Transient stimulated raman excited fluorescence spectroscopy method and system

Abstract

The present application relates to the field of spectral detection and optical imaging technology, and provides a kind of transient stimulated raman excitation fluorescence spectrum method and system, the method includes based on stimulated raman excitation pulse pair, generates two stimulated raman excitation pulse pairs with relative delay and the relative delay adjustable;Based on two stimulated raman excitation pulse pairs, excite the sample to be measured to generate transient stimulated raman scattering, and based on probe pulse, excite the sample to be measured to generate fluorescence signal;Collect the fluorescence signal generated by the sample to be measured, and the fluorescence signal is Fourier transformed, and the Raman spectrum corresponding to the sample to be measured is obtained.Due to the introduction of two stimulated raman excitation pulse pairs with adjustable relative delay, transient stimulated raman scattering is generated by exciting the sample to be measured, so that the sample to be measured generates fluorescence signal containing Raman resonance frequency in time domain when being excited by probe pulse, and then clear Raman spectrum without fluorescence background is seen in frequency domain after Fourier transform, to ensure the quality of Raman spectrum.

Description

Technical Field

[0001] This invention relates to the fields of spectral detection and optical imaging technology, and in particular to a transient stimulated Raman excitation fluorescence spectroscopy method and system. Background Technology

[0002] Raman spectroscopy is a powerful tool for analyzing molecular structures, containing rich information about the molecular structure of samples. It can also be used to analyze molecular dynamics and interactions with the solvent environment, and is widely applied in the study of molecular reactions in biology, chemistry, and other fields. However, Raman spectroscopy has a very small scattering cross section, resulting in low detection sensitivity. Furthermore, it is susceptible to interference from fluorescence background.

[0003] Based on this, stimulated Raman excited fluorescence (SREF) spectroscopy was developed. However, the Raman spectra obtained by SREF spectroscopy have a strong, inherent fluorescence background, which current excitation techniques cannot effectively suppress.

[0004] Therefore, there is an urgent need to provide a transient stimulated Raman excitation fluorescence spectroscopy method. Summary of the Invention

[0005] This invention provides a transient stimulated Raman excitation fluorescence spectroscopy method and system to address the shortcomings of existing excitation techniques in effectively suppressing fluorescence background.

[0006] This invention provides a transient stimulated Raman excitation fluorescence spectroscopy method, comprising:

[0007] Based on the stimulated Raman excitation pulse pair, two stimulated Raman excitation pulse pairs with relative delay and adjustable relative delay are generated; the stimulated Raman excitation pulse pair includes a pump pulse and a Stokes pulse that are aligned in time and space dimensions based on femtosecond pulsed laser;

[0008] Based on the two pairs of stimulated Raman excitation pulses, the sample under test is excited to generate transient stimulated Raman scattering, and based on the probe pulse, the sample under test is excited to generate a fluorescence signal.

[0009] The fluorescence signal is acquired, and the fluorescence signal is subjected to Fourier transform to obtain the Raman spectrum corresponding to the sample to be tested.

[0010] According to the present invention, a transient stimulated Raman excitation fluorescence spectroscopy method is provided, wherein the femtosecond pulsed laser is generated based on a femtosecond laser;

[0011] There is degeneracy information between the stimulated Raman excitation pulse pair and the detection pulse. The degeneracy information is determined based on the bandwidth of the femtosecond laser, the Raman shift of the excited Raman mode of the sample under test, and the energy level structure.

[0012] According to the present invention, a transient stimulated Raman excitation fluorescence spectroscopy method is provided, wherein performing a Fourier transform on the fluorescence signal to obtain the Raman spectrum corresponding to the sample to be tested includes:

[0013] Filter out background light of wavelengths other than the fluorescence signal and select the fluorescent photons at the focal position in the fluorescence signal;

[0014] The time-domain fluorescence signal corresponding to the fluorescent photon is recorded, and the time-domain fluorescence signal is subjected to Fourier transform to obtain the Raman spectrum.

[0015] According to the present invention, a transient stimulated Raman excitation fluorescence spectroscopy method is provided, wherein the step of exciting the sample to generate transient stimulated Raman scattering based on two stimulated Raman excitation pulse pairs includes:

[0016] The bandwidth of the Stokes pulse is adjusted so that the bandwidth difference between the Stokes pulse and the pump pulse is within a preset range.

[0017] This invention provides a transient stimulated Raman excitation fluorescence spectroscopy system, comprising: a femtosecond laser source, a time-delay scanning device, and a signal acquisition device;

[0018] The femtosecond laser source is used to generate stimulated Raman excitation pulse pairs and probe pulses. The stimulated Raman excitation pulse pairs include pump pulses and Stokes pulses that are aligned in the time and space dimensions based on femtosecond pulsed lasers.

[0019] The time-delay scanning device is used to generate two stimulated Raman excitation pulse pairs with a relative delay and an adjustable relative delay based on the stimulated Raman excitation pulse pair. The two stimulated Raman excitation pulse pairs are used to excite the sample to generate transient stimulated Raman scattering, so that the sample to be tested is excited by the probe pulse to generate a fluorescence signal.

[0020] The signal acquisition device is used to acquire the fluorescence signal and perform a Fourier transform on the fluorescence signal to obtain the Raman spectrum corresponding to the sample to be tested.

[0021] According to the present invention, a transient stimulated Raman excitation fluorescence spectroscopy system is provided, wherein the femtosecond laser source includes a femtosecond laser, and the femtosecond laser is used to generate the femtosecond pulsed laser;

[0022] The degeneracy information between the stimulated Raman excitation pulse pair and the detection pulse is determined based on the bandwidth of the femtosecond laser, the Raman shift of the excited Raman mode of the sample under test, and the energy level structure.

[0023] According to the transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention, there is no degeneracy between the stimulated Raman excitation pulse pair and the detector pulse, or the pump pulse and the detector pulse are degenerate; accordingly,

[0024] The femtosecond laser source includes a femtosecond laser, a polarization beam splitter, a first dichroic mirror, a first light source branch, and a second light source branch; the femtosecond laser is used to generate the femtosecond pulsed laser; the polarization beam splitter is used to split the femtosecond pulsed laser into a first beam and a second beam.

[0025] The first light source branch includes a first dispersion compensation device, which is used to perform dispersion compensation on the first part of the beam to obtain the Stokes pulse;

[0026] The second light source branch includes a frequency doubler, an optical parametric oscillator, a second dispersion compensation device, and an adjustable first optical delay line; the frequency doubler is used to double the frequency of the second portion of the beam to obtain a frequency-doubled laser; the optical parametric oscillator is driven by the frequency-doubled laser and outputs the pump pulse; the first optical delay line is used to align the pump pulse with the Stokes pulse in the time dimension; the second dispersion compensation device is used to perform dispersion compensation on the pump pulse;

[0027] The first dichroic mirror is used to align the pump pulse with the Stokes pulse in spatial dimensions.

[0028] According to the transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention, the first light source branch further includes a spectral filtering device;

[0029] The spectral filtering device is used to adjust the bandwidth of the Stokes pulse so that the bandwidth difference between the Stokes pulse and the pump pulse is within a preset range.

[0030] According to the transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention, there is no degeneracy between the stimulated Raman excitation pulse pair and the detection pulse; correspondingly,

[0031] The femtosecond laser source also includes a probe laser and a second optical delay line and a second dichroic mirror arranged along the optical path;

[0032] The detection laser is used to generate the detection pulse;

[0033] The second optical delay line is used to control the delay between the probe pulse and the target pulse pair, wherein the target pulse pair is the later stimulated Raman excitation pulse pair among the two stimulated Raman excitation pulse pairs;

[0034] The second dichroic mirror is used to align the two stimulated Raman excitation pulse pairs with the detection pulse in spatial dimension.

