 5 As shown, the forward scattering detection method is adopted. That is, the incident detection laser interacts with the above-mentioned thermo-optical effect to generate forward scattering detection laser, and the subsequent detection principle is the same as the backscattering detection method, which will not be repeated.

[0130] Third, if Figure 6 As shown, the present application proposes a new Bessel beam-Mach-Zehnder interference method for the detection of the above-mentioned thermo-optical effect. The detection laser is first divided into a reference arm detection laser and a measuring arm detection laser. The reference arm detection laser passes through a space with a fixed optical path. The measuring arm detection laser is transformed into a Bessel beam through the first Bessel beam transformation component and the objective lens to be incident on the sample to be measured and interact with the above-mentioned thermo-optical effect. At this time, the optical path of the measuring arm detection laser is coded and modulated by the Raman oscillation information to be measured. The measuring arm detection laser is then restored to the original beam form through the collecting mirror and the second Bessel beam transformation component, and returns to the original path through the reflector, and again undergoes the above-mentioned beam transformation-interaction with the thermo-optical effect-beam transformation mirror-symmetric process. Because the measuring arm detection laser interacts with the above-mentioned thermo-optical effect twice in succession, the optical path modulation introduced thereby is twice that of a single path. Finally, the reference arm detection laser and the measuring arm detection laser are combined again to interfere, and the interference signal is detected and Fourier transformed to obtain a broadband Raman spectrum.

[0131] The following is a detailed description of the above three solutions.

[0132] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Figure 7 As shown, the system includes a light source component 10, a scanning component 11 and a detection component 12; the light source component 10 is used to generate a first excitation pulse, a first delayed detection laser and a first detection laser; the scanning component 11 is used to copy and delay the first excitation pulse to obtain a second excitation pulse and a third excitation pulse with a relative delay, and use the first delayed detection laser to monitor the relative delay between the second excitation pulse and the third excitation pulse; the detection component 12 is used to use the second excitation pulse and the third excitation pulse to perform coherent Raman excitation on the sample to be tested so that the sample to be tested produces a thermo-optical effect; it is also used to adjust or transform the first detection laser to detect the thermo-optical effect, and determine the coherent Raman scattering spectrum of the sample to be tested based on the thermo-optical effect and the relative delay.

[0133] Wherein, the light source component 10 is used to generate a first excitation pulse, a first delayed detection laser and a first detection laser, wherein the first excitation pulse is a conversion limit femtosecond pulse with adjustable absolute energy and its spectrum meets the requirements of exciting the Raman wave number to be detected, the first delayed detection laser is a single-wavelength continuous laser used to detect and monitor the relative delay between the excitation pulses, and the first detection laser is a single-wavelength continuous laser used to detect the thermo-optical effect induced by the relaxation attenuation of the coherent Raman oscillation of the sample to be tested. After the first excitation pulse and the first delayed detection laser are combined, they enter the scanning component 11, and the first detection laser enters the detection component 12;

[0134] The scanning component 11 is used to replicate and delay scan the first excitation pulse and the first delayed detection laser to obtain the second excitation pulse and the third excitation pulse with relative delay, and the second delayed detection laser and the third delayed detection laser with completely consistent relative delay, and measure the interference signal of the second delayed detection laser and the third delayed detection laser through the principle of single-frequency laser interference, so as to monitor the relative delay between the second excitation pulse and the third excitation pulse in real time;

[0135] The detection component 12 is used to use the second excitation pulse and the third excitation pulse to perform coherent Raman excitation on the sample to be tested, and the coherent Raman oscillation relaxation decay induces the sample to be tested to produce a thermal effect and then a thermo-optical effect. After the first detection laser is adjusted or transformed, the above-mentioned thermo-optical effect is detected to determine the detection result of the sample to be tested. Among them, the detection result is the intensity information of the thermo-optical effect, and the intensity information of the thermo-optical effect can reflect the coherent Raman scattering spectrum information.

[0136] In the above embodiment, the system includes a light source component 10, a scanning component 11 and a detection component 12; the light source component 10 is used to generate a first excitation pulse, a first delayed detection laser and a first detection laser; the scanning component 11 is used to perform a copy process and a delay process on the first excitation pulse to obtain a second excitation pulse and a third excitation pulse with a relative delay, and use the first delayed detection laser to monitor the relative delay between the second excitation pulse and the third excitation pulse; the detection component 12 is used to use the second excitation pulse and the third excitation pulse to perform coherent Raman excitation on the sample to be tested, so that the sample to be tested produces a thermo-optical effect; it is also used to adjust or transform the first detection laser to detect the thermo-optical effect, and determine the coherent Raman scattering spectrum of the sample to be tested based on the thermo-optical effect and the relative delay. Since the system uses a combination of time domain excitation and photothermal detection to perform coherent Raman excitation, signal collection and detection on the sample to be tested, thereby obtaining the coherent Raman spectrum information of the biochemical substance in the sample to be tested; therefore, the non-resonance-induced exothermic effect can be suppressed by removing the data near the zero delay, thereby improving the accuracy of the coherent Raman scattering spectrum measurement. In addition, thanks to the characteristics of time-domain detection, this method can completely scan the relaxation decay curve of Raman oscillation in the time domain, so that it can theoretically measure the Raman peak at the natural broadening limit, thereby improving the resolution of the spectrum. At the same time, this technology is compatible with the advantages of photothermal detection, ensuring high detection sensitivity and spatial resolution.

