Time-domain excitation coherent raman photothermal spectroscopy microscopic probing system

Abstract

The application relates to a time-domain excitation coherent Raman photothermal spectrum microscopic detection system, which comprises a light source assembly, a scanning assembly and a detection assembly. The light source assembly is used for generating a first excitation pulse, a first delay detection laser and a first detection laser. The scanning assembly is used for carrying out copying and delay processing on the first excitation pulse to obtain a second excitation pulse and a third excitation pulse with a relative delay, and the relative delay amount between the second excitation pulse and the third excitation pulse is monitored by using the first delay detection laser. The detection assembly is used for carrying out coherent Raman excitation on a sample to be measured by using the second excitation pulse and the third excitation pulse, so that the sample to be measured generates a thermal light effect. The detection assembly is also used for adjusting or transforming the first detection laser to detect the thermal light effect, and determining a coherent Raman scattering spectrum of the sample to be measured based on the thermal light effect and the relative delay amount. The system can improve the measurement accuracy and the spectral resolution of the coherent Raman spectrum.

Description

TECHNICAL FIELD

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

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

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

[0004] However, this method can introduce non-resonant induced exothermic effect (for example, relaxation decay exothermic of two-photon absorption), and the spectral resolution is limited (the linewidth of the natural broadening limit cannot be measured), thereby affecting the measurement of coherent Raman scattering spectrum. SUMMARY

[0005] Therefore, it is necessary to provide a time-domain excitation coherent Raman photothermal spectral microscopic detection system capable of improving the accuracy and spectral resolution of coherent Raman photothermal spectral measurement in view of the above technical problems.

[0006] In a first aspect, the present application provides a time-domain excitation coherent Raman photothermal spectral microscopic detection system, which comprises a light source assembly, a scanning assembly and a detection assembly.

[0007] The light source assembly is configured to generate a first excitation pulse, a first delay detection laser and a first detection laser.

[0008] The scanning assembly is configured to perform copying and delay processing on the first excitation pulse to obtain a second excitation pulse and a third excitation pulse with a relative delay, and to monitor the relative delay amount between the second excitation pulse and the third excitation pulse by using the first delay detection laser.

[0009] The detection assembly is configured to perform coherent Raman excitation on the sample to be measured by using the second excitation pulse and the third excitation pulse, so as to cause the sample to be measured to generate a thermal light effect; and is further configured to adjust or transform the first detection laser to detect the thermal light effect, and to determine the coherent Raman scattering spectrum of the sample to be measured based on the thermal light effect and the relative delay amount.

[0010] In one embodiment, the detection assembly comprises a backscattering detection assembly, which comprises a first detection laser adjusting member, a first detection laser beam splitting member, a first excitation assembly and a detection member.

[0011] The first detection laser adjusting member is configured to adjust the axial focal point position of the first detection laser at the sample to be measured to obtain a second detection laser.

[0012] a first probe laser beam splitting element, configured to direct the second probe laser beam to the first excitation assembly;

[0013] a first excitation assembly, configured to focus the second excitation pulse and the third excitation pulse to the sample to be measured, so that the sample to be measured generates a thermal light effect, and the second probe laser beam is used to probe the thermal light effect to obtain a third probe laser beam;

[0014] a detection element, configured to determine the coherent Raman scattering spectrum of the sample to be measured based on the third probe laser beam.

[0015] In one of the embodiments, the first excitation assembly comprises a first objective lens, a first collection lens, a first filter and a first mirror;

[0016] the first mirror is configured to reflect the second probe laser beam so that the second probe laser beam passes through the first filter and enters the first collection lens;

[0017] the first collection lens is configured to collect and tightly focus the second probe laser beam on the sample to be measured to obtain a fourth probe laser beam;

[0018] the first objective lens is configured to collect and tightly focus the second excitation pulse and the third excitation pulse on the sample to be measured to obtain 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 measured, so that the sample to be measured generates a thermal light effect; the fourth probe laser beam interacts with the thermal light effect to obtain a fifth probe laser beam;

[0019] the first collection lens is further configured to collect the fifth probe laser beam to obtain a sixth probe laser beam;

[0020] the first filter is further configured to filter out the residual excitation pulse in the sixth probe laser beam to obtain the third probe laser beam;

[0021] the first mirror is further configured to reflect the third probe laser beam to the detection element.

[0022] In one of the embodiments, the first probe laser beam adjusting element comprises a second mirror, a first lens and a second lens;

[0023] the second mirror is configured to reflect the first probe laser beam 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 configured to adjust the axial focal point position of the first probe laser beam at the sample to be measured to obtain the second probe laser beam.

[0025] In one of the embodiments, the detection assembly comprises a forward scattering detection assembly, and the forward scattering detection assembly comprises a second probe laser beam adjusting element, a second excitation assembly and a detection element;

[0026] a second probe laser adjusting component configured to adjust an axial focal position of the first probe laser at the sample under test to obtain a second probe laser;

[0027] a second excitation component configured to focus the second excitation pulse and the third excitation pulse to the sample under test to cause the sample under test to generate thermal light effect, and detect the thermal light effect based on the second probe laser to obtain a third probe laser;

[0028] a detection component configured to determine the coherent Raman scattering spectrum of the sample under test based on the third probe laser.

[0029] In one embodiment, the second excitation component comprises a second objective lens, a second collection lens, a second filter and a third mirror.

[0030] the second objective lens is configured to collect and tightly focus the second probe laser, the second excitation pulse and the third excitation pulse to the sample under test to obtain a fourth probe laser, a fourth excitation pulse and a fifth excitation pulse respectively, the fourth excitation pulse and the fifth excitation pulse excite coherent Raman oscillation of the sample under test to cause the sample under test to generate thermal light effect, and the fourth probe laser interacts with the thermal light effect to obtain a fifth probe laser;

[0031] the second collection lens is configured to collect the fifth probe laser to obtain a sixth probe laser;

[0032] the second filter is configured to filter out residual excitation pulses in the sixth probe laser to obtain the third probe laser;

[0033] the third mirror is further configured to reflect the third probe laser to the detection component.

[0034] In one embodiment, the second probe laser adjusting component comprises a third lens and a fourth lens.

[0035] the third lens and the fourth lens are configured to adjust the axial focal position of the first probe laser at the sample under test to obtain the second probe laser.

[0036] In one embodiment, the detection component comprises a Bessel beam detection component, and the Bessel beam detection component comprises a second probe laser splitting component, a first Bessel beam transforming component, a third excitation component, a fourth mirror and the detection component.

[0037] the second probe laser splitting component is configured to split the first probe laser to obtain a first reference arm probe laser and a first measurement arm probe laser;

[0038] the first Bessel beam transforming component is configured to transform the first measurement arm probe laser into a Bessel beam, and adjust an axial focal position of the first measurement arm probe laser at the sample under test to obtain a second measurement arm probe laser;

[0039] a third excitation component configured to focus the second measurement arm probe laser, the second excitation pulse and the third excitation pulse to the sample to be measured to cause the sample to be measured to generate a thermal light effect, and to detect the thermal light effect based on the second measurement arm probe laser to obtain a third measurement arm probe laser;

[0040] a fourth mirror configured to reflect the first reference arm probe laser to return the first reference arm probe laser to the second probe laser beam splitting component to obtain a second reference arm probe laser;

[0041] the second probe laser beam splitting component is further configured to combine the third measurement arm probe laser and the second reference arm probe laser to obtain a combined probe laser;

[0042] a detection component configured to determine a coherent Raman scattering spectrum of the sample to be measured based on the combined probe laser.