[0035] According to the present invention, a transient stimulated Raman excitation fluorescence spectroscopy system is provided, wherein the time-delay scanning device includes an interferometer, and one arm of the interferometer includes an adjustable third optical delay line.

[0036] According to the present invention, a transient stimulated Raman excitation fluorescence spectroscopy system is provided, wherein the third optical delay line is fixed on a displacement stage;

[0037] The signal acquisition device is triggered based on the position information of the displacement stage.

[0038] According to a transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention, the interferometer further includes a first beam splitter, a second beam splitter, and a reference signal detection device;

[0039] The first beam splitter is used to divide the target beam of the stimulated Raman excitation pulse pair generated by the femtosecond laser source into a third beam and a fourth beam. The third beam is transmitted along the first arm of the interferometer, and the fourth beam is transmitted along the second arm of the interferometer.

[0040] The second beam splitter is used to combine the beam output from the first arm and the beam output from the second arm to obtain a fifth beam and a sixth beam, respectively. The fifth beam is used to excite the sample under test to generate transient stimulated Raman scattering and the fluorescence signal.

[0041] The reference signal detection device is used to receive the sixth part of the beam and convert the sixth part of the beam into a reference signal with a coherence time greater than a preset threshold. The reference signal is used to calibrate the position information of the displacement stage.

[0042] According to a transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention, the reference signal detection device includes a grating, a lens, and a slit;

[0043] The sixth beam passes sequentially through the grating, the lens, and the slit to obtain the reference signal.

[0044] According to a transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention, the signal acquisition device includes a filter, a screening element, a photodetector, and a data acquisition card arranged along the optical path;

[0045] The filter is used to filter out background light of wavelengths other than the fluorescence signal;

[0046] The filtering element is used to select fluorescent photons that are located at the focal position in the fluorescence signal;

[0047] The photodetector is used to record the time-domain fluorescence signal corresponding to the fluorescent photon;

[0048] The data acquisition card is used to perform Fourier transform on the time-domain fluorescence signal to obtain the Raman spectrum.

[0049] The present invention provides a transient stimulated Raman excitation fluorescence spectroscopy method and system. The method includes: generating two stimulated Raman excitation pulse pairs with adjustable relative delays; exciting a sample to produce transient stimulated Raman scattering based on the two stimulated Raman excitation pulse pairs, and exciting the sample to produce a fluorescence signal based on a probe pulse; acquiring the fluorescence signal and performing a Fourier transform on the fluorescence signal to obtain the Raman spectrum corresponding to the sample. By introducing two stimulated Raman excitation pulse pairs with adjustable relative delays to excite transient stimulated Raman scattering in the sample, the sample can produce a fluorescence signal containing the Raman resonance frequency in the time domain when excited by the probe pulse. Therefore, after Fourier transform, a clear Raman spectrum without fluorescence background can be observed in the frequency domain, ensuring the quality of the Raman spectrum and avoiding the influence of fluorescence background on the Raman spectrum. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on the drawings described below without creative effort.

[0051] Figure 1 This is a schematic flowchart of the transient stimulated Raman excitation fluorescence spectroscopy method provided by the present invention;

[0052] Figure 2 This is the excitation energy level diagram of the sample under test in the transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention, under the condition that the stimulated Raman excitation pulse pair and the detector pulse are not degenerate;

[0053] Figure 3 This is the excitation energy level diagram of the sample under test in the transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention, under the condition that the pump pulse and the detector pulse are degenerate.

[0054] Figure 4This is the excitation energy level diagram of the sample under test in the transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention, under the condition that the stimulated Raman excitation pulse pair and the detector pulse are degenerate;

[0055] Figure 5 This is one of the structural schematic diagrams of the transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention;

[0056] Figure 6 This is the second schematic diagram of the transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention;

[0057] Figure 7 This is the third schematic diagram of the transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention;

[0058] Figure 8 This is the fourth schematic diagram of the transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention;

[0059] Figure 9 This is the fifth schematic diagram of the transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention;

[0060] Figure 10 This is a complete technical roadmap of the T-SREF spectroscopy technology used in the transient stimulated Raman excitation fluorescence spectroscopy system provided by this invention;

[0061] Figure 11 This is a schematic diagram of the time-domain fluorescence signal of ATTO740 dye molecules in the transient stimulated Raman excitation fluorescence spectroscopy system provided by the present invention, which is excited by two stimulated Raman excitation pulses to produce transient stimulated Raman scattering and excited by a probe pulse to produce a C=C skeleton mode.

[0062] Figure 12 The spectrum is obtained by performing a Fourier transform on the time-domain fluorescence signal using the signal acquisition device in the transient stimulated Raman excitation fluorescence spectroscopy system provided by this invention. Detailed Implementation

[0063] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0064] Because existing SREF spectroscopy techniques produce Raman spectra with a strong, inherent fluorescence background, current excitation techniques cannot effectively suppress this background. Therefore, this invention provides a transient stimulated Raman excitation fluorescence spectroscopy method to generate Raman spectra without fluorescence background.

[0065] Figure 1 This is a schematic flowchart of a transient stimulated Raman excitation fluorescence spectroscopy method provided in an embodiment of the present invention, as shown below. Figure 1 As shown, the method includes:

[0066] S1, based on the stimulated Raman excitation pulse pair, generate two stimulated Raman excitation pulse pairs with relative delay and adjustable relative delay; the stimulated Raman excitation pulse pair includes a pump pulse and a Stokes pulse that are aligned in time and space dimensions based on a femtosecond pulsed laser;

[0067] S2, based on the two pairs of stimulated Raman excitation pulses, the sample under test is excited to generate transient stimulated Raman scattering, and based on the probe pulse, the sample under test is excited to generate a fluorescence signal;

[0068] S3. Acquire the fluorescence signal, perform Fourier transform on the fluorescence signal, and obtain the Raman spectrum corresponding to the sample to be tested.

[0069] Specifically, the transient stimulated Raman excitation fluorescence spectroscopy method provided in this embodiment of the invention can be implemented using a transient stimulated Raman excitation fluorescence spectroscopy system.

[0070] First, step S1 is performed to generate two stimulated Raman excitation pulse pairs with a relative delay that is adjustable. The stimulated Raman excitation pulse pairs can be generated using a femtosecond laser source and may include pump pulses and Stokes pulses aligned in both time and spatial dimensions based on femtosecond pulsed laser generation. The femtosecond laser source may include a femtosecond laser that can be used to generate femtosecond pulsed lasers.

[0071] Here, the two stimulated Raman excitation pulse pairs can be generated by a time-delay scanning device, which can be an interferometer or other similar equipment; no specific limitation is made here. Alignment means overlap. Temporal alignment means that the pump pulse and the Stokes pulse are in the same position at a certain moment, i.e., they can meet. Spatial alignment means that the pump pulse and the Stokes pulse merge into a single beam.

[0072] Then, step S2 is executed, using the generated two pairs of stimulated Raman excitation pulses to excite the sample to produce transient stimulated Raman scattering. Subsequently, a probe pulse is used to excite the sample to produce a fluorescence signal. That is, the sample will be further excited to an excited electronic energy level by the probe pulse to produce a fluorescence signal. This fluorescence signal contains the Raman resonance frequency in the time domain.