[0137] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Figure 8 As shown, the detection component 12 includes a backscatter detection component 121, and the backscatter detection component 121 includes a first detection laser adjustment component 1211, a first detection laser beam splitter 1212, a first excitation component 1213 and a detection component 1214; the first detection laser adjustment component 1211 is used to adjust the axial focus position of the first detection laser at the sample to be tested to obtain a second detection laser; the first detection laser beam splitter 1212 is used to make the second detection laser incident on the first excitation component 1213; the first excitation component 1213 is used to focus the second excitation pulse and the third excitation pulse to the sample to be tested, so that the sample to be tested generates a thermo-optical effect, and detects the thermo-optical effect based on the second detection laser to obtain a third detection laser; the detection component 1214 is used to determine the coherent Raman scattering spectrum of the sample to be tested based on the third detection laser.

[0138] The first detection laser adjustment member 1211 adjusts the axial focus position of the first detection laser at the sample to be tested to obtain the second detection laser, so as to obtain the best thermo-optical effect detection signal.

[0139] The first detection laser beam splitter 1212 injects the second detection laser into the first excitation component 1213. The first excitation component 1213 focuses the second excitation pulse and the third excitation pulse onto the sample to be tested, so that the sample to be tested generates a thermo-optical effect, and detects the thermo-optical effect based on the second detection laser to obtain a third detection laser; then, the detection component 1214 is used to determine the coherent Raman scattering spectrum of the sample to be tested based on the third detection laser.

[0140] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Fig. 9 As shown, the first excitation component 1213 includes a first objective lens 12131, a first collecting mirror 12132, a first filter 12133 and a first reflecting mirror 12134; the first reflecting mirror 12134 is used to reflect the second detection laser so that the second detection laser passes through the first filter 12133 and enters the first collecting mirror 12132; the first collecting mirror 12132 is used to collect and tightly focus the second detection laser onto the sample to be tested to obtain the fourth detection laser; the first objective lens 12131 is used to collect and tightly focus the second excitation pulse and the third excitation pulse onto the sample to be tested A fourth excitation pulse and a fifth excitation pulse are obtained respectively, and the fourth excitation pulse and the fifth excitation pulse excite the coherent Raman oscillation of the sample to be tested, so that the sample to be tested produces a thermo-optical effect; the fourth detection laser interacts with the thermo-optical effect to obtain a fifth detection laser; the first collecting mirror 12132 is also used to collect the fifth detection laser to obtain a sixth detection laser; the first filter 12133 is also used to filter out the residual excitation pulses in the sixth detection laser to obtain a third detection laser; the first reflecting mirror 12134 is also used to reflect the third detection laser to the detection element 1214.

[0141] Among them, the first detection laser beam splitter 1212 is first used to reflect the second detection laser to make it incident on the first reflection mirror 12134; the first reflection mirror 12134 is first used to reflect the second detection laser reflected by the first detection laser beam splitter 1212 to make it enter the first collecting mirror 12132 through the first filter 12133; the first collecting mirror 12132 is first used to collect and tightly focus the second detection laser onto the sample to be tested to obtain the fourth detection laser; the first objective lens 12131 is used to collect and tightly focus the second excitation pulse and the third excitation pulse onto the sample to be tested to obtain the fourth excitation pulse and the fifth excitation pulse respectively, and the fourth excitation pulse and the fifth excitation pulse excite the coherent Raman oscillation of the sample to be tested; the sample to be tested is the coherent Raman spectrum signal to be measured. The above-mentioned coherent Raman oscillation and the thermo-optical effect induced by the relaxation attenuation of the coherent Raman oscillation occur here, and the fourth detection laser interacts with the thermo-optical effect to obtain the backscattered fifth detection laser encoded by the thermo-optical effect; the first collecting mirror 12132 is also used to collect the fifth detection laser to obtain the sixth detection laser; the first filter 12133 is used to filter out the residual excitation pulse in the sixth detection laser to obtain the third detection laser; the first reflector 12134 is also used to reflect the third detection laser to the first detection laser beam splitter 1212; then, the first detection laser beam splitter 1212 is also used to transmit the third detection laser reflected by the first reflector 12134, and the third detection laser is then incident on the detection component 12.

[0142] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Fig.10 As shown, the first detection laser adjustment component 1211 includes a second reflector 12111, a first lens 12112 and a second lens 12113; the second reflector 12111 is used to reflect the first detection laser to the first lens 12112; the optical axes of the first lens 12112 and the second lens 12113 are located in the same straight line; the first lens 12112 and the second lens 12113 are used to adjust the axial focal position of the first detection laser at the sample to be measured to obtain the second detection laser.

[0143] Among them, the second reflector 12111 is used to reflect the first detection laser to the first lens 12112; the optical axes of the first lens 12112 and the second lens 12113 are located in the same straight line and coincide with the first detection laser beam; the first lens 12112 and the second lens 12113 are used to adjust the axial focal position of the first detection laser at the sample to be tested to obtain the second detection laser, so as to obtain the best thermal-optical effect detection signal.