[0043] In one embodiment, the third excitation component comprises a third objective lens, a third collection lens, a third filter, a second Bessel beam transformation component and a fifth mirror;

[0044] the third objective lens is configured to collect and tightly focus the second excitation pulse, the third excitation pulse and the second measurement arm probe laser to the sample to be measured to obtain a fourth excitation pulse, a fifth excitation pulse and a fourth measurement arm probe laser respectively, the fourth excitation pulse and the fifth excitation pulse excite coherent Raman oscillation of the sample to be measured to cause the sample to be measured to generate a thermal light effect; the fourth measurement arm probe laser interacts with the thermal light effect to obtain a fifth measurement arm probe laser;

[0045] the third collection lens is configured to collect the fifth measurement arm probe laser to obtain a sixth measurement arm probe laser;

[0046] the third filter is configured to filter out residual excitation pulses in the sixth measurement arm probe laser to obtain a seventh measurement arm probe laser;

[0047] the second Bessel beam transformation component is configured to transform the seventh measurement arm probe laser into a Bessel beam to obtain an eighth measurement arm probe laser, and is further configured to transform the eighth measurement arm probe laser to restore the eighth measurement arm probe laser to an original beam shape to obtain a ninth measurement arm probe laser;

[0048] the fifth mirror is configured to fold back the ninth measurement arm probe laser to the original path, so that the ninth measurement arm probe laser again undergoes a mirror-symmetrical process of the first measurement arm probe laser to the ninth measurement arm probe laser to obtain the third measurement arm probe laser in front of the second probe laser beam splitting component.

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

[0050] a first conical lens for transforming the first measurement arm probe laser into a Bessel beam to obtain an intermediate measurement arm probe laser;

[0051] a fifth lens for transforming the intermediate measurement arm probe laser to form a ring-shaped line light source on the front focal plane of the third objective lens to obtain a second measurement arm probe laser.

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

[0053] a sixth lens for transforming the seventh measurement arm probe laser into a Bessel beam to obtain an eighth measurement arm probe laser;

[0054] a second conical lens for transforming the eighth measurement arm probe laser to restore it to the original beam shape to obtain a ninth measurement arm probe laser.

[0055] In one embodiment, the probe assembly comprises a seventh lens and a first photodetector;

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

[0057] a first photodetector for detecting the signal intensity of the third probe laser or the combined probe laser and using Fourier transform to analyze the coherent Raman scattering spectrum of the sample to be measured.

[0058] In one embodiment, the light source assembly comprises 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] the femtosecond pulse generating device is configured to generate an original pulse;

[0060] the femtosecond pulse splitting device is configured to split and control the energy of the original pulse to obtain a redundant pulse and a first excitation pulse;

[0061] the single-wavelength continuous laser generating device is configured to generate an original laser;

[0062] the first beam splitter is configured to split the original laser to obtain a first delay probe laser and a first probe laser;

[0063] the first dichroic mirror is configured to combine the first excitation pulse and the first delay probe laser; wherein the first excitation pulse and the first delay probe laser subsequently enter the scanning assembly, and the first probe laser subsequently enters the probe assembly.

[0064] In one of the embodiments, the scanning assembly comprises a second beam splitter, a first time delay assembly, a second time delay assembly, a second dichroic mirror and a time delay detection assembly.

[0065] The second beam splitter is configured to replicate the first excitation pulse and the first time delay probe laser to obtain a first replicated excitation pulse, a second replicated excitation pulse, a first replicated time delay probe laser and a second replicated time delay probe laser.

[0066] The first time delay assembly is configured to reflect the first replicated excitation pulse and the first replicated time delay probe laser to obtain the second excitation pulse and the second time delay probe laser.

[0067] The second time delay assembly is configured to reflect and time delay scan the second replicated excitation pulse and the second replicated time delay probe laser to obtain a third excitation pulse and a third time delay probe laser.

[0068] The second beam splitter is further configured to combine the second excitation pulse and the third excitation pulse; and further configured to combine the second time delay probe laser and the third time delay probe laser.

[0069] The second dichroic mirror is configured to separate the excitation pulse and the time delay probe laser, and further configured to combine the excitation pulse and the probe laser in the detection assembly.

[0070] The time delay detection assembly is configured to detect the relative time delay between the second excitation pulse and the third excitation pulse; and the relative time delay between the second time delay probe laser and the third time delay probe laser is used to represent the relative time delay between the second excitation pulse and the third excitation pulse.

[0071] The time-domain excitation coherent Raman photothermal spectrum microscopic detection 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, a first delay detection laser and a first detection laser. The scanning assembly is used for copying and delaying the first excitation pulse to obtain a second excitation pulse and a third excitation pulse with a 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 coherently exciting the sample to be measured by using the second excitation pulse and the third excitation pulse to make the sample to be measured generate a thermal light effect, and is also used for adjusting or transforming the first detection laser to detect the thermal light effect, and determining the coherent Raman scattering spectrum of the sample to be measured based on the thermal light effect and the relative delay amount. Since the system combines time-domain excitation and photothermal detection to coherently excite, collect and detect the sample to be measured, the coherent Raman spectrum information of the biochemical substance in the sample to be measured is obtained. Therefore, the non-resonant 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, so that the accuracy and spectral resolution of the coherent Raman photothermal spectrum measurement are improved while the high sensitivity and spatial resolution are ensured. BRIEF DESCRIPTION OF DRAWINGS

[0072] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the related art, the drawings needed to be used in the description of the embodiments of the present application or the related art will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other related drawings can be obtained by those skilled in the art without creative labor.

[0073] Figure 1 A frequency-domain schematic diagram of a variable-transform limit femtosecond pulse excitation coherent Raman oscillation in an embodiment;

[0074] Figure 2 A time-domain schematic diagram of a single variable-transform limit femtosecond pulse excitation coherent Raman oscillation relaxation decay inducing a thermal light effect in an embodiment;

[0075] Figure 3 A time-domain schematic diagram of a time-domain excitation coherent Raman photothermal spectrum microscopic detection system in an embodiment;

[0076] Figure 4 A schematic diagram of a backscattering detection laser detecting a thermal light effect in an embodiment;

[0077] Figure 5 A schematic diagram of a forward scattering detection laser detecting a thermal light effect in an embodiment;

[0078] Figure 6 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0079] Figure 7 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0080] Figure 8 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0081] Figure 9 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0082] Figure 10 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0083] Figure 11 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0084] Figure 12 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0085] Figure 13 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0086] Figure 14 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0087] Figure 15 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0088] Figure 16 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0089] Figure 17 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0090] Figure 18 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0091] Figure 19 Schematic diagram of a system for detecting thermal light effect by using a Bessel beam-Mach-Zehnder interferometer in an embodiment;

[0092] Figure 20 Structure diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0093] Figure 21 Structure diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0094] Figure 22 Structure diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0095] Figure 23 Structure diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0096] Figure 24 Structure diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0097] Figure 25 Structure diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0098] Figure 26 Structure diagram of a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system in another embodiment;

[0099] Explanation of reference numerals:

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

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

[0102] Single-wavelength continuous laser generating device 103, first beam splitter 104, first dichroic mirror 105;

[0103] Scanning assembly 11, second beam splitter 111, first time-delay assembly 112;

[0104] Second time-delay assembly 113, sixth mirror 1131, curved mirror 1132;