[0073] If the relative delay between two stimulated Raman excitation pulse pairs is τ, then after the sample under test undergoes two stimulated Raman excitations with a time interval of τ, the vibrational quantum state wave packets generated by the two excitations are χ(t) and χ(t), respectively. χ(t) and The propagation time difference is τ. For each Raman mode |ν within the vibrational quantum state wave packet, i The final activation probability P i For each excitation, a coherent superposition occurs, proportional to cos(Ω). i The quantum interference beat frequency term of τ). Where, Ω i τ is the phase difference accumulated with relative delay τ, Ω i This is the resonance frequency of the Raman mode. The quantum interference beat frequency term can be mapped to the excited state of the electronic energy level by the probe pulse, which is ultimately reflected in the intensity of the readout fluorescence signal.

[0074] Here, the stimulated Raman excitation pulse pair and the probe pulse can be generated by one laser or by two different lasers, depending on the bandwidth of the lasers generated, which is not specifically limited here.

[0075] Finally, step S3 is executed to acquire the fluorescence signal and perform a Fourier transform on the fluorescence signal to obtain the Raman spectrum corresponding to the sample. This process can be achieved using a signal acquisition device, which may include a connected single-photon counter and a data acquisition card. The single-photon counter can detect and record the intensity of the fluorescence signal in the time domain; the data acquisition card can perform a Fourier transform on the time-domain fluorescence signal to obtain the Raman spectrum. The data acquisition card can be connected to a display to display the Raman spectrum.

[0076] Here, since the fluorescence signal generated by the probe pulse on the sample contains Raman resonance frequencies in the time domain, a clear vibrational spectrum without fluorescence background can be observed in the frequency domain after Fourier transform; this is the Raman spectrum, also known as transient stimulated Raman fluorescence spectrum. Furthermore, in this embodiment of the invention, the fluorescence background can be completely eliminated by windowing the time-domain fluorescence signal.

[0077] Since it uses a femtosecond pulsed laser, this transient stimulated Raman excitation fluorescence spectroscopy method employs ultrafast time-domain spectroscopy, namely transient stimulated Raman excitation fluorescence (T-SREF) spectroscopy. T-SREF spectroscopy uses the relative delay between two stimulated Raman excitation pulse pairs containing pump pulses and Stokes pulses to excite the sample to generate a fluorescence signal in the time domain that contains the Raman resonance frequency. After Fourier transform, a clear Raman spectrum without fluorescence background can be seen in the frequency domain.

[0078] The transient stimulated Raman excitation fluorescence spectroscopy method provided by this invention includes: generating two stimulated Raman excitation pulse pairs with a relatively delayed and tunable delay based on stimulated Raman excitation pulse pairs; exciting the sample to generate transient stimulated Raman scattering based on the two stimulated Raman excitation pulse pairs, and exciting the sample to generate a fluorescence signal based on a probe pulse; acquiring the fluorescence signal, and performing a Fourier transform on the fluorescence signal to obtain the Raman spectrum corresponding to the sample. By introducing two stimulated Raman excitation pulse pairs with tunable relative delays to excite transient stimulated Raman scattering in the sample, the sample can generate a fluorescence signal containing the Raman resonance frequency in the time domain when excited by the probe pulse. Therefore, after Fourier transform, a clear Raman spectrum without fluorescence background can be observed in the frequency domain, ensuring the quality of the Raman spectrum and avoiding the influence of fluorescence background on the Raman spectrum.

[0079] Based on the above embodiments, the transient stimulated Raman excitation fluorescence spectroscopy method provided in this embodiment of the invention, wherein the femtosecond pulsed laser is generated based on a femtosecond laser;

[0080] There is degeneracy information between the stimulated Raman excitation pulse pair and the detection pulse. The degeneracy information is determined based on the bandwidth of the femtosecond laser, the Raman shift of the excited Raman mode of the sample under test, and the energy level structure.

[0081] Specifically, the degeneracy information between stimulated Raman excitation pulse pairs and detector pulses can include three cases: no degeneracy between stimulated Raman excitation pulse pairs and detector pulses, degeneracy between pump pulses and detector pulses, and degeneracy between stimulated Raman excitation pulse pairs and detector pulses. Which case it is depends on the bandwidth of the femtosecond laser, the Raman shift of the Raman mode excited by the sample under test, and the energy level structure.

[0082] In this case, the stimulated Raman excitation pulse pair and the detector pulse are not degenerate, thus avoiding noise caused by degeneracy. The excitation energy level diagram of the sample under test in this situation is as follows: Figure 2As shown. Where τ2=τ, τ1 is the time delay between the probe pulse and the later stimulated Raman excitation pulse pair in the two stimulated Raman excitation pulse pairs. |e0> is the electronic ground state, and |g> is the vibrational ground state.

[0083] Pump pulse and probe pulse degeneracy refers to a situation where the pump pulse and probe pulse are the same pulse, covering the frequency components of both the pump pulse and the probe pulse, thus achieving the functions of both. In this case, the excitation energy level diagram of the sample under test is as follows: Figure 3 As shown. Where τ2=τ, τ1=0.

[0084] Stimulated Raman excitation pulse degeneracy refers to a situation where the pump pulse, Stokes pulse, and probe pulse are the same pulse. This pulse covers the frequency components of the pump pulse, Stokes pulse, and probe pulse, thus achieving the functions of a pump pulse, a Stokes pulse, and a probe pulse. In this case, the excitation energy level diagram of the sample is as follows: Figure 4 As shown.

[0085] In this embodiment of the invention, the degeneracy information between the stimulated Raman excitation pulse pair and the detector pulse can be determined based on the bandwidth of the femtosecond laser, the Raman shift of the Raman mode excited by the sample under test, and the energy level structure, which helps to simplify the structure of the femtosecond laser source.

[0086] Based on the above embodiments, the transient stimulated Raman excitation fluorescence spectroscopy method provided in this embodiment of the invention, wherein the generation of two stimulated Raman excitation pulse pairs with relative delays that are tunable, based on stimulated Raman excitation pulse pairs, includes the following prior steps:

[0087] The femtosecond pulse laser is divided into a first beam and a second beam. Dispersion compensation is performed on the first beam to obtain the Stokes pulse.

[0088] The second portion of the beam is frequency doubled to obtain a frequency-doubled laser, and an optical parametric oscillator is driven based on the frequency-doubled laser to output the pump pulse;

[0089] Align the pump pulse with the Stokes pulse in both the time and spatial dimensions.

[0090] Specifically, when generating two stimulated Raman excitation pulse pairs, the femtosecond pulse laser is first split into a first beam and a second beam. Dispersion compensation is then performed on the first beam to obtain a Stokes pulse. The femtosecond pulse laser can be split into the first beam and the second beam using beam splitting devices such as polarization beam splitters in a femtosecond laser source.

[0091] Then, dispersion compensation devices can be used to compensate for the dispersion of the first portion of the beam to obtain a Stokes pulse. A frequency doubler (SHG) can be used to double the frequency of the second portion of the beam to obtain a frequency-doubled laser.

[0092] Subsequently, a frequency-doubled laser can be used to drive an optical parametric oscillator (OPO), causing the OPO to output pump pulses.

[0093] Finally, optical delay lines can be used to align pump pulses with Stokes pulses in both time and space dimensions.

[0094] In this embodiment of the invention, by splitting the femtosecond pulsed laser to generate pump pulses and Stokes pulses respectively, a light source can be provided for the subsequent excitation of the sample under test to generate transient stimulated Raman scattering, avoiding interference caused by pump pulses and Stokes pulses generated by different light sources.