[0144] In some embodiments, the distance between the first lens 12112 and the second lens 12113 is adjusted to approximately the sum of their focal lengths.

[0145] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Fig.11 As shown, the detection component 12 includes a forward scattering detection component 122, and the forward scattering detection component 122 includes a second detection laser adjustment component 1221, a second excitation component 1222 and a detection component 1214; the second detection laser adjustment component 1221 is used to adjust the axial focus position of the first detection laser at the sample to be tested to obtain a second detection laser; the second excitation component 1222 is used to focus the second excitation pulse and the third excitation pulse to the sample to be tested so that the sample to be tested produces a thermo-optical effect, and detects the thermo-optical effect based on the second detection laser to obtain a third detection laser; the detection component 1214 is used to determine the coherent Raman scattering spectrum of the sample to be tested based on the third detection laser.

[0146] Among them, the second detection laser adjustment component 1221 is used to adjust the axial focus position of the first detection laser at the sample to be tested to obtain the second detection laser to obtain the best thermo-optical effect detection signal; then, the second dichroic mirror 114 in the scanning component 11 is used to combine the second and third excitation pulses with the second detection laser; the second excitation component 1222 is used to focus the second excitation pulse and the third excitation pulse to the sample to be tested so that the sample to be tested produces a thermo-optical effect, and the thermo-optical effect is detected based on the second detection laser to obtain the third detection laser; the detection component 1214 is used to determine the coherent Raman scattering spectrum of the sample to be tested based on the third detection laser.

[0147] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Fig.12 As shown, the second excitation component 1222 includes a second objective lens 12221, a second collecting mirror 12222, a second filter 12223 and a third reflecting mirror 12224;

[0148] The second objective lens 12221 is used to collect and focus the second detection laser, the second excitation pulse and the third excitation pulse onto the sample to be tested, and obtain the fourth detection laser, the fourth excitation pulse and the fifth excitation pulse respectively. The fourth excitation pulse and the fifth excitation pulse excite the coherent Raman oscillation of the sample to be tested, so that the sample to be tested produces a thermo-optical effect; the fourth detection laser interacts with the thermo-optical effect to obtain the fifth detection laser; the second collecting mirror 12222 is used to collect the fifth detection laser to obtain the sixth detection laser; the second filter 12223 is used to filter out the residual excitation pulses in the sixth detection laser to obtain the third detection laser; the third reflecting mirror 12224 is also used to reflect the third detection laser to the detection element 1214.

[0149] Among them, the second objective lens 12221 is used to collect and focus the second excitation pulse, the third excitation pulse and the second detection laser onto the sample to be tested, and obtain the fourth excitation pulse, the fifth excitation pulse and the fourth detection laser respectively. The fourth excitation pulse and the fifth excitation pulse excite the coherent Raman oscillation of the sample to be tested; the sample to be tested is a biochemical substance sample whose coherent Raman spectrum information is to be measured, and the above-mentioned coherent Raman oscillation and the thermo-optical effect induced by the relaxation attenuation of the coherent Raman oscillation occur here. The fourth detection laser interacts with the thermo-optical effect to obtain the fifth detection laser forward scattered by the thermo-optical effect; the second collecting mirror 12222 is used to collect the fifth detection laser to obtain the sixth detection laser; the second filter 12223 is used to filter out the residual excitation pulses in the sixth detection laser to obtain the third detection laser; the third reflecting mirror 12224 is used to reflect the third detection laser to the detection element 1214.

[0150] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Fig.13 As shown, the second detection laser adjustment member 1221 includes a third lens 12211 and a fourth lens 12212; the third lens 12211 and the fourth lens 12212 are used to adjust the axial focal position of the first detection laser at the sample to be measured to obtain the second detection laser.

[0151] Among them, the optical axes of the third lens 12211 and the fourth lens 12212 are located in the same straight line and coincide with the first detection laser beam; the third lens 12211 and the fourth lens 12212 are used to adjust the axial focal position of the first detection laser at the sample to be tested to obtain the second detection laser to obtain the best thermal-optical effect detection signal.

[0152] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Fig.14As shown, the detection component 12 includes a Bessel beam detection component 123, and the Bessel beam detection component 123 includes a second detection laser beam splitter 1231, a first Bessel beam transformation component 1232, a third excitation component 1233, a fourth reflector 1234 and a detection component 1214; the second detection laser beam splitter 1231 is used to split the first detection laser to obtain a first reference arm detection laser and a first measuring arm detection laser; the first Bessel beam transformation component 1232 is used to transform the first measuring arm detection laser into a Bessel beam, and adjust the axial focus position of the first measuring arm detection laser at the sample to be measured to obtain the second measuring arm detection laser; the third excitation component 1233, It is used to focus the second measuring arm detection laser, the second excitation pulse and the third excitation pulse onto the sample to be measured, so that the sample to be measured produces a thermo-optical effect, and detects the thermo-optical effect based on the second measuring arm detection laser to obtain the third measuring arm detection laser; the fourth reflector 1234 is used to reflect the first reference arm detection laser, so that the first reference arm detection laser returns to the second detection laser beam splitter 1231 to obtain the second reference arm detection laser; the second detection laser beam splitter 1231 is also used to combine the third measuring arm detection laser and the second reference arm detection laser to obtain the combined detection laser; the detection element 1214 is used to determine the coherent Raman scattering spectrum of the sample to be measured based on the combined detection laser.