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

[0106] Eighth lens 1151, second photodetector 1152, detection assembly 12;

[0107] Backscattering detection assembly 121, first detection laser adjusting member 1211;

[0108] The second mirror 12111, the first lens 12112, the second lens 12113;

[0109] The first probe laser beam splitter 1212, the first excitation component 1213;

[0110] The first objective lens 12131, the first collection mirror 12132, the first filter 12133;

[0111] The first mirror 12134, the forward scattering probe component 122;

[0112] The second probe laser beam splitter 1221, the third lens 12211, the fourth lens 12212;

[0113] The second excitation component 1222, the second objective lens 12221, the second collection mirror 12222;

[0114] The second filter 12223, the third mirror 12224, the Bessel beam probe component 123;

[0115] The second probe laser beam splitter 1231, the first Bessel beam transformation component 1232;

[0116] The first conical lens 12321, the fifth lens 12322, the third excitation component 1233;

[0117] The third objective lens 12331, the third collection mirror 12332, the third filter 12333;

[0118] The second Bessel beam transformation component 12334, the sixth lens 123341;

[0119] The second conical lens 123342, the fifth mirror 12335, the fourth mirror 1234;

[0120] The probe component 1214, the seventh lens 12141, the first photodetector 12142, the sample to be measured 13. DETAILED DESCRIPTION

[0121] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the 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 spectral 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, material science and the like. In the traditional technology, the coherent Raman photothermal microscopic detection technology can be realized based on spectral focusing and photothermal microscopic imaging.

[0123] However, the above coherent Raman photothermal spectroscopy technology is based on the spectral focusing excitation strategy to scan and obtain broadband spectrum, which has the following technical limitations. 1) It is difficult to obtain high spectral resolution by spectral focusing, even if the excitation light is strictly equal linear chirp without high-order dispersion, the Raman linewidth of the natural broadening limit cannot be measured; 2) The spectral focusing excitation mode may introduce non-resonant induced heat release effect (such as two-photon absorption relaxation decay heat release), which interferes with the measured spectral signal.

[0124] Therefore, it is necessary to provide a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system to improve the spectral resolution (theoretically, the Raman peak width of the natural broadening limit can be measured), and to avoid the influence of non-resonant induced heat release effect, and in one embodiment, based on the principle of Bessel beam-Mach-Zehnder interference, a new thermo-optic 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 microscopic system.

[0125] Firstly, the technical principle of the time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system involved in the embodiments of the present application is described. The embodiments of the present application use transform-limited femtosecond pulse to excite the coherent Raman oscillation of the sample to be measured, and the frequency domain principle diagram is as shown in Figure 1 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 the coherent Raman oscillation.

[0126] In the time domain, as shown in (a) of Figure 2 After a single excitation pulse is incident on the sample to be measured, the sample molecules are excited to produce Raman oscillation Q1 (assuming the oscillation angular frequency is Ω0), and the intensity of Q1 will relax and decay exponentially with time (the decay constant is about picosecond) after being excited, while releasing heat. As shown in (b) of Figure 2 (b), where r represents the distance from the center of the excitation pulse focus, the heat will cause the local temperature distribution near the excitation pulse focus to rise, thereby triggering the change of the local refractive index distribution, i.e. 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 microscopic detection system is as shown in Figure 3 The fourth excitation pulse and the fifth excitation pulse after time delay τ are incident on the sample to be measured in turn to excite the coherent Raman oscillation Q1 and Q2 respectively, Q1 and Q2 interfere after the fifth excitation pulse excites Q2, the coherent Raman oscillation of the molecules continues in the form of Q1+Q2, and the relaxation decay and heat release of the coherent Raman oscillation are always performed at the same time. Finally, the size of the released heat depends on the constructive or destructive interference of Q1+Q2, i.e. depends on the relative time delay τ between the fourth excitation pulse and the fifth excitation pulse. By scanning the relative time delay τ,Figure 3 (c) as shown in (c) of FIG. 1, the heat release curve modulated by the Raman oscillation information to be detected can be obtained. Meanwhile, as shown in (d, e) of FIG. 1, the thermal-optic effect near the focal point of the excitation pulse is also encoded by the heat release curve, and finally carries the Raman oscillation information to be detected along with the scanning of the relative delay τ. The thermal-optic effect encoded by the Raman oscillation information to be detected is detected by using the probe laser, and the Raman oscillation information to be detected can be demodulated by Fourier transform, and the broadband Raman spectrum can be obtained. Figure 3

[0127] For the detection of the above thermal-optic effect, three schemes are provided in the embodiments of the present application.

[0128] First, as shown in FIG. 2, a backscattering detection method is adopted. That is, the incident probe laser interacts with the above thermal-optic effect to generate backscattering probe laser. Since the above thermal-optic effect carries the Raman oscillation information to be detected along with the scanning of the relative delay τ, the backscattering probe laser also carries the information. The relative intensity change of the backscattering probe laser is detected, and Fourier transform is performed on the change, and the broadband Raman spectrum can be obtained. It should be noted that the probe laser can be a continuous laser with a shorter wavelength than the excitation pulse, so as to obtain a smaller diffraction-limited spot and improve the spatial resolution. Figure 4 Second, as shown in FIG. 3, a forward scattering detection method is adopted. That is, the incident probe laser interacts with the above thermal-optic effect to generate forward scattering probe laser, and the subsequent detection principle is the same as that of the backscattering detection method, which will not be described herein.

[0129] Figure 5 Third, as shown in FIG. 4, a new Bessel beam-Mach-Zehnder interference method is provided by the present application for the detection of the above thermal-optic effect. The probe laser is first divided into reference arm probe laser and measurement arm probe laser. The reference arm probe laser passes through a space with a fixed optical path, and the measurement arm probe laser is converted into a Bessel beam by a first Bessel beam transformation assembly and an objective lens, and then is incident on the sample to be detected and interacts with the above thermal-optic effect. At this time, the optical path of the measurement arm probe laser is modulated and encoded by the Raman oscillation information to be detected. The measurement arm probe laser is then restored to the original beam shape by a collecting mirror and a second Bessel beam transformation assembly, and is folded back through a mirror, and then passes through the above mirror-symmetric process of beam transformation-interaction with the thermal-optic effect-beam transformation again. Because the measurement arm probe laser interacts with the above thermal-optic effect twice, the optical path modulation introduced thereby is twice the single-path. Finally, the reference arm probe laser and the measurement arm probe laser are combined again to interfere, the interference signal is detected, and Fourier transform is performed on the signal, and the broadband Raman spectrum can be obtained.

[0130] The above three schemes will be described in detail below. Figure 6

[0131] ​​​​

[0132] The embodiment of the present application provides a time-domain excitation coherent Raman photothermal spectrum microscopic detection system, as shown in the figure, the system comprises a light source assembly 10, a scanning assembly 11 and a detection assembly 12; the light source assembly 10 is used for generating a first excitation pulse, a first delay detection laser and a first detection laser; the scanning assembly 11 is used for carrying out copying processing and delay processing on the first excitation pulse, obtaining a second excitation pulse and a third excitation pulse with a 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 12 is used for carrying out coherent Raman excitation on a to-be-detected sample by using the second excitation pulse and the third excitation pulse, so that the to-be-detected sample generates a thermal light effect; and is also used for adjusting or transforming the first detection laser to detect the thermal light effect, and determining a coherent Raman scattering spectrum of the to-be-detected sample based on the thermal light effect and the relative delay amount. Figure 7 The light source assembly 10 is used for generating a first excitation pulse, a first delay detection laser and a first detection laser, wherein the first excitation pulse is a transform-limited femtosecond pulse with adjustable absolute energy and its spectrum meets the requirement of exciting a to-be-detected Raman wave number, the first delay detection laser is a single-wavelength continuous laser used to detect and monitor the relative delay between excitation pulses, and the first detection laser is a single-wavelength continuous laser used to detect a thermal light effect induced by coherent Raman oscillation relaxation decay of the to-be-detected sample; the first excitation pulse and the first delay detection laser enter the scanning assembly 11 after being combined, and the first detection laser enters the detection assembly 12.