[0095] Based on the above embodiments, the transient stimulated Raman excitation fluorescence spectroscopy method provided in this embodiment of the invention, wherein the step of performing a Fourier transform on the fluorescence signal to obtain the Raman spectrum corresponding to the sample to be tested includes:

[0096] Filter out background light of wavelengths other than the fluorescence signal and select the fluorescent photons at the focal position in the fluorescence signal;

[0097] The time-domain fluorescence signal corresponding to the fluorescent photon is recorded, and the time-domain fluorescence signal is subjected to Fourier transform to obtain the Raman spectrum.

[0098] Specifically, in the process of obtaining the Raman spectrum corresponding to the sample to be tested, the background light of wavelengths other than the fluorescence signal can be filtered out first, and the fluorescent photons at the focal position in the fluorescence signal can be selected, which can improve the signal-to-noise ratio of the Raman spectrum.

[0099] Subsequently, the time-domain fluorescence signal corresponding to the fluorescent photon was recorded, and the time-domain fluorescence signal was Fourier transformed to obtain a clear Raman spectrum without fluorescence background in the frequency domain.

[0100] Based on the above embodiments, the transient stimulated Raman excitation fluorescence spectroscopy method provided in this embodiment of the invention, wherein the step of exciting the sample to be tested to generate transient stimulated Raman scattering based on two stimulated Raman excitation pulse pairs, includes the following prior steps:

[0101] The bandwidth of the Stokes pulse is adjusted so that the bandwidth difference between the Stokes pulse and the pump pulse is within a preset range.

[0102] Specifically, after generating the Stokes pulse, a spectral filtering device can be used to adjust the bandwidth of the Stokes pulse so that the bandwidth difference between the Stokes pulse and the pump pulse is within a preset range, so that the bandwidth of the Stokes pulse and the pump pulse are the same or close, thereby improving the signal-to-noise ratio of the fluorescence signal.

[0103] Figure 5 This is a schematic diagram of the transient stimulated Raman excitation fluorescence spectroscopy system provided in an embodiment of the present invention, as shown below. Figure 5 As shown, the transient stimulated Raman excitation fluorescence spectroscopy system includes a femtosecond laser source 1, a time-delay scanning device 2, and a signal acquisition device 3;

[0104] The femtosecond laser source 1 is used to generate stimulated Raman excitation pulse pairs and probe pulses. The stimulated Raman excitation pulse pairs include pump pulses and Stokes pulses that are aligned in both time and space dimensions.

[0105] The time-delay scanning device 2 is used to generate two stimulated Raman excitation pulse pairs with relative delay and adjustable relative delay based on the stimulated Raman excitation pulse pairs. The two stimulated Raman excitation pulse pairs are used to excite the test sample 4 to generate transient stimulated Raman scattering, so that the test sample 4 is excited by the detection pulse to generate a fluorescence signal.

[0106] The signal acquisition device 3 is used to acquire the fluorescence signal and perform Fourier transform on the fluorescence signal to obtain the Raman spectrum corresponding to the sample 4 to be tested.

[0107] Specifically, the transient stimulated Raman excitation fluorescence spectroscopy system provided in this embodiment of the invention uses a femtosecond laser source to generate stimulated Raman excitation pulse pairs and probe pulses. The stimulated Raman excitation pulse pairs may include pump pulses and Stokes pulses aligned in both time and space dimensions.

[0108] The stimulated Raman excitation pulse pair and the probe pulse can be generated by one laser or by two different lasers, depending on the bandwidth of the lasers generated, which is not specifically limited here.

[0109] The stimulated Raman excitation pulse pair, consisting of a pump pulse and a Stokes pulse, is used to pump the molecules of the analyte sample to a vibrational excited state located in the electronic ground state. The pump pulse can be a femtosecond pulse with a tunable wavelength in the range of 800 nm to 900 nm. The Stokes pulse can have a wavelength range of 1020 nm to 1050 nm. The probe pulse is used to excite the molecules of the analyte sample from a vibrational excited state to an electronic excited state. Its photon energy can be the energy of the molecule's first electronic excited state minus the energy of the vibrational excited state of the electronic ground state.

[0110] The delay scanning device 2 can generate two stimulated Raman excitation pulse pairs with a relative delay that is adjustable, using stimulated Raman excitation pulse pairs. The delay scanning device 2 can be an interferometer, which may include two arms, a first arm and a second arm, each arm producing one stimulated Raman excitation pulse pair. By introducing an optical delay line in the first or second arm, a relative delay can be created between the two stimulated Raman excitation pulse pairs, and this relative delay can be adjusted by controlling the optical delay line.

[0111] The sample 4 to be tested can be fixed on a stage or placed in a cuvette. The sample 4 can be positioned in the transmission optical path of two stimulated Raman excitation pulse pairs and a probe pulse. The two stimulated Raman excitation pulse pairs excite the sample 4 to produce transient stimulated Raman scattering. The sample is further excited to an excited electronic energy level by the probe pulse, generating a fluorescence signal. This fluorescence signal contains a Raman resonance frequency in the time domain.

[0112] In this embodiment of the invention, the relative delay τ between two stimulated Raman excitation pulse pairs, i.e., the delay scanning range of the delay scanning device 2, can be set to be greater than 5 ps. This ensures that the resolution of the subsequently determined Raman spectrum is higher than 8 cm⁻¹. -1 This resolution is 1.2 / 5 ps under the box-car apodization function, which is higher than the linewidth of a typical Raman mode. The scan step size of the delay scan can be set to less than 1 fs, which can capture the fluorescence intensity oscillations caused by the transient absorption of the second harmonic of the laser wavelength, i.e., the transient absorption spectra of single-photon and multi-photon signals.

[0113] The transient stimulated Raman excitation fluorescence spectroscopy system also includes a signal acquisition device, which may include a connected single-photon counter and a data acquisition card. The single-photon counter can detect and record the intensity of the fluorescence signal in the time domain; the data acquisition card can perform a Fourier transform on the time-domain fluorescence signal to obtain the Raman spectrum. The data acquisition card can be connected to a display to display the Raman spectrum.

[0114] Here, since the fluorescence signal generated by the probe pulse on the sample contains Raman resonance frequencies in the time domain, a clear vibrational spectrum without fluorescence background can be observed in the frequency domain after Fourier transform; this is the Raman spectrum, also known as transient stimulated Raman fluorescence spectrum. Furthermore, in this embodiment of the invention, the fluorescence background can be completely eliminated by windowing the time-domain fluorescence signal.

[0115] The transient stimulated Raman excitation fluorescence spectroscopy system provided in this embodiment of the invention includes: a femtosecond laser source, a time-delay scanning device, and a signal acquisition device; the femtosecond laser source is used to generate stimulated Raman excitation pulse pairs and a probe pulse, the stimulated Raman excitation pulse pairs including a pump pulse and a Stokes pulse aligned in the time and spatial dimensions; the time-delay scanning device is used to generate two stimulated Raman excitation pulse pairs with a relative delay that is adjustable, based on the stimulated Raman excitation pulse pairs, the two stimulated Raman excitation pulse pairs are used to excite the sample to generate transient stimulated Raman scattering, so that the sample to be tested is excited by the probe pulse to generate a fluorescence signal; the signal acquisition device is used to acquire the fluorescence signal and perform a Fourier transform on the fluorescence signal to obtain the Raman spectrum corresponding to the sample to be tested. By introducing two stimulated Raman excitation pulse pairs with adjustable relative delays, the sample under test is excited to produce transient stimulated Raman scattering. This allows the sample to generate a fluorescence signal in the time domain containing the Raman resonance frequency when excited by the probe pulse. After Fourier transform, a clear Raman spectrum without fluorescence background can be seen in the frequency domain, which can ensure the quality of the Raman spectrum and avoid the influence of fluorescence background on the Raman spectrum.