[0153] The second detection laser beam splitter 1231 is first used to split the first detection laser to obtain the first reference arm detection laser and the first measuring arm detection laser; the first Bessel beam conversion component 1232 is used to convert the first measuring arm detection laser into a Bessel beam. At the same time, the geometric structure of the first Bessel beam conversion component 1232 can also adjust the axial focus position of the measuring arm detection laser at the sample to be measured to obtain the best thermal-optical effect detection signal; then, the second dichroic mirror 114 in the scanning component 11 is used to combine the second excitation pulse, the third excitation pulse and the second measuring arm detection laser; the third excitation component 1233 is used to convert the second measuring arm detection laser into a Bessel beam. The detection laser, the second excitation pulse and the third excitation pulse are focused on the sample to be tested to generate a thermo-optical effect on the sample to be tested, and the thermo-optical effect is detected based on the second measuring arm detection laser to obtain the third measuring arm detection laser; the fourth reflector 1234 is used to reflect the first reference arm detection laser to return the first reference arm detection laser to the second detection laser beam splitter 1231 to obtain the second reference arm detection laser; the second detection laser beam splitter 1231 is also used to combine the third measuring arm detection laser and the second reference arm detection laser to obtain a combined detection laser; the detection element 1214 is used to determine the coherent Raman scattering spectrum of the sample to be tested based on the combined detection laser.

[0154] In the above embodiment, the first detection laser is divided into two paths, one path passes through a space with a constant optical path as a reference arm, and the other path is first transformed into a Bessel beam as a measuring arm to enter the sample and interact with the above-mentioned thermo-optical effect, that is, the optical path is modulated by the thermo-optical effect, and then transformed into the original beam form to interfere with the reference arm detection laser beam, and the modulation information of the thermo-optical effect is obtained by analyzing the interference signal. Thanks to the anti-interference, anti-scattering and strong penetration capabilities of the Bessel beam, the random phase noise introduced by the random scattering when the detection laser interacts with the sample to be measured will be greatly suppressed. This advantage is particularly significant when the sample to be measured is a thick sample with complex tissue components. In addition, using a Bessel beam detection laser of the same wavelength can further compress the size of the focal spot of the detection laser at the sample to be measured to improve the spatial resolution of the spectral microscopy system.

[0155] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Fig.15 As shown, the third excitation component 1233 includes a third objective lens 12331, a third collecting mirror 12332, a third filter 12333, a second Bessel beam transformation component 12334 and a fifth reflecting mirror 12335; the third objective lens 12331 is used to collect and tightly focus the second excitation pulse, the third excitation pulse and the second measuring arm detection laser onto the sample to be measured, and obtain the fourth excitation pulse, the fifth excitation pulse and the fourth measuring arm detection laser respectively, and the fourth excitation pulse and the fifth excitation pulse excite the coherent Raman oscillation of the sample to be measured, so that the sample to be measured produces a thermo-optical effect; the fourth measuring arm detection laser interacts with the thermo-optical effect to obtain the fifth measuring arm detection laser; the third collecting mirror 12332 is used to collect the fifth measuring arm detection laser to obtain the sixth measuring arm detection laser The measuring arm detection laser is a third filter 12333, which is used to filter out the residual excitation pulse in the sixth measuring arm detection laser to obtain the seventh measuring arm detection laser; the second Bessel beam transformation component 12334 is used to transform the seventh measuring arm detection laser into a Bessel beam to obtain the eighth measuring arm detection laser; it is also used to transform the eighth measuring arm detection laser to restore the eighth measuring arm detection laser to the original beam form to obtain the ninth measuring arm detection laser; the fifth reflector 12335 is used to retrace the ninth measuring arm detection laser so that the ninth measuring arm detection laser again passes through the mirror-symmetrical process experienced by the first measuring arm detection laser to the ninth measuring arm detection laser in turn, so as to obtain the third measuring arm detection laser before the second detection laser beam splitter 1231.

[0156] The third objective lens 12331 is used to collect and focus the second excitation pulse, the third excitation pulse and the second measuring arm detection laser onto the sample to be tested, and obtain the fourth excitation pulse, the fifth excitation pulse and the fourth measuring arm detection laser respectively. The fourth excitation pulse and the fifth excitation pulse excite the coherent Raman oscillation of the sample to be tested. The sample to be tested is a biochemical substance sample whose coherent Raman spectrum information is to be measured. The above-mentioned coherent Raman oscillation and the thermo-optical effect induced by the relaxation attenuation of the coherent Raman oscillation both occur here. The fourth measuring arm detection laser interacts with the thermo-optical effect to obtain the fifth measuring arm detection laser of the forward transmission of the optical path encoded by the thermo-optical effect. The third collecting mirror 12332 is used to collect the fifth measuring arm detection laser to obtain the sixth The measuring arm detection laser is provided with a third filter 12333, which is used to filter out the residual excitation pulse in the sixth measuring arm detection laser to obtain the seventh measuring arm detection laser; the second Bessel beam transformation component 12334 is used to first transform the seventh measuring arm detection laser into a Bessel beam to obtain the eighth measuring arm detection laser, and then transform the eighth measuring arm detection laser to restore it to the original beam form to obtain the ninth measuring arm detection laser; the fifth reflector 12335 is used to return the ninth measuring arm detection laser to the original path, so that the measuring arm detection laser again passes through the mirror-symmetrical process experienced by the first measuring arm detection laser to the ninth measuring arm detection laser in sequence, and finally obtains the third measuring arm detection laser in front of the second detection laser beam splitter 1231.