[0133] The scanning assembly 11 is used for carrying out copying and delay scanning on the first excitation pulse and the first delay detection laser, obtaining a second excitation pulse and a third excitation pulse with a relative delay, and a second delay detection laser and a third delay detection laser with a completely consistent relative delay; by measuring an interference signal of the second delay detection laser and the third delay detection laser through a single-frequency laser interference principle, the relative delay amount between the second excitation pulse and the third excitation pulse can be monitored in real time. The detection assembly 12 is used for carrying out coherent Raman excitation on the to-be-detected sample by using the second excitation pulse and the third excitation pulse, and the thermal effect of the to-be-detected sample is induced by coherent Raman oscillation relaxation decay, and then the thermal light effect is generated; after the first detection laser is adjusted or transformed, the thermal light effect is detected, and the detection result of the to-be-detected sample is determined. The detection result is intensity information of the thermal light effect, and the intensity information of the thermal light effect can reflect coherent Raman scattering spectrum information.

[0134] The detection assembly 12 is used for carrying out coherent Raman excitation on the to-be-detected sample by using the second excitation pulse and the third excitation pulse, and the thermal effect of the to-be-detected sample is induced by coherent Raman oscillation relaxation decay, and then the thermal light effect is generated; after the first detection laser is adjusted or transformed, the thermal light effect is detected, and the detection result of the to-be-detected sample is determined. The detection result is intensity information of the thermal light effect, and the intensity information of the thermal light effect can reflect coherent Raman scattering spectrum information. The detection assembly 12 is used for carrying out coherent Raman excitation on the to-be-detected sample by using the second excitation pulse and the third excitation pulse, and the thermal effect of the to-be-detected sample is induced by coherent Raman oscillation relaxation decay, and then the thermal light effect is generated; after the first detection laser is adjusted or transformed, the thermal light effect is detected, and the detection result of the to-be-detected sample is determined. The detection result is intensity information of the thermal light effect, and the intensity information of the thermal light effect can reflect coherent Raman scattering spectrum information.

[0135] The detection assembly 12 is used for carrying out coherent Raman excitation on the to-be-detected sample by using the second excitation pulse and the third excitation pulse, and the thermal effect of the to-be-detected sample is induced by coherent Raman oscillation relaxation decay, and then the thermal light effect is generated; after the first detection laser is adjusted or transformed, the thermal light effect is detected, and the detection result of the to-be-detected sample is determined. The detection result is intensity information of the thermal light effect, and the intensity information of the thermal light effect can reflect coherent Raman scattering spectrum information. The detection assembly 12 is used for carrying out coherent Raman excitation on the to-be-detected sample by using the second excitation pulse and the third excitation pulse, and the thermal effect of the to-be-detected sample is induced by coherent Raman oscillation relaxation decay, and then the thermal light effect is generated; after the first detection laser is adjusted or transformed, the thermal light effect is detected, and the detection result of the to-be-detected sample is determined. The detection result is intensity information of the thermal light effect, and the intensity information of the thermal light effect can reflect coherent Raman scattering spectrum information.

[0136] In the above embodiments, the system includes a light source assembly 10, a scanning assembly 11, and a detection assembly 12. The light source assembly 10 is used to generate a first excitation pulse, a first delayed detection laser, and a first detection laser. The scanning assembly 11 is used to replicate and delay the first excitation pulse to obtain a second and third excitation pulse with relative delays, and to monitor the relative delay between the second and third excitation pulses using the first delayed detection laser. The detection assembly 12 is used to coherently Raman excite the sample under test using the second and third excitation pulses to induce a thermo-optical effect in the sample. 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 under test based on the thermo-optical effect and the relative delay. Because the system uses a combination of time-domain excitation and photothermal detection to coherently Raman excite, collect, and detect the sample under test, thereby obtaining the coherent Raman spectral information of biochemical substances in the sample under test, the non-resonance-induced exothermic effect can be suppressed by removing data near zero delay, thus improving the accuracy of coherent Raman scattering spectroscopy measurement. Furthermore, thanks to the characteristics of time-domain detection, this method can completely scan the relaxation decay curve of Raman oscillations in the time domain, thus theoretically enabling the measurement of the Raman peak in the natural broadening limit, thereby improving spectral resolution. Simultaneously, this technique incorporates the advantages of photothermal detection, ensuring high detection sensitivity and spatial resolution.

[0137] This application provides a time-domain excited coherent Raman photothermal spectroscopy microscopy detection system, such as... Figure 8 As shown, the detection component 12 includes a backscattering detection component 121, which includes a first detection laser adjustment component 1211, a first detection laser beam splitter 1212, a first excitation component 1213, and a detector 1214. The first detection laser adjustment component 1211 is used to adjust the axial focal 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 incident the second detection laser onto the first excitation component 1213. The first excitation component 1213 is used to focus the second excitation pulse and the third excitation pulse onto the sample to be tested to generate a thermo-optical effect, and to detect the thermo-optical effect based on the second detection laser to obtain a third detection laser. The detector 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 component 1211 adjusts the axial focal position of the first detection laser at the sample to be tested, thereby obtaining the second detection laser to obtain the optimal thermo-optical effect detection signal.

[0139] The first probe laser beam splitter 1212 is configured to direct the second probe laser beam to the first excitation assembly 1213. The first excitation assembly 1213 is configured to focus the second excitation pulse and the third excitation pulse to the sample to be measured, so that the sample to be measured generates thermal light effect, and the second probe laser beam is used to probe the thermal light effect to obtain the third probe laser beam. Then, the probe assembly 1214 is configured to determine the coherent Raman scattering spectrum of the sample to be measured based on the third probe laser beam.

[0140] The embodiment of the present application provides a time-domain excitation coherent Raman photothermal spectrum microscopic detection system, as shown in the figure. Figure 9 The first excitation assembly 1213 includes a first objective lens 12131, a first collection lens 12132, a first filter 12133 and a first mirror 12134. The first mirror 12134 is configured to reflect the second probe laser beam, so that the second probe laser beam passes through the first filter 12133 and enters the first collection lens 12132. The first collection lens 12132 is configured to collect and tightly focus the second probe laser beam on the sample to be measured to obtain the fourth probe laser beam. The first objective lens 12131 is configured to collect and tightly focus the second excitation pulse and the third excitation pulse on the sample to be measured to obtain 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 measured, so that the sample to be measured generates thermal light effect. The fourth probe laser beam interacts with the thermal light effect to obtain the fifth probe laser beam. The first collection lens 12132 is further configured to collect the fifth probe laser beam to obtain the sixth probe laser beam. The first filter 12133 is further configured to filter out the residual excitation pulse in the sixth probe laser beam to obtain the third probe laser beam. The first mirror 12134 is further configured to reflect the third probe laser beam to the probe assembly 1214.