[0116] Based on the above embodiments, the transient stimulated Raman excitation fluorescence spectroscopy system provided in this embodiment of the invention includes a femtosecond laser source comprising a femtosecond laser, which is used to generate the stimulated Raman excitation pulse pair; the degeneracy information between the stimulated Raman excitation pulse pair and the detection pulse is determined based on the bandwidth of the femtosecond laser, the Raman shift of the excited Raman mode of the sample under test, and the energy level structure.

[0117] Specifically, in this embodiment of the invention, the femtosecond laser source includes a femtosecond laser, such as a femtosecond fiber laser, which can be a high-power ytterbium-doped femtosecond fiber laser. This femtosecond laser is used to generate stimulated Raman excitation pulse pairs. When generating stimulated Raman excitation pulse pairs, the femtosecond laser can be combined with auxiliary structures. Here, the auxiliary structures can be selected as needed, and no specific limitations are imposed.

[0118] Depending on the bandwidth of the femtosecond laser, the Raman shift of the excited Raman mode of the sample under test, and the energy level structure, there may or may not be degeneracy between the stimulated Raman excitation pulse pair and the detector pulse. That is, the degeneracy information between the stimulated Raman excitation pulse pair and the detector pulse can include three cases: no degeneracy between the stimulated Raman excitation pulse pair and the detector pulse, degeneracy between the pump pulse and the detector pulse, and degeneracy between the stimulated Raman excitation pulse pair and the detector pulse.

[0119] In this case, the stimulated Raman excitation pulse pair and the probe pulse are not degenerate, thus avoiding noise caused by degeneracy. The femtosecond laser source also includes a probe laser for generating the probe pulse. The excitation energy level diagram of the sample under test in this situation is as follows: Figure 2 As shown.

[0120] When the pump pulse and probe pulse are degenerate, a laser for generating the probe pulse is not required, simplifying the structure of the femtosecond laser source. However, this introduces noise due to degeneracy. The excitation energy level diagram of the sample under test in this case is as follows: Figure 3 As shown.

[0121] When the stimulated Raman excitation pulse pair is degenerate with the probe pulse, the excitation energy level diagram of the sample under test is as follows: Figure 4 As shown. At this point, only a femtosecond laser is needed, and no auxiliary structure is required, which can further simplify the structure of the femtosecond laser source, but it will also increase the noise caused by degeneracy.

[0122] Furthermore, the bandwidth of a femtosecond laser is maximized when the stimulated Raman excitation pulse pair and the probe pulse are degenerate. That is, only when the femtosecond laser is an ultrafast femtosecond laser with an ultra-wide bandwidth can it completely cover the frequency components of the pump pulse, the Stokes pulse, and the probe pulse, thus achieving degeneracy of the stimulated Raman excitation pulse pair and the probe pulse.

[0123] Because existing SREF spectroscopy techniques use narrowband picosecond laser pulses for single-frequency point excitation, they cannot excite multiple Raman labels simultaneously, resulting in limited throughput.

[0124] Based on this, and building upon the above embodiments, the transient stimulated Raman excitation fluorescence spectroscopy system provided in this embodiment of the invention has no degeneracy between the stimulated Raman excitation pulse pair and the detector pulse, or the pump pulse and the detector pulse are degenerate; correspondingly,

[0125] The femtosecond laser source includes a femtosecond laser, a polarization beam splitter, a first dichroic mirror, a first light source branch, and a second light source branch; the femtosecond laser is used to generate femtosecond pulsed laser; the polarization beam splitter is used to split the femtosecond pulsed laser into a first beam and a second beam.

[0126] The first light source branch includes a first dispersion compensation device, which is used to perform dispersion compensation on the first part of the light beam to obtain the Stokes pulse;

[0127] The second light source branch includes a frequency doubler, an optical parametric oscillator, a second dispersion compensation device, and an adjustable first optical delay line; the frequency doubler is used to double the frequency of the second portion of the beam to obtain a frequency-doubled laser; the optical parametric oscillator is driven by the frequency-doubled laser and outputs the pump pulse; the first optical delay line is used to align the pump pulse with the Stokes pulse in the time dimension; the second dispersion compensation device is used to perform dispersion compensation on the pump pulse;

[0128] The first dichroic mirror is used to align the pump pulse with the Stokes pulse in spatial dimensions.

[0129] Specifically, in cases where there is no degeneracy between the stimulated Raman excitation pulse pair and the probe pulse, or where the pump pulse and the probe pulse are degenerate, such as Figure 6 As shown, each femtosecond laser source includes a femtosecond laser 11, a polarizing beam splitter 12, a first dichroic mirror 13, a first light source branch, and a second light source branch.

[0130] The parameters of the femtosecond laser 11 may include: a center wavelength of 1030 nm, a repetition rate of 100 MHz, and a pulse width of 80 fs. The femtosecond laser 11 is used to generate femtosecond pulsed lasers.

[0131] The polarization beam splitter 12 can split a femtosecond pulse laser into a first part of the beam with a first polarization state and a second part of the beam with a second polarization state.

[0132] The first light source branch includes a first dispersion compensation device 141, which can perform dispersion compensation on the first part of the beam so that the pulse width of the first part of the beam is close to the Fourier transform limit, thereby obtaining a Stokes pulse.

[0133] To shorten the optical path length and reduce the size of the transient stimulated Raman excitation fluorescence spectroscopy system, a first reflecting mirror 142 can be introduced into the first light source branch. The height of the first reflecting mirror 142 can be lower than the transmission optical path of the first part of the beam, so that the first part of the beam can be transmitted to the first dispersion compensation device 141 above the first reflecting mirror.

[0134] The first dispersion compensation device 141 may include two prisms and a reflector. The first portion of the light beam passes through the prisms in sequence and is then reflected by the reflector. The reflected beam then passes through the two prisms in sequence and is reflected by the first reflector 142 to obtain a Stokes pulse beam. The wavelength range of the Stokes pulse may be 1020nm-1050nm.

[0135] The second light source branch includes a frequency multiplier 151, an optical parametric oscillator 152, a second dispersion compensation device 153, and an adjustable first optical delay line (ODL) 154.

[0136] Frequency doubler 151 doubles the frequency of the second portion of the beam, increasing its frequency to twice its original value, thus obtaining a frequency-doubled laser. Optical parametric oscillator 152, driven by the frequency-doubled laser, outputs a pump pulse. The wavelength of this pump pulse can be adjusted within the range of 800nm-900nm.

[0137] The first optical delay line 154 can control the transmission delay of the pump pulse, thereby aligning the pump pulse with the Stokes pulse in the time dimension.

[0138] To shorten the optical path length and reduce the size of the transient stimulated Raman excitation fluorescence spectroscopy system, a second reflector 155 can be introduced into the second light source branch. The height of the second reflector 155 can be lower than the transmission optical path of the pump pulse, so that the pump pulse can be transmitted to the second dispersion compensation device 153 above the second reflector 155.

[0139] The second dispersion compensation device 153 may also include two prisms and a reflector. The pump pulse beam passes through the prisms in sequence and is reflected by the reflector. The reflected beam passes through the prisms in sequence and is reflected by the second reflector 155 to obtain the dispersion-compensated pump pulse beam. The pulse width of the pump pulse is close to the Fourier transform limit.