[0157] Since the measuring arm detection laser passes through the sample to be measured twice and interacts with the above-mentioned thermo-optical effect, the optical path modulation introduced thereby is expanded to twice that of a single path.

[0158] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Fig.16 As shown, the first Bessel beam transformation component 1232 includes a first conical lens 12321 and a fifth lens 12322; the first conical lens 12321 is used to transform the first measuring arm detection laser into a Bessel beam to obtain the middle measuring arm detection laser; the fifth lens 12322 is used to transform the middle measuring arm detection laser to form an annular line light source on the front focal plane of the third objective lens 12331 to obtain the second measuring arm detection laser.

[0159] Among them, the first conical lens 12321 is used to transform the first measuring arm detection laser into a Bessel beam to obtain the middle measuring arm detection laser, wherein the beam propagation direction of the first measuring arm detection laser is perpendicular to and facing the plane surface of the first conical lens 12321, and its beam center is to pass through the conical vertex of the first conical lens 12321. In addition, adjusting the front and rear positions of the first conical lens 12321 can adjust the axial focal position of the first measuring arm detection laser at the sample to be measured to obtain the best thermal-optical effect detection signal; the fifth lens 12322 is used to transform the middle measuring arm detection laser to form a ring-shaped line light source on the front focal plane of the third objective lens 12331 (which is also the back focal plane of the fifth lens 12322) to obtain the second measuring arm detection laser.

[0160] In some embodiments, the fifth lens 12322 and the third objective lens 12331 form a 4F system, which conjugates the middle measuring arm detection laser to the fourth measuring arm detection laser.

[0161] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Fig.17 As shown, the second Bessel beam transformation component 12334 includes a sixth lens 123341 and a second conical lens 123342; the sixth lens 123341 is used to transform the seventh measuring arm detection laser into a Bessel beam to obtain the eighth measuring arm detection laser; the second conical lens 123342 is used to transform the eighth measuring arm detection laser to restore it to the original beam shape to obtain the ninth measuring arm detection laser.

[0162] Among them, the sixth lens 123341 is used to transform the seventh measuring arm detection laser into a Bessel beam to obtain the eighth measuring arm detection laser; the second conical lens 123342 is used to transform the eighth measuring arm detection laser to restore it to the original beam shape to obtain the ninth measuring arm detection laser, wherein the beam propagation direction of the eighth measuring arm detection laser must be perpendicular to and facing the conical surface of the second conical lens 123342, and the center of its beam must pass through the conical vertex of the second conical lens 123342.

[0163] In some embodiments, the third collecting mirror 12332 and the sixth lens 123341 form a 4F system to conjugate the fifth measuring arm detection laser to the eighth measuring arm detection laser.

[0164] In some embodiments, the middle measuring arm detection laser, the fourth measuring arm detection laser, the fifth measuring arm detection laser and the eighth measuring arm detection laser are all Bessel beams.

[0165] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Fig.18As shown, the detection element 1214 includes a seventh lens 12141 and a first photodetector 12142; the seventh lens 12141 is used to focus the third detection laser or the combined detection laser on the detection target surface of the first photodetector;

[0166] The first photoelectric detector is used to detect the signal intensity of the third detection laser or the combined beam detection laser, and to analyze the coherent Raman scattering spectrum of the sample to be tested by Fourier transform.

[0167] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Fig.19 As shown, the light source assembly 10 includes a femtosecond pulse generating device 101, a femtosecond pulse splitter 102, a single-wavelength continuous laser generating device 103, a first beam splitter 104 and a first dichroic mirror 105; the femtosecond pulse generating device 101 is used to generate an original pulse; the femtosecond pulse splitter 102 is used to perform splitting and energy control processing on the original pulse to obtain a redundant pulse and a first excitation pulse; the single-wavelength continuous laser generating device 103 is used to generate an original laser; the first beam splitter 104 is used to split the original laser to obtain a first delayed detection laser and a first detection laser; the first dichroic mirror 105 is used to combine the first excitation pulse and the first delayed detection laser; wherein the first excitation pulse and the first delayed detection laser then enter the scanning assembly 11, and the first detection laser then enters the detection assembly 12.