[0141] The first probe laser beam splitter 1212 is used to reflect the second probe laser beam to the first mirror 12134; the first mirror 12134 is used to reflect the second probe laser beam reflected by the first probe laser beam splitter 1212 to pass through the first filter 12133 to the first collection lens 12132; the first collection lens 12132 is used to collect and tightly focus the second probe laser beam on the sample to be measured to obtain the fourth probe laser beam; the first objective lens 12131 is used to collect and tightly focus the second excitation pulse and the third excitation pulse on the sample to be measured 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 measured; the sample to be measured is a biochemical sample to be measured for coherent Raman spectrum information, and the coherent Raman oscillation and the thermal-optic effect induced by the relaxation and attenuation of the coherent Raman oscillation both occur here; the fourth probe laser beam interacts with the thermal-optic effect to obtain the backscattered fifth probe laser beam encoded by the thermal-optic effect; the first collection lens 12132 is also used to collect the fifth probe laser beam to obtain the sixth probe laser beam; the first filter 12133 is used to filter out the residual excitation pulse in the sixth probe laser beam to obtain the third probe laser beam; the first mirror 12134 is also used to reflect the third probe laser beam to the first probe laser beam splitter 1212; then, the first probe laser beam splitter 1212 is also used to transmit the third probe laser beam reflected by the first mirror 12134, and the third probe laser beam is subsequently incident on the probe assembly 12.

[0142] The embodiment of the present application provides a time-domain excitation coherent Raman photothermal spectrum microscopic detection system, as shown in the figure. Figure 10 The first probe laser adjusting component 1211 comprises a second mirror 12111, a first lens 12112 and a second lens 12113; the second mirror 12111 is used to reflect the first probe laser beam to the first lens 12112; the optical axes of the first lens 12112 and the second lens 12113 are located on the same straight line; the first lens 12112 and the second lens 12113 are used to adjust the axial focal point position of the first probe laser beam at the sample to be measured to obtain the second probe laser beam.

[0143] The second mirror 12111 is used to reflect the first probe laser beam to the first lens 12112; the optical axes of the first lens 12112 and the second lens 12113 are located on the same straight line and coincide with the first probe laser beam; the first lens 12112 and the second lens 12113 are used to adjust the axial focal point position of the first probe laser beam at the sample to be measured to obtain the second probe laser beam, so that the best thermal-optic effect detection signal can be obtained.

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

[0145] The embodiment of the present application provides a time-domain excitation coherent Raman photothermal spectroscopy microscopic detection system, as shown in Figure 11 The detection component 12 includes a forward scattering detection component 122, the forward scattering detection component 122 includes a second detection laser adjusting part 1221, a second excitation component 1222 and a detection part 1214; the second detection laser adjusting part 1221 is used for adjusting the axial focal point position of the first detection laser at the sample to be detected, and obtaining the second detection laser; the second excitation component 1222 is used for focusing the second excitation pulse and the third excitation pulse to the sample to be detected, so that the sample to be detected generates a thermal light effect, and the thermal light effect is detected based on the second detection laser to obtain the third detection laser; and the detection part 1214 is used for determining the coherent Raman scattering spectrum of the sample to be detected based on the third detection laser.

[0146] The second detection laser adjusting part 1221 is used for adjusting the axial focal point position of the first detection laser at the sample to be detected, and obtaining the second detection laser, so that the best thermal light effect detection signal is obtained; then, the second dichroic mirror 114 in the scanning component 11 is used for combining the second excitation pulse, the third excitation pulse and the second detection laser; the second excitation component 1222 is used for focusing the second excitation pulse and the third excitation pulse to the sample to be detected, so that the sample to be detected generates a thermal light effect, and the thermal light effect is detected based on the second detection laser to obtain the third detection laser; and the detection part 1214 is used for determining the coherent Raman scattering spectrum of the sample to be detected based on the third detection laser.

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

[0148] The second objective lens 12221 is used for collecting and tightly focusing the second detection laser, the second excitation pulse and the third excitation pulse on the sample to be detected, to 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 detected, so that the sample to be detected generates a thermal light effect; the fourth detection laser interacts with the thermal light effect to obtain the fifth detection laser; the second collection lens 12222 is used for collecting the fifth detection laser to obtain the sixth detection laser; the second filter 12223 is used for filtering out the residual excitation pulse in the sixth detection laser to obtain the third detection laser; and the third mirror 12224 is also used for reflecting the third detection laser to the detection part 1214.

[0149] The second objective 12221 is configured to collect and tightly focus the second excitation pulse, the third excitation pulse and the second probe laser on the sample to be measured to obtain the fourth excitation pulse, the fifth excitation pulse and the fourth probe laser, respectively, wherein the fourth excitation pulse and the fifth excitation pulse excite coherent Raman oscillation of the sample to be measured; the sample to be measured is a biochemical sample to be measured for coherent Raman spectrum information, and the coherent Raman oscillation and thermal-optic effect induced by relaxation and attenuation of the coherent Raman oscillation both occur on the sample to be measured; the fourth probe laser interacts with the thermal-optic effect to obtain the fifth probe laser which is forward scattered and encoded by the thermal-optic effect; the second collection lens 12222 is configured to collect the fifth probe laser to obtain the sixth probe laser; the second filter 12223 is configured to filter out the excitation pulse in the sixth probe laser to obtain the third probe laser; and the third mirror 12224 is configured to reflect the third probe laser to the detection member 1214.

[0150] The embodiment of the present application provides a time-domain excitation coherent Raman photothermal spectrum microscopic detection system, as shown in the figure. Figure 13 The second probe laser adjusting member 1221 comprises a third lens 12211 and a fourth lens 12212; the third lens 12211 and the fourth lens 12212 are configured to adjust the axial focal point position of the first probe laser on the sample to be measured to obtain the second probe laser.

[0151] The optical axes of the third lens 12211 and the fourth lens 12212 are located on the same straight line and coincide with the first probe laser beam; the third lens 12211 and the fourth lens 12212 are configured to adjust the axial focal point position of the first probe laser on the sample to be measured to obtain the second probe laser, so that the best thermal-optic effect detection signal can be obtained.

[0152] The embodiment of the present application provides a time-domain excitation coherent Raman photothermal spectrum microscopic detection system, as shown in the figure. Figure 14As shown, the probe assembly 12 comprises a Bessel beam probe assembly 123, the Bessel beam probe assembly 123 comprising a second probe laser beam splitting element 1231, a first Bessel beam transforming assembly 1232, a third excitation assembly 1233, a fourth mirror 1234 and a probe element 1214; the second probe laser beam splitting element 1231 is configured to split the first probe laser beam to obtain a first reference arm probe laser beam and a first measurement arm probe laser beam; the first Bessel beam transforming assembly 1232 is configured to transform the first measurement arm probe laser beam into a Bessel beam and adjust the axial focal point position of the first measurement arm probe laser beam at the sample under test to obtain a second measurement arm probe laser beam; the third excitation assembly 1233 is configured to focus the second measurement arm probe laser beam, the second excitation pulse and the third excitation pulse to the sample under test to make the sample under test generate a thermal light effect, and detect the thermal light effect based on the second measurement arm probe laser beam to obtain a third measurement arm probe laser beam; the fourth mirror 1234 is configured to reflect the first reference arm probe laser beam to make the first reference arm probe laser beam return to the second probe laser beam splitting element 1231 to obtain a second reference arm probe laser beam; the second probe laser beam splitting element 1231 is further configured to combine the third measurement arm probe laser beam and the second reference arm probe laser beam to obtain a combined probe laser beam; and the probe element 1214 is configured to determine the coherent Raman scattering spectrum of the sample under test based on the combined probe laser beam.