[0140] One side of the first dichroic mirror 13 receives and reflects the beam of the Stokes pulse, while the other side receives and transmits the beam of the pump pulse, so that the pump pulse and the Stokes pulse are combined through the wavelength difference, thereby aligning the two in spatial dimensions.

[0141] In this embodiment of the invention, a first light source branch and a second light source branch are introduced into the femtosecond laser light source, which can generate pump pulses and Stokes pulses respectively. The pump pulses and Stokes pulses are aligned in the time and space dimensions through the first optical delay line and the first dichroic mirror, providing a light source for the subsequent excitation of the sample under test to generate transient stimulated Raman scattering.

[0142] Due to the working principle of femtosecond lasers, the bandwidth of the pump pulse generated by the first light source branch and the Stokes pulse generated by the second light source branch may be significantly different, which will result in a low signal-to-noise ratio of the subsequently generated fluorescence signal.

[0143] Based on this, and building upon the above embodiments, the transient stimulated Raman excitation fluorescence spectroscopy system provided in this embodiment of the invention further includes a spectral filtering device in the first light source branch.

[0144] The spectral filtering device is used to adjust the bandwidth of the Stokes pulse so that the bandwidth difference between the Stokes pulse and the pump pulse is within a preset range.

[0145] Specifically, such as Figure 7 As shown, the first light source branch also includes a spectral filtering device 143, which can adjust the bandwidth of the Stokes pulse so that the bandwidth difference between the Stokes pulse and the pump pulse is within a preset range, even if the bandwidths of the Stokes pulse and the pump pulse are the same or close.

[0146] Here, the spectral filtering device 143 can be a 4f optical system, which may include a first grating 1431, a first lens 1432, a first slit 1433, and a mirror.

[0147] To shorten the optical path length and reduce the size of the transient stimulated Raman excitation fluorescence spectroscopy system, a third mirror 144 and a fourth mirror 145 can be introduced into the first light source branch. The height of the third mirror 144 can be lower than the transmission optical path of the Stokes pulse, so that the Stokes pulse can be transmitted to the spectral filter device 143 above the third mirror 144.

[0148] The Stokes pulse beam passes sequentially through the first grating 1431, the first lens 1432, and the first slit 1433. After being reflected by the mirror, it passes sequentially through the first slit 1433, the first lens 1432, and the first grating 1431 to output the spectrum filter device 143.

[0149] The beam of Stokes pulse output by the spectral filter 143 is reflected by the third mirror 144 and then by the fourth mirror 145 on one side of the first dichroic mirror 13.

[0150] Based on the above embodiments, the transient stimulated Raman excitation fluorescence spectroscopy system provided in this embodiment of the invention has no degeneracy between the stimulated Raman excitation pulse pair and the detection pulse; correspondingly,

[0151] The femtosecond laser source also includes a probe laser and a second optical delay line and a second dichroic mirror arranged along the optical path;

[0152] The detection laser is used to generate the detection pulse;

[0153] The second optical delay line is used to control the delay between the probe pulse and the target pulse pair, wherein the target pulse pair is the later stimulated Raman excitation pulse pair among the two stimulated Raman excitation pulse pairs;

[0154] The second dichroic mirror is used to align the two stimulated Raman excitation pulse pairs with the detection pulse in spatial dimension.

[0155] Specifically, such as Figure 8 As shown, in the absence of degeneracy between the stimulated Raman excitation pulse pair and the probe pulse, the femtosecond laser source also includes a probe laser 16, a second optical delay line 17 and a second dichroic mirror 18 arranged along the optical path.

[0156] The probe laser is used to generate probe pulses. The type of probe laser can be selected as needed, and there is no specific limitation here, as long as it can generate probe pulses.

[0157] The second optical delay line 17 controls the delay τ1 between the probe pulse and the target pulse pair, specifically the delay between the probe pulse and the later stimulated Raman excitation pulse pair in the two stimulated Raman excitation pulse pairs. The delay τ1 can range from 0 to 5 ps and is used to detect the lifetime of the electronic energy levels in the sample. A larger delay τ1 results in a weaker fluorescence signal, while a delay τ1 of 0 results in the strongest fluorescence signal.

[0158] The second dichroic mirror 18 is used to align the two stimulated Raman excitation pulse pairs with the probe pulse in the spatial dimension, that is, to combine the beams by utilizing the wavelength difference.

[0159] Based on the above embodiments, the transient stimulated Raman excitation fluorescence spectroscopy system provided in this embodiment of the invention includes an interferometer in which one arm of the interferometer includes an adjustable third optical delay line.

[0160] Specifically, the time-delay scanning device can be implemented using an interferometer, which can be a Mach-Zehnder interferometer, a Michelson interferometer, etc., without specific limitations here. The interferometer may include two arms, each used to output a pair of stimulated Raman excitation pulses. One of the two arms of the interferometer includes an adjustable third optical delay line, used to control the magnitude of the relative delay between the two pairs of stimulated Raman excitation pulses output by the interferometer.

[0161] Based on the above embodiments, the transient stimulated Raman excitation fluorescence spectroscopy system provided in this embodiment of the invention has the third optical delay line fixed on the displacement stage;

[0162] The signal acquisition device is triggered based on the position information of the displacement stage.

[0163] Specifically, the third optical delay line can be fixed on the displacement stage. The movement of the displacement stage drives the movement of the third optical delay line, thereby achieving control over the relative delay between the two stimulated Raman excitation pulse pairs.

[0164] The signal acquisition device can be connected to the time-delay scanning device, specifically to the displacement stage in the time-delay scanning device. When the position information of the displacement stage changes, the signal acquisition device is triggered to acquire the fluorescence signal and perform a Fourier transform on it. This enables the synchronous acquisition of the relative delay of the two stimulated Raman excitation pulse pairs and the fluorescence signal intensity, improving the accuracy and precision of the signal and Raman spectrum.

[0165] Since the movement of the translation stage is not completely uniform, this will lead to deviations in the control of relative delay, thereby affecting the accurate acquisition of fluorescence signals and the accuracy of Raman spectroscopy. Based on this, the transient stimulated Raman excitation fluorescence spectroscopy system provided in this embodiment of the invention further includes a first beam splitter, a second beam splitter, and a reference signal detection device.

[0166] The first beam splitter is used to divide the target beam of the stimulated Raman excitation pulse pair generated by the femtosecond laser source into a third beam and a fourth beam. The third beam is transmitted along the first arm of the interferometer, and the fourth beam is transmitted along the second arm of the interferometer.

[0167] The second beam splitter is used to combine the beam output from the first arm and the beam output from the second arm to obtain a fifth beam and a sixth beam, respectively. The fifth beam is used to excite the sample under test to generate transient stimulated Raman scattering and the fluorescence signal.

[0168] The reference signal detection device is used to receive the sixth part of the beam and convert the sixth part of the beam into a reference signal with a coherence time greater than a preset threshold. The reference signal is used to calibrate the position information of the displacement stage.

[0169] Specifically, such as Figure 9 As shown, the interferometer also includes a first beam splitter 21, a second beam splitter 22, and a reference signal detection device. Both the first beam splitter 21 and the second beam splitter 22 can be semi-transparent and semi-reflective beam splitters with a transmittance of around 50%.

[0170] The first beam splitter 21 is used to divide the target beam of the stimulated Raman excitation pulse pair generated by the femtosecond laser source into a third beam and a fourth beam. The third beam is transmitted along the first arm of the interferometer, and the fourth beam is transmitted along the second arm of the interferometer. Figure 8 In the interferometer, the first arm has an adjustable third optical delay line 23, and the second arm has a fixed fourth optical delay line 24.