[0168] Among them, Fig. 20 As shown, the femtosecond pulse spectrometer 102 includes a half-wave plate 1021, a polarization beam splitter 1022 and a light block 1023. The original pulse is a transformation-limited femtosecond pulse whose spectrum meets the requirements for exciting the Raman oscillation wave number to be measured; the original laser is a single-wavelength continuous laser

[0169] In the above embodiment, the absolute energy of the first excitation pulse can be controlled and adjusted through the combination of the half-wave plate, the polarization beam splitter and the light block.

[0170] The present application embodiment provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, such as Fig.21As shown, the scanning component 11 includes a second beam splitter 111, a first delay component 112, a second delay component 113, a second dichroic mirror 114 and a delay detection component 115; the second beam splitter 111 is used to replicate the first excitation pulse and the first delayed detection laser to obtain a first replicated excitation pulse, a second replicated excitation pulse, a first replicated delayed detection laser and a second replicated delayed detection laser; the first delay component 112 is used to reflect the first replicated excitation pulse and the first replicated delayed detection laser to obtain a second excitation pulse and a second delayed detection laser; the second delay component 113 is used to reflect the second replicated excitation pulse and the second replicated delayed detection laser The second beam splitter 111 is also used for beam combining the second excitation pulse and the third excitation pulse; it is also used for performing the same beam combining process on the second delayed detection laser and the third delayed detection laser; the second dichroic mirror 114 is used for separating the excitation pulse and the delayed detection laser, and is also used for beam combining the excitation pulse and the detection laser in the detection component 12; the delay detection component 115 is used for detecting the relative delay between the second excitation pulse and the third excitation pulse; the relative delay between the second delayed detection laser and the third delayed detection laser is used to characterize the relative delay between the second excitation pulse and the third excitation pulse.

[0171] The second beam splitter 111 is used to perform a replication process on the first excitation pulse and the first delayed detection laser to obtain a first replicated excitation pulse and a second replicated excitation pulse, and simultaneously obtain a first replicated delayed detection laser and a second replicated delayed detection laser; the first delay component 112 is used to perform a reflection process on the first replicated excitation pulse and the first replicated delayed detection laser to obtain a second excitation pulse and a second delayed detection laser; the second delay component 113 is used to perform a reflection and delay scanning process on the second replicated excitation pulse and the second replicated delayed detection laser to obtain a third excitation pulse and a third delayed detection laser; the second beam splitter 111 is used to perform a replication process on the first replicated excitation pulse and the first replicated delayed detection laser to obtain a first replicated excitation pulse and a second delayed detection laser; the second delay component 113 is used to perform a reflection and delay scanning process on the second replicated excitation pulse and the second replicated delayed detection laser to obtain a third excitation pulse and a third delayed detection laser; The beam splitter 111 is also used for combining the second excitation pulse and the third excitation pulse, and is also used for performing the same combining process on the second delayed detection laser and the third delayed detection laser; the second dichroic mirror 114 is used for separating the excitation pulse from the delayed detection laser, and is also used for combining the excitation pulse and the detection laser in the detection component 12; the delay detection component 115 is used for detecting the relative delay between the second delayed detection laser and the third delayed detection laser, thereby detecting the relative delay between the second excitation pulse and the third excitation pulse; wherein the second excitation pulse and the third excitation pulse subsequently enter the detection component 12.

[0172] In one embodiment, Fig. 22As shown, the second delay component 113 includes a sixth reflector 1131, a curved mirror 1132 and a resonant scanning mirror 1133; the second replica excitation pulse and the second replica delayed detection laser are respectively reflected by the sixth reflector 1131, the curved mirror 1132, the resonant scanning mirror 1133, the curved mirror 1132 and the sixth reflector 1131 to obtain a third excitation pulse and a third delayed detection laser and return to the second beam splitter 111.

[0173] In the above embodiment, the relative delay of the two groups of excitation pulses is constantly changing through the scanning of the resonant scanning mirror 1133; thus, a prerequisite is provided for scanning the interference state of the two groups of Raman oscillations Q1 and Q2 with relative delay, and Fourier transform spectrum detection can be performed.

[0174] In one embodiment, Fig.23 As shown, the delay detection component 115 includes an eighth lens 1151 and a second photodetector 1152; the eighth lens 1151 is used to focus the combined and interfered delay detection laser onto the detection target surface of the second photodetector 1152; the second photodetector 1152 is used to detect the interference signal intensity of the delay detection laser to resolve the relative delay amount.

[0175] In the embodiments of the present application, structural schematic diagrams of time-domain excitation coherent Raman photothermal spectroscopy microscopy detection systems are provided for the above three detection components. Fig.24 , which is a schematic diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system (backscatter detection); Fig.25 , which is a schematic diagram of the structure of a time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system (forward scattering detection); Fig.26 , which is a schematic diagram of the structure of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system (Bessel beam-Mach-Zehnder interferometry detection) in one embodiment.

[0176] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this application.

[0177] The above embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system, characterized in that: The system includes a light source component, a scanning component and a detection component; The light source assembly is used to generate a first excitation pulse, a first delayed detection laser and a first detection laser; The scanning component is used to perform a copy process and a delay process on the first excitation pulse to obtain a second excitation pulse and a third excitation pulse with relative delay, and use the first delay detection laser to monitor the relative delay between the second excitation pulse and the third excitation pulse; The detection component is used to use the second excitation pulse and the third excitation pulse to perform coherent Raman excitation on the sample to be tested so that the sample to be tested produces a thermo-optical effect; it is also used to adjust or transform the first detection laser to detect the thermo-optical effect, and determine the coherent Raman scattering spectrum of the sample to be tested based on the thermo-optical effect and the relative delay.

2. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to claim 1, characterized in that: The detection assembly includes a backscatter detection assembly, and the backscatter detection assembly includes a first detection laser adjustment component, a first detection laser beam splitter, a first excitation component and a detection component; The first detection laser adjustment member is used to adjust the axial focus position of the first detection laser at the sample to be measured to obtain a second detection laser; The first detection laser beam splitter is used to inject the second detection laser into the first excitation component; The first excitation component is used to focus the second excitation pulse and the third excitation pulse onto the sample to be tested, so that the sample to be tested generates the thermo-optical effect, and detect the thermo-optical effect based on the second detection laser to obtain the third detection laser; The detection element is used to determine the coherent Raman scattering spectrum of the sample to be tested based on the third detection laser.

3. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to claim 2, characterized in that: The first excitation assembly includes a first objective lens, a first collecting lens, a first filter and a first reflecting mirror; The first reflector is used to reflect the second detection laser so that the second detection laser passes through the first filter and enters the first collecting mirror; The first collecting mirror is used to collect and tightly focus the second detection laser onto the sample to be tested to obtain a fourth detection laser; The first objective lens is used to collect and tightly focus the second excitation pulse and the third excitation pulse onto the sample to be tested, respectively obtaining a fourth excitation pulse and a fifth excitation pulse, wherein the fourth excitation pulse and the fifth excitation pulse excite coherent Raman oscillation of the sample to be tested, so that the sample to be tested generates a thermo-optical effect; the fourth detection laser interacts with the thermo-optical effect to obtain a fifth detection laser; The first collecting mirror is further used to collect the fifth detection laser to obtain a sixth detection laser; The first filter is further used to filter out residual excitation pulses in the sixth detection laser to obtain the third detection laser; The first reflecting mirror is further used to reflect the third detection laser to the detection element.

4. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to claim 2, characterized in that: The first detection laser adjustment member includes a second reflector, a first lens, and a second lens; The second reflector is used to reflect the first detection laser to the first lens; the optical axes of the first lens and the second lens are located on the same straight line; The first lens and the second lens are used to adjust the axial focus position of the first detection laser at the sample to be measured to obtain the second detection laser.

5. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to claim 1, characterized in that: The detection assembly includes a forward scattering detection assembly, and the forward scattering detection assembly includes a second detection laser adjustment component, a second excitation component and a detection component; The second detection laser adjustment member is used to adjust the axial focus position of the first detection laser at the sample to be measured to obtain a second detection laser; The second excitation component is used to focus the second excitation pulse and the third excitation pulse onto the sample to be tested, so that the sample to be tested generates the thermo-optical effect, and detects the thermo-optical effect based on the second detection laser to obtain a third detection laser; The detection element is used to determine the coherent Raman scattering spectrum of the sample to be tested based on the third detection laser.

6. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to claim 5, characterized in that: The second excitation assembly includes a second objective lens, a second collecting lens, a second filter and a third reflecting mirror; The second objective lens is used to collect and tightly focus the second detection laser, the second excitation pulse and the third excitation pulse onto the sample to be tested, and obtain a fourth detection laser, a fourth excitation pulse and a fifth excitation pulse respectively. The fourth excitation pulse and the fifth excitation pulse excite the coherent Raman oscillation of the sample to be tested, so that the sample to be tested generates a thermo-optical effect; the fourth detection laser interacts with the thermo-optical effect to obtain a fifth detection laser; The second collecting mirror is used to collect the fifth detection laser to obtain a sixth detection laser; The second filter is used to filter out residual excitation pulses in the sixth detection laser to obtain the third detection laser; The third reflecting mirror is further used to reflect the third detection laser to the detection element.

7. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to claim 6, characterized in that: The second detection laser adjustment member includes a third lens and a fourth lens; The third lens and the fourth lens are used to adjust the axial focus position of the first detection laser at the sample to be measured to obtain the second detection laser.

8. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to claim 1, characterized in that: The detection assembly comprises a Bessel beam detection assembly, and the Bessel beam detection assembly comprises a second detection laser beam splitter, a first Bessel beam transformation assembly, a third excitation assembly, a fourth reflector and a detection element; The second detection laser beam splitter is used to split the first detection laser to obtain a first reference arm detection laser and a first measurement arm detection laser; The first Bessel beam conversion component is used to convert the first measuring arm detection laser into a Bessel beam, and adjust the axial focus position of the first measuring arm detection laser at the sample to be measured to obtain the second measuring arm detection laser; The third excitation component is used to focus the second measuring arm detection laser, the second excitation pulse and the third excitation pulse onto the sample to be measured, so that the sample to be measured generates the thermo-optical effect, and detects the thermo-optical effect based on the second measuring arm detection laser to obtain the third measuring arm detection laser; The fourth reflector is used to reflect the first reference arm detection laser so that the first reference arm detection laser returns to the second detection laser beam splitter to obtain the second reference arm detection laser; The second detection laser beam splitter is further used to combine the third measurement arm detection laser and the second reference arm detection laser to obtain a combined detection laser; The detection element is used to determine the coherent Raman scattering spectrum of the sample to be tested based on the combined beam detection laser.

9. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to claim 8, characterized in that: The third excitation component includes a third objective lens, a third collecting lens, a third filter, a second Bessel beam transformation component and a fifth reflecting mirror; The third objective lens is used to collect and tightly focus the second excitation pulse, the third excitation pulse and the second measuring arm detection laser onto the sample to be tested, and obtain a fourth excitation pulse, a fifth excitation pulse and a fourth measuring arm detection laser respectively. The fourth excitation pulse and the fifth excitation pulse excite the coherent Raman oscillation of the sample to be tested, so that the sample to be tested generates a thermo-optical effect; The fourth measuring arm detection laser interacts with the thermal-optical effect to obtain a fifth measuring arm detection laser; The third collecting mirror is used to collect the detection laser of the fifth measuring arm to obtain the detection laser of the sixth measuring arm; The third filter is used to filter out the residual excitation pulse in the sixth measuring arm detection laser to obtain the seventh measuring arm detection laser; The second Bessel beam transformation component is used to transform the seventh measuring arm detection laser into a Bessel beam to obtain an eighth measuring arm detection laser; and is also used to transform the eighth measuring arm detection laser to restore the eighth measuring arm detection laser to an original beam form to obtain a ninth measuring arm detection laser; The fifth reflector is used to retrace the ninth measuring arm detection laser along its original path, so that the ninth measuring arm detection laser again passes through the mirror-symmetrical process experienced by the first measuring arm detection laser to the ninth measuring arm detection laser in sequence, so as to obtain the third measuring arm detection laser before the second detection laser beam splitter.

10. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to claim 8, characterized in that: The first Bessel beam transformation assembly includes a first conical lens and a fifth lens; The first conical lens is used to transform the first measuring arm detection laser into a Bessel beam to obtain the intermediate measuring arm detection laser; The fifth lens is used to transform the intermediate measuring arm detection laser to form an annular line light source on the front focal plane of the third objective lens to obtain the second measuring arm detection laser.

11. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to claim 9, characterized in that: The second Bessel beam transformation assembly includes a sixth lens and a second conical lens; The sixth lens is used to transform the seventh measuring arm detection laser into a Bessel beam to obtain the eighth measuring arm detection laser; The second conical lens is used to transform the eighth measuring arm detection laser to restore it to the original light beam form to obtain the ninth measuring arm detection laser.

12. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to claim 2, 5 or 8, characterized in that: The detection element includes a seventh lens and a first photoelectric detector; The seventh lens is used to focus the third detection laser or the combined-beam detection laser onto the detection target surface of the first photodetector; The first photoelectric detector is used to detect the signal intensity of the third detection laser or the combined-beam detection laser, and to analyze the coherent Raman scattering spectrum of the sample to be tested by Fourier transform.

13. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to any one of claims 1 to 11, characterized in that: The light source assembly includes a femtosecond pulse generating device, a femtosecond pulse light splitting device, a single-wavelength continuous laser generating device, a first beam splitter and a first dichroic mirror; The femtosecond pulse generating device is used to generate original pulses; The femtosecond pulse spectrometer is used to perform spectrometry and energy control processing on the original pulse to obtain a redundant pulse and the first excitation pulse; The single-wavelength continuous laser generating device is used to generate original laser; The first beam splitter is used to split the original laser to obtain the first delayed detection laser and the first detection laser; The first dichroic mirror is used to combine the first excitation pulse and the first delayed detection laser; wherein the first excitation pulse and the first delayed detection laser then enter the scanning component, and the first detection laser then enters the detection component.

14. The time-domain excitation coherent Raman photothermal spectroscopy microscopy detection system according to any one of claims 1 to 11, characterized in that: The scanning assembly includes a second beam splitter, a first delay assembly, a second delay assembly, a second dichroic mirror and a delay detection assembly; The second beam splitter is used to perform replication processing on the first excitation pulse and the first delayed detection laser to obtain a first replicated excitation pulse, a second replicated excitation pulse, a first replicated delayed detection laser, and a second replicated delayed detection laser; The first delay component is used to reflect the first replica excitation pulse and the first replica delayed detection laser to obtain the second excitation pulse and the second delayed detection laser; The second delay component is used to reflect and delay scan the second replica excitation pulse and the second replica delayed detection laser to obtain the third excitation pulse and the third delayed detection laser; The second beam splitter is further used to perform beam combining processing on the second excitation pulse and the third excitation pulse; and is also used to perform the same beam combining processing on the second delayed detection laser and the third delayed detection laser; The second dichroic mirror is used to separate the excitation pulse and the delayed detection laser, and is also used to combine the excitation pulse and the detection laser in the detection component; The delay detection component is used to detect the relative delay between the second excitation pulse and the third excitation pulse; The relative delay between the second delayed detection laser and the third delayed detection laser is used to characterize the relative delay between the second excitation pulse and the third excitation pulse.

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