[0153] The second probe laser beam splitting element 1231 is configured to split the first probe laser beam to obtain a first reference arm probe laser beam and a first measurement arm probe laser beam; the first Bessel beam transforming assembly 1232 is configured to transform the first measurement arm probe laser beam into a Bessel beam, and adjusting the geometric structure of the first Bessel beam transforming assembly 1232 can also adjust the axial focal point position of the measurement arm probe laser beam at the sample under test to obtain the best thermal light effect detection signal; then, the second dichroic mirror 114 in the scanning assembly 11 is used to combine the second excitation pulse, the third excitation pulse and the second measurement arm probe laser beam; the third excitation assembly 1233 is configured to focus the second measurement arm probe laser beam, the second excitation pulse and the third excitation pulse to the sample under test to make the sample under test generate a thermal light effect, and detect the thermal light effect based on the second measurement arm probe laser beam to obtain a third measurement arm probe laser beam; the fourth mirror 1234 is configured to reflect the first reference arm probe laser beam to make the first reference arm probe laser beam return to the second probe laser beam splitting element 1231 to obtain a second reference arm probe laser beam; the second probe laser beam splitting element 1231 is further configured to combine the third measurement arm probe laser beam and the second reference arm probe laser beam to obtain a combined probe laser beam; and the probe element 1214 is configured to determine the coherent Raman scattering spectrum of the sample under test based on the combined probe laser beam.

[0154] In the above embodiment, the first probe laser is divided into two paths, one path is a reference arm through a space with constant optical path, and the other path is a measurement arm which is first transformed into a Bessel beam to be incident on the sample and interacts with the above-mentioned thermo-optic effect, that is, the optical path is modulated by the thermo-optic effect, and then is transformed into the original beam mode to interfere with the reference arm probe laser, and the modulation information of the thermo-optic effect is obtained by analyzing the interference signal. Thanks to the anti-interference, anti-scattering and strong penetration ability of the Bessel beam, the random phase noise introduced by the random scattering of the probe laser and the sample under test is greatly suppressed. This advantage is particularly significant when the sample under test is a thick sample with complex tissue composition. In addition, using the Bessel beam probe laser with the same wavelength can further compress the size of the focal spot of the probe laser on the sample under test, so as to improve the spatial resolution of the spectral microscopic system.

[0155] The embodiment of the present application provides a time-domain excitation coherent Raman photo-thermal spectral microscopic detection system, as shown in the figure. Figure 15 The third excitation component 1233 includes a third objective lens 12331, a third collection lens 12332, a third filter 12333, a second Bessel beam transformation component 12334 and a fifth 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 measurement arm probe laser onto the sample under test, to obtain the fourth excitation pulse, the fifth excitation pulse and the fourth measurement arm probe laser respectively, the fourth excitation pulse and the fifth excitation pulse excite the coherent Raman oscillation of the sample under test, so that the sample under test generates a thermo-optic effect; the fourth measurement arm probe laser interacts with the thermo-optic effect to obtain the fifth measurement arm probe laser; the third collection lens 12332 is used to collect the fifth measurement arm probe laser to obtain the sixth measurement arm probe laser; the third filter 12333 is used to filter out the residual excitation pulse in the sixth measurement arm probe laser to obtain the seventh measurement arm probe laser; the second Bessel beam transformation component 12334 is used to transform the seventh measurement arm probe laser into a Bessel beam to obtain the eighth measurement arm probe laser; and is also used to transform the eighth measurement arm probe laser to restore the original beam mode of the eighth measurement arm probe laser to obtain the ninth measurement arm probe laser; the fifth mirror 12335 is used to fold back the ninth measurement arm probe laser to the original path, so that the ninth measurement arm probe laser again undergoes the mirror-symmetrical process of the first measurement arm probe laser to the ninth measurement arm probe laser, to obtain the third measurement arm probe laser in front of the second probe laser beam splitter 1231.

[0156] The third objective lens 12331 is used for collecting and tightly focusing the second excitation pulse, the third excitation pulse and the second measurement arm probe laser onto the sample to be measured, so as to obtain the fourth excitation pulse, the fifth excitation pulse and the fourth measurement arm probe laser respectively, and the fourth excitation pulse and the fifth excitation pulse excite the coherent Raman oscillation of the sample to be measured; the sample to be measured is a biochemical sample to be measured for coherent Raman spectrum information, and the coherent Raman oscillation and the thermal-optic effect induced by the relaxation and attenuation of the coherent Raman oscillation both occur here; the fourth measurement arm probe laser interacts with the thermal-optic effect to obtain the fifth measurement arm probe laser which is forward transmitted and encoded by the optical path of the thermal-optic effect; the third collection lens 12332 is used for collecting the fifth measurement arm probe laser to obtain the sixth measurement arm probe laser; the third filter 12333 is used for filtering out the residual excitation pulse in the sixth measurement arm probe laser to obtain the seventh measurement arm probe laser; the first Bessel beam transformation assembly 1232 is used for transforming the seventh measurement arm probe laser into a Bessel beam to obtain the eighth measurement arm probe laser, and then transforming the eighth measurement arm probe laser to restore the original beam shape to obtain the ninth measurement arm probe laser; and the fifth mirror 12335 is used for folding back the ninth measurement arm probe laser to make the measurement arm probe laser pass through the mirror image symmetric process of the first measurement arm probe laser to the ninth measurement arm probe laser again, and finally obtain the third measurement arm probe laser in front of the second probe laser beam splitter 1231.

[0157] Because the measurement arm probe laser passes through the sample to be measured twice and interacts with the thermal-optic effect, the optical path modulation introduced thereby is doubled.

[0158] The embodiment of the present application provides a time-domain excitation coherent Raman photo-thermal spectrum microscopic detection system. Figure 16 As shown in the figure, the first Bessel beam transformation assembly 1232 includes a first conical lens 12321 and a fifth lens 12322; the first conical lens 12321 is used for transforming the first measurement arm probe laser into a Bessel beam to obtain an intermediate measurement arm probe laser; and the fifth lens 12322 is used for transforming the intermediate measurement arm probe laser to form a ring-shaped line light source on the front focal plane of the third objective lens 12331 to obtain the second measurement arm probe laser.

[0159] The first conical lens 12321 is used to transform the first measurement arm probe laser into a Bessel beam to obtain intermediate measurement arm probe laser, wherein the beam propagation direction of the first measurement arm probe laser is perpendicular to and faces the planar surface of the first conical lens 12321, and the beam center thereof passes through the conical vertex of the first conical lens 12321, and in addition, adjusting the front and back positions of the first conical lens 12321 can adjust the axial focal point position of the first measurement arm probe laser at the sample to be measured to obtain the best thermal light effect detection signal; the fifth lens 12322 is used to transform the intermediate measurement arm probe 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 measurement arm probe laser.

[0160] In some embodiments, the fifth lens 12322 and the third objective lens 12331 form a 4F system to conjugate the intermediate measurement arm probe laser to the fourth measurement arm probe laser.