[0171] The second beam splitter 22 is used to combine the beam output from the first arm and the beam output from the second arm to obtain the fifth beam and the sixth beam, respectively. The fifth beam can be incident on the sample 4 under test through the reflector.

[0172] Figure 9 In order to shorten the optical path length and reduce the size of the transient stimulated Raman excitation fluorescence spectroscopy system, a third dichroic mirror 5 and a fifth reflecting mirror 6 are introduced into the transient stimulated Raman excitation fluorescence spectroscopy system. The fifth part of the beam is reflected by the third dichroic mirror 5 and then by the fifth reflecting mirror 6. After being expanded by two lenses, it is reflected by a reflecting mirror to the objective lens 7. Through the objective lens 7, two stimulated Raman excitation pulse pairs and a detection pulse are incident on the sample 4 to be tested on the stage and excited the sample 4.

[0173] Subsequently, the fluorescence signal generated by the detection pulse on the sample 4 is also received by the objective lens 7 and collected by the signal acquisition device 3 through the third dichroic mirror 5.

[0174] The reference signal detection device can receive the sixth portion of the beam and convert it into a reference signal with a coherence time greater than a preset threshold. This preset threshold can be set as needed, for example, to 10 ps. This reference signal is a narrowband, highly coherent light. The position information of the translation stage can be calibrated using the interference fringes of the reference signal.

[0175] In this embodiment of the invention, the introduction of a reference signal detection device can avoid deviations in the control of relative delay caused by the non-uniform movement of the translation stage, thereby enabling accurate acquisition of fluorescence signals and improving the accuracy of Raman spectroscopy.

[0176] Based on the above embodiments, the transient stimulated Raman excitation fluorescence spectroscopy system provided in this embodiment of the invention may include a reference signal detection device comprising a grating, a lens, a slit, and a photodiode.

[0177] The sixth beam passes sequentially through the grating, the lens, and the slit to obtain the reference signal;

[0178] The photodiode is used to detect the reference signal.

[0179] Specifically, such as Figure 9 As shown, in order to distinguish the grating, lens and slit in the reference signal detection device from the spectral filtering device 143, the grating in the reference signal detection device is referred to as the second grating 231, the lens as the second lens 232, and the slit as the second slit 233.

[0180] The sixth beam passes sequentially through the second beam 231, the second lens 232, and the second slit 233 to achieve spectral filtering, thereby obtaining a reference signal. This reference signal can be detected by a photodiode (PD) 234.

[0181] Based on the above embodiments, the transient stimulated Raman excitation fluorescence spectroscopy system provided in this embodiment of the invention includes a signal acquisition device comprising a filter, a screening element, a photodetector, and a data acquisition card arranged along the optical path.

[0182] The filter is used to filter out background light of wavelengths other than the fluorescence signal;

[0183] The filtering element is used to select fluorescent photons that are located at the focal position in the fluorescence signal;

[0184] The photodetector is used to record the time-domain fluorescence signal corresponding to the fluorescent photon;

[0185] The data acquisition card is used to perform Fourier transform on the time-domain fluorescence signal to obtain the Raman spectrum.

[0186] Specifically, the screening element may include a confocal aperture and / or multimode fiber, and the photodetector may include a single-photon counter or other photodetectors. For example... Figure 9 As shown, the signal acquisition device 3 may include a filter 31, a screening element 32, a photodetector 33, and a data acquisition card 34 arranged along the optical path. The filter 31 can be used to filter out noise signals in the fluorescence signal, and the screening element 32 can be used to select fluorescent photons located at the focal position in the fluorescence signal. In this embodiment of the invention, the introduction of the filter and the screening element can improve the signal-to-noise ratio of the Raman spectrum.

[0187] The photodetector 33 can have high quantum efficiency and is used to record the time-domain fluorescence signal corresponding to the fluorescent photon. The data acquisition card 34 can be used to perform Fourier transform on the time-domain fluorescence signal to obtain the Raman spectrum.

[0188] In summary, the transient stimulated Raman excitation fluorescence spectroscopy system provided in this embodiment of the invention, using T-SREF spectroscopy, can achieve a detection sensitivity of no less than 40 nM for the Raman mode of the sample to be tested. In addition to acquiring Raman spectra, T-SREF spectroscopy can also observe a large number of transient spectra with multiphoton absorption. Compared to traditional SREF spectroscopy, this transient stimulated Raman excitation fluorescence spectroscopy system, while maintaining the same sensitivity, can eliminate the inherent fluorescence background and achieve high-spectral data acquisition covering hundreds of wavenumbers in a single excitation, thus solving the main technical bottlenecks faced by traditional SREF spectroscopy in one fell swoop.

[0189] T-SREF spectroscopy can be used in many fields, including ultra-multicolor multiplexed fluorescence microscopy, spatial transcriptional imaging, spatial protein compositional imaging, transient Raman spectroscopy imaging, and electric field sensing imaging. This T-SREF spectroscopy technique can simultaneously acquire Raman spectra as well as single-photon and multi-photon transient absorption spectra.

[0190] Figure 10 A complete technology roadmap for T-SREF spectroscopy, such as Figure 10 As shown, in the absence of degeneracy between the stimulated Raman excitation pulse pair and the detector pulse, the pump pulse and Stokes pulse, after being aligned in both time and spatial dimensions, generate two stimulated Raman excitation pulse pairs with adjustable relative delays via a time-delay scanning device. On one hand, the relative delay generated by the time-delay scanning device is triggered synchronously with the acquisition action of the signal acquisition device; on the other hand, the detector pulse has a time delay relative to the later stimulated Raman excitation pulse pair generated by the time-delay scanning device, but they are aligned in the spatial dimension. Finally, the sample under test is excited using a beam containing the pump pulse, Stokes pulse, and detector pulse. The fluorescence signal generated by the sample is acquired using the signal acquisition device, and a Fourier transform is performed on the fluorescence signal to obtain the Raman spectrum.

[0191] Taking the ATTO740 dye as an example, the ATTO740 dye molecule is excited by two stimulated Raman excitation pulses, producing transient stimulated Raman scattering, and is excited by a probe pulse to produce a time-domain fluorescence signal in the C=C backbone mode, as shown below. Figure 11 As shown. Figure 11 Taking a rectangular window width of 4.5 ps as an example, the width of the rectangular window is the relative delay τ between two stimulated Raman excitation pulse pairs.

[0192] Figure 12 For signal acquisition devices Figure 11 The spectrum obtained by performing Fourier transform on the time-domain fluorescence signal. Figure 12 Middle, 1642cm -1 The spectral line at 1642 cm⁻¹ is the Raman spectrum of the C=C skeleton mode of the ATTO740 dye molecule. -1 Other spectral lines are the transient absorption spectra of the electronic energy levels of captured single-photon and multi-photon electrons.

[0193] The device embodiments described above are merely illustrative. The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the modules can be selected to achieve the purpose of this embodiment according to actual needs. Those skilled in the art can understand and implement this without any creative effort.

[0194] Through the above description of the embodiments, those skilled in the art can clearly understand that each embodiment can be implemented by means of software plus necessary general-purpose hardware platforms, and of course, it can also be implemented by hardware. Based on this understanding, the above technical solutions, in essence or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product can be stored in a computer-readable storage medium, such as ROM / RAM, magnetic disk, optical disk, etc., and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute the methods described in the various embodiments or some parts of the embodiments.