[0161] The embodiment of the present application provides a time-domain excitation coherent Raman photothermal spectrum microscopic detection system, as shown in the figure, Figure 17 The second Bessel beam transformation assembly 12334 includes a sixth lens 123341 and a second conical lens 123342; the sixth lens 123341 is used to transform the seventh measurement arm probe laser into a Bessel beam to obtain the eighth measurement arm probe laser; and the second conical lens 123342 is used to transform the eighth measurement arm probe laser to restore it to the original beam shape to obtain the ninth measurement arm probe laser.

[0162] The sixth lens 123341 is used to transform the seventh measurement arm probe laser into a Bessel beam to obtain the eighth measurement arm probe laser; and the second conical lens 123342 is used to transform the eighth measurement arm probe laser to restore it to the original beam shape to obtain the ninth measurement arm probe laser, wherein the beam propagation direction of the eighth measurement arm probe laser is perpendicular to and faces the conical surface of the second conical lens 123342, and the beam center thereof passes through the conical vertex of the second conical lens 123342.

[0163] In some embodiments, the third collection lens 12332 and the sixth lens 123341 form a 4F system to conjugate the fifth measurement arm probe laser to the eighth measurement arm probe laser.

[0164] In some embodiments, the intermediate measurement arm probe laser, the fourth measurement arm probe laser, the fifth measurement arm probe laser and the eighth measurement arm probe laser are all Bessel beams.

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

[0166] The first photodetector is used to detect the signal intensity of the third probe laser or the combined probe laser, and analyze the coherent Raman scattering spectrum of the sample to be measured by using Fourier transform.

[0167] The embodiment of the present application provides a time-domain excitation coherent Raman photothermal spectrum microscopic detection system, which comprises a light source assembly 10, a scanning assembly 11, a detection assembly 12 and a signal processing assembly 13. Figure 19 As shown, the light source assembly 10 comprises a femtosecond pulse generating device 101, a femtosecond pulse splitting device 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 splitting device 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 delay probe laser and a first probe laser; the first dichroic mirror 105 is used to combine the first excitation pulse and the first delay probe laser; wherein the first excitation pulse and the first delay probe laser subsequently enter the scanning assembly 11, and the first probe laser subsequently enters the detection assembly 12.

[0168] As shown in the figure, Figure 20 As shown, the femtosecond pulse splitting device 102 comprises a half-wave plate 1021, a polarization beam splitter 1022 and a light block 1023. The original pulse is a transform-limited femtosecond pulse whose spectrum meets the excitation requirement of 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 by the combination of the half-wave plate, the polarization beam splitter and the light block.

[0170] The embodiment of the present application provides a time-domain excitation coherent Raman photothermal spectrum microscopic detection system, which comprises a light source assembly 10, a scanning assembly 11, a detection assembly 12 and a signal processing assembly 13. Figure 21As shown, the scanning assembly 11 comprises a second beam splitter 111, a first delay assembly 112, a second delay assembly 113, a second dichroic mirror 114 and a delay detection assembly 115; the second beam splitter 111 is configured to replicate the first excitation pulse and the first delay probe laser to obtain a first replicated excitation pulse, a second replicated excitation pulse, a first replicated delay probe laser and a second replicated delay probe laser; the first delay assembly 112 is configured to reflect the first replicated excitation pulse and the first replicated delay probe laser to obtain the second excitation pulse and the second delay probe laser; the second delay assembly 113 is configured to reflect and delay scan the second replicated excitation pulse and the second replicated delay probe laser to obtain the third excitation pulse and the third delay probe laser; the second beam splitter 111 is further configured to combine the second excitation pulse and the third excitation pulse; and is further configured to combine the second delay probe laser and the third delay probe laser in the same way; the second dichroic mirror 114 is configured to separate the excitation pulse and the delay probe laser, and is further configured to combine the excitation pulse and the probe laser in the detection assembly 12; the delay detection assembly 115 is configured to detect the relative delay between the second excitation pulse and the third excitation pulse; and the relative delay between the second delay probe laser and the third delay probe laser is used to represent the relative delay between the second excitation pulse and the third excitation pulse.

[0171] The second beam splitter 111 is configured to replicate the first excitation pulse and the first delay probe laser to obtain the first replicated excitation pulse and the second replicated excitation pulse, and to obtain the first replicated delay probe laser and the second replicated delay probe laser; the first delay assembly 112 is configured to reflect the first replicated excitation pulse and the first replicated delay probe laser to obtain the second excitation pulse and the second delay probe laser; the second delay assembly 113 is configured to reflect and delay scan the second replicated excitation pulse and the second replicated delay probe laser to obtain the third excitation pulse and the third delay probe laser; the second beam splitter 111 is further configured to combine the second excitation pulse and the third excitation pulse, and is further configured to combine the second delay probe laser and the third delay probe laser in the same way; the second dichroic mirror 114 is configured to separate the excitation pulse and the delay probe laser, and is further configured to combine the excitation pulse and the probe laser in the detection assembly 12; the delay detection assembly 115 is configured to detect the relative delay between the second delay probe laser and the third delay probe laser, thereby detecting the relative delay between the second excitation pulse and the third excitation pulse; and the second excitation pulse and the third excitation pulse subsequently enter the detection assembly 12.

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

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

[0174] In one embodiment, such as Figure 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 delayed detection laser beam that is combined and interferes onto the detection target surface of the second photodetector 1152; the second photodetector 1152 is used to detect the interference signal intensity of the delayed detection laser to resolve the relative delay amount.

[0175] In the embodiments of this application, schematic diagrams of time-domain excited coherent Raman photothermal spectroscopy microscopy detection systems are provided for the above three detection components. Figure 24 The diagram shown is a schematic of a time-domain excited coherent Raman photothermal spectroscopy microscopic detection system (backscattering detection); Figure 25 The diagram shown is a schematic of the structure of a time-domain excited coherent Raman photothermal spectroscopy microscopic detection system (forward scattering detection); Figure 26 The diagram shown is a schematic representation of a time-domain excited coherent Raman photothermal spectroscopy microscopic detection system (Bessel beam-Mach-Zehnder interferometer) in one embodiment.

[0176] The technical features of the above embodiments can be combined in any way. For the sake of brevity, 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 are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A time-domain excited coherent Raman photothermal spectroscopy microscopic detection system, characterized in that, The system includes a light source assembly, a scanning assembly, and a detection assembly; 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 copying and delay processing on the first excitation pulse to obtain a second excitation pulse and a third excitation pulse with relative delay, and to 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 perform coherent Raman excitation on the sample under test using the second excitation pulse and the third excitation pulse to induce a thermo-optic effect in the sample under test; it is also used to adjust or transform the first detection laser to detect the thermo-optic effect, and determine the coherent Raman scattering spectrum of the sample under test based on the thermo-optic effect and the relative delay.

2. The time-domain excited coherent Raman photothermal spectroscopy microscopy detection system according to claim 1, characterized in that, The detection assembly includes a backscattering detection assembly, which 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 component is used to adjust the axial focal position of the first detection laser at the sample to be tested, so as to obtain the second detection laser. The first detection laser beam splitter is used to direct the second detection laser onto 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-optic effect, and to detect the thermo-optic effect based on the second detection laser to obtain the third detection laser; The detector is used to determine the coherent Raman scattering spectrum of the sample under test based on the third detection laser.

3. The time-domain excited 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 focus the second detection laser onto the sample to be tested, thereby obtaining the fourth detection laser; The first objective lens is used to collect and focus the second and third excitation pulses onto the sample under test to obtain a fourth and a fifth excitation pulse, respectively. The fourth and fifth excitation pulses excite coherent Raman oscillations in the sample under test to generate a thermo-optic effect. The fourth probe laser interacts with the thermo-optic effect to obtain a fifth probe laser. The first collecting mirror is also used to collect the fifth detection laser to obtain the sixth detection laser; The first filter is also used to filter out residual excitation pulses in the sixth detection laser to obtain the third detection laser; The first reflector is also used to reflect the third detection laser onto the detector.