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A transient stimulated Raman excitation fluorescence spectroscopy method, characterized in that, include: Based on the stimulated Raman excitation pulse pair, two stimulated Raman excitation pulse pairs with relative delay and adjustable relative delay are generated by a time delay scanning device; The stimulated Raman excitation pulse pair includes pump pulses and Stokes pulses that are aligned in the time and space dimensions based on femtosecond pulsed lasers. Based on the two pairs of stimulated Raman excitation pulses, the sample under test is excited to generate transient stimulated Raman scattering, and based on the probe pulse, the sample under test is excited to generate a fluorescence signal. The fluorescence signal is acquired, and the fluorescence signal is subjected to Fourier transform to obtain the Raman spectrum corresponding to the sample to be tested.

2. The transient stimulated Raman excitation fluorescence spectroscopy method according to claim 1, characterized in that, The femtosecond pulsed laser is generated based on a femtosecond laser; There is degeneracy information between the stimulated Raman excitation pulse pair and the detection pulse. The degeneracy information is determined based on the bandwidth of the femtosecond laser, the Raman shift of the excited Raman mode of the sample under test, and the energy level structure.

3. The transient stimulated Raman excitation fluorescence spectroscopy method according to claim 1, characterized in that, The step of performing a Fourier transform on the fluorescence signal to obtain the Raman spectrum corresponding to the sample to be tested includes: Filter out background light of wavelengths other than the fluorescence signal and select the fluorescent photons at the focal position in the fluorescence signal; The time-domain fluorescence signal corresponding to the fluorescent photon is recorded, and the time-domain fluorescence signal is subjected to Fourier transform to obtain the Raman spectrum.

4. The transient stimulated Raman excitation fluorescence spectroscopy method according to any one of claims 1-3, characterized in that, The process of exciting the sample to produce transient stimulated Raman scattering based on the two stimulated Raman excitation pulse pairs includes the following prior steps: The bandwidth of the Stokes pulse is adjusted so that the bandwidth difference between the Stokes pulse and the pump pulse is within a preset range.

5. A transient stimulated Raman excitation fluorescence spectroscopy system, characterized in that, include: Femtosecond laser source, time-delay scanning device, and signal acquisition device; The femtosecond laser source is used to generate stimulated Raman excitation pulse pairs and probe pulses. The stimulated Raman excitation pulse pairs include pump pulses and Stokes pulses that are aligned in the time and space dimensions based on femtosecond pulsed lasers. The time-delay scanning device is used to generate two stimulated Raman excitation pulse pairs with a relative delay and an adjustable relative delay based on the stimulated Raman excitation pulse pair. The two stimulated Raman excitation pulse pairs are used to excite the sample to generate transient stimulated Raman scattering, so that the sample to be tested is excited by the probe pulse to generate a fluorescence signal. The signal acquisition device is used to acquire the fluorescence signal and perform a Fourier transform on the fluorescence signal to obtain the Raman spectrum corresponding to the sample to be tested.

6. The transient stimulated Raman excitation fluorescence spectroscopy system according to claim 5, characterized in that, The femtosecond laser source includes a femtosecond laser, which is used to generate the femtosecond pulsed laser. The degeneracy information between the stimulated Raman excitation pulse pair and the detection pulse is determined based on the bandwidth of the femtosecond laser, the Raman shift of the excited Raman mode of the sample under test, and the energy level structure.

7. The transient stimulated Raman excitation fluorescence spectroscopy system according to claim 5, characterized in that, There is no degeneracy between the stimulated Raman excitation pulse pair and the probe pulse, or the pump pulse and the probe pulse are degenerate; accordingly, The femtosecond laser source includes a femtosecond laser, a polarization beam splitter, a first dichroic mirror, a first light source branch, and a second light source branch; the femtosecond laser is used to generate the femtosecond pulsed laser; the polarization beam splitter is used to split the femtosecond pulsed laser into a first beam and a second beam. The first light source branch includes a first dispersion compensation device, which is used to perform dispersion compensation on the first part of the beam to obtain the Stokes pulse; The second light source branch includes a frequency multiplier, an optical parametric oscillator, a second dispersion compensation device, and an adjustable first optical delay line; The frequency doubler is used to double the frequency of the second part of the beam to obtain a frequency-doubled laser; the optical parametric oscillator is driven by the frequency-doubled laser and outputs the pump pulse; The first optical delay line is used to align the pump pulse with the Stokes pulse in the time dimension; the second dispersion compensation device is used to perform dispersion compensation on the pump pulse. The first dichroic mirror is used to align the pump pulse with the Stokes pulse in spatial dimensions.

8. The transient stimulated Raman excitation fluorescence spectroscopy system according to claim 7, characterized in that, The first light source branch also includes a spectral filtering device; The spectral filtering device is used to adjust the bandwidth of the Stokes pulse so that the bandwidth difference between the Stokes pulse and the pump pulse is within a preset range.

9. The transient stimulated Raman excitation fluorescence spectroscopy system according to claim 7, characterized in that, There is no degeneracy between the stimulated Raman excitation pulse pair and the detection pulse; correspondingly, The femtosecond laser source also includes a probe laser and a second optical delay line and a second dichroic mirror arranged along the optical path; The detection laser is used to generate the detection pulse; The second optical delay line is used to control the delay between the probe pulse and the target pulse pair, wherein the target pulse pair is the later stimulated Raman excitation pulse pair among the two stimulated Raman excitation pulse pairs; The second dichroic mirror is used to align the two stimulated Raman excitation pulse pairs with the detection pulse in spatial dimension.

10. The transient stimulated Raman excitation fluorescence spectroscopy system according to any one of claims 5-9, characterized in that, The time-delay scanning device includes an interferometer, one arm of which includes an adjustable third optical delay line.

11. The transient stimulated Raman excitation fluorescence spectroscopy system according to claim 10, characterized in that, The third optical delay line is fixed on the displacement stage; The signal acquisition device is triggered based on the position information of the displacement stage.

12. The transient stimulated Raman excitation fluorescence spectroscopy system according to claim 11, characterized in that, The interferometer also includes a first beam splitter, a second beam splitter, and a reference signal detection device; The first beam splitter is used to divide the target beam of the stimulated Raman excitation pulse pair generated by the femtosecond laser source into a third beam and a fourth beam. The third beam is transmitted along the first arm of the interferometer, and the fourth beam is transmitted along the second arm of the interferometer. The second beam splitter is used to combine the beam output from the first arm and the beam output from the second arm to obtain a fifth beam and a sixth beam, respectively. The fifth beam is used to excite the sample under test to generate transient stimulated Raman scattering and the fluorescence signal. The reference signal detection device is used to receive the sixth part of the beam and convert the sixth part of the beam into a reference signal with a coherence time greater than a preset threshold. The reference signal is used to calibrate the position information of the displacement stage.

13. The transient stimulated Raman excitation fluorescence spectroscopy system according to claim 12, characterized in that, The reference signal detection device includes a grating, a lens, a slit, and a photodiode; The sixth beam passes sequentially through the grating, the lens, and the slit to obtain the reference signal; The photodiode is used to detect the reference signal.

14. The transient stimulated Raman excitation fluorescence spectroscopy system according to any one of claims 5-9, characterized in that, The signal acquisition device includes a filter, a screening element, a photodetector, and a data acquisition card arranged along the optical path; The filter is used to filter out background light of wavelengths other than the fluorescence signal; The filtering element is used to select fluorescent photons that are located at the focal position in the fluorescence signal; The photodetector is used to record the time-domain fluorescence signal corresponding to the fluorescent photon; The data acquisition card is used to perform Fourier transform on the time-domain fluorescence signal to obtain the Raman spectrum.

Images