4. The time-domain excited coherent Raman photothermal spectroscopy microscopy detection system according to claim 2, characterized in that, The first detection laser adjustment component includes a second reflector, a first lens, and a second lens; The second reflector is used to reflect the first probe laser back to the first lens; the optical axes of the first lens and the second lens are on the same straight line; The first lens and the second lens are used to adjust the axial focal position of the first probe laser at the sample to be tested, so as to obtain the second probe laser.

5. The time-domain excited coherent Raman photothermal spectroscopy microscopy detection system according to claim 1, characterized in that, The detection component includes a forward scattering detection component, which includes a second detection laser adjustment component, a second excitation component, and a detector component; The second detection laser adjustment component is used to adjust the axial focal position of the first detection laser at the sample to be tested, thereby obtaining the 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-optic effect, and to detect the thermo-optic effect based on the second detection laser to obtain the third detection laser; The detector is used to determine the coherent Raman scattering spectrum of the sample under test based on the third detection laser.

6. The time-domain excited 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 focus the second detection laser, the second excitation pulse, and the third excitation pulse onto the sample under test, thereby obtaining a fourth detection laser, a fourth excitation pulse, and a fifth excitation pulse. The fourth and fifth excitation pulses excite coherent Raman oscillations in the sample under test to induce a thermo-optic effect. The fourth detection laser interacts with the thermo-optic effect to generate the fifth detection laser. The second collecting mirror is used to collect the fifth detection laser to obtain the sixth detection laser; The second filter is used to filter out the residual excitation pulse in the sixth detection laser to obtain the third detection laser; The third reflector is also used to reflect the third detection laser back to the detector.

7. The time-domain excited coherent Raman photothermal spectroscopy microscopy detection system according to claim 6, characterized in that, The second detection laser adjustment component includes a third lens and a fourth lens; The third lens and the fourth lens are used to adjust the axial focal position of the first detection laser at the sample to be tested, so as to obtain the second detection laser.

8. The time-domain excited coherent Raman photothermal spectroscopy microscopy detection system according to claim 1, characterized in that, 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 detector. The second detection laser beam splitter is used to split the first detection laser beam to obtain the first reference arm detection laser and the first measurement arm detection laser; The first Bessel beam conversion component is used to convert the first measuring arm probe laser into a Bessel beam and adjust the axial focal position of the first measuring arm probe laser at the sample to be tested to obtain the second measuring arm probe laser; The third excitation component is used to focus the second measuring arm probe laser, 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-optic effect, and to obtain the third measuring arm probe laser by detecting the thermo-optic effect based on the second measuring arm probe 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 also used to combine the detection laser of the third measuring arm and the detection laser of the second reference arm to obtain a combined detection laser. The detector is used to determine the coherent Raman scattering spectrum of the sample under test based on the combined detection laser beam.

9. The time-domain excited coherent Raman photothermal spectroscopy microscopy detection system according to claim 8, characterized in that, The third excitation assembly includes a third objective lens, a third collecting lens, a third filter, a second Bessel beam conversion assembly, and a fifth reflecting mirror; The third objective lens is used to collect and focus the second excitation pulse, the third excitation pulse, and the second measuring arm probe laser onto the sample to be tested, thereby obtaining a fourth excitation pulse, a fifth excitation pulse, and a fourth measuring arm probe laser. The fourth excitation pulse and the fifth excitation pulse excite coherent Raman oscillations in the sample to be tested, so that the sample to be tested produces a thermo-optic effect. The fourth measuring arm probe laser interacts with the thermo-optic effect to obtain the fifth measuring arm probe 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 detection laser of the sixth measuring arm to obtain the detection laser of the seventh measuring arm; The second Bessel beam conversion component is used to convert the seventh measuring arm probe laser into a Bessel beam to obtain the eighth measuring arm probe laser; it is also used to convert the eighth measuring arm probe laser to restore the original beam shape to obtain the ninth measuring arm probe laser. The fifth reflector is used to reverse the original path of the ninth measuring arm probe laser, so that the ninth measuring arm probe laser passes through the mirror-symmetric process experienced by the first measuring arm probe laser to the ninth measuring arm probe laser again, so as to obtain the third measuring arm probe laser in front of the second probe laser beam splitter.

10. The time-domain excited coherent Raman photothermal spectroscopy microscopy detection system according to claim 9, characterized in that, The first Bessel beam conversion component includes a first conical lens and a fifth lens; The first conical lens is used to convert the probe laser of the first measuring arm into a Bessel beam to obtain the probe laser of the middle measuring arm; The fifth lens is used to transform the intermediate measuring arm probe laser to form a ring-shaped line light source on the front focal plane of the third objective lens, thereby obtaining the second measuring arm probe laser.

11. The time-domain excited coherent Raman photothermal spectroscopy microscopy detection system according to claim 9, characterized in that, The second Bessel beam conversion assembly includes a sixth lens and a second conical lens; The sixth lens is used to convert the probe laser of the seventh measuring arm into a Bessel beam to obtain the probe laser of the eighth measuring arm; The second conical lens is used to transform the detection laser of the eighth measuring arm to restore it to its original beam shape, thus obtaining the detection laser of the ninth measuring arm.

12. The time-domain excited coherent Raman photothermal spectroscopy microscopic detection system according to claim 2, 5, or 8, characterized in that, The detector includes a seventh lens and a first photodetector; The seventh lens is used to focus the third detection laser or the combined detection laser onto the detection target surface of the first photodetector; The first photodetector is used to detect the signal intensity of the third detection laser or the combined detection laser, and to analyze the coherent Raman scattering spectrum of the sample under test using Fourier transform.

13. The time-domain excited coherent Raman photothermal spectroscopy microscopic detection system according to any one of claims 1-11, characterized in that, The light source assembly includes a femtosecond pulse generating device, a femtosecond pulse beam splitter, 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 the raw pulse; The femtosecond pulse beam splitter is used to perform beam splitting 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 raw laser light; The first beam splitter is used to split the original laser beam to obtain the first delayed probe laser and the first probe 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 excited coherent Raman photothermal spectroscopy microscopic detection system according to any one of claims 1-11, characterized in that, The scanning component includes a second beam splitter, a first delay component, a second delay component, a second dichroic mirror, and a delay detection component; The second beam splitter is used to copy the first excitation pulse and the first delayed probe laser to obtain a first copied excitation pulse, a second copied excitation pulse, a first copied delayed probe laser, and a second copied delayed probe laser. The first delay component is used to reflect the first replicated excitation pulse and the first replicated delayed probe laser to obtain the second excitation pulse and the second delayed probe laser; The second delay component is used to perform reflection and delay scanning processing on the second replicated excitation pulse and the second replicated delayed detection laser to obtain the third excitation pulse and the third delayed detection laser; The second beam splitter is also used to perform beam combining on the second excitation pulse and the third excitation pulse; and to perform the same beam combining on the second delayed probe laser and the third delayed probe laser. The second dichroic mirror is used to separate the excitation pulse and the delayed probe laser, and also to combine the excitation pulse and the probe 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 and third delayed probe lasers is used to characterize the relative delay between the second and third excitation pulses.

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